U.S. patent application number 14/988387 was filed with the patent office on 2016-06-09 for methods of treatment for alzheimer's disease and huntington's disease.
The applicant listed for this patent is Annexon, Inc., Children's Medical Center Corporation. Invention is credited to Soyon Hong, Michael Leviten, Arnon Rosenthal, Beth A. Stevens, Daniel Wilton.
Application Number | 20160159890 14/988387 |
Document ID | / |
Family ID | 52280583 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160159890 |
Kind Code |
A1 |
Rosenthal; Arnon ; et
al. |
June 9, 2016 |
METHODS OF TREATMENT FOR ALZHEIMER'S DISEASE AND HUNTINGTON'S
DISEASE
Abstract
This invention relates generally to methods of treatment for
neurodegenerative diseases such as Alzheimer's disease,
Alzheimer's-related diseases, and Huntington's disease, and more
specifically to methods involving the inhibition of the classical
pathway of complement activation.
Inventors: |
Rosenthal; Arnon; (Woodside,
CA) ; Leviten; Michael; (Palo Alto, CA) ;
Stevens; Beth A.; (Boston, MA) ; Hong; Soyon;
(Boston, MA) ; Wilton; Daniel; (Boston,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Annexon, Inc.
Children's Medical Center Corporation |
South San Francisco
Boston |
CA
MA |
US
US |
|
|
Family ID: |
52280583 |
Appl. No.: |
14/988387 |
Filed: |
January 5, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2014/046045 |
Jul 9, 2014 |
|
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14988387 |
|
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61871813 |
Aug 29, 2013 |
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61844369 |
Jul 9, 2013 |
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Current U.S.
Class: |
424/136.1 ;
424/133.1; 424/139.1 |
Current CPC
Class: |
A61P 25/16 20180101;
A61P 3/10 20180101; C07K 2317/31 20130101; C07K 2317/34 20130101;
A61P 25/00 20180101; A61P 7/00 20180101; C07K 2317/94 20130101;
C07K 16/40 20130101; A61P 13/12 20180101; A61P 27/02 20180101; A61P
27/06 20180101; C07K 2317/33 20130101; A61K 2039/505 20130101; A61P
1/04 20180101; C07K 2317/56 20130101; A61P 17/00 20180101; A61P
37/06 20180101; C07K 2317/54 20130101; C07K 2317/92 20130101; A61P
11/00 20180101; A61P 11/06 20180101; C07K 2317/20 20130101; C07K
2317/734 20130101; A61P 29/00 20180101; A61K 49/0004 20130101; C07K
2317/14 20130101; C07K 16/18 20130101; A61P 25/14 20180101; A61P
7/06 20180101; C07K 2317/76 20130101; G01N 33/56966 20130101; A61P
9/10 20180101; A61P 3/00 20180101; A61P 21/00 20180101; A61P 37/02
20180101; A61P 3/04 20180101; A61K 2039/507 20130101; A61P 21/04
20180101; C07K 2317/55 20130101; G01N 2333/4716 20130101; A61P
25/28 20180101; C07K 2317/24 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C07K 16/40 20060101 C07K016/40 |
Claims
1. A method of treating or preventing Alzheimer's disease or
Huntington's disease comprising administering an anti-C1q
antibody.
2. (canceled)
3. The method of claim 1, wherein the anti-C1q antibody is: a) an
anti-C1q antibody comprising a light chain variable domain and a
heavy chain variable domain, wherein 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 with ATCC Accession
Number PTA-120399 or progeny thereof; and/or wherein 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 with
ATCC Accession Number PTA-120399 or progeny thereof; b) an isolated
anti-C1q antibody which binds essentially the same C1q epitope as
the antibody M1 produced by the hybridoma cell line with ATCC
Accession Number PTA-120399 or anti-C1q binding fragments thereof;
or c) an murine anti-human C1q monoclonal antibody M1 produced by a
hybridoma cell line with ATCC Accession Number PTA-120399, or
progeny thereof.
4. The method of claim 1, wherein the anti-C1q antibody binds to a
C1q protein and binds to one or more amino acids of the C1q protein
within amino acid residues selected from: a) amino acid residues
196-226 of SEQ ID NO:1 (SEQ ID NO:6), or amino acid residues of a
C1q protein chain A (C1qA) corresponding to amino acid residues
196-226 (GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1 (SEQ ID
NO:6); b) amino acid residues 196-221 of SEQ ID NO:1 (SEQ ID NO:7),
or amino acid residues of a C1qA corresponding to amino acid
residues 196-221 (GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1 (SEQ
ID NO:7); c) amino acid residues 202-221 of SEQ ID NO:1 (SEQ ID
NO:8), or amino acid residues of a C1qA corresponding to amino acid
residues 202-221 (SGGMVLQLQQGDQVWVEKDP) of SEQ ID NO:1 (SEQ ID
NO:8); d) amino acid residues 202-219 of SEQ ID NO:1 (SEQ ID NO:9),
or amino acid residues of a C1qA corresponding to amino acid
residues 202-219 (SGGMVLQLQQGDQVWVEK) of SEQ ID NO:1 (SEQ ID NO:9);
and e) amino acid residues Lys 219 and/or Ser 202 of SEQ ID NO:1,
or amino acid residues of a C1qA corresponding Lys 219 and/or Ser
202 of SEQ ID NO:1.
5. The method of claim 1, wherein the anti-C1q antibody binds to
one or more amino acids of the C1q protein within amino acid
residues selected from: a) amino acid residues 218-240 of SEQ ID
NO:3 (SEQ ID NO:10) or amino acid residues of a C1q protein chain C
(C1qC) corresponding to amino acid residues 218-240 (WLAVNDYYDMVGI
QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:10); b) amino acid residues
225-240 of SEQ ID NO:3 (SEQ ID NO:11) or amino acid residues of a
C1qC corresponding to amino acid residues 225-240 (YDMVGI
QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:11); c) amino acid residues
225-232 of SEQ ID NO:3 (SEQ ID NO:12) or amino acid residues of a
C1qC corresponding to amino acid residues 225-232 (YDMVGIQG) of SEQ
ID NO:3 (SEQ ID NO:12); d) amino acid residue Tyr 225 of SEQ ID
NO:3 or an amino acid residue of a C1qC 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:13) or amino acid residues of a C1qC
corresponding to amino acid residues 174-196
(HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3 (SEQ ID NO:13); f) amino
acid residues 184-192 of SEQ ID NO:3 (SEQ ID NO:14) or amino acid
residues of a C1qC corresponding to amino acid residues 184-192
(RSGVKVVTF) of SEQ ID NO:3 (SEQ ID NO:14); g) amino acid residues
185-187 of SEQ ID NO:3 or amino acid residues of a C1qC
corresponding to amino acid residues 185-187 (SGV) of SEQ ID NO:3;
and h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acid
residue of a C1qC corresponding to amino acid residue Ser 185 of
SEQ ID NO:3.
6. The method of claim 1, wherein the anti-C1q antibody binds to a
C1q protein and binds to one or more amino acids of the C1q protein
chain A (C1qA) within amino acid residues selected from: a) amino
acid residues 196-226 of SEQ ID NO:1 (SEQ ID NO:6), or amino acid
residues of a C1q protein chain A (C1qA) corresponding to amino
acid residues 196-226 (GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID
NO:1 (SEQ ID NO:6); b) amino acid residues 196-221 of SEQ ID NO:1
(SEQ ID NO:7), or amino acid residues of a C1qA corresponding to
amino acid residues 196-221 (GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID.
NO:1 (SEQ ID NO:7); c) amino acid residues 202-221 of SEQ ID NO:1
(SEQ ID NO:8), or amino acid residues of a C1qA corresponding to
amino acid residues 202-221 (SGGMVLQLQQGDQVWVEKDP) of SEQ ID NO:1
(SEQ ID NO:8); d) amino acid residues 202-219 of SEQ ID NO:1 (SEQ
ID NO:9), or amino acid residues of a C1qA corresponding to amino
acid residues 202-219 (SGGMVLQLQQGDQVWVEK) of SEQ ID NO:1 (SEQ ID
NO:9); and e) amino acid residue Lys 219 of SEQ ID NO:1, or an
amino acid residue of a C1qA corresponding Lys 219 of SEQ ID NO:1;
and wherein the anti-C1q antibody binds to one or more amino acids
of the C1q protein chain C (C1qC) within amino acid residues
selected from: a) amino acid residues 174-196 of SEQ ID NO:3 (SEQ
ID NO:13) or amino acid residues of a C1qC corresponding to amino
acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3 (SEQ
ID NO:13); b) amino acid residues 184-192 of SEQ ID NO:3 (SEQ ID
NO:14) or amino acid residues of a C1qC corresponding to amino acid
residues 184-192 (RSGVKVVTF) of SEQ ID NO:3 (SEQ ID NO:14); c)
amino acid residues 185-187 of SEQ ID NO:3 or amino acid residues
of a C1qC corresponding to amino acid residues 185-187 (SGV) of SEQ
ID NO:3; and d) amino acid residue Ser 185 of SEQ ID NO:3 or an
amino acid residue of a C1qC corresponding to amino acid residue
Ser 185 of SEQ ID NO:3.
7. The method of claim 1, wherein the anti-C1q antibody binds
specifically to rat C1q, both human C1q and mouse C1q, or human
C1q, mouse C1q, and rat C1q.
8-9. (canceled)
10. The method of claim 1, wherein the anti-C1q antibody has a
dissociation constant (K.sub.D) for human C1q and mouse C1q less
than about 30 nM.
11-16. (canceled)
17. The method claim 1, wherein the anti-C1q antibody specifically
binds to and inhibits a biological activity of C1q.
