Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR TREATING FRONTOTEMPORAL
DEMENTIA
RELATED APPLICATIONS
This Application claims the benefit of U.S. Provisional Application
62/364,013, filed
July 19th, 2016, the entire contents of which are incorporated herein by
reference.
BACKGROUND
Neurodegenerative diseases are debilitating disorders of the nervous system
that
affect approximately 30 million individuals worldwide. Neurodegenerative
diseases are
challenging to treat and are also a growing health concern, both in terms of
mortality and the
cost of care for the afflicted. The nervous system is a fragile element of the
body and has a
limited capacity to regenerate from both acute injuries, such as stroke and
spinal cord injury,
or degenerative diseases. Neurodegenerative diseases can be characterized by
progressive
loss of neuronal subtypes in the brain and spinal cord and may be either
sporadic or familial.
Symptoms of neurodegenerative diseases commonly appear during middle or old
age. Given
the increasing life expectancy of the population, the incidence of these
diseases will increase.
New therapies are needed to treat neurodegenerative diseases.
Frontotemporal dementia (FTD) is a neurodegenerative disorder characterized by
progressive deficits in either behavior and personality changes or language
disturbance. FTD
is an umbrella term for a broad spectrum of diseases including Progressive
Supranuclear
Palsy, Corticobasal Degeneration, and Amyotrophic Lateral Sclerosis (ALS). FTD
is often
misdiagnosed in the early stage, either as a psychiatric disorder, or as a
different type of
dementia such as Alzheimer's disease (AD). Because of the close similarity of
behavioral
changes in patients with frontotemporal dementia to those seen in patients
with psychiatric
disorders, diagnosis is challenging. Various underlying neuropathological
changes lead to
the FTD phenotype, all of which are characterized by selective degradation of
the frontal and
temporal cortices.
FTD is commonly used for the description of a group of early onset dementias,
and is
the second most common dementing disorder among people under 65 years of age.
However,
in 25% of the cases FTD presents in old age. The estimated prevalence of FTD
is 15-
22/100,000 and population studies indicate an equal gender distribution. The
World Health
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Organization estimates that dementia rates will double nearly every 20 years,
reaching an
estimated 135.5 million people in 2050.
No approved disease-modifying drugs are available for the treatment of FTD.
Treatment is focused on management of behavioral symptoms. Severity of
compulsion,
agitation, aggressiveness, impulsivity, and aberrant eating behavior can
improve with the use
of selective serotonin reuptake inhibitors. Behavioral abnormalities can be
managed with
low doses of atypical antipsychotics. Cholinesterase inhibitors are not
beneficial and can
worsen behavioral abnormalities seen in patients with FTD. Memantine does not
improve or
delay progression of FTD symptoms.
Despite the recent discovery of two genes associated with FTD, available
treatment
options are not efficacious enough to prevent or treat FTD, and FTD remains
difficult to
eradicate completely. Thus, there is a need for new therapies to prevent,
reduce the risk of
developing, and treat FTD.
SUMMARY
The present disclosure is generally directed to methods of preventing,
reducing risk of
developing, or treating frontotemporal dementia (FTD) or a variant of FTD,
comprising
administering to a subject an inhibitor of the complement pathway.
Although there are varied etiologies among neurodegenerative diseases, one
cellular
commonality which exists among all neurons is the synapse. Degeneration of
functional
synapses is of crucial importance to understanding the primary mechanisms of
overall
neurodegeneration. Evidence suggests that synapse loss precedes neuron loss,
such as in early
Alzheimer's Disease (AD). Synapse loss is inhibited by contacting neurons with
inhibitors or
antagonists of the complement pathway. For example, inhibitors may block
activation of the
complement cascade, can block the expression of specific complement proteins
in neurons,
interfere with signaling molecules that induce complement activation,
upregulate expression
of complement inhibitors in neurons, or otherwise interfere with the role of
complement in
synapse loss. The ability to prevent synapse loss, e.g. in adult brains, has
important
implications for maintaining normal neuronal function in a variety of
neurodegenerative
conditions.
Accordingly, inhibition of complement activation pathways may be a promising
therapeutic strategy for preventing, reducing risk of developing, or treating
frontotemporal
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dementia, e.g., using antibodies to inhibit the early stages of complement
activation,
including the complement activation pathway. Specifically, anti-C 1 q, anti-
Clr, and anti-Cis
antibodies may prevent autoantibodies from triggering the classical pathway of
complement
activation and prevent synapse loss resulting from the neuronal expression of
complement
factors.
The present disclosure is generally directed to methods of preventing,
reducing risk of
developing, or treating frontotemporal dementia (FTD) by inhibiting complement
activation,
e.g., by inhibiting complement factor Clq, Clr, or Cls, e.g., through the
administration of
antibodies, such as monoclonal, chimeric, humanized antibodies, antibody
fragments, etc.,
which bind to one or more of these complement factors.
Human complement was originally defined as the heat-labile component of plasma
that "complemented" the humoral system and aided antibody-dependent killing of
bacteria.
Complement is now known to be a tightly regulated proteolytic network of more
than 30
proteins circulating in the blood or attached to membrane surfaces that
coordinate crucial
roles in mammalian innate immunity, especially as it relates to inflammation
and the body's
defense against invading organisms. Complement proteins are produced by many
cell types
and have diverse cooperative functions. For example, complement is involved in
the
clearance of self-antigens and apoptotic cells, forms a bridge to adaptive
immunity, and also
plays a significant role in tissue regeneration and tumor growth. To exercise
these functions,
the complement system relies on an interplay of soluble and cell-surface-bound
proteins that
interact with pathogen cell surfaces to mark them for destruction by
phagocytes. The
complement system is made up of a large number of distinct plasma proteins,
primarily
produced by the liver. A number of these proteins are a class of proteases
known as
zymogens, which are themselves activated by proteolytic cleavage. These
zymogens may be
widely distributed in an inactive form until an invading pathogen is detected.
The
complement system thus is activated through a triggered enzyme cascade.
Complement activation is initiated through three pathways: classical,
alternative and
lectin pathways. All three pathways are initiated by detection of surface
structures by pattern
recognition proteins. In addition, all three pathways merge through a common
intersection,
complement C3. C3 is an acute phase reactant. The liver is the main site of
synthesis,
although small amounts are also produced by activated monocytes and
macrophages. A
single chain precursor (pro-C3) of approximately 200 kD is found
intracellularly; the cDNA
shows that it comprises 1,663 amino acids. This is processed by proteolytic
cleavage into
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alpha and beta subunits, which in the mature protein are linked by disulfide
bonds. Pro-C3
contains a signal peptide of 22 amino acid residues, the beta chain (645
residues) and the
alpha chain (992 residues). The 2 chains are joined by 4 arginine residues
that are not present
in the mature protein.
The classical pathway is activated by the binding of the complement protein
Clq
directly to patches of surface-bound antibodies (IgM and IgG), and also to C-
reactive protein,
serum amyloid P, pentraxin 3, and other known and unknown binding sites on the
surfaces of
cells, synapses, and microbes.
Clq is a large multimeric protein of 460 kDa consisting of 18 polypeptide
chains (6
Clq A chains, 6 Clq B chains, and 6 Clq C chains). Clr and Cis complement
proteins bind
to the Clq tail region to form the Cl complex (C1qr2s2). Binding of the Clq
complex to the
surface of a cell or to the complement binding domain of an antibody Fc region
induces a
conformational change in Clq that leads to activation of an autocatalytic
enzymatic activity
in Clr, which then cleaves Cis to generate an active serine protease. Once
activated, Cis
cleaves C4 resulting in C4b, which in turn binds to C2. C2 is cleaved by Cis,
resulting in the
activated form, C2a, bound to C4b and forming the C3 convertase (C4b2a) of the
classical
pathway. Ultimately, this pathway leads to the formation of a membrane attack
complex,
which lyses and kills the affected cell.
The lectin pathway is activated by the binding of mannose-binding lectin (MBL)
through two serum serine proteases designated MASP-1 and MASP-2. Similar to
the
classical complement pathway, the lectin complement pathway also requires C4
and C2 for
activation of C3 and other terminal components further downstream in the
cascade.
Activation of the complement pathway generates biologically active fragments
of
complement proteins, e.g., C3a, C4a and C5a anaphylatoxins and sC5b-9 membrane
attack
complex (MAC), which mediates inflammatory activities involving leukocyte
chemotaxis,
activation of macrophages, neutrophils, platelets, mast cells and endothelial
cells, increased
vascular permeability, cytolysis, and tissue injury. Antibody bound to a cell
surface antigen
can also activate the complement system, creating pores in the membrane of a
foreign cell, or
it can mediate cell destruction by antibody-dependent cell-mediated
cytotoxicity (ADCC). In
this process, cytotoxic cells with Fc receptors bind to the Fc region of
antibodies on target
cells and promote killing of the cells. Antibody bound to a foreign cell also
can serve as an
opsonin, enabling phagocytic cells with Fc or C3b receptors to bind and
phagocytose the
antibody-coated cell.
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Complement is nonspecific in that it can attack both foreign invaders and host
cells.
Under normal conditions, host cells, including neurons, are protected from
potential
complement-mediated damage by various fluid-phase and membrane-bound
complement
regulatory proteins, such as Cl inhibitor (C1-Inh). C1-INH dissociates Clr and
Cis from the
active Cl complex, which protects host cells from lysis or damage from the
membrane attack
complex. Other proteins that protect from potential complement-mediated damage
include
C4b-binding protein (C4BP), factor H (FH), complement receptor 1 (CR1; CD35),
complement receptor Ig (CRIg), decay accelerating factor (DAF; CD55), membrane
cofactor
protein (MCP; CD46), and CD59. However, deficiencies of these components or
excessive
activation of complement in response to certain pathological conditions can
overwhelm this
protective mechanism. Such unbalanced activation has been associated with a
growing
number of diseases and pathological disorders.
For example, various complement components are expressed by neurons and glial
cells in vitro and in vivo. While their function in the brain is unknown, the
expression of
many of these complement proteins is upregulated by serum or inflammatory
cytokines after
brain injury or during the course of neurodegenerative disease pathology.
Astrocytes in
culture have been reported to express Clq, Clr, Cis, C4, C2, and C3, as well
as the more
terminal complement proteins. Neurons have been reported to express C4 and C3.
Clq was
shown to be expressed in neuronal synapses and to mark these synapses for
elimination. See,
e.g., U.S. Patent Publication Nos. 2012/0195880 and 2012/328601. While
selective synapse
loss is an essential aspect of normal brain development ("synaptic pruning"),
excessive
synapse loss, especially in a mature or aging brain, results in
neurodegeneration and
cognitive decline. Elevated synaptic complement expression led to contribute
to synaptic loss
in normal aging and in neurodegenerative disease progression. Conversely,
lowering
complement expression was neuroprotective. Neurons affected by synapse loss
may be
central nervous system neurons, or peripheral nervous system neurons.
Neutralizing the activity of complement factors such as Clq, Clr, or Cis can
block
complement activity, prevent synapse loss, and slow neurodegenerative disease
progression
in disorders like FTD. Methods related to neutralizing complement factors such
as Clq, Clr,
or Cis in FTD are disclosed herein.
All sequences mentioned in the present disclosure are incorporated by
reference from
WO 2015/006504, U.S. Provisional Pat. App. No. 62/075793, U.S. Provisional
Pat. App. No.
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62/261376, WO 2014/186599, U.S. Pat. No. 8,877,197, each of which is hereby
incorporated
by reference for the antibodies and related compositions that it discloses.
In certain aspects, disclosed herein is a method of preventing, reducing risk
of
developing, or treating frontotemporal dementia (FTD) or a variant of FTD,
comprising
administering to a subject an inhibitor of the complement pathway. A variant
of FTD may
include a behavioral variant, a semantic variant, a non-fluent variant, and/or
primary
progressive aphasia. The subject may have social symptoms, emotional symptoms,
eating and
oral symptoms, repetitive or compulsive symptoms, sensory symptoms, motor
symptoms,
executive symptoms, language symptoms, neuropsychiatric symptoms, and/or other
symptoms.
Disclosed herein is a method of inhibiting synapse loss in FTD, comprising
administering to a patient suffering from adverse synapse loss an antibody,
such as an anti-
Clq antibody, an anti-Clr antibody, or an anti-Cls antibody. The method may
further
comprise administration of neural progenitors, or a neurogenesis enhancer. In
certain
preferred embodiments, the antibody binds to Clq, Clr, or Cis and inhibits
complement
activation.
Full-length antibodies may be prepared by the use of recombinant DNA
engineering
techniques. Such engineered versions include those created, for example, from
natural
antibody variable regions by insertions, deletions or changes in or to the
amino acid
sequences of the natural antibodies. Particular examples of this type include
those engineered
variable region domains containing at least one CDR and optionally one or more
framework
amino acids from one antibody and the remainder of the variable region domain
from a
second antibody. The DNA encoding the antibody may be prepared by deleting all
but the
desired portion of the DNA that encodes the full length antibody. DNA encoding
chimerized
antibodies may be prepared by recombining DNA substantially or exclusively
encoding
human constant regions and DNA encoding variable regions derived substantially
or
exclusively from the sequence of the variable region of a mammal other than a
human. DNA
encoding humanized antibodies may be prepared by recombining DNA encoding
constant
regions and variable regions other than the complementarity determining
regions (CDRs)
derived substantially or exclusively from the corresponding human antibody
regions and
DNA encoding CDRs derived substantially or exclusively from a mammal other
than a
human.
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Suitable sources of DNA molecules that encode antibodies include cells, such
as
hybridomas, that express the full length antibody. For example, the antibody
may be isolated
from a host cell that expresses an expression vector that encodes the heavy
and/or light chain
of the antibody.
Antibody fragments may also be prepared by the use of recombinant DNA
engineering techniques involving the manipulation and re-expression of DNA
encoding
antibody variable and constant regions. Standard molecular biology techniques
may be used
to modify, add or delete further amino acids or domains as desired. Any
alterations to the
variable or constant regions are still encompassed by the terms 'variable' and
'constant'
regions as used herein. In some instances, PCR is used to generate an antibody
fragment by
introducing a stop codon immediately following the codon encoding the
interchain cysteine
of CH1, such that translation of the CH1 domain stops at the interchain
cysteine. Methods for
designing suitable PCR primers are well known in the art and the sequences of
antibody CH1
domains are readily available. In some embodiments, stop codons may be
introduced using
site-directed mutagenesis techniques.
An antibody of the present disclosure may be derived from any antibody isotype
("class") including for example IgG, IgM, IgA, IgD and IgE and subclasses
thereof,
including for example IgGl, IgG2, IgG3 and IgG4. In certain preferred
embodiments, the
heavy and light chains of the antibody are from murine IgGl.
In some embodiments, the inhibitor is an antibody, such as an anti-Clq
antibody, an
anti-Clr antibody, or an anti-Cls antibody. The anti-Clq antibody may inhibit
the interaction
between Clq and an autoantibody, or between Clq and Clr, or between Clq and
Cis. The
anti-Clr antibody may inhibit the interaction between Clr and Clq, or between
Clr and Cis.
The anti-Clr antibody may inhibit the catalytic activity of Clr, or the anti-
Clr antibody may
inhibit the processing of pro-Clr to an active protease. The anti-Cis antibody
may inhibit the
interaction between Cis and Clq, or between Cis and Clr, or between Cis and C2
or C4, or
the anti-Cis antibody may inhibit the catalytic activity of Cls, or it may
inhibit the
processing of pro-Cis to an active protease. In some instances, the anti-Clq,
anti-Clr, or
anti-Cis antibody causes clearance of Clq, Clr or Cis from the circulation or
a tissue.
The antibody disclosed herein may be a monoclonal antibody, e.g., that binds
mammalian Clq, Clr, or Cis, preferably human Clq, Clr, or Cis. The antibody
may be a
mouse antibody, a human antibody, a humanized antibody, a chimeric antibody,
or an
antibody fragment. The antibodies disclosed herein may also cross the blood
brain barrier
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(BBB). The antibody may activate a BBB receptor-mediated transport system,
such as a
system that utilizes the insulin receptor, transferrin receptor, leptin
receptor, LDL receptor, or
IGF receptor. The antibody can be a chimeric antibody with sufficient human
sequence that is
suitable for administration to a human. The antibody can be glycosylated or
nonglycosylated;
in some embodiments, the antibody is glycosylated, e.g., in a glycosylation
pattern produced
by post-translational modification in a CHO cell.
The antibodies of the present disclosure may also be covalently linked to a
therapeutic
agent, such as an anti-inflammatory protein, neurotherapeutic agent, anti-
viral, anti-parasitic,
anti-bacterial, endocrine drug, metabolic drug, mitotoxin, chemotherapy drug,
or siRNA, for
which transport across the BBB is desired. The covalent linkage between the
antibody and,
for example, the neurotherapeutic agent may be a linkage between any suitable
portion of the
antibody and the therapeutic agent, as long as it allows the antibody-agent
fusion to cross the
blood brain barrier and the therapeutic agent to retain a therapeutically
useful portion of its
activity within the central nervous system. For example, the covalent link may
be between
one or more light chains of the antibody and the therapeutic agent. In the
case of a peptide
neurotherapeutic agent (e.g., a neurotrophin such as brain derived
neurotrophic factor,
BDNF), the peptide can be covalently linked by its carboxy or amino terminus
to the carboxy
or amino terminus of the light chain (LC) or heavy chain (HC) of the antibody.
Other neurotherapeutic agents that can be linked to antibodies of the present
disclosure include a neurotrophin selected from brain derived neurotrophic
factor (BDNF),
nerve growth factor (NGF), neurotrophin-4/5, fibroblast growth factor (FGF)-2
and other
FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor
(HGF),
epidermal growth factor (EGF), transforming growth factor (TGF)-a, TGF-f3,
vascular
endothelial growth factor (VEGF), interleukin-1 receptor antagonist (IL-lra),
ciliary
neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF),
neurturin, platelet-
derived growth factor (PDGF), heregulin, neuregulin, artemin, persephin,
interleukins,
granulocyte-colony stimulating factor (C SF), granulocyte-macrophage-CSF,
netrins,
cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF), midkine,
pleiotrophin, bone
morphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stem cell
factor (SCF).
The antibody may be a bispecific antibody, recognizing a first and a second
antigen,
e.g., the first antigen is selected from Clq, Clr, and Cis and/or the second
antigen is an
antigen that allows the antibody to cross the blood-brain-barrier, such as an
antigen selected
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from transferrin receptor (TR), insulin receptor (HIR), Insulin-like growth
factor receptor
(IGFR), low-density lipoprotein receptor related proteins 1 and 2 (LPR-1 and
2),
diphtheria toxin receptor, CRM197, a llama single domain antibody, TMEM 30(A),
a
protein transduction domain, TAT, Syn-B, penetratin, a poly-arginine peptide,
an angiopep
peptide, or ANG1005.
An antibody of the present disclosure may bind to and inhibit a biological
activity of
Clq, Clr, or Cl. For example, (1) Clq binding to an autoantibody, (2) Clq
binding to Clr,
(3) Clq binding to Cis, (4) Clq binding to phosphatidylserine, (5) Clq binding
to pentraxin-
3, (6) Clq binding to C-reactive protein (CRP), (7) Clq binding to globular
Clq receptor
(gClqR), (8) Clq binding to complement receptor 1 (CR1), (9) Clq binding to B-
amyloid, or
(10) Clq binding to calreticulin. In other embodiments, the biological
activity of Clq is (1)
activation of the classical complement activation pathway, (2) activation of
antibody and
complement dependent cytotoxicity, (3) CH50 hemolysis, (4) synapse loss, (5) B-
cell
antibody production, (6) dendritic cell maturation, (7) T-cell proliferation,
(8) cytokine
production (9) microglia activation, (10) Arthus reaction, (11) phagocytosis
of synapses or
nerve endings or (12) activation of complement receptor 3 (CR3/C3) expressing
cells.
In some embodiments, CH50 hemolysis comprises human, mouse, and/or rat CH50
hemolysis. In some embodiments, the antibody is capable of neutralizing from
at least about
50%, to at least about 95% of CH50 hemolysis. The antibody may also be capable
of
neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng,
less than 100 ng,
less than 50 ng, or less than 20 ng.
Other in vitro assays to measure complement activity include ELISA assays for
the
measurement of split products of complement components or complexes that form
during
complement activation. Complement activation via the classical pathway can be
measured by
following the levels of C4d and C4 in the serum. Activation of the alternative
pathway can be
measured in an ELISA by assessing the levels of Bb or C3bBbP complexes in
circulation. An
in vitro antibody-mediated complement activation assay may also be used to
evaluate
inhibition of C3a production.