18. The method of claim 17, wherein the biological activity is (1)
C1q binding to an autoantibody, (2) C1q binding to C1r , (3) C1q
binding to C1s, (4) C1q binding to phosphatidylserine, (5) C1q
binding to pentraxin-3, (6) C1q binding to C-reactive protein
(CRP), (7) C1q binding to globular C1q receptor (gC1qR), (8) C1q
binding to complement receptor 1 (CR1), (9) C1q binding to
beta-amyloid, (10) C1q binding to calreticulin, (11) activation of
the classical complement activation pathway, (12) activation of
antibody and complement dependent cytotoxicity, (13) CH50
hemolysis, (14) synapse loss, (15) B-cell antibody production, (16)
dendritic cell maturation, (17) T-cell proliferation, (18) cytokine
production (19) microglia activation, (20) Arthus reaction, (21)
phagocytosis of synapses or nerve endings, or (22) activation of
complement receptor 3 (CR3/C3) expressing cells.
19. (canceled)
20. The method of claim 18, wherein CH50 hemolysis comprises human,
mouse, and/or rat CH50 hemolysis.
21. The method of 18, wherein the anti-C1q antibody is capable of
neutralizing at least 50%, at least 80%, or at least 90% of CH50
hemolysis.
22. The method of claim 18, wherein the anti-C1q antibody is
capable of neutralizing at least 50% of CH50 hemolysis at a dose of
less than 200 ng/ml, less than 100 ng/ml, less than 50 ng/ml, or
less than 20 ng/ml.
23. The method of claim 1, wherein the anti-C1q antibody is a
murine, a humanized, a chimeric, or a human antibody.
24. (canceled)
25. The method of claim 23, wherein the anti-C1q antibody binds to
amino acid residue Lys 219 and Ser 202 of the human C1qA as shown
in SEQ ID NO:1 or amino acids of a human C1qA corresponding to Lys
219 and Ser 202 as shown in SEQ ID NO:1, and amino acid residue Tyr
225 of the human C1qC as shown in SEQ ID NO:3 or an amino acid
residue of a human C1qC corresponding to Tyr 225 as shown in SEQ ID
NO:3.
26. The method of claim 23, wherein the anti-C1q antibody binds to
amino acid residue Lys 219 of the human C1qA as shown in SEQ ID
NO:1 or an amino acid residue of a human C1qA corresponding to Lys
219 as shown in SEQ ID NO:1, and amino acid residue Ser 185 of the
human C1qC as shown in SEQ ID NO:3 or an amino acid residue of a
human C1qC corresponding to Ser 185 as shown in SEQ ID NO:3.
27. The method of claim 1, wherein the anti-C1q antibody is a
multivalent antibody or a bispecific antibody.
28-34. (canceled)
35. The method of claim 1, wherein the anti-C1q antibody is an
antibody fragment, wherein the fragment is a Fab, F(ab').sub.2 or
Fab ' fragment.
36-38. (canceled)
39. The method of claim 35, wherein the antibody fragment has a
shorter half-life as compared to its corresponding full-length
antibody.
40-41. (canceled)
42. The method of claim 1, wherein the anti-C1q antibody inhibits
complement-dependent cell-mediated cytotoxicity (CDCC) activation
pathway by an amount from at least 30% to at least 99.9%.
43. The method of claim 1, wherein the anti-C1q antibody does not
inhibit the lectin complement activation pathway.
44. The method of claim 1, wherein the anti-C1q antibody having a
dissociation constant (K.sub.D) for its corresponding antigen from
100 nM to 0.005 nM or less than 0.005 nM.
45. The method of claim 1, wherein the anti-C1q antibody inhibits
autoantibody-dependent and complement-dependent cytotoxicity
(CDC).
46. The method of claim 1, wherein the anti-C1q antibody prevents
amplification of the alternative complement activation pathway
initiated by C1q binding.
47. (canceled)
48. The method of claim 1, wherein the anti-C1q antibody does not
inhibit autoantibody-dependent cellular cytotoxicity (ADCC).
49. The method of claim 1, comprising administering a
therapeutically effective amount of two antibodies, wherein the two
antibodies are selected from an anti-C1q antibody, an anti-C1r
antibody, and an anti-C1s antibody.
50-54. (canceled)
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/844,369, filed Jul. 9, 2013, and U.S.
Provisional Application No. 61/871,813, filed Aug. 29, 2013, each
of which is hereby incorporated by reference in its entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
717192000840SeqList.txt, date recorded: Jul. 9, 2014, size: 13
KB).
BACKGROUND
[0003] 1. Field
[0004] This invention relates generally to methods of treatment for
neurodegenerative diseases such as Alzheimer's disease,
Alzheimer's-related diseases, and Huntington's disease, and more
specifically to methods involving the inhibition of the classical
pathway of complement activation.
[0005] 2. Description of Related Art
[0006] A neurodegenerative disease is a disease involving cognitive
disorders, such as Alzheimer's disease (AD) and Huntington's
disease (HD), to name but a few. These cognitive disorders are
caused by an increase in cell death processes that results in a
great reduction of neuron number, behavioral changes and a general,
gradual degeneration that leads to the patient's death. Currently,
approximately 5 million Americans suffer from Alzheimer's disease
(Thies et al. 2013), and because neurodegenerative diseases strike
primarily in mid-to-late life, the incidence of Alzheimer's disease
and other neurodegenerative disorders is expected to soar as the
population ages. Accordingly, there is a continuing need for new
treatments and cures for such neurodegenerative diseases.
[0007] Alzheimer's disease is characterized by a progressive loss
of cognitive function associated with an excessive number of senile
plaques in the cerebral cortex and subcortical gray matter.
Alzheimer's disease accounts for more than 65% of dementias in the
elderly. Pathologically, Alzheimer's disease is characterized by
two brain lesions, called senile plaques and neurofibrillary
tangles (NFT), which accumulate, respectively, ABeta (A.beta.)
peptides and the microtubule-associated protein tau (MAPT). These
neuropathological hallmarks have given rise to two corresponding
hypothesis of the neurodegenerative process: the .beta./amyloid
cascade hypothesis (Hardy and Selkoe 2002; Karran et al. 2011), and
the Tau-phosphorylation/NFT hypothesis (Vishnu, 2013). In the
A.beta./amyloid cascade hypothesis, the A.beta. peptide evolves
from the cleavage of the amyloid precursor protein (APP) by
.beta./.gamma.-secretase complexes, which associate presenilins-1
or -2 (PS-1 or PS-2), leading typically to the formation of a 38,
40, or 42 amino acid peptide (A.beta.38, 40 or 42). Among the most
abundant A.beta. species, A.beta.42 is the predominant form that
aggregates outside the cells forming the characteristic amyloid
plaques (Yan and Wang 2008). The longer the A.beta. peptide (i.e.,
the more C-terminal within the .beta.-CTF transmembrane domain
where g-secretase cleaves), the greater its hydrophobicity and
propensity to oligomerize (Haass and Selkoe 2007). Dyshomeostasis
of A.beta., in particular, A.beta.42, is currently thought to be
one of the earliest biomarkers of Alzheimer's disease in humans,
even before clinical symptoms emerge (Sperling et al., 2011); the
lowering of the A.beta.42 peptide precedes the rise of levels of
tau and phosphorylated tau in the CSF of humans (Craig-Schapiro et
al., 2009; Golde et al., 2011). Moreover, several lines of
evidence, such as genetics, support the role of A.beta.
dyshomeostasis as the principal driving force in Alzheimer's
disease (Tanzi 2012). Current evidence also suggests that soluble
low-n A.beta. oligomers, but not A.beta. monomers, mediate
synaptotoxicity (Walsh and Selkoe 2007). Several studies suggest
that small oligomeric morphologies of A.beta. are the primary toxic
species in Alzheimer's disease (Glabe et al. 2006; Walsh et al.
2002).
[0008] Huntington's disease is an inherited neurodegenerative
disease, characterized by motor, cognitive, behavioral, and
psychological dysfunction. Similar to Alzheimer's disease, where
protein aggregates are associated with disease, Huntington's
disease belongs to a family of neurodegenerative diseases caused by
mutations in which an expanded CAG repeat tract results in long
stretches of polyglutamine (polyQ) in the encoded protein (i.e.,
mutant Huntington) which in Huntington's disease forms aggregates
within the cell (Martindale et al., 1998).
[0009] One of the earliest events in both Alzheimer's disease and
Huntington's disease pathology is dysfunction and loss of synapses
(Scheff et al., 2006; Graveland et al., 1995; Ferrante et al.,
1991; Selkoe 2002; Mallucci 2009). Dramatic synapse loss is seen in
mesiotemporal regions of Alzheimer's disease brains (Davies et al.,
1987; DeKosky and Scheff, 1990; Terry et al., 1991; Masliah et al.,
2001) and the functional significance of this synaptic deficiency
is supported by multiple studies showing that synapse loss is
actually the best pathological correlate of cognitive dysfunction
(Terry et al., 1991; DeKosky et al., 1996; Coleman et al., 2003).
Synaptic dysfunction and loss are early manifestations of disease
in human Huntington's disease with significant deficits in synaptic
protein levels (a-tubulin, neurofilament, MAP-2) and subcellular
distribution (neurofilament, PACSIN 1) beginning at the
pre-symptomatic stage of disease (DiProspero et al., 2004). This
synaptic dysfunction has been explored in mouse transgenic models
of Huntington's disease, confirming that decreased synapse function
is an early manifestation of disease. Multiple studies have shown
that long-term potentiation (LTP), a measure of synaptic
plasticity, is significantly reduced in mutant Huntingtin
transgenic mice, with synapses unable to respond normally to
repetitive stimuli, potentially accounting for some of the
cognitive decline associated with the disease (Usdin et al., 1999;
Lynch et al., 2007). In addition, the association of disease with
synapse dysfunction caused by excitotoxicity has been demonstrated.
Surgical and chemical damage to the corticostriatal and
nigrostriatal pathways mediating excitotoxicity attenuate
neuropathological and clinical phenotypes in the R6/2 transgenic
model of Huntington's disease (Stack et al., 2007). Similar
disturbances in corticostriatal synapse function were reported in
independent studies using additional Huntingtin transgenics
(Milnerwood and Raymond, 2007).
[0010] There is currently no disease modifying therapy for
Alzheimer's disease and existing drugs only provide modest
symptomatic relief. These drugs fall into two classes based on
mechanism of action: 1) N-methyl-D-asparate receptor antagonists
(e.g., Ebixa, Namenda); and 2) acetylcholinesterase inhibitors
(e.g., Razadyne, Exelon, Aricept and Cognex) (Ballard et al 2011).
There are currently over 25 drugs in clinical trials targeting a
number of distinct pathways. The mechanisms being targeted include
clearance of A.beta. plaques, reduction of inflammation, prevention
of neuronal death, and preservation of mitochondrial function.
However, one problem is that, to date, antibodies designed to
facilitate clearance of plaques and drugs designed to preserve
mitochondrial function have failed to meet primary endpoints of
mid-stage clinical trials (Delrieu et al., 2012). These
disappointing results have stimulated a reevaluation of the
mechanisms underlying the disease, particularly the role of
plaques. Accordingly, there is a continued need to develop
alternative therapeutic strategies for treating neurodegenerative
diseases, such as Alzheimer's disease and Huntington's disease. The
C1q protein and other components of the C1 complex may be
attractive therapeutic targets for preventing early synapse loss
characteristic of such neurodegenerative diseases.
[0011] All references cited herein, including patent applications
and publications, are hereby incorporated by reference in their
entirety.
BRIEF SUMMARY
[0012] Certain aspects of the present disclosure provide anti-C1q
antibodies and methods of using such antibodies for treating or
preventing Alzheimer's disease, Alzheimer's-related diseases, and
Huntington's disease.
[0013] Certain aspects of the present disclosure are directed to
methods and kits for treating or preventing Alzheimer's disease
and/or Huntington's disease that include inhibiting the classical
pathway of complement activation by neutralizing complement factor
C1q, e.g., through the administration of antibodies, such as
monoclonal, chimeric, humanized antibodies, antibody fragments,
etc., which bind to C1q.
[0014] In certain aspects, the present disclosure provides a method
of treating or preventing Alzheimer's disease in an individual in
need of such treatment, the method comprising the step of
administering a therapeutically effective dose of an anti-C1q
antibody. In other aspects, the present disclosure provides an
anti-C1q antibody for use in treating or preventing Alzheimer's
disease in an individual in need of such treatment. In other
aspects, the present disclosure provides use of an anti-C1q
antibody in the manufacture of a medicament for treating or
preventing Alzheimer's disease. In other aspects, the present
disclosure provides a kit comprising an anti-C1q antibody and a
package insert comprising instructions for using the antibody to
treat or prevent Alzheimer's disease in an individual in need of
such treatment.
[0015] In other aspects, the present disclosure provides a method
of treating or preventing Huntington's disease in an individual in
need of such treatment, the method comprising the step of
administering a therapeutically effective dose of an anti-C1q
antibody. In other aspects, the present disclosure provides an
anti-C1q antibody for use in treating or preventing Huntington's
disease in an individual in need of such treatment. In other
aspects, the present disclosure provides use of an anti-C1q
antibody in the manufacture of a medicament for treating or
preventing Huntington's disease. In other aspects, the present
disclosure provides a kit comprising an anti-C1q antibody and a
package insert comprising instructions for using the antibody to
treat or prevent Huntington's disease in an individual in need of
such treatment.
[0016] In certain embodiments that may be combined with any of the
preceding embodiments, the anti-C1q antibody is: i) an isolated
anti-C1q antibody comprising a light chain variable domain and a
heavy chain variable domain, wherein 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 with ATCC Accession
Number PTA-120399 or progeny thereof; and/or wherein 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 with
ATCC Accession Number PTA-120399 or progeny thereof; ii) an
isolated anti-C1q antibody which binds essentially the same C1q
epitope as the antibody M1 produced by the hybridoma cell line with
ATCC Accession Number PTA-120399 or anti-C1q binding fragments
thereof; or iii) an isolated murine anti-human C1q monoclonal
antibody M1 produced by a hybridoma cell line with ATCC Accession
Number PTA-120399, or progeny thereof. In certain embodiments that
may be combined with any of the preceding embodiments, the anti-C1q
antibody binds to a C1q protein and binds to one or more amino
acids of the C1q protein within amino acid residues selected from
the group consisting of: i) amino acid residues 196-226 of SEQ ID
NO:1 (SEQ ID NO:6), or amino acid residues of a C1q protein chain A
(C1qA) corresponding to amino acid residues 196-226
(GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1 (SEQ ID NO:6); ii)
amino acid residues 196-221 of SEQ ID NO:1 (SEQ ID NO:7), or amino
acid residues of a C1qA corresponding to amino acid residues
196-221 (GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1 (SEQ ID NO:7);
iii) amino acid residues 202-221 of SEQ ID NO:1 (SEQ ID NO:8), or
amino acid residues of a C1qA corresponding to amino acid residues
202-221 (SGGMVLQLQQGDQVWVEKD) of SEQ ID NO:1 (SEQ ID NO:8); iv)
amino acid residues 202-219 of SEQ ID NO:1 (SEQ ID NO:9), or amino
acid residues of a C1qA corresponding to amino acid residues
202-219 (SGGMVLQLQQGDQVWVEK) of SEQ ID NO:1 (SEQ ID NO:9); and v)
amino acid residues Lys 219 and/or Ser 202 of SEQ ID NO:1, or amino
acid residues of a C1qA corresponding Lys 219 and/or Ser 202 of SEQ
ID NO:1. In certain embodiments that may be combined with any of
the preceding embodiments, the anti-C1q antibody binds to one or
more amino acids of the C1q protein within amino acid residues
selected from the group consisting of: i) amino acid residues
218-240 of SEQ ID NO:3 (SEQ ID NO:10) or amino acid residues of a
C1q protein chain C (C1qC) corresponding to amino acid residues
218-240 (WLAVNDYYDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:10);
ii) amino acid residues 225-240 of SEQ ID NO:3 (SEQ ID NO:11) or
amino acid residues of a C1qC corresponding to amino acid residues
225-240 (YDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:11); iii)
amino acid residues 225-232 of SEQ ID NO:3 (SEQ ID NO:12) or amino
acid residues of a C1qC corresponding to amino acid residues
225-232 (YDMVGIQG) of SEQ ID NO:3 (SEQ ID NO:12); iv) amino acid
residue Tyr 225 of SEQ ID NO:3 or an amino acid residue of a C1qC
corresponding to amino acid residue Tyr 225 of SEQ ID NO:3; v)
amino acid residues 174-196 of SEQ ID NO:3 (SEQ ID NO:13) or amino
acid residues of a C1qC corresponding to amino acid residues
174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3 (SEQ ID NO:13);
vi) amino acid residues 184-192 of SEQ ID NO:3 (SEQ ID NO:14) or
amino acid residues of a C1qC corresponding to amino acid residues
184-192 (RSGVKVVTF) of SEQ ID NO:3 (SEQ ID NO:14); vii) amino acid
residues 185-187 of SEQ ID NO:3 or amino acid residues of a C1qC
corresponding to amino acid residues 185-187 (SGV) of SEQ ID NO:3;
and viii) amino acid residue Ser 185 of SEQ ID NO:3 or an amino
acid residue of a C1qC corresponding to amino acid residue Ser 185
of SEQ ID NO:3. In certain embodiments that may be combined with
any of the preceding embodiments, the anti-C1q antibody binds to a
C1q protein and binds to one or more amino acids of the C1q protein
chain A (C1qA) within amino acid residues selected from the group
consisting of: i) amino acid residues 196-226 of SEQ ID NO:1 (SEQ
ID NO:6), or amino acid residues of a C1q protein chain A (C1qA)
corresponding to amino acid residues 196-226
(GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1 (SEQ ID NO:6); ii)
amino acid residues 196-221 of SEQ ID NO:1 (SEQ ID NO:7), or amino
acid residues of a C1qA corresponding to amino acid residues
196-221 (GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1 (SEQ ID NO:7);
iii) amino acid residues 202-221 of SEQ ID NO:1 (SEQ ID NO:8), or
amino acid residues of a C1qA corresponding to amino acid residues
202-221 (SGGMVLQLQQGDQVWVEKD) of SEQ ID NO:1 (SEQ ID NO:8); iv)
amino acid residues 202-219 of SEQ ID NO:1 (SEQ ID NO:9), or amino
acid residues of a C1qA corresponding to amino acid residues
202-219 (SGGMVLQLQQGDQVWVEK) of SEQ ID NO:1 (SEQ ID NO:9); and v)
amino acid residue Lys 219 of SEQ ID NO:1, or an amino acid residue
of a C1qA corresponding Lys 219 of SEQ ID NO:1; and wherein the
isolated anti-C1q antibody binds to one or more amino acids of the
C1q protein chain C (C1qC) within amino acid residues selected from
the group consisting of: i) amino acid residues 174-196 of SEQ ID
NO:3 (SEQ ID NO:13) or amino acid residues of a C1qC corresponding
to amino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID
NO:3 (SEQ ID NO:13); ii) amino acid residues 184-192 of SEQ ID NO:3
(SEQ ID NO:14) or amino acid residues of a C1qC corresponding to
amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3 (SEQ ID
NO:14); iii) amino acid residues 185-187 of SEQ ID NO:3 or amino
acid residues of a C1qC corresponding to amino acid residues
185-187 (SGV) of SEQ ID NO:3; and iv) amino acid residue Ser 185 of
SEQ ID NO:3 or an amino acid residue of a C1qC corresponding to
amino acid residue Ser 185 of SEQ ID NO:3. In certain embodiments
that may be combined with any of the preceding embodiments, the
anti-C1q antibody binds to amino acid residue Lys 219 and Ser 202
of the human C1qA as shown in SEQ ID NO:1 or amino acids of a human
C1qA corresponding to Lys 219 and Ser 202 as shown in SEQ ID NO:1,
and amino acid residue Tyr 225 of the human C1qC as shown in SEQ ID
NO:3 or an amino acid residue of a human C1qC corresponding to Tyr
225 as shown in SEQ ID NO:3. In certain embodiments that may be
combined with any of the preceding embodiments, the anti-C1q
antibody binds to amino acid residue Lys 219 of the human C1qA as
shown in SEQ ID NO:1 or an amino acid residue of a human C1qA
corresponding to Lys 219 as shown in SEQ ID NO:1, and amino acid
residue Ser 185 of the human C1qC as shown in SEQ ID NO:3 or an
amino acid residue of a human C1qC corresponding to Ser 185 as
shown in SEQ ID NO:3.