An antibody of the present disclosure may be a monoclonal antibody, a
polyclonal
antibody, a recombinant antibody, a humanized antibody, a chimeric antibody, a
multispecific antibody, or an antibody fragment thereof.
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The antibodies of the present disclosure may also be an antibody fragment,
such as a
Fab fragment, a Fab' fragment, a F(ab')2 fragment, a FIT fragment, a diabody,
or a single chain
antibody molecule.
Disclosed herein are methods of administering to the subject a second agent,
such as
a second antibody or a second inhibitor. The antibody may be an anti-Clq
antibody, an
anti-Clr antibody, or an anti-Cis antibody. The inhibitor may be an inhibitor
of antibody-
dependent cellular cytotoxicity, alternative complement activation pathway;
and/or an
inhibitor of the interaction between the autoantibody and an autoantigen.
In some embodiments, a method is provided of determining a subject's risk of
developing frontotemporal dementia, comprising: (a) administering an antibody
to the subject
(i.e. an anti-Clq, anti-Clr, or anti-Cis antibody) , wherein the antibody is
coupled to a
detectable label; (b) detecting the detectable label to measure the amount or
location of Clq,
Clr, or Cis in the subject; and (c) comparing the amount or location of one or
more of Clq,
Clr, or Cis to a reference, wherein the risk of developing frontotemporal
dementia is
characterized based on a the comparison of the amount or location of one or
more of Cl q,
Clr, or Cis to the reference. The detectable label may comprise a nucleic
acid,
oligonucleotide, enzyme, radioactive isotope, biotin or a fluorescent label.
In some instances,
the antibody may be labeled with a coenzyme such as biotin using the process
of
biotinylation. When biotin is used as a label, the detection of the antibody
is accomplished by
addition of a protein such as avidin or its bacterial counterpart
streptavidin, either of which
can be bound to a detectable marker such as the aforementioned dye, a
fluorescent marker
such as fluorescein, a radioactive isotope or an enzyme such as peroxidase. In
some
embodiments, the antibody is an antibody fragment (e.g., Fab, Fab'-SH, Fv,
scFv, or F(ab')2
fragments).
The antibodies disclosed herein may also be coupled to a labeling group, e.g.,
an radioisotope, radionuclide, an enzymatic group, biotinyl group, a nucleic
acid,
oligonucleotide, enzyme, or a fluorescent label. A labeling group may be
coupled to the
antibody via a spacer arm of any suitable length to reduce potential steric
hindrance. Various
methods for labeling proteins are known in the art and can be used to prepare
such labeled
antibodies.
Various routes of administration are contemplated. Such methods of
administration
include but are not limited to, topical, parenteral, subcutaneous,
intraperitoneal,
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intrapulmonary, intrathecal, intranasal, and intralesional administration.
Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal, or
subcutaneous
administration. For treatment of central nervous system conditions, the
antibody may be
adapted to cross the blood-brain barrier following a non-invasive peripheral
route of
administration such as intravenous intramuscular, subcutaneous,
intraperitoneal, or even oral
administration.
The present disclosure also provides a method of detecting synapses in an
individual,
by a) administering an antibody from any of the embodiments to the subject,
wherein the
antibody is coupled to a detectable label; (b) detecting the detectable label
to measure the
amount or location of the antibody in the subject; and (c) comparing the
amount or location
of the antibody to a reference, wherein the risk of developing a disease
associated with
complement activation is characterized based on the comparison of the amount
of antibody
as compared to the reference. For example, the detectable label may comprise a
nucleic acid,
oligonucleotide, enzyme, radioactive isotope, biotin, or a fluorescent label
(e.g., fluorescein,
rhodamine, cyanine dyes or BODIPY). The detectable label may be detected using
an
imaging agent for x-ray, CT, MM, ultrasound, PET and SPECT.
It is to be understood that one, some, or all of the properties of the various
embodiments described herein may be combined to form other embodiments of the
compositions and methods provided herein. All combinations of the embodiments
pertaining
to the invention are specifically embraced by the present invention and are
disclosed herein
just as if each and every combination was individually and explicitly
disclosed. In addition,
all sub-combinations of the various embodiments and elements thereof are also
specifically
embraced by the present invention and are disclosed herein just as if each and
every such
sub-combination was individually and explicitly disclosed herein. These and
other aspects of
the compositions and methods provided herein will become apparent to one of
skill in the art.
The publications discussed herein are provided solely for their disclosure
prior to the
filing date of the present application. Nothing herein is to be construed as
an admission that
the present invention is not entitled to antedate such publication by virtue
of prior invention.
Further, the dates of publication provided can be different from the actual
publication dates,
which may need to be independently confirmed.
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DESCRIPTION OF THE FIGURES
Figure 1 depicts the amino acid sequence of Homo sapiens complement Cis
protein (SEQ ID
NO:70).
Figure 2 depicts an amino acid sequence of humanized IPN003 VH variant 1 (SEQ
ID
NO.79); and a nucleotide sequence (SEQ ID NO: 86) encoding the amino acid
sequence.
Figure 3 depicts an amino acid sequence of humanized IPN003 VH variant 2 (SEQ
ID
NO.80); and a nucleotide sequence (SEQ ID NO: 87) encoding the amino acid
sequence.
Figure 4 depicts an amino acid sequence of humanized IPN003 VH variant 3 (SEQ
ID
NO.81); and a nucleotide sequence (SEQ ID NO: 88) encoding the amino acid
sequence.
Figure 5 depicts an amino acid sequence of humanized IPN003 VH variant 4 (SEQ
ID
NO.82); and a nucleotide sequence (SEQ ID NO: 89) encoding the amino acid
sequence.
Figure 6 depicts an amino acid sequence of humanized IPN003 Vic variant 1 (SEQ
ID
NO.83); and a nucleotide sequence (SEQ ID NO: 90) encoding the amino acid
sequence.
Figure 7 depicts an amino acid sequence of humanized IPN003 Vic variant 2 (SEQ
ID
NO.84); and a nucleotide sequence (SEQ ID NO: 91) encoding the amino acid
sequence.
Figure 8 depicts an amino acid sequence of humanized IPN003 Vic variant 3 (SEQ
ID
NO.85); and a nucleotide sequence (SEQ ID NO: 92) encoding the amino acid
sequence.
Figure 9 provides Table 3, which shows the amino acid differences between
parental IPN003
VH and exemplary VH variants; and Table 4, which shows the amino acid
differences
between parental IPN003 VL and exemplary VL variants.
DETAILED DESCRIPTION
The present disclosure relates generally to methods of preventing, reducing
risk of
developing, or treating frontotemporal dementia (FTD) or a variant of FTD,
comprising
administering to a subject an inhibitor of the complement pathway.
Suitable antibodies include antibodies that bind to complement component Clq,
Clr,
or Cl s. Such antibodies include monoclonal antibodies and homologues,
analogs, and
modified or derived forms thereof, including Fab, F(a1302, Fv and single chain
antibodies.
Preferred antibodies are monoclonal antibodies, which can be raised by
immunizing
rodents (e.g., mice, rats, hamsters and guinea pigs) with either (1) the
native complement
component (e.g., Clq, Clr, or Cis) derived from enzymatic digestion of a
purified
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complement component from human plasma or serum, or (2) a recombinant
complement
component, or its derived fragment, expressed by either eukaryotic or
prokaryotic systems.
Other animals can be used for immunization, e.g., non-human primates,
transgenic mice
expressing human immunoglobulins, and severe combined immunodeficient (SCID)
mice
transplanted with human B-lymphocytes.
Polyclonal and monoclonal antibodies are naturally generated as immunoglobulin
(Ig)
molecules in the immune system's response to a pathogen. A dominating format
with a
concentration of 8 mg/ml in human serum, the ¨150-kDa IgG1 molecule is
composed of two
identical ¨50-kDa heavy chains and two identical --25-kDa light chains.
Hybridomas can be generated by conventional procedures by fusing B-lymphocytes
from the immunized animals with myeloma cells. In addition, anti -Clq, -Clr,
or ¨Cis
antibodies can be generated by screening recombinant single-chain Fv or Fab
libraries from
human B-lymphocytes in a phage-display system. The specificity of the MAbs to
human
Clq, Clr, or Cis can be tested by enzyme linked immunosorbent assay (ELISA),
Western
immunoblotting, or other immunochemical techniques.
The inhibitory activity on complement activation of antibodies identified in
the
screening process can be assessed by hemolytic assays using either
unsensitized rabbit or
guinea pig RBCs for the alternative complement pathway, or sensitized chicken
or sheep
RBCs for the classical complement pathway. Those hybridomas that exhibit an
inhibitory
activity specific for the classical complement pathway are cloned by limiting
dilution. The
antibodies are purified for characterization for specificity to human Clq,
Clr, or Cis by the
assays described above.
Based on the molecular structures of the variable regions of the anti -Clq, -
Clr, or ¨
Cls antibodies, molecular modeling and rational molecular design may be used
to generate
and screen small molecules that mimic the molecular structures of the binding
region of the
antibodies and inhibit the activities of Clq, Clr, or Cis. These small
molecules can be
peptides, peptidomimetics, oligonucleotides, or organic compounds. The
mimicking
molecules can be used as inhibitors of complement activation in inflammatory
indications
and autoimmune diseases. Alternatively, one can use large-scale screening
procedures
commonly used in the field to isolate suitable small molecules from libraries
of combinatorial
compounds.
A suitable dosage of an antibody as disclosed herein may be between 10 and 500
pg/m1 of serum. The actual dosage can be determined in clinical trials
following the
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conventional methodology for determining optimal dosages, i.e., administering
various
dosages and determining which doses provide suitable efficacy without
undesirable side-
effects.
Before the advent of recombinant DNA technology, proteolytic enzymes
(proteases)
that cleave polypeptide sequences were used to dissect the structure of
antibody molecules
and to determine which parts of the molecule are responsible for its various
functions.
Limited digestion with the protease papain cleaves antibody molecules into
three fragments.
Two fragments, known as Fab fragments, are identical and contain the antigen-
binding
activity. The Fab fragments correspond to the two identical arms of the
antibody molecule,
each of which consists of a complete light chain paired with the VH and CH1
domains of a
heavy chain. The other fragment contains no antigen binding activity but was
originally
observed to crystallize readily, and for this reason was named the Fc fragment
(Fragment
crystallizable).
A Fab molecule is an artificial ¨50-kDa fragment of the Ig molecule with a
heavy
chain lacking constant domains CH2 and CH3. Two heterophilic (VL-VH and CL-
CH1) domain
interactions underlie the two-chain structure of the Fab molecule, which is
further stabilized
by a disulfide bridge between CL and CH1. Fab and IgG have identical antigen
binding sites
formed by six complementarity-determining regions (CDRs), three each from VL
and VH
(LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3). The CDRs define the
hypervariable antigen binding site of antibodies. The highest sequence
variation is found in
LCDR3 and HCDR3, which in natural immune systems are generated by the
rearrangement
of IA and A genes or VH, DH and JH genes, respectively. LCDR3 and HCDR3
typically form
the core of the antigen binding site. The conserved regions that connect and
display the six
CDRs are referred to as framework regions. In the three-dimensional structure
of the variable
domain, the framework regions form a sandwich of two opposing antiparallel 13-
sheets that
are linked by hypervariable CDR loops on the outside and by a conserved
disulfide bridge on
the inside.
Methods are disclosed herein for protecting or treating an individual
suffering from
adverse effects of synapse loss, such as in frontotemporal dementia. It is
shown herein that
immature astrocytes in normal development produce a signal that induces
neurons to express
a specific complement protein, thus enabling a developmental window during
which synapse
elimination occurs. Expression of such a protein in development mirrors the
period of
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developmental synaptogenesis, being off in embryonic brain and adult brain but
on at high
levels in postnatal brain.
These findings have broad implications for a variety of clinical conditions,
particularly neurodegenerative conditions where synapse loss is involved, such
as
.. frontotemporal dementia. Synapse loss is inhibited by contacting neurons
with inhibitors or
antagonists of the complement pathway. For example, inhibitors can block
activation of the
complement cascade, can block the expression of specific complement proteins
in neurons,
can interfere with signaling molecules that induce complement activation, can
upregulate
expression of complement inhibitors in neurons, and otherwise interfere with
the role of
complement in synapse loss. The ability to prevent synapse loss, e.g., in
adult brains, has
important implications for maintaining normal neuronal function in a variety
of
neurodegenerative conditions.
Anti-Complement C 1 q Antibodies
Suitable inhibitors include an antibody that binds complement Clq protein
(i.e., an
anti-complement Clq antibody, also referred to herein as an anti-Clq antibody
and a Clq
antibody) and a nucleic acid molecule that encodes such an antibody for a
method of
preventing, reducing risk of developing, or treating frontotemporal dementia.
All sequences mentioned in the following fourteen paragraphs are incorporated
by
reference from WO 2015/006504 which is hereby incorporated by reference for
the
antibodies and related compositions that it discloses.
Disclosed herein are methods of administering an anti-Clq antibody comprising
a
light chain variable domain and a heavy chain variable domain. The antibody
may bind to at
least human Clq, mouse Clq, or rat Clq. The antibody may be a humanized
antibody, a
chimeric antibody, or a human antibody. The light chain variable domain
comprises the
.. HVR-L1, HVR-L2, and HVR-L3 of the monoclonal antibody M1 produced by a
hybridoma
cell line deposited with Accession Number PTA-120399. The heavy chain variable
domain
comprises the HVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody M1
produced by
a hybridoma cell line deposited with ATCC Accession Number PTA-120399.
In some embodiments, the amino acid sequence of the light chain variable
domain
.. and heavy chain variable domain comprise one or more of SEQ ID NO:5 of HVR-
L1, SEQ
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ID NO:6 of HVR-L2, SEQ ID NO:7 of HVR-L3, SEQ ID NO:9 of HVR-H1, SEQ ID NO:10
of HVR-H2, and SEQ ID NO:11 of HVR-H3.
The antibody may comprise a light chain variable domain amino acid sequence
that is
at least 85% identical to SEQ ID NO:4 and a heavy chain variable domain amino
acid
sequence that is at least 85% identical to SEQ ID NO:8.
Disclosed herein are methods of administering an anti-Clq antibody inhibits
the
interaction between Clq and an autoantibody. In some embodiments, the anti-Clq
antibody
inhibits the interaction between Clq and the synapse. In preferred
embodiments, the anti-
Clq antibody causes clearance of Clq from the circulation or tissue.
The anti-Clq antibody may bind to a Clq protein, and binds to one or more
amino
acids of the Clq protein within amino acid residues selected from (a) amino
acid residues
196-226 of SEQ ID NO:1 (SEQ ID NO:16), or amino acid residues of a Clq protein
chain A
(ClqA) corresponding to amino acid residues 196-226
(GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1 (SEQ ID NO:16);
(b) amino acid residues 196-221 of SEQ ID NO:1 (SEQ ID NO:17), or amino acid
residues
of a ClqA corresponding to amino acid residues 196-221
(GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1 (SEQ ID NO:17); (c) amino
acid residues 202-221 of SEQ ID NO:1 (SEQ ID NO:18), or amino acid residues of
a ClqA
corresponding to amino acid residues 202-221 (SGGMVLQLQQGDQVWVEKDP) of SEQ
ID NO:1 (SEQ ID NO:18); (d) amino acid residues 202-219 of SEQ ID NO:1 (SEQ ID
NO:19), or amino acid residues of a ClqA corresponding to amino acid residues
202-219
(SGGMVLQLQQGDQVWVEK) of SEQ ID NO:1 (SEQ ID NO:19); and (e) amino acid
residues Lys 219 and/or Ser 202 of SEQ ID NO:1, or amino acid residues of a
ClqA
corresponding Lys 219 and/or Ser 202 of SEQ ID NO: 1.
In some embodiments, the antibody further binds to one or more amino acids of
the
Clq protein within amino acid residues selected from: (a) amino acid residues
218-240 of
SEQ ID NO:3 (SEQ ID NO:20) or amino acid residues of a Clq protein chain C
(ClqC)
corresponding to amino acid residues 218-240 (WLAVNDYYDMVGI QGSDSVFSGF) of
SEQ ID NO:3 (SEQ ID NO:20); (b) amino acid residues 225-240 of SEQ ID NO:3
(SEQ ID
NO:21) or amino acid residues of a ClqC corresponding to amino acid residues
225-240
(YDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:21); (c) amino acid residues 225-
232 of SEQ ID NO:3 (SEQ ID NO:22) or amino acid residues of a ClqC
corresponding to
amino acid residues 225-232 (YDMVGIQG) of SEQ ID NO:3 (SEQ ID NO:22); (d)
amino
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acid residue Tyr 225 of SEQ ID NO:3 or an amino acid residue of a ClqC
corresponding to
amino acid residue Tyr 225 of SEQ ID NO:3; (e) amino acid residues 174-196 of
SEQ ID
NO:3 (SEQ ID NO:23) or amino acid residues of a ClqC corresponding to amino
acid
residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3 (SEQ ID NO:23);
.. (f) amino acid residues 184-192 of SEQ ID NO:3 (SEQ ID NO:24) or amino acid
residues of
a ClqC corresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3
(SEQ ID NO:24); (g) amino acid residues 185-187 of SEQ ID NO:3 or amino acid
residues
of a ClqC corresponding to amino acid residues 185-187 (SGV) of SEQ ID NO:3;
(h) amino
acid residue Ser 185 of SEQ ID NO:3 or an amino acid residue of a ClqC
corresponding to
amino acid residue Ser 185 of SEQ ID NO:3.
In certain embodiments, the anti-Clq antibody binds to amino acid residue Lys
219
and Ser 202 of the human ClqA as shown in SEQ ID NO:1 or amino acids of a
human ClqA
corresponding to Lys 219 and Ser 202 as shown in SEQ ID NO:1, and amino acid
residue
Tyr 225 of the human ClqC as shown in SEQ ID NO:3 or an amino acid residue of
a human
ClqC corresponding to Tyr 225 as shown in SEQ ID NO:3. In certain embodiments,
the
anti-Clq antibody binds to amino acid residue Lys 219 of the human ClqA as
shown in SEQ
ID NO:1 or an amino acid residue of a human ClqA corresponding to Lys 219 as
shown in
SEQ ID NO:1, and amino acid residue Ser 185 of the human ClqC as shown in SEQ
ID
NO:3 or an amino acid residue of a human ClqC corresponding to Ser 185 as
shown in SEQ
ID NO:3.
In some embodiments, the anti-Clq antibody binds to a Clq protein and binds to
one
or more amino acids of the Clq protein within amino acid residues selected
from: (a) amino
acid residues 218-240 of SEQ ID NO:3 (SEQ ID NO:20) or amino acid residues of
a ClqC
corresponding to amino acid residues 218-240 (WLAVNDYYDMVGI QGSDSVFSGF) of
SEQ ID NO:3 (SEQ ID NO:20); (b) amino acid residues 225-240 of SEQ ID NO:3
(SEQ ID
NO:21) or amino acid residues of a ClqC corresponding to amino acid residues
225-240
(YDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:21); (c) amino acid residues 225-
232 of SEQ ID NO:3 (SEQ ID NO:22) or amino acid residues of a ClqC
corresponding to
amino acid residues 225-232 (YDMVGIQG) of SEQ ID NO:3 (SEQ ID NO:22); (d)
amino
acid residue Tyr 225 of SEQ ID NO:3 or an amino acid residue of a ClqC
corresponding to
amino acid residue Tyr 225 of SEQ ID NO:3; (e) amino acid residues 174-196 of
SEQ ID
NO:3 (SEQ ID NO:23) or amino acid residues of a ClqC corresponding to amino
acid
residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3 (SEQ ID NO:23);
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(f) amino acid residues 184-192 of SEQ ID NO:3 (SEQ ID NO:24) or amino acid
residues of
a ClqC corresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3
(SEQ ID NO:24); (g) amino acid residues 185-187 of SEQ ID NO:3 or amino acid
residues
of a ClqC corresponding to amino acid residues 185-187 (SGV) of SEQ ID NO:3;
(h) amino
acid residue Ser 185 of SEQ ID NO:3 or an amino acid residue of a ClqC
corresponding to
amino acid residue Ser 185 of SEQ ID NO:3.
In some embodiments, the anti-Clq antibody of this disclosure inhibits the
interaction
between Clq and Cls. In some embodiments, the anti-Clq antibody inhibits the
interaction
between Clq and Clr. In some embodiments the anti-Clq antibody inhibits the
interaction
between Clq and Cls and between Clq and Clr. In some embodiments, the anti-Clq
antibody inhibits the interaction between Clq and another antibody, such as an
autoantibody.