[0017] In certain embodiments that may be combined with any of the
preceding embodiments, the anti-C1q antibody binds specifically to
both human C1q and mouse C1q. In certain embodiments that may be
combined with any of the preceding embodiments, the anti-C1q
antibody binds specifically to rat C1q. In certain embodiments that
may be combined with any of the preceding embodiments, the anti-C1q
antibody binds specifically to human C1q, mouse C1q, and rat C1q.
In certain embodiments that may be combined with any of the
preceding embodiments, the anti-C1q has dissociation constant
(K.sub.D) for human C1q and mouse C1q that ranges from less than
about 30 nM to less than about 100 pM. In certain embodiments that
may be combined with any of the preceding embodiments, the anti-C1q
antibody has dissociation constant (K.sub.D) for human C1q and
mouse C1q of less than about 30 nM. In certain embodiments that may
be combined with any of the preceding embodiments, the anti-C1q
antibody has dissociation constant (K.sub.D) for human C1q and
mouse C1q of less than about 20 nM. In certain embodiments that may
be combined with any of the preceding embodiments, the anti-C1q
antibody has dissociation constant (K.sub.D) for human C1q and
mouse C1q of less than about 10 nM. In certain embodiments that may
be combined with any of the preceding embodiments, the anti-C1q
antibody has dissociation constant (K.sub.D) for human C1q and
mouse C1q of less than about 5 nM. In certain embodiments that may
be combined with any of the preceding embodiments, the anti-C1q
antibody has dissociation constant (K.sub.D) for human C1q and
mouse C1q of less than about 1 nM. In certain embodiments that may
be combined with any of the preceding embodiments, the anti-C1q
antibody has dissociation constant (K.sub.D) for human C1q and
mouse C1q of less than about 100 pM. In certain embodiments that
may be combined with any of the preceding embodiments, the anti-C1q
antibody specifically binds to and neutralizes a biological
activity of C1q. In certain embodiments that may be combined with
any of the preceding embodiments, the biological activity is (1)
C1q binding to an autoantibody, (2) C1q binding to C1r, (3) C1q
binding to C1s, (4) C1q binding to phosphatidylserine, (5) C1q
binding to pentraxin-3, (6) C1q binding to C-reactive protein
(CRP), (7) C1q binding to globular C1q receptor (gC1qR), (8) C1q
binding to complement receptor 1 (CR1), (9) C1q binding to
beta-amyloid, or (10) C1q binding to calreticulin. In certain
embodiments that may be combined with any of the preceding
embodiments, the biological activity 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 certain embodiments that
may be combined with any of the preceding embodiments, CH50
hemolysis comprises human, mouse, and/or rat CH50 hemolysis. In
certain embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody is capable of neutralizing at
least 50%, at least 80%, or at least 90% of CH50 hemolysis. In
certain embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody is capable of neutralizing at
least 50% of CH50 hemolysis at a dose of less than 200 ng/ml, less
than 100 ng/ml, less than 50 ng/ml, or less than 20 ng/ml. In
certain embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody is a murine antibody. In certain
embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody is a humanized, a chimeric
antibody, or a human antibody.
[0018] In certain embodiments that may be combined with any of the
preceding embodiments, the anti-C1q antibody is a multivalent
antibody. In certain embodiments that may be combined with any of
the preceding embodiments, the anti-C1q antibody is a bispecific
antibody. In certain embodiments that may be combined with any of
the preceding embodiments, the anti-C1q antibody has been
engineered to increase brain penetration. In certain embodiments
that may be combined with any of the preceding embodiments, the
anti-C1q antibody is a bispecific antibody recognizing a first
antigen and a second antigen. In certain embodiments that may be
combined with any of the preceding embodiments, the first antigen
is a C1q protein and the second antigen is an antigen facilitating
transport across the blood-brain-barrier. In certain embodiments
that may be combined with any of the preceding embodiments, the
second antigen is selected from the group consisting of 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, and ANG1005. In certain embodiments that may be combined
with any of the preceding embodiments, the anti-C1q antibody is of
the IgG class. In certain embodiments that may be combined with any
of the preceding embodiments, the anti-C1q antibody has an
IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4 isotype. In certain
embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody is an antibody fragment. In
certain embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody is a Fab, F(ab').sub.2 or Fab'
fragment. In certain embodiments that may be combined with any of
the preceding embodiments, the antibody fragment specifically binds
to and neutralizes a biological activity of C1q. In certain
embodiments that may be combined with any of the preceding
embodiments, the antibody fragment has better brain penetration as
compared to its corresponding full-length antibody. In certain
embodiments that may be combined with any of the preceding
embodiments, the antibody fragment has a shorter half-life as
compared to its corresponding full-length antibody. In certain
embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody inhibits C3 deposition. In
certain embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody inhibits synapse loss. In
certain embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody inhibits complement-dependent
cell-mediated cytotoxicity (CDCC) activation pathway by an amount
that ranges from at least 30% to at least 99.9%. In certain
embodiments that may be combined with any of the preceding
embodiments, the anti-C1q antibody does not inhibit the lectin
complement activation pathway. In certain embodiments that may be
combined with any of the preceding embodiments, the anti-C1q
antibody comprises a dissociation constant (K.sub.D) for its
corresponding antigen that ranges from 100 nM to 0.005 nM or less
than 0.005 nM. In certain embodiments that may be combined with any
of the preceding embodiments, the anti-C1q antibody inhibits
autoantibody-dependent and complement-dependent cytotoxicity (CDC).
In certain embodiments that may be combined with any of the
preceding embodiments, the anti-C1q antibody prevents amplification
of the alternative complement activation pathway initiated by C1q
binding. In certain embodiments that may be combined with any of
the preceding embodiments, the anti-C1q antibody comprises an
EC.sub.50 that ranges from 3 .mu.g/ml to 0.05 .mu.g/ml, or less
than 0.05 .mu.g/ml. In certain embodiments that may be combined
with any of the preceding embodiments, the anti-C1q antibody does
not inhibit autoantibody-dependent cellular cytotoxicity (ADCC). In
certain embodiments that may be combined with any of the preceding
embodiments, the method further comprises administering to the
individual a therapeutically effective amount of a second anti-C1q
antibody. In certain embodiments that may be combined with any of
the preceding embodiments, the method further comprises
administering to the individual a therapeutically effective amount
of a second antibody, wherein the second antibody is selected from
the group consisting of an anti-C1r antibody, an anti-C1s antibody
and an anti-C1 complex antibody. In certain embodiments that may be
combined with any of the preceding embodiments, the method further
comprises administering to the individual a therapeutically
effective amount of an inhibitor of antibody-dependent cellular
cytotoxicity (ADCC). In certain embodiments that may be combined
with any of the preceding embodiments, the method further comprises
administering to the individual a therapeutically effective amount
of an inhibitor of the alternative complement activation pathway.
In certain embodiments that may be combined with any of the
preceding embodiments, the method further comprises administering
to the individual an inhibitor of the interaction between an
autoantibody and an autoantigen. In certain embodiments that may be
combined with any of the preceding embodiments, the anti-C1q
antibody binds a C1q antigen with a binding stoichiometry that
ranges from 20:1 to 1.0:1 or less than 1.0:1.
[0019] In certain aspects, the present disclosure provides for a
diagnostic kit comprising an anti-C1q antibody of any of the
preceding embodiments for treating or preventing Alzheimer's
disease. In other aspects, the present disclosure provides for a
diagnostic kit comprising an anti-C1q antibody of any of the
preceding embodiments for treating or preventing Huntington's
disease.
[0020] 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. These and other aspects of the compositions and
methods provided herein will become apparent to one of skill in the
art.
DESCRIPTION OF THE FIGURES
[0021] FIG. 1 illustrates the results of an ELISA screen for
antibodies specifically binding human C1q. Hybridoma supernatants
containing anti-C1q antibodies 1C7, 2A1, 3A2, or 5A3 respectively
were tested. Left columns (grey) represent signals for anti-C1q
antibody-binding to human C1q protein. Right columns (black)
represent signals for anti-C1q antibody-binding to human
transferrin (HT).
[0022] FIG. 2 illustrates the C1q-neutralizing activities of
anti-C1q antibodies 1C7, 2A1, 3A2, and 5A3 in a human CH50
hemolytic assay in a single-dose format.