In preferred embodiments, the anti-Clq antibody causes clearance of Clq from
the
circulation or tissue. In some embodiments, the anti-Clq antibody inhibits the
respective
interactions, at a stoichiometry of less than 2.5:1; 2.0:1; 1.5:1; or 1.0:1.
In some
embodiments, the Clq antibody inhibits an interaction, such as the Clq-Cls
interaction, at
approximately equimolar concentrations of Clq and the anti-Clq antibody. In
other
embodiments, the anti-Clq antibody binds to Clq with a stoichiometry of less
than 20:1; less
than 19.5:1; less than19:1; less than 18.5:1; less than 18:1; less than
17.5:1; less than 17:1;
less than 16.5:1; less than 16:1; less than 15.5:1; less than 15:1; less than
14.5:1; less than
14:1; less than 13.5:1; less than 13:1; less than 12.5:1; less than 12:1; less
than 11.5:1; less
than 11:1; less than 10.5:1; less than 10:1; less than 9.5:1; less than 9:1;
less than 8.5:1; less
than 8:1; less than 7.5:1; less than 7:1; less than 6.5:1; less than 6:1; less
than 5.5:1; less than
5:1; less than 4.5:1; less than 4:1; less than 3.5:1; less than 3:1; less than
2.5:1; less than
2.0:1; less than 1.5:1; or less than 1.0:1. In certain embodiments, the anti-
Clq antibody binds
Clq with a binding stoichiometry that ranges from 20:1 to 1.0:1 or less
than1.0:1. In certain
embodiments, the anti-Clq antibody binds Clq with a binding stoichiometry that
ranges from
6:1 to 1.0:1 or less than1.0:1. In certain embodiments, the anti-Clq antibody
binds Clq with
a binding stoichiometry that ranges from 2.5:1 to 1.0:1 or less than1.0:1. In
some
embodiments, the anti-Clq antibody inhibits the interaction between Clq and
Clr, or
between Clq and Cls, or between Clq and both Clr and Cls. In some embodiments,
the
anti-Clq antibody inhibits the interaction between Clq and Clr, between Clq
and Cls,
and/or between Clq and both Clr and Cls. In some embodiments, the anti-Clq
antibody
binds to the Clq A-chain. In other embodiments, the anti-Clq antibody binds to
the Clq B-
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chain. In other embodiments, the anti-Clq antibody binds to the Clq C-chain.
In some
embodiments, the anti-Clq antibody binds to the Clq A-chain, the Clq B-chain
and/or the
Clq C-chain. In some embodiments, the anti-Clq antibody binds to the globular
domain of
the Clq A-chain, B-chain, and/or C-chain. In other embodiments, the anti-Clq
antibody
binds to the collagen-like domain of the Clq A-chain, the Clq B-chain, and/or
the Clq C-
chain.
Where antibodies of this disclosure inhibit the interaction between two or
more
complement factors, such as the interaction of Clq and Cls, or the interaction
between Clq
and Clr, the interaction occurring in the presence of the antibody may be
reduced by at least
10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least
80%, at least 90%, at least 95%, or at least 99% relative to a control wherein
the antibodies of
this disclosure are absent. In certain embodiments, the interaction occurring
in the presence
of the antibody is reduced by an amount that ranges from at least 30% to at
least 99% relative
to a control wherein the antibodies of this disclosure are absent.
In some embodiments, the antibodies of this disclosure inhibit C2 or C4-
cleavage by
at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%,
at least 90%, at least 95%, or at least 99%, or by an amount that ranges from
at least 30% to
at least 99%, relative to a control wherein the antibodies of this disclosure
are absent.
Methods for measuring C2 or C4-cleavage are well known in the art. The ECso
values for
antibodies of this disclosure with respect C2 or C4-cleavage may be less than
3 ug/m1; 2.5
ug/m1; 2.0 ug/m1; 1.5 ug/m1; 1.0 ug/m1; 0.5 ug/m1; 0.25 ug/m1; 0.1 ug/m1; 0.05
ug/ml. In
some embodiments, the antibodies of this disclosure inhibit C2 or C4-cleavage
at
approximately equimolar concentrations of Clq and the respective anti-Clq
antibody.
In some embodiments, the antibodies of this disclosure inhibit autoantibody-
dependent and complement-dependent cytotoxicity (CDC) by at least 20%, at
least 30%, at
least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%,
or at least 99%, or by an amount that ranges from at least 30% to at least
99%, relative to a
control wherein the antibodies of this disclosure are absent. The ECso values
for antibodies
of this disclosure with respect to inhibition of autoantibody-dependent and
complement-
dependent cytotoxicity may be less than 3 ug/m1; 2.5 ug/m1; 2.0 ug/m1; 1.5
ug/m1; 1.0 ug/m1;
0.5 ug/m1; 0.25 ug/m1; 0.1 ug/m1; 0.05 ug/ml.
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In some embodiments, the antibodies of this disclosure inhibit complement-
dependent
cell-mediated cytotoxicity (CDCC) by at least 20%, at least 30%, at least 40%,
at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at
least 99%, or by an
amount that ranges from at least 30% to at least 99%, relative to a control
wherein the
antibodies of this disclosure are absent. Methods for measuring CDCC are well
known in the
art. The ECso values for antibodies of this disclosure with respect CDCC
inhibition may be 1
less than 3 g/m1; 2.5 g/m1; 2.0 g/m1; 1.5 g/m1; 1.0 g/m1; 0.5 g/m1; 0.25
g/m1; 0.1
g/m1; 0.05 g/ml. In some embodiments, the antibodies of this disclosure
inhibit CDCC but
not antibody-dependent cellular cytotoxicity (ADCC).
Humanized anti-complement C lq Antibodies
Humanized antibodies of the present disclosure specifically bind to a
complement
factor Clq and/or Clq protein in the Cl complex of the classical complement
pathway. The
humanized anti-Clq antibody may specifically bind to human Clq, human and
mouse Clq,
to rat Clq, or human Clq, mouse Clq, and rat Clq.
All sequences mentioned in the following sixteen paragraphs are incorporated
by
reference from U.S. Provisional Pat. App. No. 62/075793, which is hereby
incorporated by
reference for the antibodies and related compositions that it discloses.
In some embodiments, the human heavy chain constant region is a human IgG4
heavy
chain constant region comprising the amino acid sequence of SEQ ID NO:47, or
with at least
70%, at least about 75%, at least about 80%, at least about 85%, at least
about 90%
homology to SEQ ID NO: 37. The human IgG4 heavy chain constant region may
comprise
an Fc region with one or more modifications and/or amino acid substitutions
according to
Kabat numbering. In such cases, the Fc region comprises a leucine to glutamate
amino acid
substitution at position 248, wherein such a substitution inhibits the Fc
region from
interacting with an Fc receptor. In some embodiments, the Fc region comprises
a serine to
proline amino acid substitution at position 241, wherein such a substitution
prevents arm
switching in the antibody.
The amino acid sequence of human IgG4 (5241P L248E) heavy chain constant
domain is:
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEG
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GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE
EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 47).
The antibody may comprise a heavy chain variable domain and a light chain
variable
domain, wherein the heavy chain variable domain comprises an amino acid
sequence selected
from any one of SEQ ID NOs: 31-34, or an amino acid sequence with at least
about 90%
homology to the amino acid sequence selected from any one of SEQ ID NOs: 31-
34. In
certain such embodiments, the light chain variable domain comprises an amino
acid sequence
selected from any one of SEQ ID NOs: 35-38, or an amino acid sequence with at
least about
90% homology to the amino acid sequence selected from any one of SEQ ID NOs:
35-38.
The amino acid sequence of heavy chain variable domain variant 1 (VH1) is:
QVQLVQSGAELKKPGASVKVSCKSSGYHFTSYWMHWVKQAPGQGLEWIGVIHPN
SGSINYNEKFESKATITVDKST STAYMQL S SLTSEDSAVYYCAGERDSTEVLPMDY
WGQGTSVTVSS (SEQ ID NO: 31). The hyper variable regions (HVRs) of VH1 are
depicted in bolded and underlined text.
The amino acid sequence of heavy chain variable domain variant 2 (VH2) is:
QVQLVQSGAELKKPGASVKVSCKSSGYHFTSYWMHWVKQAPGQGLEWIGVIHPN
SGSINYNEKFESRATITVDKST STAYMEL SSLRSEDTAVYYCAGERDSTEVLPMDY
WGQGTTVTVSS (SEQ ID NO: 32). The hyper variable regions (HVRs) of VH2 are
depicted in bolded and underlined text.
The amino acid sequence of heavy chain variable domain variant 3 (VH3) is:
QVQLVQSGAELKKPGASVKVSCKSSGYHFTSYWMHWVKQAPGQGLEWIGVIHPN
SGSINYNEKFESRVTITVDKST STAYMEL SSLRSEDTAVYYCAGERDSTEVLPMDY
WGQGTTVTVSS (SEQ ID NO: 33). The hyper variable regions (HVRs) of VH3 are
depicted in bolded and underlined text.
The amino acid sequence of heavy chain variable domain variant 4 (VH4) is:
QVQLVQSGAELKKPGASVKVSCKSSGYHFTSYWMHWVRQAPGQGLEWIGVIHPN
SGSINYNEKFESRVTITVDKST STAYMEL SSLRSEDTAVYYCAGERDSTEVLPMDY
WGQGTTVTVSS (SEQ ID NO: 34). The hyper variable regions (HVRs) of VH4 are
depicted in bolded and underlined text.
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The amino acid sequence of kappa light chain variable domain variant 1 (W1)
is:
DVQITQSPSYLAASLGERATINCRASKSINKYLAWYQQKPGKTNKLLIYSGSTLQSGI
PARF SGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGQGTKLEIK (SEQ ID NO:
35). The hyper variable regions (HVRs) of W1 are depicted in bolded and
underlined text.
The amino acid sequence of kappa light chain variable domain variant 2 (Vic2)
is:
DVQITQSPSSLSASLGERATINCRASKSINKYLAWYQQKPGKANKLLIYSGSTLQSGI
PARF SGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGQGTKLEIK (SEQ ID NO:
36). The hyper variable regions (HVRs) of Vic2 are depicted in bolded and
underlined text.
The amino acid sequence of kappa light chain variable domain variant 3 (Vic3)
is:
DVQITQSPSSLSASLGERATINCRASKSINKYLAWYQQKPGKAPKWYSGSTLQSGI
PARF SGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGQGTKLEIK (SEQ ID NO:
37). The hyper variable regions (HVRs) of Vic3 are depicted in bolded and
underlined text.
The amino acid sequence of kappa light chain variable domain variant 4 (Vic4)
is:
DIQLTQSPSSLSASLGERATINCRASKSINKYLAWYQQKPGKAPKWYSGSTLQSGIP
ARF SGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTF GQGTKLEIK (SEQ ID NO:
38). The hyper variable regions (HVRs) of Vic4 are depicted in bolded and
underlined text.
In some embodiments, humanized anti-Clq antibodies of the present disclosure
include a heavy chain variable region that contains an Fab region and a heavy
chain constant
regions that contains an Fc region, where the Fab region specifically binds to
a Clq protein
of the present disclosure, but the Fc region is incapable of binding the Clq
protein. In some
embodiments, the Fc region is from a human IgGl, IgG2, IgG3, or IgG4 isotype.
In some
embodiments, the Fc region is incapable of inducing complement activity and/or
incapable of
inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments,
the Fc
region comprises one or more modifications, including, without limitation,
amino acid
substitutions. In certain embodiments, the Fc region of humanized anti-Clq
antibodies of the
present disclosure comprise an amino acid substitution at position 248
according to Kabat
numbering convention or a position corresponding to position 248 according to
Kabat
numbering convention, and/or at position 241 according to Kabat numbering
convention or a
position corresponding to position 241 according to Kabat numbering
convention. In some
embodiments, the amino acid substitution at position 248 or a positions
corresponding to
position 248 inhibits the Fc region from interacting with an Fc receptor. In
some
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embodiments, the amino acid substitution at position 248 or a positions
corresponding to
position 248 is a leucine to glutamate amino acid substitution. In some
embodiments, the
amino acid substitution at position 241 or a positions corresponding to
position 241prevent5
arm switching in the antibody. In some embodiments, the amino acid
substitution at position
241 or a positions corresponding to position 241 is a serine to proline amino
acid
substitution. In certain embodiments, the Fc region of humanized anti-Clq
antibodies of the
present disclosure comprises the amino acid sequence of SEQ ID NO: 37, or an
amino acid
sequence with at least about 70%, at least about 75%, at least about 80% at
least about 85%
at least about 90%, or at least about 95% homology to the amino acid sequence
of SEQ ID
.. NO: 47.
In some embodiments, humanized anti-Clq antibodies of the present disclosure
may
bind to a Clq protein and binds to one or more amino acids of the Clq protein
within amino
acid residues selected from (a) amino acid residues 196-226 of SEQ ID NO: 39
(SEQ ID
NO:42), or amino acid residues of a Clq protein chain A (C lqA) corresponding
to amino
acid residues 196-226 (GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:
39 (SEQ ID NO:42); (b) amino acid residues 196-221 of SEQ ID NO: 39 (SEQ ID
NO:43),
or amino acid residues of a ClqA corresponding to amino acid residues 196-221
(GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO: 39 (SEQ ID NO:43); (c)
amino acid residues 202-221 of SEQ ID NO:39 (SEQ ID NO:44), or amino acid
residues of a
.. ClqA corresponding to amino acid residues 202-221 (SGGMVLQLQQGDQVWVEKDP) of
SEQ ID NO: 39 (SEQ ID NO:44); (d) amino acid residues 202-219 of SEQ ID NO: 39
(SEQ
ID NO:45), or amino acid residues of a ClqA corresponding to amino acid
residues 202-219
(SGGMVLQLQQGDQVWVEK) of SEQ ID NO: 39 (SEQ ID NO:45); and (e) amino acid
residues Lys 219 and/or Ser 202 of SEQ ID NO: 39, or amino acid residues of a
ClqA
corresponding Lys 219 and/or Ser 202 of SEQ ID NO: 39.
In some embodiments, the humanized anti-Clq antibodies may further binds to
one or
more amino acids of the Clq protein within amino acid residues selected from:
(a) amino
acid residues 218-240 of SEQ ID NO: 41 (SEQ ID NO:46) or amino acid residues
of a Clq
protein chain C (C1qC) corresponding to amino acid residues 218-240
(WLAVNDYYDMVGI QGSDSVFSGF) of SEQ ID NO: 41 (SEQ ID NO:46); (b) amino
acid residues 225-240 of SEQ ID NO: 41 (SEQ ID NO:48) or amino acid residues
of a ClqC
corresponding to amino acid residues 225-240 (YDMVGI QGSDSVFSGF) of SEQ ID NO:
41 (SEQ ID NO:48); (c) amino acid residues 225-232 of SEQ ID NO: 41 (SEQ ID
NO:49) or
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amino acid residues of a ClqC corresponding to amino acid residues 225-232
(YDMVGIQG)
of SEQ ID NO: 41 (SEQ ID NO:49); (d) amino acid residue Tyr 225 of SEQ ID NO:
41 or an
amino acid residue of a ClqC corresponding to amino acid residue Tyr 225 of
SEQ ID NO:
41; (e) amino acid residues 174-196 of SEQ ID NO: 41 (SEQ ID NO:50) or amino
acid
residues of a ClqC corresponding to amino acid residues 174-196
(HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO: 41 (SEQ ID NO:50); (f) amino acid
residues 184-192 of SEQ ID NO: 41 (SEQ ID NO:30) or amino acid residues of a
ClqC
corresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO: 41 (SEQ
ID
NO:30); (g) amino acid residues 185-187 of SEQ ID NO: 41 or amino acid
residues of a
ClqC corresponding to amino acid residues 185-187 (SGV) of SEQ ID NO: 41; (h)
amino
acid residue Ser 185 of SEQ ID NO: 41 or an amino acid residue of a ClqC
corresponding to
amino acid residue Ser 185 of SEQ ID NO: 41.
In certain embodiments, humanized anti-Clq antibodies of the present
disclosure may
bind to amino acid residue Lys 219 and Ser 202 of the human ClqA as shown in
SEQ ID
NO: 39 or amino acids of a human ClqA corresponding to Lys 219 and Ser 202 as
shown in
SEQ ID NO: 39, and amino acid residue Tyr 225 of the human ClqC as shown in
SEQ ID
NO: 41 or an amino acid residue of a human ClqC corresponding to Tyr 225 as
shown in
SEQ ID NO: 41. In certain embodiments, the anti-Clq antibody binds to amino
acid residue
Lys 219 of the human ClqA as shown in SEQ ID NO: 39 or an amino acid residue
of a
human ClqA corresponding to Lys 219 as shown in SEQ ID NO: 39, and amino acid
residue
Ser 185 of the human ClqC as shown in SEQ ID NO: 41 or an amino acid residue
of a
human ClqC corresponding to Ser 185 as shown in SEQ ID NO: 41.
In some embodiments, humanized anti-Clq antibodies of the present disclosure
may
bind to a Clq protein and binds to one or more amino acids of the Clq protein
within amino
acid residues selected from: (a) amino acid residues 218-240 of SEQ ID NO: 41
(SEQ ID
NO:46) or amino acid residues of a ClqC corresponding to amino acid residues
218-240
(WLAVNDYYDMVGI QGSDSVFSGF) of SEQ ID NO: 41 (SEQ ID NO:46); (b) amino
acid residues 225-240 of SEQ ID NO: 41 (SEQ ID NO:48) or amino acid residues
of a ClqC
corresponding to amino acid residues 225-240 (YDMVGI QGSDSVFSGF) of SEQ ID NO:
41 (SEQ ID NO:48); (c) amino acid residues 225-232 of SEQ ID NO: 41 (SEQ ID
NO:49) or
amino acid residues of a ClqC corresponding to amino acid residues 225-232
(YDMVGIQG)
of SEQ ID NO: 41 (SEQ ID NO:49); (d) amino acid residue Tyr 225 of SEQ ID NO:
41 or an
amino acid residue of a ClqC corresponding to amino acid residue Tyr 225 of
SEQ ID NO:
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41; (e) amino acid residues 174-196 of SEQ ID NO: 41 (SEQ ID NO:50) or amino
acid
residues of a ClqC corresponding to amino acid residues 174-196
(HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO: 41 (SEQ ID NO:50); (f) amino acid
residues 184-192 of SEQ ID NO: 41 (SEQ ID NO:50) or amino acid residues of a
ClqC
.. corresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO: 41
(SEQ ID
NO:50); (g) amino acid residues 185-187 of SEQ ID NO: 41 or amino acid
residues of a
ClqC corresponding to amino acid residues 185-187 (SGV) of SEQ ID NO: 41; (h)
amino
acid residue Ser 185 of SEQ ID NO: 41 or an amino acid residue of a ClqC
corresponding to
amino acid residue Ser 185 of SEQ ID NO: 41.
Anti-Complement Cis Antibodies
Suitable inhibitors include an antibody that binds complement Cis protein
(i.e., an
anti-complement Cis antibody, also referred to herein as an anti-Cis antibody
and a Cis
antibody) and a nucleic acid molecule that encodes such an antibody.
Complement Cis is an
attractive target as it is upstream in the complement cascade and has a narrow
range of
substrate specificity. Furthermore it is possible to obtain antibodies (for
example, but not
limited to, monoclonal antibodies) that specifically bind the activated form
of Cls.
All sequences mentioned in the following two paragraphs are incorporated by
reference from WO 2014/186599, which is hereby incorporated by reference for
the
antibodies and related compositions that it discloses.
In certain aspects, disclosed herein are methods of administering an anti-Cis
antibody. The antibody may be a murine, humanized, or chimeric antibody. In
some
embodiments, the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-
L3,
and the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3 of a murine anti-
human
Cis monoclonal antibody 5A1 produced by a hybridoma cell line deposited with
ATCC on
5/15/2013 or progeny thereof (ATCC Accession No. PTA-120351). In other
embodiments,
the light chain variable domain comprises the HVR-L1, HVR-L2, and HVR-L3 and
the
heavy chain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3 of a
murine
anti-human Cis monoclonal antibody 5C12 produced by a hybridoma cell line
deposited with
ATCC on 5/15/2013, or progeny thereof (ATCC Accession No. PTA-120352).
In some embodiments, antibodies specifically bind to and inhibit a biological
activity
of Cis or the Cis proenzyme, such as Cis binding to Clq, Cis binding to Clr,
or Cis
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binding to C2 or C4. The biological activity may be a proteolytic enzyme
activity of Cl s, the
conversion of the Cis proenzyme to an active protease, or proteolytic cleavage
of C2 or C4.
In certain embodiments, the biological activity is activation of the classical
complement
activation pathway, activation of antibody and complement dependent
cytotoxicity, or C1F
.. hemolysis.
All sequences in the following sixty-two paragraphs are incorporated by
reference
from Van Vlasselaer, U.S. Pat. No. 8,877,197, which is hereby incorporated by
reference for
the antibodies and related compositions that it discloses.
Disclosed herein are methods of administering a humanized monoclonal antibody
.. that specifically binds an epitope within a region encompassing domains IV
and V of
complement component Cis. In some cases, the antibody inhibits binding of Cis
to
complement component 4 (C4) and/or does not inhibit protease activity of Cl s.
In some
embodiments, the method comprises administering a humanized monoclonal
antibody that
binds complement component Cis in a Cl complex with high avidity.
Disclosed herein are methods of administering an anti-Cis antibody with one or
more
of the complementarity determining regions (CDRs) of an antibody light chain
variable
region comprising amino acid sequence SEQ ID NO:57 and/or one or more of the
CDRs of
an antibody heavy chain variable region comprising amino acid sequence SEQ ID
NO:58.
The anti-Cis antibody may bind a human or rat complement Cls protein. In some
embodiments, an anti-Cis antibody inhibits cleavage of at least one substrate
cleaved by
complement Cls protein.
In certain embodiments, the antibody comprises: a) a complementarity
determining
region (CDR) having an amino acid sequence selected from SEQ ID NO:51, SEQ ID
NO:52,
SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56; and/ orb) a CDR
having an amino acid sequence selected from SEQ ID NO:62, SEQ ID NO:63, SEQ ID
NO:53, SEQ ID NO:64, SEQ ID NO:65: and SEQ ID NO:66.
The antibody may comprise a CDR-L1 having amino acid sequence SEQ ID NO:51, a
CDR-L2 having amino acid sequence SEQ ID NO:52, a CDR-L3 having amino acid
sequence SEQ ID NO:53, a CDR-H1 having amino acid sequence SEQ ID NO:54, a CDR-
H2 having amino acid sequence SEQ ID NO:55, and a CDR-H3 having amino acid
sequence
SEQ ID NO:56.
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In other embodiments, the antibody may comprise light chain CDRs of a variable
region with an amino acid sequence of SEQ ID NO:67, and/or heavy chain CDRs of
a
variable region with an amino acid sequence of SEQ ID NO:68.
The antibody can be a humanized antibody that specifically binds complement
component Cis, wherein the antibody competes for binding the epitope with an
antibody that
comprises one or more of the CDRs of an antibody light chain variable region
comprising
amino acid sequence SEQ ID NO:57 or SEQ ID NO:67, and/or one or more of the
CDRs of
an antibody heavy chain variable region comprising amino acid sequence SEQ ID
NO:58 or
SEQ ID NO:68.
In other instances, the antibody can be a humanized antibody that specifically
binds
complement Cis, wherein the antibody is selected from: a) a humanized antibody
that
specifically binds an epitope within the complement Cls protein, wherein the
antibody
competes for binding the epitope with an antibody that comprises a CDR having
an amino
acid sequence selected from SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID
NO:54, SEQ ID NO:55, and SEQ ID NO:56; and b) a humanized antibody that
specifically
binds an epitope within the complement Cls protein, wherein the antibody
competes for
binding the epitope with an antibody that comprises a CDR having an amino acid
sequence
selected from SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64, SEQ ID
NO:65, and SEQ ID NO:66. In some cases, the antibody competes for binding the
epitope
with an antibody that comprises heavy and light chain CDRs comprising: a) SEQ
ID NO:51,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:69, SEQ ID NO:55, and SEQ ID NO:56; orb)
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64, SEQ ID NO:65, and SEQ
ID NO:66.
The antibody may comprise a light chain region and a heavy chain region that
are
present in separate polypeptides. The antibody may comprise an Fc region.
Disclosed herein is an anti-Cis antibody comprising a light chain variable
region of
an amino acid sequence that is 90% identical to amino acid sequence SEQ ID
NO:57, and a
heavy chain variable region comprising an amino acid sequence that is 90%
identical to
amino acid sequence SEQ ID NO:58.
The anti-Cis antibody may be selected from an antigen binding fragment, Ig
monomer, a Fab fragment, a F(ab1)2fragment, a Fd fragment, a scFv, a scAb, a
dAb, a Fv, a
single domain heavy chain antibody, a single domain light chain antibody, a
mono-specific
antibody, a bi-specific antibody, or a multi-specific antibody.
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Disclosed herein are methods of administering an antibody that competes for
binding
the epitope bound by antibody IPN003 (also referred to herein as "IPN-M34" or
"M34" or
"TNT003"), e.g., an antibody comprising a variable domain of antibody IPN003,
such as
antibody IPN003.
In some embodiments, the method comprises administering an antibody that
specifically binds an epitope within a complement Cis protein. In some
embodiments, the
isolated anti-Cis antibody binds an activated Cis protein. In some
embodiments, the isolated
anti-Cis antibody binds an inactive form of Cis. In other instances, the
isolated anti-Cis
antibody binds both an activated Cls protein and an inactive form of Cl s.
In some embodiments, the method comprises administering a monoclonal antibody
that inhibits cleavage of C4, where the isolated monoclonal antibody does not
inhibit
cleavage of C2. In some embodiments, the method comprises administering a
monoclonal
antibody that inhibits cleavage of C2, where the isolated monoclonal antibody
does not
inhibit cleavage of C4. In some cases, the isolated monoclonal antibody is
humanized. In
some cases, the antibody inhibits a component of the classical complement
pathway. In some
cases, the component of the classical complement pathway that is inhibited by
the antibody is
Cis. The present disclosure also provides methods of treating a complement-
mediated
disease or disorder, by administering to an individual in need thereof an
isolated monoclonal
antibody that inhibits cleavage of C4, or a pharmaceutical composition
comprising the
isolated monoclonal antibody, where the isolated monoclonal antibody does not
inhibit
cleavage of C2.
In some embodiments, the method comprises administering a monoclonal antibody
that inhibits cleavage of C2 or C4 by Cis, i.e., inhibits Cls-mediated
proteolytic cleavage of
C2 or C4. In some cases, the monoclonal antibody is humanized. In some cases,
the antibody
inhibits cleavage of C2 or C4 by Cis by inhibiting binding of C2 or C4 to Cis;
for example,
in some cases, the antibody inhibits Cls-mediated cleavage of C2 or C4 by
inhibiting binding
of C2 or C4 to a C2 or C4 binding site of Cls. Thus, in some cases, the
antibody functions as
a competitive inhibitor.
The present disclosure also provides methods of treating
frontotemporal dementia, by administering to an individual in need thereof an
isolated
monoclonal antibody that inhibits cleavage of C2 or C4 by Cis, i.e., inhibits
Cis-mediated
proteolytic cleavage of C2 or C4.
In some embodiments, the method comprises administering a monoclonal antibody
that inhibits cleavage of C4 by C is, where the antibody does not inhibit
cleavage of
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complement component C2 by Cis; i.e., the antibody inhibits Cls-mediated
cleavage of C4,
but does not inhibit Cls-mediated cleavage of C2. In some cases, the
monoclonal antibody is
humanized. In some cases, the monoclonal antibody inhibits binding of C4 to
Cis, but does
not inhibit binding of C2 to Cis. In some embodiments, the method comprises
treating a
.. complement-mediated disease or disorder, by administering to an individual
in need thereof
an isolated monoclonal antibody that inhibits cleavage of C4 by Cis, where the
antibody
does not inhibit cleavage of complement component C2 by Cis; i.e., the
antibody inhibits
Cis-mediated cleavage of C4, but does not inhibit Cis-mediated cleavage of C2.
In some
embodiments of the method, the antibody is humanized.
In some embodiments, the method comprises administering a humanized monoclonal
antibody that specifically binds an epitope within a region encompassing
domains IV and V
of Cis. For example, the humanized monoclonal antibody specifically binds an
epitope
within amino acids 272-422 of the amino acid sequence depicted in FIG. 1 and
set forth in
SEQ ID NO:70. In some cases, the humanized monoclonal antibody specifically
binds an
epitope within amino acids 272-422 of the amino acid sequence depicted in FIG.
1 and set
forth in SEQ ID NO:70, and inhibits binding of C4 to Cis. In some embodiments,
the
method comprises treating a complement-mediated disease or disorder, by
administering to
an individual in need thereof a humanized monoclonal antibody that
specifically binds an
epitope within amino acids 272-422 of the amino acid sequence depicted in FIG.
1 and set
forth in SEQ ID NO:70, and inhibits binding of C4 to Cis.
In some embodiments, the method comprises administering a humanized monoclonal
antibody that specifically binds a conformational epitope within a region
encompassing
domains IV and V of Cis. For example, the humanized monoclonal antibody that
specifically
binds a conformational epitope within amino acids 272-422 of the amino acid
sequence
depicted in FIG. 1 and set forth in SEQ ID NO:70. In some cases, the humanized
monoclonal
antibody specifically binds a conformational epitope within amino acids 272-
422 of the
amino acid sequence depicted in FIG. 1 and set forth in SEQ ID NO:70, and
inhibits binding
of C4 to Cis. In some embodiments, the method comprises frontotemporal
dementia, the
method comprising administering to an individual in need thereof a humanized
monoclonal
antibody that specifically binds a conformational epitope within amino acids
272-422 of the
amino acid sequence depicted in FIG. 1 and set forth in SEQ ID NO:70, and
inhibits binding
of C4 to Cis.
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In some embodiments, the method comprises administering a monoclonal antibody
that binds complement component Cis in a Cl complex. The Cl complex is
composed of 6
molecules of Clq, 2 molecules of Clr, and 2 molecules of Cis. In some cases,
the
monoclonal antibody is humanized. Thus, in some cases, the humanized
monoclonal
antibody that binds complement component Cis in a Cl complex. In some cases,
the
antibody binds Cis present in a Cl complex with high avidity.
In some embodiments, the anti-CI s antibody (e.g., a subject antibody that
specifically
binds an epitope in a complement Cis protein) comprises: a) a light chain
region comprising
one, two, or three VL CDRs of an IPN003 antibody; and b) a heavy chain region
comprising
one, two, or three VH CDRs of an IPN003 antibody; where the VH and VL CDRs are
as
defined by Kabat (see, e.g., Table 1; and Kabat 1991).
In other embodiments, the anti-Cis antibody (e.g., a subject antibody that
specifically
binds an epitope in a complement Cis protein) comprises: a) a light chain
region comprising
one, two, or three VL CDRs of an IPN003 antibody; and b) a heavy chain region
comprising
one, two, or three VH CDRs of an IPN003 antibody; where the VH and VL CDRs are
as
defined by Chothia (see, e.g., Table 1, and Chothia 1987).
CDR amino acid sequences, and VL and VH amino acid sequences, of IPN003
antibody are provided in Table 2. Table 2 also provides the SEQ ID NOs
assigned to each of
the amino acid sequences.
In some embodiments, the anti-CI s antibody (e.g., a subject antibody that
specifically
binds an epitope in a complement Cis protein) comprises: a) a light chain
region comprising
one, two, or three CDRs selected from SEQ ID NO:51, SEQ ID NO:52, and SEQ ID
NO:53;
and b) a heavy chain region comprising one, two, or three CDRs selected from
SEQ ID
NO:54, SEQ ID NO:55, and SEQ ID NO:56. In some of these embodiments, the anti-
Cis
antibody includes a humanized VH and/or VL framework region.
SEQ ID NO. 51: SSVS S SYLHWYQ;
SEQ ID NO. 52: STSNLASGVP;
SEQ ID NO. 53: HQYYRLPPIT;
SEQ ID NO. 54: GF TF SNYAMSWV;
SEQ ID NO. 55: IS SGGSHTYY;
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SEQ ID NO. 56: ARLFTGYAMDY.
In some embodiments, the anti-C is antibody comprises a CDR having an amino
acid
sequence selected from SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54,
SEQ ID NO:55, and SEQ ID NO:56.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising amino acid sequences SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.
In some embodiments, the anti-C is antibody comprises a heavy chain variable
region
comprising amino acid sequences SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.
In some embodiments, the anti-Cis antibody comprises a CDR-L1 having amino
acid
sequence SEQ ID NO:51, a CDR-L2 having amino acid sequence SEQ ID NO:52, a CDR-
L3
having amino acid sequence SEQ ID NO:53, a CDR-H1 having amino acid sequence
SEQ ID
NO:54, a CDR-H2 having amino acid sequence SEQ ID NO:55, and a CDR-H3 having
amino acid sequence SEQ ID NO:56.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set
forth in
SEQ ID NO:57.
SEQ ID NO. 57:
DIVMTQTTAIMSASLGERVTMTCTASS SVS SSYLHWYQQKPGS SPKLWIYSTSNLAS
GVPARF SGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK.
In some embodiments, the anti-C is antibody comprises a heavy chain variable
region
comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set
forth in
SEQ ID NO. 58.
SEQ ID NO. 58:
QVKLEE SGGALVKP GGSLKL S CAA S GF TF SNYAMSWVRQIPEKRLEWVATIS SGGSH
TYYLDSVKGRFTISRDNARDTLYLQMS SLRSED TALYYC ARLF T GYAMDYWGQ GT S
VT.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising an amino acid sequence that is 90% identical to amino acid sequence
SEQ ID
NO:57.
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In some embodiments, the anti-CI s antibody comprises a heavy chain variable
region
comprising an amino acid sequence that is 90% identical to amino acid sequence
SEQ ID
NO:58.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising amino acid sequence SEQ ID NO:57.
In some embodiments, the anti-CI s antibody comprises a heavy chain variable
region
comprising amino acid sequence SEQ ID NO:58.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising an amino acid sequence that is 90% identical to amino acid sequence
SEQ ID
NO:57 and a heavy chain variable region comprising an amino acid sequence that
is 90%
identical to amino acid sequence SEQ ID NO:58.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising amino acid sequence SEQ ID NO:57 and a heavy chain variable region
comprising amino acid sequence SEQ ID NO:58.
In some embodiments, the anti-Cis antibody specifically binds an epitope
within the
complement C is protein, wherein the antibody competes for binding the epitope
with an
antibody that comprises light chain CDRs of an antibody light chain variable
region
comprising amino acid sequence SEQ ID NO:57 and heavy chain CDRs of an
antibody heavy
chain variable region comprising amino acid sequence SEQ ID NO:58.
In some embodiments, the anti-Cis antibody comprises light chain CDRs of an
antibody light chain variable region comprising amino acid sequence SEQ ID
NO:57 and
heavy chain CDRs of an antibody heavy chain variable region comprising amino
acid
sequence SEQ ID NO:58.
In some embodiments, the anti-CI s antibody (e.g., a subject antibody that
specifically
binds an epitope in a complement Cis protein) comprises: a) a light chain
region comprising
one, two, or three CDRs selected from SEQ ID NO:62, SEQ ID NO:63, and SEQ ID
NO:53;
and b) a heavy chain region comprising one, two, or three CDRs selected from
SEQ ID
NO:64, SEQ ID NO:65, and SEQ ID NO:66.
SEQ ID NO.62: TASSSVSSSYLH;
SEQ ID NO. 63: STSNLAS;
SEQ ID NO.53: HQYYRLPPIT;
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SEQ ID NO.64: NYAMS;
SEQ ID NO.65: TISSGGSHTYYLDSVKG;
SEQ ID NO.66: LFTGYAMDY
In some embodiments, the anti-C is antibody comprises a CDR having an amino
acid
sequence selected from SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64,
SEQ ID NO:65, and SEQ ID NO:66.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising amino acid sequences SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:53.
In some embodiments, the anti-C is antibody comprises a heavy chain variable
region
comprising amino acid sequences SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.
In some embodiments, the anti-Cis antibody comprises a CDR-L1 having amino
acid
sequence SEQ ID NO:62, a CDR-L2 having amino acid sequence SEQ ID NO:63, a CDR-
L3
having amino acid sequence SEQ ID NO:53, a CDR-H1 having amino acid sequence
SEQ ID
NO:64, a CDR-H2 having amino acid sequence SEQ ID NO:65, and a CDR-H3 having
amino acid sequence SEQ ID NO:66.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set
forth in
SEQ ID NO:67.
SEQ ID NO. 67:
QIVLTQSPAIMSASLGERVTMTCTAS S SVS S SYLHWYQQKPGS SPKLWIYSTSNLASG
VPARF SGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK.
In some embodiments, the anti-C is antibody comprises a heavy chain variable
region
comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set
forth in
SEQ ID NO:68.
SEQ ID NO. 68:
EVMLVESGGALVKPGGSLKL S CAA S GF TF SNYAMSWVRQIPEKRLEWVATIS SGGSH
TYYLD SVKGRF TI SRDNARD TLYL QM S SLRSED TALYYC ARLF T GYAMDYWGQ GT S
VTVSS.
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In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising an amino acid sequence that is 90% identical to amino acid sequence
SEQ ID
NO:67.
In some embodiments, the anti-C is antibody comprises a heavy chain variable
region
comprising an amino acid sequence that is 90% identical to amino acid sequence
SEQ ID
NO:68.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising amino acid sequence SEQ ID NO:67.
In some embodiments, the anti-C is antibody comprises a heavy chain variable
region
comprising amino acid sequence SEQ ID NO:68.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising an amino acid sequence that is 90% identical to amino acid sequence
SEQ ID
NO:67 and a heavy chain variable region comprising an amino acid sequence that
is 90%
identical to amino acid sequence SEQ ID NO:68.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising an amino acid sequence that is 95% identical to amino acid sequence
SEQ ID
NO:67 and a heavy chain variable region comprising an amino acid sequence that
is 95%
identical to amino acid sequence SEQ ID NO:68.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising amino acid sequence SEQ ID NO:67 and a heavy chain variable region
comprising amino acid sequence SEQ ID NO:68.
In some embodiments, the anti-CI s antibody specifically binds an epitope
within the
complement C is protein, wherein the antibody competes for binding the epitope
with an
antibody that comprises light chain CDRs of an antibody light chain variable
region
comprising amino acid sequence SEQ ID NO:67 and heavy chain CDRs of an
antibody heavy
chain variable region comprising amino acid sequence SEQ ID NO:68.
In some embodiments, the anti-Cis antibody comprises light chain CDRs of an
antibody light chain variable region comprising amino acid sequence SEQ ID
NO:67 and
heavy chain CDRs of an antibody heavy chain variable region comprising amino
acid
sequence SEQ ID NO:68.
In some embodiments, the anti-Cis antibody comprises a light chain variable
region
comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%,
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93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set
forth in
SEQ ID NO:67.
In some embodiments, the anti-CI s antibody comprises a heavy chain variable
region
comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set
forth in
SEQ ID NO:68.
An anti-Cis antibody can comprise a heavy chain variable region comprising an
amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in
SEQ ID
i0 NO:79 and depicted in FIG. 2 (VH variant 1).
An anti-Cis antibody can comprise a heavy chain variable region comprising an
amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in
SEQ ID
NO:80 and depicted in FIG. 3 (VH variant 2).
An anti-Cis antibody can comprise a heavy chain variable region comprising an
amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in
SEQ ID
NO:81 and depicted in FIG. 4 (VH variant 3).
An anti-Cis antibody can comprise a heavy chain variable region comprising an
amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in
SEQ ID
NO:82 and depicted in FIG. 5 (VH variant 4).
An anti-Cis antibody can comprise a light chain variable region comprising an
amino
acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%,
97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ
ID NO:83
and depicted in FIG. 6 (VK variant 1).
An anti-Cis antibody can comprise a light chain variable region comprising an
amino
acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%,
97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ
ID NO:84
and depicted in FIG. 7 (VK variant 2).
An anti-Cis antibody can comprise a light chain variable region comprising an
amino
acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%,
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97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ
ID NO:85
and depicted in FIG. 8 (VK variant 3).
An anti-Cis antibody can comprise a heavy chain variable region comprising 1,
2, 3,
4, 5, 6, 7, 8, 9, 10, 11, or 12 of the framework (FR) amino acid
substitutions, relative to the
IPN003 parental antibody FR amino acid sequences, depicted in Table 3 (FIG.
9).