[0023] FIG. 3 illustrates the C1q-neutralizing activities of
anti-C1q antibodies 1C7, 3A2, and 4A4B 11 in a human CH50 hemolytic
assay in a dose-response format.
[0024] FIG. 4 illustrates the C1q-neutralizing activities of
anti-C1q antibodies M1 and 4A4B11 in human, mouse, and rat CH50
hemolytic assays in a dose-response format. FIG. 4A illustrates
results from a human CH50 hemolytic assay. FIG. 4B illustrates
results from a mouse CH50 hemolytic assay. FIG. 4C illustrates
results from a rat CH50 hemolytic assay.
[0025] FIG. 5 illustrates mass spectrometry characterization of C1q
antibody complexes. FIG. 5A shows a mixture of ANN-001 (4A4B 11)
and C1q shows that ANN-001 monomer at the predicted mass of
.about.150 kDa, C1q monomer at the expected mass of .about.460 kDa,
and the C1q/ANN-001 1:1 complex at the predicted mass of .about.600
kDa. FIG. 5B shows a mixture of ANN-005 (M1) and C1q shows that
ANN-005 monomer at the predicted mass of .about.150 kDa, C1q
monomer at the expected mass of .about.460 kDa, and the C1q/ANN-005
1:1 complex at the predicted mass of .about.600 kDa.
[0026] FIG. 6 illustrates C1q peptides do not compete with intact
C1q for binding to monoclonal antibody ANN-005 (M1). FIG. 6A
depicts C1q and ANN-005 mixed in equimolar concentrations and
incubated in the absence of a mixture of C1q peptides. FIG. 6B
depicts C1q and ANN-005 mixed in equimolar concentrations and
incubated in the presence of a mixture of C1q peptides generated by
pepsin digestion of C1q and analyzed by mass spectrometry. In each
case, a portion of the unbound antibody and antigen (ANN-005 and
C1q) can be identified at the expected masses for monomers
(.about.150 kDa and .about.460kDa respectively) and a 1:1 complex
is present at a mass of .about.615 kDa.
[0027] FIG. 7A illustrates a general schematic representation of
the complement cascade, including the three complement activation
pathways and the terminal pathway. FIG. 7B illustrated a schematic
of the C1 complex. The C1s and C1r dimers are seen in a complex
with the C1q hexamer.
[0028] FIG. 8 illustrates that A.beta. oligomers induce C1q
deposition and localization to synapses in wild-type brain. 18
hours post ICV injection of A.beta., brains were harvested for IHC.
FIG. 8A shows high-resolution images of C1q deposition in the
hippocampus of wild-type (WT) mice. The left frame depicts C1q
deposition in the hippocampus of control mice, middle frame depicts
C1q deposition in the hippocampus of wild-type mice injected with
A.beta. monomers, and right frame depicts C1q deposition in the
hippocampus of wild-type mice injected with A.beta. oligomers. FIG.
8B shows C1q deposition induced by A.beta. oligomer injection and
co-localization with the synaptic marker PSD95 (left frame), and a
quantitative comparison of the percentage of PSD95 (% PSD95)
co-localized with C1q between A.beta. oligomer-injected mice and
A.beta. monomer-injected mice. N=2 mice per treatment.
[0029] FIG. 9 illustrates how C1q deficiency suppresses the
hippocampal synapse loss induced by soluble A.beta. oligomers. ICV
injection of either soluble A.beta. oligomers or A.beta. monomers
was performed in healthy adult (2-3 mo) wild-type (WT) mice. After
72 hours, brains were harvested for IHC using synapsin as a
pre-synaptic marker and PSD95 as a post-synaptic marker. FIG. 9A
shows the labeling of synapses in control mice (left frame), mice
injected with A.beta. monomers (middle frame), and mice injected
with A.beta. oligomers (right frame). FIG. 9B shows a plot of the
number of structural synapses as defined by number of co-localized
puncta (i.e., synapsin-co-immunoreactive and
PSD95-co-immunoreactive) using ImageJ (N=3-5 mice per group.
Values=Ave.+-.SEM. **P=0.0032 by 1-way ANOVA). FIG. 9C shows a plot
of co-localized synaptic puncta in C1qA knock-out (KO) mice 72
hours post ICV injection of A.beta. oligomers and monomers, as
compared to no A.beta. injection (None). N=3 mice per group.
[0030] FIG. 10 depicts high-resolution images illustrating that
A.beta. oligomers fail to induce C3 in C1q knock-out (KO) mice. 18
hours post ICV injection of A.beta., brains were harvested for IHC.
Representative images of N=3 mice per group.
[0031] FIG. 11 illustrates that anti-C1q antibodies can suppress
complement deposition and synapse loss in an acute model of
A.beta.-induced synaptotoxicity. FIG. 11A shows photomicrographs of
C3 staining in the hippocampus of wild-type (WT) mice co-injected
with A.beta. oligomers and either a C1q blocking antibody or an IgG
control (5 ng ICV plus 20 mg/kg IP). FIG. 11B shows photomicrogaphs
of PSD95 and Synapsin staining in the hippocampus of WT mice
co-injected with A.beta. oligomers and either a C1q blocking
antibody or an IgG control.
[0032] FIG. 12 depicts fluorescence photomicrographs illustrating
that the anti-C1q antibody M1 can prevent complement deposition in
a transgenic mouse model of Huntington's disease (HD). 20 mg/kg C1q
blocking antibody and IgG control were injected IP (twice over a 48
hr period) into 5mo wild-type (WT) mice and zQ175 mice.
DETAILED DESCRIPTION OF THE INVENTION
General Techniques
[0033] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, 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 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. Calos, 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 (V. T. DeVita et al., eds., J.
B. Lippincott Company, 1993).
Definitions
[0034] As used herein, the term "preventing" includes providing
prophylaxis with respect to occurrence or recurrence of a
particular disease, disorder, or condition in an individual. An
individual may be predisposed to, susceptible to a particular
disease, disorder, or condition, or at risk of developing such a
disease, disorder, or condition, but has not yet been diagnosed
with the disease, disorder, or condition.
[0035] 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.
[0036] As used herein, the term "treatment" refers to clinical
intervention designed to alter the natural course of the individual
being treated during the course of clinical pathology. Desirable
effects of treatment include decreasing the rate of progression,
ameliorating or palliating the pathological state, and remission or
improved prognosis of a particular disease, disorder, or condition.
An individual is successfully "treated", for example, if one or
more symptoms associated with a particular disease, disorder, or
condition are mitigated or eliminated.
[0037] An "effective amount" refers to at least an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic or prophylactic result. An effective amount
can be provided in one or more administrations.
[0038] A "therapeutically effective amount" is at least the minimum
concentration required to effect a measurable improvement of a
particular disease, disorder, or condition. 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 anti-C1q antibody to elicit a desired response in the
individual. A therapeutically effective amount may also be one in
which any toxic or detrimental effects of the anti-C1q antibody are
outweighed by the therapeutically beneficial effects.
[0039] 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 consecutively done without interruption, but rather is
cyclic in nature.
[0040] As used herein, administration "in conjunction" 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.
[0041] An "individual" for purposes of treatment, prevention, or
reduction of risk, refers to any animal classified as a mammal,
including humans, domestic and farm animals, and zoo, sport, or pet
animals, such as dogs, horses, rabbits, cattle, pigs, hamsters,
gerbils, mice, ferrets, rats, cats, and the like. In some
embodiments, the individual is human.
[0042] As used herein, "autoantibody" means any antibody that
recognizes a host antigen.
[0043] 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, multispecific antibodies (e.g. bispecific
antibodies) formed from at least two intact antibodies, and
antibody fragments so long as they exhibit the desired biological
activity.
[0044] 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 V.sub.H and V.sub.L
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,
Conn., 1994, page 71 and Chapter 6.
[0045] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa (".kappa.") and
lambda (".lamda."), 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 designated alpha (".alpha."), delta (".delta."), epsilon
(".epsilon."), gamma (".gamma.") and mu (".mu."), respectively. The
.gamma. and .alpha. 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: IgG1, IgG2, IgG3, IgG4, IgA1, 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 al., Cellular and Molecular Immunology,
4.sup.th ed. (W.B. Saunders Co., 2000).
[0046] "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 (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) 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.
[0047] An "isolated" antibody, such as an anti-C1q antibody of the
present disclosure, is one that has been identified, separated
and/or recovered from a component of its production environment
(e.g., naturally or recombinantly). In some embodiments, 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 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 some 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 silver stain. 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 at least one purification step.
[0048] The "variable region" or "variable domain" of an antibody,
such as an anti-C1q antibody of the present disclosure, 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 "V.sub.H" and "V.sub.L", 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.
[0049] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies, such as anti-C1q antibodies of the present disclosure.
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.
[0050] The term "monoclonal antibody" as used herein refers to an
antibody, such as an anti-C1q antibody of the present disclosure,
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising 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, the monoclonal antibodies are advantageous in that
they are synthesized by the hybridoma culture, uncontaminated by
other immunoglobulins. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies to be used in accordance with
the present invention may be made by 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, e.g., U.S.
Pat. No. 4,816,567), phage-display technologies (see, e.g.,
Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol.
Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):
299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004);
Fellouse, Proc. Nat'l Acad. Sci. USA 101(34):12467-472 (2004); and
Lee et al., J. 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
al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol.
7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology
10:779-783 (1992); Lonberg et al., 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).
[0051] The terms "full-length antibody," "intact antibody" or
"whole antibody" are used interchangeably to refer to an antibody,
such as and anti-C1q antibody of the present disclosure, 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.
[0052] An "antibody fragment" comprises a portion of an intact
antibody, the antigen binding and/or the variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2 and Fv fragments; diabodies; linear antibodies (see
U.S. Pat. 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.
[0053] Papain digestion of antibodies, such as anti-C1q antibodies
of the present disclosure, produces two identical antigen-binding
fragments, called "Fab" fragments, and a residual "Fc" 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 (V.sub.H), and the first constant
domain of one heavy chain (C.sub.H1). 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').sub.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 fragments by having a few additional
residues at the carboxy terminus of the C.sub.H1 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').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0054] 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.
[0055] "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.
[0056] "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. In some embodiments, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L 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).
[0057] "Functional fragments" of antibodies, such as anti-C1q
antibodies of the present disclosure, 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.
[0058] 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 V.sub.H and
V.sub.L 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 V.sub.H and V.sub.L domains of the two
antibodies are present on different polypeptide chains. Diabodies
are described in greater detail in, for example, EP 404,097; WO
93/11161; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48
(1993).
[0059] As used herein, a "chimeric antibody" refers to an antibody
(immunoglobulin), such as an anti-C1q antibody of the present
disclosure, 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. Pat. No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci.
USA, 81:6851-55 (1984)). Chimeric antibodies of interest herein
include PRIMATIZED.RTM. 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 used a subset of "chimeric
antibodies."
[0060] "Humanized" forms of non-human (e.g., murine) antibodies,
such as anti-C1q antibodies of the present disclosure, are chimeric
antibodies that contain minimal sequence derived from non-human
immunoglobulin. In one embodiment, 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. Pat. Nos. 6,982,321 and 7,087,409.
[0061] A "human antibody" is one that possesses an amino-acid
sequence corresponding to that of an antibody, such as an anti-C1q
antibody of the present disclosure, 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, J. Mol. Biol., 227:381 (1991);
Marks et al., J. 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., J. 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
produce such antibodies in response to antigenic challenge, but
whose endogenous loci have been disabled, e.g., immunized xenomice
(see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding
XENOMOUSE.TM. 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.
[0062] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody-variable domain, such
as that of an anti-C1q antibody of the present disclosure, 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).
[0063] 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. J. Mol. 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.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 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
[0064] 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 (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.
[0065] "Framework" or "FR" residues are those variable-domain
residues other than the HVR residues as herein defined.
[0066] 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.
[0067] 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).
[0068] 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 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 less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, where pre-existing amino
acid changes are present in a VH, 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 one embodiment, the VL acceptor human framework is identical in
sequence to the VL human immunoglobulin framework sequence or human
consensus framework sequence.
[0069] 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 al., supra. Additionally, for the VH,
the subgroup may be subgroup I, subgroup II, or subgroup III as in
Kabat et al., supra.
[0070] An "amino-acid modification" at a specified position, e.g.,
of an anti-C1q antibody of the present disclosure, 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. In some embodiments, the amino
acid modification herein is a substitution.
[0071] An "affinity-matured" antibody, such as an anti-C1q antibody
of the present disclosure, 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 one embodiment, 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. J. Immunol. 155:1994-2004 (1995);
Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et
al, J. Mol. Biol. 226:889-896 (1992).
[0072] 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, such as an anti-C1q antibody of the present disclosure,
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, such as an anti-C1q antibody
of the present disclosure, 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 by reading this
definition 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 10.sup.3 M.sup.-1 or
10.sup.4 M.sup.-1, sometimes about 10.sup.5 M.sup.-1 or 10.sup.6
M.sup.-1, in other instances about 10.sup.6 M.sup.-1 or 10.sup.7
M.sup.-1, about 10.sup.8 M.sup.-1 to 10.sup.9 M.sup.-1, or about
10.sup.10 M to 10.sup.11 M.sup.-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.
[0073] As used herein, an "interaction" between a complement
protein, such as complement factor C1 q, 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.
[0074] A "blocking" antibody, an "antagonist" antibody, an
"inhibitory" antibody, or a "neutralizing" antibody is an antibody,
such as an anti-C1q antibody of the present disclosure 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.
[0075] 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.
[0076] 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 invention include human IgG1, IgG2,
IgG3 and IgG4.
[0077] 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.
[0078] 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. In some embodiments, the variant
Fc region differs in one or more amino acid substitution(s). In
some embodiments, 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, in some embodiments, from about one
to about five amino acid substitutions in a native sequence Fc
region or in the Fc region of the parent polypeptide. The variant
Fc region herein will, in some embodiments, possess at least about
80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide, and, in some embodiments, at least
about 90% homology therewith, and, in some embodiments, at least
about 95% homology therewith.
[0079] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. In some embodiments, the FcR is a
native sequence human FcR. Moreover, in some embodiments, a FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and FcyR.gamma.III
subclasses, including allelic variants and alternatively spliced
forms of these receptors, Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif ("ITAM") in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB 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., J. Lab. Clin. 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.
[0080] 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 Fc
region are administered. WO 2004/42072 (Presta) describes antibody
variants with improved or diminished binding to FcRs. See also,
e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).
[0081] The term "k.sub.on", as used herein, is intended to refer to
the rate constant for association of an antibody to an antigen.
[0082] The term "k.sub.off", as used herein, is intended to refer
to the rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0083] The term "K.sub.D", as used herein, is intended to refer to
the equilibrium dissociation constant of an antibody-antigen
interaction.
[0084] 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 MEGALIGN.TM. (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.
[0085] 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. In some embodiments, 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-C1q antibody of the present disclosure. In other embodiments,
the isolated cell is a host cell or hybridoma cell producing an
anti-C1q antibody of the present disclosure.
[0086] An "isolated" nucleic acid molecule encoding an antibody,
such as an anti-C1q antibody of the present disclosure, 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.
In some embodiments, 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 acid
encoding the polypeptides and antibodies herein existing naturally
in cells.
[0087] 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 of
utility 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.
[0088] "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'-O-methyl-, 2'-O-allyl-, 2'-fluoro-
or 2'-azido-ribose, carbocyclic sugar analogs, .alpha.-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(O)S
("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P
(O)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 (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0089] 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 invention.
[0090] "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
TWEEN.TM., polyethylene glycol (PEG), and PLURONIC S.TM..
[0091] The term "about" as used herein refers to the usual error
range for the respective value readily known to the skilled person
in this technical field. Reference to "about" a value or parameter
herein includes (and describes) embodiments that are directed to
that value or parameter per se.
[0092] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly indicates otherwise. For example, reference to an
"antibody" is a reference to from one to many antibodies, such as
molar amounts, and includes equivalents thereof known to those
skilled in the art, and so forth.
[0093] It is understood that aspect and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially" of aspects and embodiments.
Overview
[0094] In certain aspects, the present disclosure provides methods
for treating, preventing, or reducing risk of Alzheimer's disease
and/or Huntington's disease. Without wishing to be bound by theory,
it is believed that inhibition of the classical pathway of
complement activation is an effective therapeutic strategy for the
treatment of Alzheimer's disease and Hntington's disease (FIG. 7A).
It is further believed that effective strategies for inhibiting the
classical pathway include inhibiting the interaction between C1q
and autoantibodies, such as anti-ganglioside autoantibodies,
inhibiting the interaction between C1q and C1r or C1s, blocking the
catalytic activity of C1r or C1s, and blocking the interactions
between C1r or C1s and their respective substrates (FIG. 7A). It is
also believed that effective agents for the inhibition of the
classical complement pathway include neutralizing antibodies for
C1q that inhibit the interaction between C1q and autoantibodies,
such as anti-ganglioside autoantibodies, and/or inhibit the
interaction between C1q and C1r or C1q and C1s (FIG. 7B). As
disclosed herein, an autoantibody of the present disclosure
includes, without limitation, an antibody that recognizes a host
antigen and activates the classical pathway of complement
activation. In the first step of this activation process complement
factor C1q binds to the autoantibody-autoantigen-immune
complex.
[0095] Accordingly, certain aspects of the present disclosure
relate to anti-C1q antibodies for use in treating, preventing, or
reducing risk of Alzheimer's disease and/or Huntinton disease, in
individuals in need thereof.
[0096] In one aspect, the present disclosure provides methods for
treating or preventing Alzheimer s disease and/or Huntington's
disease, in an individual in need of such treatment, by
administering to the individual a therapeutically effective dose of
an anti-C1q antibody. In some embodiments, the anti-C1q antibody is
a C1q neutralizing antibody. In some embodiments, the anti-C1q
antibody binds to C1 complex. In some embodiments, the anti-C1q
antibody inhibits the interaction between C1q and an autoantibody,
between C1q and C1r, and/or between C1q and C1s. In some
embodiments, the individual has Alzheimer's disease or Huntington's
disease. In certain preferred embodiments, the individual is a
human.
[0097] Further aspects of the present disclosure provide anti-C1q
antibodies and uses thereof. The anti-C1q antibodies of this
disclosure specifically bind a C1q protein of this disclosure. In
some embodiments, the anti-C1q antibodies are C1q neutralizing
antibodies. In some embodiments, the anti-C1q antibodies of this
disclosure may bind to C1 complex.
[0098] In certain aspects, the present disclosure provides murine
monoclonal antibody M1, which is produced by a hybridoma cell line
referred to as mouse hybridoma C1q-M1 7788-1(M) 051613 and which
was deposited with ATCC on Jun. 6, 2013 with ATCC Accession Number
PTA-120399.
[0099] In certain aspects, the present disclosure provides an
anti-C1q antibody comprising a light chain variable domain and a
heavy chain variable domain, wherein the light chain variable
domain comprises the light chain variable domain sequence of
antibody M1; and/or wherein the heavy chain comprises the heavy
chain variable domain sequence of antibody M1.