Definitions
As used herein the specification, "a" or "an" may mean one or more. As used
herein
in the claim(s), when used in conjunction with the word "comprising", the
words "a" or "an"
may mean one or more than one. For example, reference to an "antibody" is a
reference from
one to many antibodies. As used herein "another" may mean at least a second or
more.
As used herein, administration "conjointly" with another compound or
composition
includes simultaneous administration and/or administration at different times.
Administration in conjunction also encompasses administration as a co-
formulation or
administration as separate compositions, including at different dosing
frequencies or
intervals, and using the same route of administration or different routes of
administration.
The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.
The
term "antibody" herein is used in the broadest sense and specifically covers
monoclonal
antibodies, polyclonal antibodies, multi specific 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 basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of
two
identical light (L) chains and two identical heavy (H) chains. The pairing of
a VH and VL
together forms a single antigen-binding site. For the structure and properties
of the different
classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed.,
Daniel P. Stites,
Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT,
1994, page 71
and Chapter 6.
The L chain from any vertebrate species can be assigned to one of two clearly
distinct
types, called kappa ("x") and lambda ("k"), based on the amino acid sequences
of their
constant domains. Depending on the amino acid sequence of the constant domain
of their
heavy chains (CH), immunoglobulins can be assigned to different classes or
isotypes. There
are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy
chains
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designated alpha ("a"), delta ("6"), epsilon ("c"), gamma ("y") and mu CO,
respectively.
The y and a classes are further divided into subclasses (isotypes) on the
basis of relatively
minor differences in the CH sequence and function, e.g., humans express the
following
subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The subunit structures and
three
dimensional configurations of different classes of immunoglobulins are well
known and
described generally in, for example, Abbas et at., Cellular and Molecular
Immunology, 4th
ed. (W.B. Saunders Co., 2000).
"Native antibodies" are usually heterotetrameric glycoproteins of about
150,000
daltons, composed of two identical light (L) chains and two identical heavy
(H) chains. Each
light chain is linked to a heavy chain by one covalent disulfide bond, while
the number of
disulfide linkages varies among the heavy chains of different immunoglobulin
isotypes. Each
heavy and light chain also has regularly spaced intrachain disulfide bridges.
Each heavy
chain has at one end a variable domain (VH) followed by a number of constant
domains.
Each light chain has a variable domain at one end (VI) and a constant domain
at its other end;
the constant domain of the light chain is aligned with the first constant
domain of the heavy
chain, and the light chain variable domain is aligned with the variable domain
of the heavy
chain. Particular amino acid residues are believed to form an interface
between the light
chain and heavy chain variable domains.
An "isolated" molecule or cell is a molecule or a cell that is identified and
separated
from at least one contaminant molecule or cell with which it is ordinarily
associated in the
environment in which it was produced. Preferably, the isolated molecule or
cell is free of
association with all components associated with the production environment.
The isolated
molecule or cell is in a form other than in the form or setting in which it is
found in nature.
Isolated molecules therefore are distinguished from molecules existing
naturally in cells;
isolated cells are distinguished from cells existing naturally in tissues,
organs, or individuals.
In some embodiments, the isolated molecule is an anti-Cis, anti-Clq, or anti-
Clr antibody of
the present disclosure. In other embodiments, the isolated cell is a host cell
or hybridoma cell
producing an anti-Cis, anti-Clq, or anti-Clr antibody of the present
disclosure.
An "isolated' antibody is one that has been identified, separated and/or
recovered
from a component of its production environment (e.g., naturally or
recombinantly).
Preferably, the isolated polypeptide is free of association with all other
contaminant
components from its production environment. Contaminant components from its
production
environment, such as those resulting from recombinant transfected cells, are
materials that
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would typically interfere with research, diagnostic or therapeutic uses for
the antibody, and
may include enzymes, hormones, and other proteinaceous or non-proteinaceous
solutes. In
certain preferred embodiments, the polypeptide will be purified: (1) to
greater than 95% by
weight of antibody as determined by, for example, the Lowry method, and in
some
embodiments, to greater than 99% by weight; (2) to a degree sufficient to
obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a spinning
cup sequenator,
or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions
using
Coomassie blue or, preferably, silver stain. An isolated antibody includes the
antibody in situ
within recombinant T-cells since at least one component of the antibody's
natural
environment will not be present. Ordinarily, however, an isolated polypeptide
or antibody
will be prepared by a process including at least one purification step.
The "variable region" or "variable domain" of an antibody refers to the amino-
terminal domains of the heavy or light chain of the antibody. The variable
domains of the
heavy chain and light chain may be referred to as "VH" and "VL", respectively.
These
domains are generally the most variable parts of the antibody (relative to
other antibodies of
the same class) and contain the antigen binding sites.
The term "variable" refers to the fact that certain segments of the variable
domains
differ extensively in sequence among antibodies. The V domain mediates antigen
binding
and defines the specificity of a particular antibody for its particular
antigen. However, the
variability is not evenly distributed across the entire span of the variable
domains. Instead, it
is concentrated in three segments called hypervariable regions (HVRs) both in
the light-chain
and the heavy chain variable domains. The more highly conserved portions of
variable
domains are called the framework regions (FR). The variable domains of native
heavy and
light chains each comprise four FR regions, largely adopting a beta-sheet
configuration,
connected by three HVRs, which form loops connecting, and in some cases
forming part of,
the beta-sheet structure. The HVRs in each chain are held together in close
proximity by the
FR regions and, with the HVRs from the other chain, contribute to the
formation of the
antigen binding site of antibodies (see Kabat et al., Sequences of
Immunological Interest,
Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The
constant domains are
not involved directly in the binding of antibody to an antigen, but exhibit
various effector
functions, such as participation of the antibody in antibody-dependent-
cellular toxicity.
As used herein, the term "CDR" or "complementarity determining region" is
intended
to mean the non-contiguous antigen binding sites found within the variable
region of both
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heavy and light chain polypeptides. CDRs have been described by Kabat et al.,
J. Biol. Chem.
252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services,
"Sequences
of proteins of immunological interest" (1991) (also referred to herein as
Kabat 1991); by
Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as
Chothia 1987);
and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions
include
overlapping or subsets of amino acid residues when compared against each
other.
Nevertheless, application of either definition to refer to a CDR of an
antibody or grafted
antibodies or variants thereof is intended to be within the scope of the term
as defined and
used herein. The amino acid residues, which encompass the CDRs, as defined by
each of the
above cited references are set forth below in Table 1 as a comparison. The
CDRs listed in
Table 2 were defined in accordance with Kabat 1991.
Table 1
CDR Definitions
Kabat Chothia2 iviacCallum3
VH CDR-1 31_35 26-32 30-35
VH CDR-2 50-65 53-55 47-58
Vi-1 CDR-3 95-102 96-101 93-101
VL CDR-1 24-34 26-32 30-36
VL CDR-2 50-56 50-52 46-55
VL CDR-3 89-97 91-96 89-96
'Residue numbering follows the nomenclature of Kabat et al., supra
2Residue numbering follows the nomenclature of Chothia et al., supra
3Residue numbering follows the nomenclature of MacCallum et al., supra
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Tattle 2
I ,Arttibody CM:Z-1 CDR-2 CDR-3 rogion
11Pt44134 VI SE0 ID N051 SW 0 NO.52 ..SEO ID NO .53 SEC: ID NO.57
=
DSVSSSYLHWY0 SISNLASGVP HOYYRLPPIT IDIVMICRTAIMSASIDERVTNITCTASSSVSSSYL
IHWYOQKPGSSPKLWIYSTSNLASGVPARFSDSGi
ISGITYSITMSMEAEDDATYYCHWYRIPMFG
MITKLEIK
11PN-M34 VH SE0 10 NO.54 SEG 0 NO .55 5E0,10 NO .56 ISEC.:? ID NO .58
GF1TSNYAMSWV ISSGGSHIYIF ARLF1GYAMOY1OVKLEESDGALVKPGGSLKISCAASOFTFSNYA
fASWVROIPEKRIEWVATISSGGSHTYYLDSVK
GRFTISRDNARDTLYLQMSSIRSEDTALYYCAR
IFIGYAMOYAKMGTSVT
As used herein, the terms "CDR-L1", "CDR-L2", and "CDR-L3" refer,
respectively,
to the first, second, and third CDRs in a light chain variable region. As used
herein, the terms
"CDR-H1", "CDR-H2", and "CDR-H3" refer, respectively, to the first, second,
and third
CDRs in a heavy chain variable region. As used herein, the terms "CDR-1", "CDR-
2", and
"CDR-3" refer, respectively, to the first, second and third CDRs of either
chain's variable
region.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies of the
population are identical except for possible naturally occurring mutations
and/or post-
translation modifications (e.g., isomerizations, amidations) that may be
present in minor
amounts. Monoclonal antibodies are highly specific, being directed against a
single antigenic
site. In contrast to polyclonal antibody preparations which typically 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,
.. monoclonal antibodies are advantageous since they are typically synthesized
by hybridoma
culture, uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates
the character of the antibody as being obtained as 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 disclosure may be made by a variety of techniques, including, for
example, the
hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo
et al.,
Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory
Manual, (Cold
Spring Harbor Laboratory Press, 2d ed. 1988); Hammerling et al., in:
Monoclonal Antibodies
and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods
(see,
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e.g., U.S. Patent No. 4,816,567), phage-display technologies (see, e.g.,
Clackson et at.,
Nature, 352:624-628 (1991); Marks et at., I Mot. Biol. 222:581-597 (1992);
Sidhu et at.,
Mot. Biol. 338(2): 299-310 (2004); Lee et al., I Mot. Biol. 340(5):1073-1093
(2004);
Fellouse, Proc. Nat? Acad. Sci. USA 101(34):12467-472 (2004); and Lee et at.,
I Immunol.
Methods 284(1-2):119-132 (2004), and technologies for producing human or human-
like
antibodies in animals that have parts or all of the human immunoglobulin loci
or genes
encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO
1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Nat'l Acad. Sci. USA
90:2551
(1993); Jakobovits et at., Nature 362:255-258 (1993); Bruggemann et at., Year
in Immunol.
7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; and
5,661,016; Marks et at., Bio/Technology 10:779-783 (1992); Lonberg et at.,
Nature 368:856-
859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature
Biotechnol.
14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg
and Huszar,
Intern. Rev. Immunol. 13:65-93 (1995).
The terms "full-length antibody," "intact antibody" and "whole antibody" are
used
interchangeably to refer to an antibody in its substantially intact form, as
opposed to an
antibody fragment. Specifically, whole antibodies include those with heavy and
light chains
including an Fc region. The constant domains may be native sequence constant
domains
(e.g., human native sequence constant domains) or amino acid sequence variants
thereof. In
some cases, the intact antibody may have one or more effector functions.
An "antibody fragment" comprises a portion of an intact antibody, preferably
the
antigen binding and/or the variable region of the intact antibody. Examples of
antibody
fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear
antibodies (see U.S.
Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062
(1995)); single-
chain antibody molecules and multispecific antibodies formed from antibody
fragments.
Papain digestion of antibodies produces two identical antigen-binding
fragments,
called "Fab" fragments, and a residual "Fe" fragment, a designation reflecting
the ability to
crystallize readily. The Fab fragment consists of an entire L chain along with
the variable
region domain of the H chain (VH), and the first constant domain of one heavy
chain (CH1).
Each Fab fragment is monovalent with respect to antigen binding, i.e., it has
a single antigen-
binding site. Pepsin treatment of an antibody yields a single large F(ab')2
fragment which
roughly corresponds to two disulfide linked Fab fragments having different
antigen-binding
activity and is still capable of cross-linking antigen. Fab' fragments differ
from Fab
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fragments by having a few additional residues at the carboxy terminus of the
CH1 domain
including one or more cysteines from the antibody hinge region. Fab'-SH is the
designation
herein for Fab' in which the cysteine residue(s) of the constant domains bear
a free thiol
group. F(ab')2 antibody fragments originally were produced as pairs of Fab'
fragments with
hinge cysteines between them. Other chemical couplings of antibody fragments
are also
known.
The Fc fragment comprises the carboxy-terminal portions of both H chains held
together by disulfides. The effector functions of antibodies are determined by
sequences in
the Fc region, the region which is also recognized by Fc receptors (FcR) found
on certain
types of cells.
The term "Fc region" herein is used to define a C-terminal region of an
immunoglobulin heavy chain, including native-sequence Fc regions and variant
Fc regions.
Although the boundaries of the Fc region of an immunoglobulin heavy chain
might vary, the
human IgG heavy-chain Fc region is usually defined to stretch from an amino
acid residue at
position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-
terminal lysine
(residue 447 according to the EU numbering system) of the Fc region may be
removed, for
example, during production or purification of the antibody, or by
recombinantly engineering
the nucleic acid encoding a heavy chain of the antibody. Accordingly, a
composition of
intact antibodies may comprise antibody populations with all K447 residues
removed,
antibody populations with no K447 residues removed, and antibody populations
having a
mixture of antibodies with and without the K447 residue. Suitable native-
sequence Fc
regions for use in the antibodies of the disclosure include human IgGl, IgG2,
IgG3 and IgG4.
A "native sequence Fc region" comprises an amino acid sequence identical to
the
amino acid sequence of an Fc region found in nature. Native sequence human Fc
regions
include a native sequence human IgG1 Fc region (non-A and A allotypes); native
sequence
human IgG2 Fc region; native sequence human IgG3 Fc region; and native
sequence human
IgG4 Fc region as well as naturally occurring variants thereof.
A "variant Fc region" comprises an amino acid sequence which differs from that
of a
native sequence Fc region by virtue of at least one amino acid modification,
preferably one or
more amino acid substitution(s). Preferably, the variant Fc region has at
least one amino acid
substitution compared to a native sequence Fc region or to the Fc region of a
parent
polypeptide, e.g., from about one to about ten amino acid substitutions, and
preferably from
about one to about five amino acid substitutions in a native sequence Fc
region or in the Fc
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region of the parent polypeptide. The variant Fe region herein will preferably
possess at least
about 80% homology with a native sequence Fe region and/or with an Fe region
of a parent
polypeptide, and most preferably at least about 90% homology therewith, more
preferably at
least about 95% homology therewith.
"Fc receptor" or "FcR" describes a receptor that binds to the Fe region of an
antibody. The preferred FcR is a native sequence human FcR. Moreover, a
preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes receptors of
the FcyRI,
FcyRII, and FcyRIII subclasses, including allelic variants and alternatively
spliced forms of
these receptors, FcyRII receptors include FcyRIIA (an "activating receptor")
and FcyRIM (an
"inhibiting receptor"), which have similar amino acid sequences that differ
primarily in the
cytoplasmic domains thereof. Activating receptor FcyRIIA contains an
immunoreceptor
tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibiting
receptor
FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in
its
cytoplasmic domain. (See, e.g., M. Daeron, Annu. Rev. Immunol. 15:203-234
(1997)). FcRs
are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel
et al.,
Immunomethods 4:25-34 (1994); and de Haas et al., I Lab. Cl/n. Med. 126: 330-
41 (1995).
Other FcRs, including those to be identified in the future, are encompassed by
the term "FcR"
herein. FcRs can also increase the serum half-life of antibodies.
Binding to FcRn in vivo and serum half-life of human FcRn high-affinity
binding
polypeptides can be assayed, e.g., in transgenic mice or transfected human
cell lines
expressing human FcRn, or in primates to which the polypeptides having a
variant Fe region
are administered. WO 2004/42072 (Presta) describes antibody variants with
improved or
diminished binding to FcRs. See also, e.g., Shields et al., I Biol. Chem.
9(2):6591-6604
(2001).
"Fv" is the minimum antibody fragment, which contains a complete antigen-
recognition and -binding site. This fragment consists of a dimer of one heavy-
and one light-
chain variable region domain in tight, non-covalent association. From the
folding of these
two domains emanate six hypervariable loops (3 loops each from the H and L
chain) that
contribute the amino acid residues for antigen binding and confer antigen
binding specificity
to the antibody. However, even a single variable domain (or half of an Fv
comprising only
three HVRs specific for an antigen) has the ability to recognize and bind
antigen, although at
a lower affinity than the entire binding site.
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"Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments
that
comprise the VH and VL antibody domains connected into a single polypeptide
chain.
Preferably, the sFv polypeptide further comprises a polypeptide linker between
the VH and VL
domains which enables the sFv to form the desired structure for antigen
binding. For a
review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies,
vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
"Functional fragments" of antibodies comprise a portion of an intact antibody,
generally including the antigen binding or variable region of the intact
antibody or the F
region of an antibody which retains or has modified FcR binding capability.
Examples of
antibody fragments include linear antibody, single-chain antibody molecules
and
multispecific antibodies formed from antibody fragments.
The term "diabodies" refers to small antibody fragments prepared by
constructing sFv
fragments (see preceding paragraph) with short linkers (about 5-10) residues)
between the VH
and VL domains such that inter-chain but not intra-chain pairing of the V
domains is
.. achieved, thereby resulting in a bivalent fragment, i.e., a fragment having
two antigen-
binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv
fragments in
which the VH and VL domains of the two antibodies are present on different
polypeptide
chains. Diabodies are described in greater detail in, for example, EP 404,097;
WO
1993/011161; WO/2009/121948; WO/2014/191493; Hollinger et al., Proc. Nat'l
Acad. Sci.
USA 90:6444-48 (1993).
As used herein, a "chimeric antibody" refers to an antibody (immunoglobulin)
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(are) 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; Morrison et
al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies of
interest herein
include PRIMATIZED antibodies wherein the antigen-binding region of the
antibody is
derived from an antibody produced by, e.g., immunizing macaque monkeys with an
antigen
of interest. As used herein, "humanized antibody" is a subset of "chimeric
antibodies."
"Humanized' forms of non-human (e.g., murine) antibodies are chimeric
antibodies
that contain minimal sequence derived from non-human immunoglobulin. In some
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embodiments, a humanized antibody is a human immunoglobulin (recipient
antibody) in
which residues from an HVR of the recipient are replaced by residues from an
HVR of a non-
human species (donor antibody) such as mouse, rat, rabbit or non-human primate
having the
desired specificity, affinity, and/or capacity. In some instances, 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 may be made to further refine
antibody
performance, such as binding affinity. In general, a humanized antibody will
comprise
substantially all of at least one, and typically two, variable domains, in
which all or
substantially all of the hypervariable loops correspond to those of a non-
human
immunoglobulin sequence, and all or substantially all of the FR regions are
those of a human
immunoglobulin sequence, although the FR regions may include one or more
individual FR
residue substitutions that improve antibody performance, such as binding
affinity,
isomerization, immunogenicity, and the like. The number of these amino acid
substitutions
in the FR is typically no more than 6 in the H chain, and in the L chain, no
more than 3. The
humanized antibody optionally will also comprise at least a portion of an
immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. For further
details, see, e.g.,
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). See also, for example,
Vaswani and
Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem.
Soc.
Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-
433 (1994);
and U.S. Patent Nos. 6,982,321 and 7,087,409.
A "human antibody" is one that possesses an amino-acid sequence corresponding
to
that of an antibody produced by a human and/or has been made using any of the
techniques
for making human antibodies as disclosed herein. This definition of a human
antibody
specifically excludes a humanized antibody comprising non-human antigen-
binding residues.
Human antibodies can be produced using various techniques known in the art,
including
phage-display libraries. Hoogenboom and Winter, I Mol. Biol., 227:381 (1991);
Marks et
al., I Mol. Biol., 222:581 (1991). Also available for the preparation of human
monoclonal
antibodies are methods described in Cole et al., Monoclonal Antibodies and
Cancer Therapy,
Alan R. Liss, p. 77 (1985); Boerner et al., I Immunol., 147(1):86-95 (1991).
See also van
Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human
antibodies can be
prepared by administering the antigen to a transgenic animal that has been
modified to
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produce such antibodies in response to antigenic challenge, but whose
endogenous loci have
been disabled, e.g., immunized xenomice (see, e.g., U.S. Patent Nos. 6,075,181
and
6,150,584 regarding XENOMOUSETm technology). See also, for example, Li et al.,
Proc.
Nat'l Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies
generated via a
human B-cell hybridoma technology.
The term "hypervariable region," "HVR," or "HV," when used herein refers to
the
regions of an antibody-variable domain that are hypervariable in sequence
and/or form
structurally defined loops. Generally, antibodies comprise six HVRs; three in
the VH (H1,
H2, H3), and three in the VL (L1, L2, L3). In native antibodies, H3 and L3
display the most
diversity of the six HVRs, and H3 in particular is believed to play a unique
role in conferring
fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45
(2000); Johnson and
Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ,
2003)).