[0100] In certain aspects, the present disclosure provides an
anti-C1q antibody comprising a light chain variable domain and a
heavy chain variable domain, wherein the light chain variable
domain comprises the HVR-L1, HVR-L2, and HVR-L3 of monoclonal
antibody M1 produced by a hybridoma cell line deposited at ATCC
with ATCC Accession Number PTA-120399 or progeny thereof; and/or
wherein the heavy chain variable domain comprises the HVR-H1,
HVR-H2, and HVR-H3 of monoclonal antibody M1 produced by a
hybridoma cell line deposited at ATCC with Accession Number
PTA-120399 or progeny thereof.
[0101] In certain aspects, the present disclosure provides an
anti-C1q antibody, which binds essentially the same C1q epitope as
(1) antibody M1 produced by the hybridoma cell line deposited with
ATCC on Jun. 6, 2013 and having ATCC Accession Number PTA-120399 or
progeny thereof, (2) an antigen binding fragment of antibody Ml, or
(3) an antibody comprising the HVR-L1, HVR-L2, HVR-L3, HVR-H1,
HVR-H2, and HVR-H3 of antibody M1.
[0102] In some embodiments, the anti-C1q antibodies of this
disclosure neutralize a biological activity of C1q. Uses for
anti-C1q antibodies include, without limitation, the detection of
complement factor C1q, e.g., in individuals having a
neurodegenerative disorder associated with complement factor 1 (CF
1)-dependent pathological synapse loss, such as Alzheimer's disease
and/or Huntington's disease. Additional non-limiting uses include
the inhibition of the classical pathway of complement activation,
e.g., in cases where the classical complement pathway is activated
by autoantibodies, such as ganglioside-specific autoantibodies.
Further non-limiting uses for anti-C1q antibodies include the
diagnosis and treatment of disorders that are associated with
elevated expression of complement factors, such as C1q, or
associated with the activation of the complement pathway. Such
disorders may include, without limitation, autoimmune disorders,
inflammatory disorders, and neurodegenerative disorders, including
neurodegenerative disorders associated with synapse loss such as
Alzheimer's disease and Huntington's disease.
[0103] In another aspect, the present disclosure provides an
isolated nucleic acid molecule encoding an antibody of this
disclosure.
[0104] The present disclosure also provides isolated host cells
containing a nucleic acid molecule that encodes an antibody of this
disclosure. In some embodiments, an isolated host cell line is
provided that produces the neutralizing monoclonal murine antibody
M1. This isolated host cell lines was deposited with ATCC and has
ATCC Accession Number PTA-120399.
[0105] Additionally, pharmaceutical compositions are provided
containing anti-C1q antibodies of this disclosure, such as C1q
neutralizing antibodies of this disclosure, in combination with
pharmaceutically acceptable carriers. The present disclosure also
provides a kit containing an anti-C1q antibody for use in any of
the methods described herein.
[0106] The present disclosure further provides methods of using the
anti-C1q antibodies of this disclosure (e.g., C1q neutralizing
antibodies of this disclosure) to treat or prevent a
neurodegenerative disease (e.g., Alzheimer's disease and
Huntington's disease) in an individual in need of such treatment,
to detect synapses in an individual having a neurodegenerative
disease, and to detect synapses in a biological sample. The present
disclosure also provides kits containing the C1q antibodies of this
disclosure (e.g., C1q neutralizing antibodies of this
disclosure).
Complement Proteins
[0107] The methods of this disclosure involve administering or
using antibodies that specifically recognizes complement factor C1q
of the classical complement activation pathway. Certain aspects of
the present disclosure further involves antibodies that
specifically recognize complement factor C1q and/or C1q in the C1
complex of the classical complement activation pathway. The
recognized complement factor may be derived, without limitation,
from any organism having a complement system, including any
mammalian organism such as human, mouse, rat, rabbit, monkey, dog,
cat, cow, horse, camel, sheep, goat, or pig.
[0108] As used herein "C1 complex" refers to a protein complex that
may include, without limitation, one C1q protein, two C1r proteins,
and two C1s proteins (e.g., C1qr.sup.2s.sup.2).
[0109] As used herein "complement factor C1q" refers to both wild
type sequences and naturally occurring variant sequences.
[0110] A non-limiting example of a complement factor C1q recognized
by antibodies of this invention is human C1q, including the three
polypeptide chains A, B, and C:
TABLE-US-00002 Clq, chain A (homo sapiens), Accession No. Protein
Data Base: NP_057075.1; GenBank No.: NM_015991:
>gi|7705753|ref|NP_057075.1| complement Clq subcomponent subunit
A precursor [Homo sapiens] (SEQ ID NO: 1)
MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGE
QGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIK
GTKGSPGNIKDQPRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGR
FVCTVPGYYYFTFQVLSQWEICLSIVSSSRGQVRRSLGFCDTTNKGLFQV
VSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA Clq, chain B (homo
sapiens), Accession No. Protein Data Base: NP_000482.3; GenBank
No.: NM_000491.3: >gi|87298828|ref|NP_000482.3| complement Clq
subcomponent subunit B precursor [Homo sapiens] (SEQ ID NO: 2)
MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPD
GQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGG
PGAPGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIRFDHVITNMN
NNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDY
AYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPD MEA Clq, chain C
(homo sapiens), Accession No. Protein Data Base: NP_001107573.1;
GenBank No.: NM_001114101.1: >gi|166235903|ref|NP_001107573.1|
complement Clq subcomponent subunit C precursor [Homo sapiens] (SEQ
ID NO: 3) MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGY
DGLPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPG
PMGIPGEPGEEGRYKQKFQSVFTVTRQTHQPPAPNSLIRFNAVLTNPQGD
YDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTN
QVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD
[0111] Accordingly, an anti-C1q antibody of the present disclosure
may bind to polypeptide chain A, polypeptide chain B, and/or
polypeptide chain C of a C1q protein. In some embodiments, an
anti-C1q antibody of the present disclosure binds to polypeptide
chain A, polypeptide chain B, and/or polypeptide chain C of human
C1q or a homolog thereof, such as mouse, rat, rabbit, monkey, dog,
cat, cow, horse, camel, sheep, goat, or pig C1q.
Anti-C1q Antibodies
[0112] The antibodies of this disclosure specifically bind to a
complement factor C1q and/or C1q in the C1 complex of the classical
complement pathway. In some embodiments, the anti-C1q antibodies
specifically bind to human C1q. In some embodiments, the anti-C1q
antibodies specifically bind to human and mouse C1q. In some
embodiments, the anti-C1q antibodies specifically bind to rat C1q.
In some embodiments, the anti-C1q antibodies specifically bind to
human C1q, mouse C1q, and rat C1q.
[0113] In some embodiments, the anti-C1q antibodies of this
disclosure neutralize a biological activity of complement factor
C1q. In some embodiments, the antibodies inhibit the interaction
between complement factor C1q and other complement factors, such as
C1r or C1s or between C1q and an antibody, such as an autoantibody.
In some embodiments, the antibodies inhibit the interaction between
complement factor C1q and a non-complement factor. A non-complement
factor may include phosphatidylserine, pentraxin-3, C-reactive
protein (CRP), globular C1q receptor (gC1qR), complement receptor 1
(CR1), .beta.-amyloid, and calreticulin. In some embodiments, the
antibodies inhibit the classical complement activation pathway. In
certain embodiments, the antibodies further inhibit the alternative
pathway. In some embodiments, the antibodies inhibit autoantibody-
and complement-dependent cytotoxicity (CDC). In some embodiments,
the antibodies inhibit complement-dependent cell-mediated
cytotoxicity (CDCC). In some embodiments, the antibodies inhibit
B-cell antibody production, dendritic cell maturation, T-cell
proliferation, cytokine production, or microglia activation. In
some embodiments, the antibodies inhibit the Arthus reaction. In
some embodiments, the antibodies inhibit phagocytosis of synapses
or nerve endings. In some embodiments, the antibodies inhibit the
activation of complement receptor 3 (CR3/C3) expressing cells.
[0114] The functional properties of the antibodies of this
invention, such as dissociation constants for antigens, inhibition
of protein-protein interactions (e.g., C1q-autoantibody
interactions), inhibition of autoantibody-dependent and
complement-dependent cytotoxicity (CDC), inhibition of
complement-dependent cell-mediated cytotoxicity (CDCC), or lesion
formation, may, without limitation, be measured in in vitro, ex
vivo, or in vivo experiments.
[0115] The dissociation constants (K.sub.D) of the anti-C1q
antibodies for C1q may be less than 100 nM, less than 90 nM, less
than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less
than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less
than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less
than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less
than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM,
less than 0.01 nM, or less than 0.005 nM. In some embodiments,
dissociation constants range from less than about 30 nM to less
than about 100 pM. In some embodiments, dissociation constants are
less than about 30 nM. In some embodiments, dissociation constants
are less than about 20 nM. In some embodiments, dissociation
constants are less than about 10 nM. In some embodiments,
dissociation constants are less than about 5 nM. In some
embodiments, dissociation constants are less than about 1 nM. In
some embodiments, dissociation constants are less than about 100
pM. In certain embodiments, the dissociation constants of the
anti-C1q antibody range from less than about 30 nM to less than
about 100 pM for human C1q, and range from less than about 30 nM to
less than about 100 pM for mouse C1q. In certain embodiments,
dissociation constants of the anti-C1q antibody are less than about
30 nM for human C1q and less than about 30 nM for mouse C1q. In
certain embodiments, dissociation constants of the anti-C1q
antibody are less than about 20 nM for human C1q and less than
about 20 nM for mouse C1q. In certain embodiments, dissociation
constants of the anti-C1q antibody are less than about 10 nM for
human C1q and less than about 10 nM for mouse C1q. In certain
embodiments, dissociation constants of the anti-C1q antibody are
less than about 5 nM for human C1q and less than about 5 nM for
mouse C1q. In certain embodiments, dissociation constants of the
anti-C1q antibody are less than about 1 nM for human C1q and less
than about 1 nM mouse C1q. In certain embodiments, the dissociation
constants of the anti-C1q antibody are less than 100 pM for human
C1q and less than 100 pM for mouse C1q. Antibody dissociation
constants for antigens other than C1q may be least 5-fold, at least
10-fold, at least 100-fold, at least 1,000-fold, at least
10,000-fold, or at least 100,000-fold higher that the dissociation
constants for C1q. For example, the dissociation constant of a C1q
antibody of this disclosure may be at least 1,000-fold higher for
C1s than for C1q. Dissociation constants may be determined through
any analytical technique, including any biochemical or biophysical
technique such as ELISA, surface plasmon resonance (SPR), bio-layer
interferometry (see, e.g., Octet System by ForteBio), isothermal
titration calorimetry (ITC), differential scanning calorimetry
(DSC), circular dichroism (CD), stopped-flow analysis, and
colorimetric or fluorescent protein melting analyses. Dissociation
constants (K.sub.D) of the anti-C1q antibodies for C1q may be
determined, e.g., using full-length antibodies or antibody
fragments, such as Fab fragments.