Indeed, naturally occurring camelid antibodies consisting of a heavy chain
only are
functional and stable in the absence of light chain. See, e.g., Hamers-
Casterman et al.,
Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736
(1996).
A number of HVR delineations are in use and are encompassed herein. The HVRs
that are Kabat complementarity-determining regions (CDRs) are based on
sequence
variability and are the most commonly used (Kabat et al., supra). Chothia
refers instead to
the location of the structural loops (Chothia and Lesk I Mot. Biol. 196:901-
917 (1987)). The
AbM HVRs represent a compromise between the Kabat CDRs and Chothia structural
loops,
and are used by Oxford Molecular's AbM antibody-modeling software. The
"contact" HVRs
are based on an analysis of the available complex crystal structures. The
residues from each
of these HVRs are noted below.
Loop Kabat AbM Chothia Contact
Li L24-L34 L24-L34 L26-L32 L30-L36
L2 L50-L56 L50-L56 L50-L52 L46-L55
L3 L89-L97 L89-L97 L91-L96 L89-L96
H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering)
H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering)
H2 H50-H65 H50-H58 H53-H55 H47-H58
H3 H95-H102 H95-H102 H96-H101 H93-H101
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HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-
56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (a
preferred
embodiment) (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable-
domain
residues are numbered according to Kabat et al., supra, for each of these
extended-HVR
definitions.
"Framework" or "FR" residues are those variable-domain residues other than the
HVR residues as herein defined.
The phrase "variable-domain residue-numbering as in Kabat" or "amino-acid-
position numbering as in Kabat," and variations thereof, refers to the
numbering system used
for heavy-chain variable domains or light-chain variable domains of the
compilation of
antibodies in Kabat et al., supra. Using this numbering system, the actual
linear amino acid
sequence may contain fewer or additional amino acids corresponding to a
shortening of, or
insertion into, a FR or HVR of the variable domain. For example, a heavy-chain
variable
domain may include a single amino acid insert (residue 52a according to Kabat)
after residue
52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc.
according to Kabat) after
heavy-chain FR residue 82. The Kabat numbering of residues may be determined
for a given
antibody by alignment at regions of homology of the sequence of the antibody
with a
"standard" Kabat numbered sequence.
The Kabat numbering system is generally used when referring to a residue in
the
variable domain (approximately residues 1-107 of the light chain and residues
1-113 of the
heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed.
Public Health
Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU
numbering system"
or "EU index" is generally used when referring to a residue in an
immunoglobulin heavy
chain constant region (e.g., the EU index reported in Kabat et al., supra).
The "EU index as
in Kabat" refers to the residue numbering of the human IgG1 EU antibody.
Unless stated
otherwise herein, references to residue numbers in the variable domain of
antibodies means
residue numbering by the Kabat numbering system. Unless stated otherwise
herein,
references to residue numbers in the constant domain of antibodies means
residue numbering
by the EU numbering system (e.g., see United States Patent Publication No.
2010-280227).
An "acceptor human framework" as used herein is a framework comprising the
amino
acid sequence of a VL or VH framework derived from a human immunoglobulin
framework
or a human consensus framework. An acceptor human framework "derived from" a
human
immunoglobulin framework or a human consensus framework may comprise the same
amino
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acid sequence thereof, or it may contain pre-existing amino acid sequence
changes. In some
embodiments, the number of pre-existing amino acid changes are 10 or fewer, 9
or fewer, 8
or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or
fewer. Where pre-
existing amino acid changes are present in a VH, preferable those changes
occur at only
.. three, two, or one of positions 71H, 73H and 78H; for instance, the amino
acid residues at
those positions may by 71A, 73T and/or 78A. In some embodiments, the VL
acceptor human
framework is identical in sequence to the VL human immunoglobulin framework
sequence or
human consensus framework sequence.
A "human consensus framework" is a framework that represents the most commonly
.. occurring amino acid residues in a selection of human immunoglobulin VL or
VH framework
sequences. Generally, the selection of human immunoglobulin VL or VH sequences
is from
a subgroup of variable domain sequences. Generally, the subgroup of sequences
is a
subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest,
5th Ed. Public
Health Service, National Institutes of Health, Bethesda, MD (1991). Examples
include for
the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV
as in Kabat et
at., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup
II, or
subgroup III as in Kabat et at., supra.
An "amino-acid modification" at a specified position refers to the
substitution or
deletion of the specified residue, or the insertion of at least one amino acid
residue adjacent
the specified residue. Insertion "adjacent" to a specified residue means
insertion within one
to two residues thereof The insertion may be N-terminal or C-terminal to the
specified
residue. The preferred amino acid modification herein is a substitution.
An "affinity-matured' antibody is one with one or more alterations in one or
more
HVRs thereof that result in an improvement in the affinity of the antibody for
antigen,
.. compared to a parent antibody that does not possess those alteration(s). In
some
embodiments, an affinity-matured antibody has nanomolar or even picomolar
affinities for
the target antigen. Affinity-matured antibodies are produced by procedures
known in the art.
For example, Marks et al., Bio/Technology 10:779-783 (1992) describes affinity
maturation
by VH- and VL-domain shuffling. Random mutagenesis of HVR and/or framework
residues
.. is described by, for example: Barbas et al. Proc Nat. Acad. Sci. USA
91:3809-3813 (1994);
Schier et al. Gene 169:147-155 (1995); Yelton et al. I Immunol. 155:1994-2004
(1995);
Jackson et al., I Immunol. 154(7):3310-9 (1995); and Hawkins et at, I Mol.
Biol. 226:889-
896 (1992).
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As use herein, the term "specifically recognizes" or "specifically binds"
refers to
measurable and reproducible interactions such as attraction or binding between
a target and
an antibody that is determinative of the presence of the target in the
presence of a
heterogeneous population of molecules including biological molecules. For
example, an
antibody that specifically or preferentially binds to a target or an epitope
is an antibody that
binds this target or epitope with greater affinity, avidity, more readily,
and/or with greater
duration than it binds to other targets or other epitopes of the target. It is
also understood
that, for example, an antibody (or a moiety) that specifically or
preferentially binds to a first
target may or may not specifically or preferentially bind to a second target.
As such,
.. "specific binding" or "preferential binding" does not necessarily require
(although it can
include) exclusive binding. An antibody that specifically binds to a target
may have an
association constant of at least about 103M-1 or 104M-1, sometimes about 105M-
1 or 106M-1,
--
in other instances about 106M-1 or 107M-1, about 108 M-1 to 109M-1, or about
1010 m 1 to 1011
M-1 or higher. A variety of immunoassay formats can be used to select
antibodies specifically
immunoreactive with a particular protein. For example, solid-phase ELISA
immunoassays
are routinely used to select monoclonal antibodies specifically immunoreactive
with a
protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual,
Cold Spring
Harbor Publications, New York, for a description of immunoassay formats and
conditions
that can be used to determine specific immunoreactivity.
"Identity", as used herein, indicates that at any particular position in the
aligned
sequences, the amino acid residue is identical between the sequences.
"Similarity", as used
herein, indicates that, at any particular position in the aligned sequences,
the amino acid
residue is of a similar type between the sequences. For example, leucine may
be substituted
for isoleucine or valine. Other amino acids which can often be substituted for
one another
include but are not limited to:
- phenylalanine, tyrosine and tryptophan (amino acids having aromatic side
chains);
- lysine, arginine and histidine (amino acids having basic side chains);
- aspartate and glutamate (amino acids having acidic side chains);
- asparagine and glutamine (amino acids having amide side chains); and
- cysteine and methionine (amino acids having sulphur-containing side chains).
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Degrees of identity and similarity can be readily calculated. (See e.g.,
Computational
Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988;
Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic
Press, New
York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and
Griffin, H.G.,
eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology,
von
Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
and
Devereux, J., eds., M Stockton Press, New York, 1991)
As used herein, an "interaction" between a complement protein and a second
protein
encompasses, without limitation, protein-protein interaction, a physical
interaction, a
chemical interaction, binding, covalent binding, and ionic binding. As used
herein, an
antibody "inhibits interaction" between two proteins when the antibody
disrupts, reduces, or
completely eliminates an interaction between the two proteins. An antibody of
the present
disclosure, or fragment thereof, "inhibits interaction" between two proteins
when the
antibody or fragment thereof binds to one of the two proteins.
A "blocking" antibody, an "antagonist" antibody, an "inhibitory" antibody, or
a
"neutralizing" antibody is an antibody that inhibits or reduces one or more
biological
activities of the antigen it binds, such as interactions with one or more
proteins. In some
embodiments, blocking antibodies, antagonist antibodies, inhibitory
antibodies, or
"neutralizing" antibodies substantially or completely inhibit one or more
biological activities
or interactions of the antigen.
Antibody "effector functions" refer to those biological activities
attributable to the Fc
region (a native sequence Fc region or amino acid sequence variant Fc region)
of an
antibody, and vary with the antibody isotype.
As used herein, the term "affinity" refers to the equilibrium constant for the
reversible
binding of two agents (e.g., an antibody and an antigen) and is expressed as a
dissociation
constant (I(D). Affinity can be at least 1-fold greater, at least 2-fold
greater, at least 3-fold
greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold
greater, at least 7-fold
greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold
greater, at least 20-fold
greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold
greater, at least 60-
fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-
fold greater, at least
100-fold greater, or at least 1,000-fold greater, or more, than the affinity
of an antibody for
unrelated amino acid sequences. Affinity of an antibody to a target protein
can be, for
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example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to
about 1
picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As
used herein,
the term "avidity" refers to the resistance of a complex of two or more agents
to dissociation
after dilution. The terms "immunoreactive" and "preferentially binds" are used
interchangeably herein with respect to antibodies and/or antigen-binding
fragments.
The term "binding" refers to a direct association between two molecules, due
to, for
example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond
interactions,
including interactions such as salt bridges and water bridges. For example, a
subject anti-Cis
antibody binds specifically to an epitope within a complement Cis protein.
"Specific
binding" refers to binding with an affinity of at least about 10-7 M or
greater, e.g., 5 x 10-7 M,
10-8 M, 5 x 10-8 M, and greater. "Non-specific binding" refers to binding with
an affinity of
less than about 10-7 M, e.g., binding with an affinity of 10' M, 10-5 M, 10'
M, etc.
The term "km", as used herein, is intended to refer to the rate constant for
association
of an antibody to an antigen.
The term "koff", as used herein, is intended to refer to the rate constant for
dissociation
of an antibody from the antibody/antigen complex.
The term "I(D", as used herein, is intended to refer to the equilibrium
dissociation
constant of an antibody-antigen interaction.
As used herein, "percent (%) amino acid sequence identity" and "homology" with
respect to a peptide, polypeptide or antibody sequence refers to the
percentage of amino acid
residues in a candidate sequence that are identical with the amino acid
residues in the specific
peptide or polypeptide 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, for instance, using publicly available computer
software such as
BLAST, BLAST-2, ALIGN or MEGALIGNTm (DNASTAR) software. Those skilled in the
art can determine appropriate parameters for measuring alignment, including
any algorithms
known in the art needed to achieve maximal alignment over the full length of
the sequences
being compared.
A "biological sample" encompasses a variety of sample types obtained from an
individual and can be used in a diagnostic or monitoring assay. The definition
encompasses
blood and other liquid samples of biological origin, solid tissue samples such
as a biopsy
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specimen or tissue cultures or cells derived therefrom and the progeny
thereof. The definition
also includes samples that have been manipulated in any way after their
procurement, such as
by treatment with reagents, solubilization, or enrichment for certain
components, such as
polynucleotides. The term "biological sample" encompasses a clinical sample,
and also
includes cells in culture, cell supernatants, cell lysates, serum, plasma,
biological fluid, and
tissue samples. The term "biological sample" includes urine, saliva,
cerebrospinal fluid,
interstitial fluid, ocular fluid, synovial fluid, blood fractions such as
plasma and serum, and
the like. The term "biological sample" also includes solid tissue samples,
tissue culture
samples, and cellular samples.
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 environment in which it was produced. Preferably, the
isolated nucleic acid
is free of association with all components associated with the production
environment. The
isolated nucleic acid molecules encoding the polypeptides and antibodies
herein is in a form
other than in the form or setting in which it is found in nature. Isolated
nucleic acid
molecules therefore are distinguished from nucleic acids encoding any
polypeptides and
antibodies herein that exist naturally in cells.
The term "vector," as used herein, is intended to refer to a nucleic acid
molecule
capable of transporting another nucleic acid to which it has been linked. One
type of vector
is a "plasmid," which refers to a circular double stranded DNA into which
additional DNA
segments may be ligated. Another type of vector is a phage vector. Another
type of vector is
a viral vector, wherein additional DNA segments may be ligated into the viral
genome.
Certain vectors are capable of autonomous replication in a host cell into
which they are
introduced (e.g., bacterial vectors having a bacterial origin of replication
and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can
be
integrated into the genome of a host cell upon introduction into the host
cell, and thereby are
replicated along with the host genome. Moreover, certain vectors are capable
of directing the
expression of genes to which they are operatively linked. Such vectors are
referred to herein
as "recombinant expression vectors," or simply, "expression vectors." In
general, expression
vectors useful in recombinant DNA techniques are often in the form of
plasmids. In the
present specification, "plasmid" and "vector" may be used interchangeably as
the plasmid is
the most commonly used form of vector.
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"Polynucleotide," or "nucleic acid," as used interchangeably herein, refer to
polymers
of nucleotides of any length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or
their analogs, or
any substrate that can be incorporated into a polymer by DNA or RNA polymerase
or by a
synthetic reaction. A polynucleotide may comprise modified nucleotides, such
as methylated
nucleotides and their analogs. If present, modification to the nucleotide
structure may be
imparted before or after assembly of the polymer. The sequence of nucleotides
may be
interrupted by non-nucleotide components. A polynucleotide may comprise
modification(s)
made after synthesis, such as conjugation to a label. Other types of
modifications include, for
example, "caps," substitution of one or more of the naturally occurring
nucleotides with an
analog, internucleotide modifications such as, for example, those with
uncharged linkages
(e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,
etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those
containing
pendant moieties, such as, for example, proteins (e.g., nucleases, toxins,
antibodies, signal
peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron, oxidative
metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha anomeric
nucleic acids, etc.),
as well as unmodified forms of the polynucleotides(s). Further, any of the
hydroxyl groups
ordinarily present in the sugars may be replaced, for example, by phosphonate
groups,
phosphate groups, protected by standard protecting groups, or activated to
prepare additional
linkages to additional nucleotides, or may be conjugated to solid or semi-
solid supports. The
5' and 3' terminal OH can be phosphorylated or substituted with amines or
organic capping
group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be
derivatized to
standard protecting groups. Polynucleotides can also contain analogous forms
of ribose or
deoxyribose sugars that are generally known in the art, including, for
example, 2'-0-methyl-,
2'-0-ally1-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, a-
anomeric sugars,
epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars,
furanose sugars,
sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl
riboside. One
or more phosphodiester linkages may be replaced by alternative linking groups.
These
alternative linking groups include, but are not limited to, embodiments
wherein phosphate is
replaced by P(0)S ("thioate"), P(S)S ("dithioate"), (0)NR2 ("amidate"), P(0)R,
P(0)OR',
CO, or CH2 ("formacetal"), in which each R or R' is independently H or
substituted or
unsubstituted alkyl (1-20 C) optionally containing an ether (-0-) linkage,
aryl, alkenyl,
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cycloalkyl, cycloalkenyl or aralkyl. Not all linkages in a polynucleotide need
be identical.
The preceding description applies to all polynucleotides referred to herein,
including RNA
and DNA.
A "host cell" includes an individual cell or cell culture that can be or has
been a
recipient for vector(s) for incorporation of polynucleotide inserts. Host
cells include progeny
of a single host cell, and the progeny may not necessarily be completely
identical (in
morphology or in genomic DNA complement) to the original parent cell due to
natural,
accidental, or deliberate mutation. A host cell includes cells transfected in
vivo with a
polynucleotide(s) of this disclosure.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or
stabilizers that are nontoxic to the cell or mammal being exposed thereto at
the dosages and
concentrations employed. Often the physiologically acceptable carrier is an
aqueous pH
buffered solution. Examples of physiologically acceptable carriers include
buffers such as
phosphate, citrate, and other organic acids; antioxidants including ascorbic
acid; low
.. molecular weight (less than about 10 residues) polypeptide; 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 TWEENTm, polyethylene
glycol (PEG),
and PLURONICSTM.
The term "neurotrophins" refers to neurotrophic factors that are
neuroprotective in
the brain. These factors are suitable for use in the compositions and methods
of the disclosure
and include brain-derived neurotrophic factor (BDNF), nerve growth factor
(NGF),
neurotrophin-4/5, fibroblast growth factor (FGF)-2 and other FGFs,
neurotrophin (NT)-3,
erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growth factor
(EGF),
transforming growth factor (TGF)-a, TGF-f3, vascular endothelial growth factor
(VEGF),
interleukin-1 receptor antagonist (IL-lra), ciliary neurotrophic factor
(CNTF), glial-derived
neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF),
heregulin,
neuregulin, artemin, persephin, interleukins, granulocyte-colony stimulating
factor (CSF),
granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs, leukemia
inhibitory
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factor (LIF), midkine, pleiotrophin, bone morphogenetic proteins (BM's),
saposins,
semaphorins, and stem cell factor (SCF).
The term "preventing" is art-recognized, and when used in relation to a
condition,
such as an FTD disease, is well understood in the art, and includes
administration of a
composition which reduces the frequency or severity, or delays the onset, of
one or more
symptoms of the medical condition in a subject relative to a subject who does
not receive the
composition. Thus, the prevention of FTD disease progression includes, for
example, slowing
the average amount of neurodegeneration in a population of patients receiving
a therapy
relative to a control population that did not receive the therapy, e.g., by a
statistically and/or
clinically significant amount. Similarly, the prevention of neurodegenerative
disease
progression includes reducing the likelihood that a patient receiving a
therapy will develop a
disability, such as cognitive decline and/or memory loss, or delaying the
onset of disability,
relative to a patient who does not receive the therapy.
The term "subject" as used herein refers to a living mammal and may be
interchangeably used with the term "patient". Examples of mammals include, but
are not
limited to, any member of the mammalian class: humans, non-human primates such
as
chimpanzees, and other apes and monkey species; farm animals such as cattle,
horses, sheep,
goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory
animals including
rodents, such as rats, mice and guinea pigs, and the like. The term does not
denote a
particular age or gender.
As used herein, the term "treating" or "treatment" includes reducing,
arresting, or
reversing the symptoms, clinical signs, or underlying pathology of a condition
to stabilize or
improve a subject's condition or to reduce the likelihood that the subject's
condition will
worsen as much as if the subject did not receive the treatment.
The term "therapeutically effective amount" of a compound with respect to the
subject method of treatment refers to an amount of the compound(s) in a
preparation which,
when administered as part of a desired dosage regimen (to a mammal, preferably
a human)
alleviates a symptom, ameliorates a condition, or slows the onset of disease
conditions
according to clinically acceptable standards for the disorder or condition to
be treated or the
cosmetic purpose, e.g., at a reasonable benefit/risk ratio applicable to any
medical treatment.
A therapeutically effective amount herein may vary according to factors such
as the disease
state, age, sex, and weight of the patient, and the ability of the antibody to
elicit a desired
response in the individual.
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As used herein, an individual "at risk" of developing a particular disease,
disorder, or
condition may or may not have detectable disease or symptoms of disease, and
may or may
not have displayed detectable disease or symptoms of disease prior to the
treatment methods
described herein. "At risk" denotes that an individual has one or more risk
factors, which are
measurable parameters that correlate with development of a particular disease,
disorder, or
condition, as known in the art. An individual having one or more of these risk
factors has a
higher probability of developing a particular disease, disorder, or condition
than an individual
without one or more of these risk factors.
"Chronic" administration refers to administration of the medicament(s) in a
continuous as opposed to acute mode, so as to maintain the initial therapeutic
effect (activity)
for an extended period of time. "Intermittent" administration refers to
treatment that is not
administered consecutively without interruption, but rather is cyclic/periodic
in nature.
As used herein, administration "conjointly" with another compound or
composition
includes simultaneous administration and/or administration at different times.
Conjoint
administration also encompasses administration as a co-formulation or
administration as
separate compositions, including at different dosing frequencies or intervals,
and using the
same route of administration or different routes of administration.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although any methods and materials similar or equivalent to those
described herein
can also be used in the practice or testing of the present invention, the
preferred methods and
materials are now described. All publications mentioned herein are
incorporated herein by
reference to disclose and describe the methods and/or materials in connection
with which the
publications are cited. such as, for example, the widely utilized
methodologies described in
Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold
Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in
Molecular Biology
(F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology
(Academic Press, Inc.):
PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds.
(1995)),
Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell
Culture
(R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984);
Methods in
Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E.
Cellis, ed.,
1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987);
Introduction to Cell
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and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and
Tissue
Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell,
eds., 1993-8) J.
Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C.
Blackwell,
eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Cabs,
eds., 1987);
PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current
Protocols in
Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular
Biology (Wiley
and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies
(P. Finch,
1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-
1989); Monoclonal
Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford
University Press,
2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold
Spring Harbor
Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds.,
Harwood
Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology
(VT. DeVita
et al., eds., J.B. Lippincott Company, 1993).
Nucleic acids, vectors and host cells
Antibodies suitable for use in the methods of the present disclosure may be
produced
using recombinant methods and compositions, e.g., as described in U.S. Patent
No.
4,816,567. In some embodiments, isolated nucleic acids having a nucleotide
sequence
encoding any of the antibodies of the present disclosure are provided. Such
nucleic acids may
encode an amino acid sequence containing the VL/CL and/or an amino acid
sequence
containing the VH/CH1 of the anti-Clq, anti-Clr or anti-Cis antibody. In some
embodiments,
one or more vectors (e.g., expression vectors) containing such nucleic acids
are provided. A
host cell containing such nucleic acid may also be provided. The host cell may
contain (e.g.,
has been transduced with): (1) a vector containing a nucleic acid that encodes
an amino acid
sequence containing the VL/CL of the antibody and an amino acid sequence
containing the
VH/CH1 of the antibody, or (2) a first vector containing a nucleic acid that
encodes an amino
acid sequence containing the VL/CL of the antibody and a second vector
containing a nucleic
acid that encodes an amino acid sequence containing the VH/CH1 of the
antibody. In some
embodiments, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO)
cell or
lymphoid cell (e.g., YO, NSO, Sp20 cell).
Methods of making an anti-Clq, anti-Clr or anti-Cis antibody are disclosed
herein.
The method includes culturing a host cell of the present disclosure containing
a nucleic acid
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encoding the anti-Clq, anti-Clr or anti-Cis antibody, under conditions
suitable for
expression of the antibody. In some embodiments, the antibody is subsequently
recovered
from the host cell (or host cell culture medium).
For recombinant production of a humanized anti-Cl q, anti-Clr or anti-Cis
antibody
of the present disclosure, a nucleic acid encoding the antibody is isolated
and inserted into
one or more vectors for further cloning and/or expression in a host cell. Such
nucleic acid
may be 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).
Suitable vectors containing a nucleic acid sequence encoding any of the
antibodies of
the present disclosure, or fragments thereof polypeptides (including
antibodies) described
herein include, without limitation, cloning vectors and expression vectors.
Suitable cloning
vectors can be constructed according to standard techniques, or may be
selected from a large
number of cloning vectors available in the art. While the cloning vector
selected may vary
according to the host cell intended to be used, useful cloning vectors
generally have the
ability to self-replicate, may possess a single target for a particular
restriction endonuclease,
and/or may carry genes for a marker that can be used in selecting clones
containing the
vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18,
pUC19,
Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9,
ColE1, pCR1,
RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many
other
cloning vectors are available from commercial vendors such as BioRad,
Stratagene, and
Invitrogen.
The vectors containing the nucleic acids of interest can be introduced into
the host
cell by any of a number of appropriate means, including electroporation,
transfection
employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-
dextran, or other
substances; microprojectile bombardment; lipofection; and infection (e.g.,
where the vector is
an infectious agent such as vaccinia virus). The choice of introducing vectors
or
polynucleotides will often depend on features of the host cell. In some
embodiments, the
vector contains a nucleic acid containing one or more amino acid sequences
encoding an anti-
Clq, anti-Clr or anti-Cis antibody of the present disclosure.
Suitable host cells for cloning or expression of antibody-encoding vectors
include
prokaryotic or eukaryotic cells. For example, an anti-Clq, anti-Clr or anti-
Cis antibody of
the present disclosure may be produced in bacteria, in particular when
glycosylation and Fc
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effector function are not needed. For expression of antibody fragments and
polypeptides in
bacteria (e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523; and
Charlton, Methods
in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ,
2003), pp. 245-
254, describing expression of antibody fragments in E. coli .). After
expression, the antibody
may be isolated from the bacterial cell paste in a soluble fraction and can be
further purified.
Antibody Screening
Candidate antibodies can be screened for the ability to modulate synapse loss.
Such
screening may be performed using an in vitro model, a genetically altered cell
or animal, or
purified protein. A wide variety of assays may be used for this purpose, such
as an in vitro
culture system. For example, an in vitro culture system may include the
addition of
microglial cells to cultures of cortical neurons, followed by counting the
number of synapses
removed from the neurons and/or ingested by the microglial cells.
Functional activity of a candidate antibody may also be tested in vivo by
assessing the
ability of the antibody to modulate synapse loss during normal aging, e.g., in
animals
challenged with intracerebral injection of amyloid-beta oligomers, or in
animals genetically
modified with human familial mutations associated with FTD or in animals with
induced
forms of FTD.
Candidate antibodies may also be identified using computer-based modeling, by
binding assays, and the like. Various in vitro models may be used to determine
whether an
antibody binds to, or otherwise affects complement activity. Such candidate
antibodies may
be tested by contacting neurons in an environment permissive for synapse loss.
Such
antibodies may be further tested in an in vivo model for an effect on synapse
loss.
Synapse loss may be quantitated by administering the candidate antibodies to
neurons
in culture, and determining the presence of synapses in the absence or
presence of the
antibodies. In some embodiments of the disclosure, the neurons are a primary
culture, e.g., of
retinal ganglion cells (RGCs). Purified populations of RGCs are obtained by
conventional
methods, such as sequential immunopanning. The cells are cultured in suitable
medium,
which will usually comprise appropriate growth factors, e.g., CNTF; BDNF; etc.
The neural
cells, e.g., RCGs, are cultured for a period of time sufficient allow robust
process outgrowth
and then cultured with a candidate antibodies for a period of about 1 day to 1
week. The
neurons may be cultured on a live astrocyte cell feeder in order to induce
signaling for
synapse loss. Methods of culturing astrocyte feeder layers are known in the
art. For example,
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cortical glia can be plated in a medium that does not allow neurons to
survive, with removal
of non-adherent cells.
For synapse quantification, cultures are fixed, blocked and washed, then
stained with
an antibody specific for synaptic proteins, e.g., synaptotagmin, etc. and
visualized with an
appropriate reagent. Analysis of the staining may be performed
microscopically. In some
embodiments, digital images of the fluorescence emission are with a camera and
image
capture software, adjusted to remove unused portions of the pixel value range
and the used
pixel values adjusted to utilize the entire pixel value range. Corresponding
channel images
may be merged to create a color (RGB) image containing the two single-channel
images as
individual color channels. Co-localized puncta can be identified using a
rolling ball
background subtraction algorithm to remove low-frequency background from each
image
channel. Number, mean area, mean minimum and maximum pixel intensities, and
mean pixel
intensities for all synaptotagmin, PSD-95, and colocalized puncta in the image
are recorded
for analysis.
Generally, a plurality of assay mixtures are run in parallel with different
antibody
concentrations to obtain a differential response to the various
concentrations. Typically one
of these concentrations serves as a negative control, i.e., at zero
concentration or below the
level of detection.
Pharmaceutical Compositions and Administration
An antibody of the present disclosure may be administered in the form of
pharmaceutical compositions.
Therapeutic formulations of an antibody of the disclosure may be prepared for
storage
by mixing the antibody having the desired degree of purity with optional
pharmaceutically
acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences 16th
edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations 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 and methionine; preservatives
(such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium
chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low
molecular weight (less than about 10 residues) polypeptides; proteins, such as
serum
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albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine, arginine, or
lysine;
monosaccharides, disaccharides, and other carbohydrates including glucose,
mannose, or
dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol,
trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-
protein
complexes); and/or non-ionic surfactants such as TWEENTm, PLUIRONICSTM or
polyethylene glycol (PEG).
Lipofections or liposomes may also be used to deliver an antibody or antibody
fragment into a cell, wherein the epitope or smallest fragment which
specifically binds to the
binding domain of the target protein is preferred.
The antibody may also be entrapped in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems (for example,
liposomes,
albumin microspheres, microemulsions, nano-particles and nanocapsules) or in
macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical
Sciences
16th edition, Osol, A. Ed. (1980).
The formulations to be used for parenteral administration must be sterile.
This is
readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations include semipermeable matrices of solid hydrophobic
polymers
containing the antibody, which matrices are in the form of shaped articles,
e.g., films, or
microcapsules. Examples of sustained-release matrices include polyesters,
hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat.
No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-
degradable
ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such
as the LUPRON
DEPOTTm (injectable microspheres composed of lactic acid-glycolic acid
copolymer and
leuprolide acetate), and poly-D-(¨)-3-hydroxybutyric acid. While polymers such
as ethylene-
vinyl acetate and lactic acid-glycolic acid enable release of molecules for
over 100 days,
certain hydrogels release proteins for shorter time periods.
The antibodies and compositions of the present disclosure are typically
administered
by various routes, including, but not limited to, topical, parenteral,
subcutaneous,
intraperitoneal, intrapulmonary, intranasal, and intralesional administration.
Parenteral routes
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of administration include intramuscular, intravenous, intra-arterial,
intraperitoneal,
intrathecal, or subcutaneous administration.
Pharmaceutical compositions may also include, depending on the formulation
desired,
pharmaceutically-acceptable, non-toxic carriers of diluents, which are defined
as vehicles
commonly used to formulate pharmaceutical compositions for animal or human
administration. The diluent is selected so as not to affect the biological
activity of the
combination. Examples of such diluents are distilled water, buffered water,
physiological
saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In
addition, the
pharmaceutical composition or formulation may include other carriers,
adjuvants, or non-
toxic, nontherapeutic, non-immunogenic stabilizers, excipients and the like.
The
compositions may also include additional substances to approximate
physiological
conditions, such as pH adjusting and buffering agents, toxicity adjusting
agents, wetting
agents and detergents.
The composition may also include any of a variety of stabilizing agents, such
as an
antioxidant for example. When the pharmaceutical composition includes a
polypeptide, the
polypeptide may be complexed with various well-known compounds that enhance
the in vivo
stability of the polypeptide, or otherwise enhance its pharmacological
properties (e.g.,
increase the half-life of the polypeptide, reduce its toxicity, enhance other
pharmacokinetic
and/or pharmacodynamic characteristics, or enhance solubility or uptake).
Examples of such
modifications or complexing agents include sulfate, gluconate, citrate and
phosphate. The
polypeptides of a composition may also be complexed with molecules that
enhance their in
vivo attributes. Such molecules include, for example, carbohydrates,
polyamines, amino
acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium,
manganese), and
lipids. Further guidance regarding formulations that are suitable for various
types of
administration may be found in Remington's Pharmaceutical Sciences, Mace
Publishing
Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for
drug delivery,
see, Langer, Science 249:1527-1533 (1990).
Toxicity and therapeutic efficacy of the active ingredient may be determined
according to standard pharmaceutical procedures in cell cultures and/or
experimental
animals, including, for example, determining the LD50 (the dose lethal to 50%
of the
population) and the ED50 (the dose therapeutically effective in 50% of the
population). The
dose ratio between toxic and therapeutic effects is the therapeutic index and
it may be
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expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic
indices are
preferred.
The data obtained from cell culture and/or animal studies may be used in
formulating
a range of dosages for humans. The dosage of the active ingredient typically
lines within a
range of circulating concentrations that include the ED50 with low toxicity.
The dosage may
vary within this range depending upon the dosage form employed and the route
of
administration utilized.
The pharmaceutical compositions described herein may be administered in a
variety
of different ways. Examples include administering a composition containing a
pharmaceutically acceptable carrier via oral, intranasal, rectal, topical,
intraperitoneal,
intravenous, intramuscular, subcutaneous, sub dermal, transdermal,
intrathecal, and
intracranial methods.
For oral administration, the active ingredient may be administered in solid
dosage
forms, such as capsules, tablets, and powders, or in liquid dosage forms, such
as elixirs,
syrups, and suspensions. The active component(s) may be encapsulated in
gelatin capsules
together with inactive ingredients and powdered carriers, such as glucose,
lactose, sucrose,
mannitol, starch, cellulose or cellulose derivatives, magnesium stearate,
stearic acid, sodium
saccharin, talcum, magnesium carbonate. Examples of additional inactive
ingredients that
may be added to provide desirable color, taste, stability, buffering capacity,
dispersion or
other known desirable features are red iron oxide, silica gel, sodium lauryl
sulfate, titanium
dioxide, and edible white ink. Similar diluents may be used to make compressed
tablets. Both
tablets and capsules may be manufactured as sustained release products to
provide for
continuous release of medication over a period of hours. Compressed tablets
may be sugar
coated or film coated to mask any unpleasant taste and protect the tablet from
the
atmosphere, or enteric-coated for selective disintegration in the
gastrointestinal tract. Liquid
dosage forms for oral administration may contain coloring and flavoring to
increase patient
acceptance.
Formulations suitable for parenteral administration include aqueous and non-
aqueous,
isotonic sterile injection solutions, which may contain antioxidants, buffers,
bacteriostats, and
solutes that render the formulation isotonic with the blood of the intended
recipient, and
aqueous and non-aqueous sterile suspensions that may include suspending
agents,
solubilizers, thickening agents, stabilizers, and preservatives.
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The components used to formulate the pharmaceutical compositions are
preferably of
high purity and are substantially free of potentially harmful contaminants
(e.g., at least
National Food (NF) grade, generally at least analytical grade, and more
typically at least
pharmaceutical grade). Moreover, compositions intended for parenteral use are
usually
sterile. To the extent that a given compound must be synthesized prior to use,
the resulting
product is typically substantially free of any potentially toxic agents,
particularly any
endotoxins, which may be present during the synthesis or purification process.
Compositions
for parental administration are also typically substantially isotonic and made
under GMP
conditions.
The compositions of the disclosure may be administered using any medically
appropriate procedure, e.g., intravascular (intravenous, intraarterial,
intracapillary)
administration, injection into the cerebrospinal fluid, intravitreal, topical,
intracavity or direct
injection in the brain. Intrathecal administration may be carried out through
the use of an
Ommaya reservoir, in accordance with known techniques. (F. Balis et al., Am J.
Pediatr.
Hematol. Oncol. 11, 74, 76 (1989).
Where the therapeutic agents are locally administered in the brain, one method
for
administration of the therapeutic compositions of the disclosure is by
deposition into or near
the site by any suitable technique, such as by direct injection (aided by
stereotaxic
positioning of an injection syringe, if necessary) or by placing the tip of an
Ommaya
reservoir into a cavity, or cyst, for administration. Alternatively, a
convection-enhanced
delivery catheter may be implanted directly into the site, into a natural or
surgically created
cyst, or into the normal brain mass. Such convection-enhanced pharmaceutical
composition
delivery devices greatly improve the diffusion of the composition throughout
the brain mass.
The implanted catheters of these delivery devices utilize high-flow
microinfusion (with flow
rates in the range of about 0.5 to 15.0 p1/minute), rather than diffusive
flow, to deliver the
therapeutic composition to the brain and/or tumor mass. Such devices are
described in U.S.
Pat. No. 5,720,720, incorporated fully herein by reference.
The effective amount of a therapeutic composition given to a particular
patient may
depend on a variety of factors, several of which may be different from patient
to patient. A
competent clinician will be able to determine an effective amount of a
therapeutic agent to
administer to a patient. Dosage of the agent will depend on the treatment,
route of
administration, the nature of the therapeutics, sensitivity of the patient to
the therapeutics, etc.
Utilizing LD50 animal data, and other information, a clinician may determine
the maximum
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safe dose for an individual, depending on the route of administration.
Utilizing ordinary skill,
the competent clinician will be able to optimize the dosage of a particular
therapeutic
composition in the course of routine clinical trials. The compositions may be
administered to
the subject in a series of more than one administration. For therapeutic
compositions, regular
periodic administration will sometimes be required, or may be desirable.
Therapeutic
regimens will vary with the agent; for example, some agents may be taken for
extended
periods of time on a daily or semi-daily basis, while more selective agents
may be
administered for more defined time courses, e.g., one, two three or more days,
one or more
weeks, one or more months, etc., taken daily, semi-daily, semi-weekly, weekly,
etc.
Formulations may be optimized for retention and stabilization in the brain.
When the
agent is administered into the cranial compartment, it is desirable for the
agent to be retained
in the compartment, and not to diffuse or otherwise cross the blood brain
barrier.
Stabilization techniques include cross-linking, multimerizing, or linking to
groups such as
polyethylene glycol, polyacrylamide, neutral protein carriers, etc., in order
to achieve an
increase in molecular weight.
Other strategies for increasing retention include the entrapment of the agent
in a
biodegradable or bioerodible implant. The rate of release of the
therapeutically active agent is
controlled by the rate of transport through the polymeric matrix, and the
biodegradation of
the implant. The transport of drug through the polymer barrier will also be
affected by
compound solubility, polymer hydrophilicity, extent of polymer cross-linking,
expansion of
the polymer upon water absorption so as to make the polymer barrier more
permeable to the
drug, geometry of the implant, and the like. The implants are of dimensions
commensurate
with the size and shape of the region selected as the site of implantation.
Implants may be
particles, sheets, patches, plaques, fibers, microcapsules and the like and
may be of any size
or shape compatible with the selected site of insertion.
The implants may be monolithic, i.e., having the active agent homogenously
distributed through the polymeric matrix, or encapsulated, where a reservoir
of active agent is
encapsulated by the polymeric matrix. The selection of the polymeric
composition to be
employed will vary with the site of administration, the desired period of
treatment, patient
tolerance, the nature of the disease to be treated and the like.
Characteristics of the polymers
will include biodegradability at the site of implantation, compatibility with
the agent of
interest, ease of encapsulation, a half-life in the physiological environment.
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Biodegradable polymeric compositions which may be employed may be organic
esters or ethers, which when degraded result in physiologically acceptable
degradation
products, including the monomers. Anhydrides, amides, orthoesters or the like,
by themselves
or in combination with other monomers, may find use. The polymers may be
condensation
polymers. The polymers may be cross-linked or non-cross-linked. Of particular
interest are
polymers of hydroxyaliphatic carboxylic acids, either homo- or copolymers, and
polysaccharides. Included among the polyesters of interest are polymers of D-
lactic acid, L-
lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and
combinations thereof. By
employing the L-lactate or D-lactate, a slowly biodegrading polymer is
achieved, while
degradation is substantially enhanced with the racemate. Copolymers of
glycolic and lactic
acid are of particular interest, where the rate of biodegradation is
controlled by the ratio of
glycolic to lactic acid. The most rapidly degraded copolymer has roughly equal
amounts of
glycolic and lactic acid, where either homopolymer is more resistant to
degradation. The ratio
of glycolic acid to lactic acid will also affect the brittleness of in the
implant, where a more
flexible implant is desirable for larger geometries. Among the polysaccharides
of interest are
calcium alginate, and functionalized celluloses, particularly
carboxymethylcellulose esters
characterized by being water insoluble, a molecular weight of about 5 kD to
500 kD, etc.
Biodegradable hydrogels may also be employed in the implants of the subject
disclosure.
Hydrogels are typically a copolymer material, characterized by the ability to
imbibe a liquid.
Exemplary biodegradable hydrogels which may be employed are described in
Heller in:
Hydrogels in Medicine and Pharmacy, N. A. Peppes ed., Vol. III, CRC Press,
Boca Raton,
Fla., 1987, pp 137-149.
Kits
The present disclosure also provides a pharmaceutical pack or kit comprising
one or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions. Associated with such container(s) may be a notice in the form
prescribed by a
governmental agency regulating the manufacture, use or sale of pharmaceuticals
or biological
products, which notice reflects approval by the agency of manufacture, use or
sale for human
administration.
Kits of the present disclosure may include one or more containers comprising a
purified anti-Clq, anti-Clr or anti-Cis antibody and instructions for use in
accordance with
methods known in the art. Generally, these instructions comprise a description
of
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administration of the inhibitor to treat or diagnose a disease, according to
any methods
known in the art. The kit may further comprise a description of selecting an
individual
suitable for treatment based on identifying whether that individual has FTD
and the stage of
the FTD.
The instructions generally include information as to dosage, dosing schedule,
and
route of administration for the intended treatment. The containers may be unit
doses, bulk
packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied
in the kits of the
present disclosure are typically written instructions on a label or package
insert (e.g., a paper
sheet included in the kit), but machine-readable instructions (e.g.,
instructions carried on a
magnetic or optical storage disk) are also acceptable.
The label or package insert indicates that the composition is used for
treating FTD.
Instructions may be provided for practicing any of the methods described
herein.
The kits of this disclosure are preferably disposed in suitable packaging.
Suitable
packaging includes, but is not limited to, vials, bottles, jars, flexible
packaging (e.g., sealed
Mylar or plastic bags), and the like. Also contemplated are packages for use
in combination
with a specific device, such as an inhaler, nasal administration device (e.g.,
an atomizer) or
an infusion device such as a minipump. A kit may have a sterile access port
(for example the
container may be an intravenous solution bag or a vial having a stopper
pierceable by a
hypodermic injection needle). The container may also have a sterile access
port (e.g., the
container may be an intravenous solution bag or a vial having a stopper
pierceable by a
hypodermic injection needle). At least one active agent in the composition is
an inhibitor of
classical complement pathway. The container may further comprise a second
pharmaceutically active agent.
Kits may optionally provide additional components such as buffers and
interpretive
information. Normally, the kit comprises a container and a label or package
insert(s) on or
associated with the container.
Inhibition of complement
A number of molecules are known that inhibit the activity of complement. In
addition
to known compounds, suitable inhibitors can be screened by methods described
herein. As
described above, normal cells can produce proteins that block complement
activity, e.g.,
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CD59, Cl inhibitor, etc. In some embodiments of the disclosure, complement is
inhibited by
upregulating expression of genes encoding such polypeptides.
Modifications of molecules that block complement activation are also known in
the
art. For example, such molecules include, without limitation, modified
complement receptors,
such as soluble CR1. The mature protein of the most common allotype of CR1
contains 1998
amino acid residues: an extracellular domain of 1930 residues, a transmembrane
region of 25
residues, and a cytoplasmic domain of 43 residues. The entire extracellular
domain is
composed of 30 repeating units referred to as short consensus repeats (SCRs)
or complement
control protein repeats (CCPRs), each consisting of 60 to 70 amino acid
residues. Recent data
indicate that Clq binds specifically to human CR1. Thus, CR1 recognizes all
three
complement opsonins, namely C3b, C4b, and Cl q. A soluble version of
recombinant human
CR1 (sCR1) lacking the transmembrane and cytoplasmic domains has been produced
and
shown to retain all the known functions of the native CR1. The
cardioprotective role of sCR1
in animal models of ischemia/reperfusion injury has been confirmed. Several
types of human
.. Clq receptors (ClqR) have been described. These include the ubiquitously
distributed 60- to
67-kDa receptor, referred to as cClqR because it binds the collagen-like
domain of Clq. This
ClqR variant was shown to be calreticulin; a 126-kDa receptor that modulates
monocyte
phagocytosis. gClqR is not a membrane-bound molecule, but rather a secreted
soluble
protein with affinity for the globular regions of Clq, and may act as a fluid-
phase regulator of
complement activation.
Decay accelerating factor (DAF) (CD55) is composed of four SCRs plus a
serine/threonine-enriched domain that is capable of extensive 0-linked
glycosylation. DAF is
attached to cell membranes by a glycosyl phosphatidyl inositol (GPI) anchor
and, through its
ability to bind C4b and C3b, it acts by dissociating the C3 and C5
convertases. Soluble
versions of DAF (sDAF) have been shown to inhibit complement activation.
Cl inhibitor, a member of the "serpin" family of serine protease inhibitors,
is a
heavily glycosylated plasma protein that prevents fluid-phase Cl activation.
Cl inhibitor
regulates the classical pathway of complement activation by blocking the
active site of Clr
and Cls and dissociating them from Clq.
Peptide inhibitors of complement activation include C5a and other inhibitory
molecules include Fucan.
Synapse loss
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Synapses are asymmetric communication junctions formed between two neurons,
or,
at the neuromuscular junction (NMJ) between a neuron and a muscle cell.
Chemical synapses
enable cell-to-cell communication via secretion of neurotransmitters, whereas
in electrical
synapses signals are transmitted through gap junctions, specialized
intercellular channels that
.. permit ionic current flow. In addition to ions, other molecules that
modulate synaptic
function (such as ATP and second messenger molecules) can diffuse through gap
junctional
pores. At the mature NMJ, pre- and postsynaptic membranes are separated by a
synaptic cleft
containing extracellular proteins that form the basal lamina. Synaptic
vesicles are clustered at
the presynaptic release site, transmitter receptors are clustered in
junctional folds at the
postsynaptic membrane, and glial processes surround the nerve terminal.
Synaptogenesis is a dynamic process. During development, more synapses are
typically established than ultimately will be retained. Therefore, the
elimination of excess
synaptic inputs is a critical step in synaptic circuit maturation. Synapse
elimination is a
competitive process that involves interactions between pre- and postsynaptic
partners. In the
CNS, as with the NMJ, a developmental, activity-dependent remodeling of
synaptic circuits
takes place by a process that may involve the selective stabilization of
coactive inputs and the
elimination of inputs with uncorrelated activity. The anatomical refinement of
synaptic
circuits occurs at the level of individual axons and dendrites by a dynamic
process that
involves rapid elimination of synapses. As axons branch and remodel, synapses
form and
dismantle with synapse elimination occurring rapidly.
In addition to the normal developmental loss, synapse loss is an early
pathological
event common to many neurodegenerative disorders, including FTD, and is the
best correlate
to the cognitive impairment. For example, studies in the brains of patients
with pre-clinical
Alzheimer's disease (AD), as well as in transgenic animal models have shown
that synaptic
damage occurs early in disease progression. This early disruption of synaptic
connections in
the brain results in neuronal dysfunction that, in turn, leads to the
characteristic symptoms of
dementia and/or motor impairment observed in several neurodegenerative
disorders.
Several molecules involved in AD and other neurodegenerative disorders play an
important role in synaptic function. For example, APPP has a preferential
localization at
central and peripheral synaptic sites. In transgenic mice, abnormal expression
of mutant
forms of APPP results not only in amyloid deposition, but also in widespread
synaptic
damage. This synaptic pathology occurs early and is associated with levels of
soluble A131-42
rather than with plaque formation. Other neurodegenerative diseases where gene
products
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have been shown to be closely associated with synaptic complexes include
Huntington's
disease (HD) and myotonic dystrophy (DM). Huntingtin (HTT) is a membrane-bound
protein
with a distribution very similar to that of synaptic vesicle protein
synaptophysin. Studies in
human brain detected HTT in perikarya of some neurons, neuropil, varicosities
and as
punctate staining likely to be nerve endings. The serine/ threonine kinase
(DMK), which is
the gene product of the DM gene, has been found to localize post-synaptically
at the
neuromuscular junction of skeletal muscle and at intercalated discs of cardiac
tissue. DMK
was also found at synaptic sites in the cerebellum, hippocampus, midbrain and
medulla.
Antibodies disclosed herein may be used to inhibit synapse loss. Inhibiting
synapse
loss results in maintenance of or reduced loss of synapses, where a decrease
would otherwise
occur.
Blood Brain Barrier
As used herein, the "blood-brain barrier" (BBB) refers to the barrier between
the
peripheral circulation and the brain and spinal cord. The BBB is formed by
tight junctions
within the brain capillary endothelial plasma membrane. The formation of such
tight
junctions creates an extremely tight barrier that restricts the transport of
molecules into the
brain, even molecules as small as urea, molecular weight of 60 Da. The blood-
brain barrier
within the brain, the blood-spinal cord barrier within the spinal cord, and
the blood-retinal
barrier within the retina, are contiguous capillary barriers within the
central nervous system
(CNS), and are collectively referred to as the blood-brain barrier or BBB. The
disclosure
provides compositions and methods that include an antibody that binds to a BBB
receptor
mediated transport system, coupled to an agent for which transport across the
BBB is desired,
e.g., a neurotherapeutic agent. The compositions and methods of the disclosure
may utilize
any suitable structure that is capable of transport by the selected endogenous
BBB receptor-
mediated transport system, and that is also capable of attachment to the
desired agent.
The BBB has been shown to have specific receptors that allow the transport
from the
blood to the brain of several macromolecules; these transporters are suitable
as transporters
for compositions of the disclosure. Endogenous BBB receptor-mediated transport
systems
useful in the disclosure include those that transport insulin, transferrin,
insulin-like growth
factors 1 and 2 (IGF1 and IGF2), leptin, and lipoproteins. In some
embodiments, the
disclosure utilizes a structure that is capable of crossing the BBB via the
endogenous insulin
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BBB receptor-mediated transport system, e.g., the human endogenous insulin BBB
receptor-
mediated transport system.
One strategy for drug delivery through the blood brain barrier (BBB) entails
disruption of the BBB, either by osmotic means such as mannitol or
leukotrienes, or
biochemically by the use of vasoactive substances such as bradykinin. The
potential for using
BBB opening to target specific agents is also an option. A BBB disrupting
agent can be co-
administered with the therapeutic compositions of the disclosure when the
compositions are
administered by intravascular injection. Other strategies to go through the
BBB may entail
the use of endogenous transport systems, including carrier-mediated
transporters such as
glucose and amino acid carriers, receptor-mediated transcytosis for insulin or
transferrin, and
active efflux transporters such as p-glycoprotein. Active transport moieties
may also be
conjugated to the antibodies of the disclosure to facilitate transport across
the epithelial wall
of the blood vessel. Alternatively, drug delivery behind the BBB may be
pursued, e.g., by
intrathecal delivery of agents directly to the cerebrospinal fluid, as through
an Ommaya
reservoir.
Frontotemporal Dementia
The antibodies of the present disclosure may be useful in the present methods
of
preventing, reducing risk of developing, or treating FTD or a variant of FTD,
comprising
administering an inhibitor of the complement pathway (e.g., an inhibitor, such
as an anti-Clq,
-Clr, or -Cis antibody). The antibodies of the present disclosure may also be
useful in
inhibiting synapse loss in FTD.
Synapse loss is a significant correlate of cognitive decline that serves as a
critical
hallmark of neurodegenerative diseases. For example, microglia prune
developing synapses
and regulate synaptic plasticity and function. Disruptions in
microglia¨synapse interactions
contribute to synapse loss and dysfunction, including cognitive impairment in
neurodegenerative diseases. Furthermore, disruption of immune-related
molecules or
receptors expressed on microglia, such as complement proteins or complement
and
fractalkine receptors, results in synaptic and wiring abnormalities in both
prenatal and
postnatal brain development implicating microglia in sculpting synaptic
connectivity.
The etiology of frontotemporal dementia remains enigmatic. Frontotemporal
lobar
degeneration (FTLD) may be used as a pathological term used for the
description of a
clinically, pathologically and genetically heterogeneous group of disorders in
which selective
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degeneration of the frontal and temporal lobes is a prominent feature. The
clinical term
frontotemporal dementia (FTD) is used for the description of early onset
dementias. FTD
may overlap with motor neuron disease/amyotrophic lateral sclerosis (MND/ALS)
(FTD-
MND), Corticobasal syndrome (CBS) and progressive palsy (PSP) syndrome. FTD is
also a
.. highly heritable disorder with approximately 30-50% of cases reporting
positive family
history, although an autosomal dominant history accounts for approximately 10%
of the
cases. Mutations in three genes, microtubule-associated protein tau (MAPT),
progranulin
(GRN) and chromosome 9 open reading frame 72 (C9orf72) genes are considered
responsible
for most of the familial cases, and about 10-20% of all cases with FTD.
Frontotemporal dementia may be classified into three clinical variants:
behavioral-
variant frontotemporal dementia, which is associated with early behavioral and
executive
deficits; non-fluent variant primary progressive aphasia, with progressive
deficits in speech,
grammar, and word output; and semantic-variant primary progressive aphasia,
which is a
progressive disorder of semantic knowledge and naming. Clinically FTD patients
can present
with one of three canonical clinical syndromes: behavioral variant FTD
(bvFTD), and two
language variants, semantic dementia and progression non-fluent aphasia
(PNFA). bvFTD is
associated with early behavioral and executive deficits, which refers to the
higher-level
cognitive skills that control and coordinate other cognitive abilities and
behaviors. Semantic
dementia, also called semantic-variant primary progressive aphasia, is a
progressive disorder
of semantic knowledge and naming. PNFA is typically characterized by
progressive deficits
in speech, grammar, and word aphasia. As FTD progresses, the symptoms of the
three
clinical variants may converge, such as when an initially focal degeneration
becomes more
diffuse and spreads to affect large regions in the frontal and temporal lobes.
Over time,
patients develop global cognitive impairment and motor deficits, including
Parkinsonism, and
.. motor neuron disease in some patients. Patents with end-stage disease
generally have
difficulty eating, moving, and swallowing. Death usually happens about 8 years
after
symptom onset and typically caused by pneumonia or other secondary infections.
The most common pronounced early symptoms of behavioral variant frontotemporal
dementia include personality changes, disinhibition, and apathy. Some
individuals who meet
diagnostic criteria for behavioral-variant frontotemporal dementia may have a
very slow
disease course (over decades) with slow progression of cognitive impairment
and often
normal MRI and PET studies. Their disease is classified as frontotemporal
dementia
phenocopy. Some of these individuals have a primary psychiatric disturbance
such as bipolar
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disorder, Asperger's syndrome, or factitious disease, whereas others might
have a slow
sporadic or genetic form of frontotemporal dementia
Patients with primary progressive aphasia typically have a progressive,
insidious
decline in linguistic skills during the initial phase of the disease. Language
dysfunction is
commonly one of the main symptoms for the first 2 years of the illness.
Deficits include
language production, object naming, syntax, or word comprehension, and are
apparent during
conversation or through speech and language assessment. Language deficit is
commonly one
of the main causes of impaired activities of daily living. Although the
underlying cause is
more often frontotemporal dementia, primary progressive aphasia can be
associated with
Alzheimer's disease.
Symptoms of semantic-variant primary progressive aphasia may result from early
asymmetrical degeneration of anterior temporal lobes and amygdala. Semantic
loss causes
anomia for people, places, and objects; word-finding difficulties; and
impaired word
comprehension. Left temporal lobe variant presents with mainly linguistic
semantic loss
(semantic-variant primary).
About 12.5% of patients with behavioral-variant frontotemporal dementia
develop
motor neuron disease, typically including upper motor neuron signs
(hyperreflexia, extensor
plantar response, spasticity), lower motor neuron signs (weakness, muscle
atrophy,
fasciculations), dysarthria, dysphagia, and pseudobulbar affect. Mild features
of motor
neuron disease can occur in up to 40% of patients with frontotemporal
dementia. Among the
frontotemporal dementia variants, motor neuron disease arises frequently in
patients with
behavioral-variant frontotemporal dementia and less often in patients with
semantic-variant
primary progressive aphasia or nonfluent variant primary progressive aphasia.
Early
parkinsonism is present in up to 20% of patients with frontotemporal dementia
and is most
often seen in patients with behavioral-variant frontotemporal dementia,
followed by those
with non-fluent variant primary progressive aphasia. Patients with
frontotemporal dementia
have features of corticobasal syndrome or progressive supranuclear palsy
syndrome.
Corticobasal syndrome is characterized by asymmetrical parkinsonism,
sensory¨motor
cortical dysfunction, alien-limb syndrome, and dystonia. Progressive
supranuclear palsy
syndrome is characterized by vertical supranuclear palsy, decreased saccade
velocity, and
early postural instability with falls. Behavioral changes, including executive
dysfunction,
apathy, and impulsivity, are common.
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Methods of Treatment
By administering agents that inhibit complement activation, synapses can be
maintained that would otherwise be lost. Such agents include an anti-Clq, anti-
Clr, or anti-
Cls antibody inhibitor. Other agents may include inhibitors that upregulate
expression of
native complement, or agents that down-regulate Clq, Clr or Cis synthesis in
neurons,
astrocytes, microglia, endothelial, or oligodendroglial cells, agents that
block complement
activation, agents that block the signal for complement activation, and the
like.
The methods promote improved maintenance of neuronal function in conditions
associated with synapse loss. The maintenance of neural connections provides
for functional
improvement in neurodegenerative disease relative to untreated patients. The
prevention of
synapse loss may comprise at least a measurable improvement relative to a
control lacking
such treatment over the period of 1, 2, 3, 4, 5, 6 days or at least one week,
for example at
least a 10% improvement in the number of synapses, at least a 20% improvement,
at least a
50% improvement, or more.
The agents of the present disclosure may be administered at a dosage that
decreases
synapse loss while minimizing any side-effects. It is contemplated that
compositions may be
obtained and used under the guidance of a physician for in vivo use. The
dosage of the
therapeutic formulation may vary widely, depending upon the nature of the
disease, the
frequency of administration, the manner of administration, the clearance of
the agent from the
host, and the like.
The effective amount of a therapeutic composition given to a particular
patient may
depend on a variety of factors, several of which may be different from patient
to patient.
Utilizing ordinary skill, the competent clinician will be able to tailor the
dosage of a
particular therapeutic or imaging composition in the course of routine
clinical trials.
Therapeutic agents, e.g., inhibitors of complement, activators of gene
expression, etc.
can be incorporated into a variety of formulations for therapeutic
administration by
combination with appropriate pharmaceutically acceptable carriers or diluents,
and may be
formulated into preparations in solid, semi-solid, liquid or gaseous forms,
such as tablets,
.. capsules, powders, granules, ointments, solutions, suppositories,
injections, inhalants, gels,
microspheres, and aerosols. As such, administration of the compounds can be
achieved in
various ways, including oral, buccal, rectal, parenteral, intraperitoneal,
intradermal,
transdermal, intrathecal, nasal, intratracheal, etc., administration. The
active agent may be
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systemic after administration or may be localized by the use of regional
administration,
intramural administration, or use of an implant that acts to retain the active
dose at the site of
implantation.
Combination Treatments
The complement inhibitors of the present disclosure may be used, without
limitation,
conjointly with any additional treatment, such as immunosuppressive therapies,
for treating
FTD.
In some embodiments, the antibodies of this disclosure may be administered in
combination with an inhibitor of the alternative pathway of complement
activation. Such
inhibitors may include, without limitation, factor B blocking antibodies,
factor D blocking
antibodies, soluble, membrane-bound, tagged or fusion-protein forms of CD59,
DAF, CR1,
CR2, Crry or Compstatin-like peptides that block the cleavage of C3, non-
peptide C3aR
antagonists such as SB 290157, Cobra venom factor or non-specific complement
inhibitors
such as nafamostat mesilate (FUTHAN; FUT-175), aprotinin, K-76 monocarboxylic
acid
(MX-1) and heparin (see, e.g., T.E. Mollnes & M. Kirschfink, Molecular
Immunology 43
(2006) 107-121). In some embodiments, the antibodies of this disclosure are
administered in
combination with an inhibitor of the interaction between the autoantibody and
its
autoantigen. Such inhibitors may include purified soluble forms of the
autoantigen, or antigen
mimetics such as peptide or RNA-derived mimotopes, including mimotopes of the
AQP4
antigen. Alternatively, such inhibitors may include blocking agents that
recognize the
autoantigen and prevent binding of the autoantibody without triggering the
classical
complement pathway. Such blocking agents may include, e.g., autoantigen-
binding RNA
aptamers or antibodies lacking functional Clq, Clr, or Cis binding sites in
their Fc domains
(e.g., Fab fragments or antibodies otherwise engineered not to bind Clq, Clr,
or Cis).
The methods of the present disclosure can find use in combination with cell or
tissue
transplantation to the central nervous system, where such grafts include
neural progenitors
such as those found in fetal tissues, neural stem cells, embryonic stem cells
or other cells and
tissues contemplated for neural repair or augmentation. Neural stem and
progenitor cells can
participate in aspects of normal development, including migration along well-
established
migratory pathways to disseminated CNS regions, differentiation into multiple
developmentally- and regionally-appropriate cell types in response to
microenvironmental
cues, and non-disruptive, non-tumorigenic interspersion with host progenitors
and their
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progeny. Human NSCs are capable of expressing foreign transgenes in vivo in
these
disseminated locations. Accordingly, these cells find use in the treatment of
FTD.
INCORPORATION BY REFERENCE
Each of the patents, published patent applications, and non-patent references
cited
herein are hereby incorporated by reference in their entirety.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following claims.
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