[0116] One exemplary way of determining binding affinity of
antibodies to C1q is by measuring binding affinity of
monofunctional Fab fragments of the antibody. To obtain
monofunctional Fab fragments, an antibody (for example, IgG) can be
cleaved with papain or expressed recombinantly. The affinity of an
Fab fragment of an antibody can be determined by surface plasmon
resonance (Biacore3000.TM. surface plasmon resonance (SPR) system,
Biacore.TM., INC, Piscataway N.J.) equipped with pre-immobilized
streptavidin sensor chips (SA) using HBS-EP running buffer (0.01M
HEPES, pH 7.4, 0.15 NaCl, 3mM EDTA, 0.005% v/v Surfactant P20).
Biotinylated human C1q (or any other C1q) can be diluted into
HBS-EP buffer to a concentration of less than 0.5 .mu.g/mL and
injected across the individual chip channels using variable contact
times, to achieve two ranges of antigen density, either 50-200
response units (RU) for detailed kinetic studies or 800-1,000 RU
for screening assays. Regeneration studies have shown that 25 mM
NaOH in 25% v/v ethanol effectively removes the bound Fab while
keeping the activity of C1q on the chip for over 200 injections.
Typically, serial dilutions (spanning concentrations of
0.1-10.times. estimated K.sub.D) of purified Fab samples are
injected for 1 min at 100 .mu.L/minute and dissociation times of up
to 2 hours are allowed. The concentrations of the Fab proteins are
determined by ELISA and/or SDS-PAGE electrophoresis using a Fab of
known concentration (as determined by amino acid analysis) as a
standard. Kinetic association rates (k.sub.on) and dissociation
rates (k.sub.off) are obtained simultaneously by fitting the data
globally to a 1:1 Langmuir binding model (Karlsson, R. Roos, H.
Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110)
using the BIAevaluation program. Equilibrium dissociation constant
(K.sub.D) values are calculated as k.sub.off/k.sub.on. This
protocol is suitable for use in determining binding affinity of an
antibody to any C1q, including human C1q, C1q of another mammal
(such as mouse C1q, rat C1q, primate C1q), as well as different
forms of C1q. Binding affinity of an antibody is generally measured
at 25.degree. C., but can also be measured at 37.degree. C.
[0117] The antibodies of this disclosure may bind to C1q antigens
derived from any organism having a complement system, including any
mammalian organism such as human, mouse, rat, rabbit, monkey, dog,
cat, cow, horse, camel, sheep, goat, or pig. In some embodiments,
the anti-C1q antibodies bind specifically to epitopes on human C1q.
In some embodiments, the anti-C1q antibodies specifically bind to
epitopes on both human and mouse C1q. In some embodiments, the
anti-C1q antibodies specifically bind to epitopes on human, mouse,
and rat C1q.
[0118] In some embodiments, provided herein is an anti-C1q antibody
that binds to an epitope of C1q that is the same as or overlaps
with the C1q epitope bound by another antibody of this disclosure.
In certain embodiments, provided herein is an anti-C1q antibody
that binds to an epitope of C1q that is the same as or overlaps
with the C1q epitope bound by anti-C1q antibody Ml. In some
embodiments, the anti-C1q antibody competes with another antibody
of this disclosure for binding to C1q. In certain embodiments, the
anti-C1q antibody competes with anti-C1q antibody M1 or an
antigen-binding fragment thereof for binding to C1q.
[0119] Methods that may be used to determine which C1q epitope of
an anti-C1q antibody binds to, or whether two antibodies bind to
the same or an overlapping epitope, may include, without
limitation, X-ray crystallography, NMR spectroscopy,
Alanine-Scanning Mutagenesis, the screening of peptide libraries
that include C1q-derived peptides with overlapping C1q sequences,
and competition assays. Competition assays are especially useful to
determine whether two antibodies bind the same epitope by
recognizing identical or sterically overlapping epitopes or whether
one antibody competitively inhibits binding of another antibody to
the antigen. These assays are known in the art. Typically, an
antigen or antigen expressing cells are immobilized on a multi-well
plate and the ability of unlabeled antibodies to block the binding
of labeled antibodies is measured. Common labels for such
competition assays are radioactive labels or enzyme labels.
[0120] Competitive antibodies encompassed herein are antibodies
that inhibit (i.e., prevent or interfere with in comparison to a
control) or reduce the binding of any anti-C1q antibody of this
disclosure (such as M1 or an antigen-binding fragment of M1) to C1q
by at least 50%, 60%, 70%, 80%, 90% and 95% at 1 .mu.M or less. For
example, the concentration competing antibody in the competition
assay may be at or below the K.sub.D of antibody M1 or an
antigen-binding fragment of Ml. Competition between binding members
may be readily assayed in vitro for example using ELISA and/or by
monitoring the interaction of the antibodies with C1q in solution.
The exact means for conducting the analysis is not critical. C1q
may be immobilized to a 96-well plate or may be placed in a
homogenous solution. In specific embodiments, the ability of
unlabeled candidate antibody(ies) to block the binding of the
labeled anti-C1q antibody, e.g. M1, can be measured using
radioactive, enzyme or other labels. In the reverse assay, the
ability of unlabeled antibodies to interfere with the interaction
of a labeled anti-C1q antibody with C1q wherein said labeled
anti-C1q antibody, e.g., M1, and C1q are already bound is
determined. The readout is through measurement of bound label. C1q
and the candidate antibody(ies) may be added in any order or at the
same time.
[0121] In some embodiments, the anti-C1q antibody inhibits the
interaction between C1q and an autoantibody. In some embodiments,
the anti-C1q antibody is murine anti-human C1q monoclonal antibody
Ml, which is produced by a hybridoma cell line deposited with ATCC
on June 6, 2013 with ATCC Accession Number PTA-120399.
[0122] In some embodiments, the anti-C1q antibody is an isolated
antibody which binds essentially the same C1q epitope as M1. In
some embodiments, the anti-C1q antibody is an isolated antibody
comprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain
variable domains of monoclonal antibody M1 produced by the
hybridoma cell line deposited with ATCC on Jun. 6, 2013 with ATCC
Accession Number PTA-120399, or progeny thereof. In some
embodiments, the anti-C1q antibody is an isolated antibody
comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain
variable domains of monoclonal antibody M1 produced by the
hybridoma cell line deposited with ATCC on Jun. 6, 2013 with ATCC
Accession Number PTA-120399, or progeny thereof. In some
embodiments, the anti-C1q antibody is an isolated antibody
comprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain
variable domains and the HVR-H1, HVR-H2, and HVR-H3 of the heavy
chain variable domains of monoclonal antibody M1 produced by the
hybridoma cell line deposited with ATCC on Jun. 6, 2013 with ATCC
Accession Number PTA-120399, or progeny thereof.
[0123] In some embodiments, the anti-C1q antibody binds to a C1q
protein and binds to one or more amino acids of the C1q protein
within amino acid residues selected from (a) amino acid residues
196-226 of SEQ ID NO:1 (SEQ ID NO:6), or amino acid residues of a
C1q protein chain A (C1qA) corresponding to amino acid residues
196-226 (GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1 (SEQ ID
NO:6); (b) amino acid residues 196-221 of SEQ ID NO:1 (SEQ ID
NO:7), or amino acid residues of a C1qA corresponding to amino acid
residues 196-221 (GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1 (SEQ
ID NO:7); (c) amino acid residues 202-221 of SEQ ID NO:1 (SEQ ID
NO:8), or amino acid residues of a C1qA corresponding to amino acid
residues 202-221 (SGGMVLQLQQGDQVWVEKD) of SEQ ID NO:1 (SEQ ID
NO:8); (d) amino acid residues 202-219 of SEQ ID NO:1 (SEQ ID
NO:9), or amino acid residues of a C1qA corresponding to amino acid
residues 202-219 (SGGMVLQLQQGDQVWVEK) of SEQ ID NO:1 (SEQ ID NO:9);
and (e) amino acid residues Lys 219 and/or Ser 202 of SEQ ID NO:1,
or amino acid residues of a C1qA corresponding Lys 219 and/or Ser
202 of SEQ ID NO:1.
[0124] In some embodiments, the antibody further binds to one or
more amino acids of the C1q protein within amino acid residues
selected from: (a) amino acid residues 218-240 of SEQ ID NO:3 (SEQ
ID NO:10) or amino acid residues of a C1q protein chain C (C1qC)
corresponding to amino acid residues 218-240 (WLAVNDYYDMVGI
QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:10); (b) amino acid residues
225-240 of SEQ ID NO:3 (SEQ ID NO:11) or amino acid residues of a
C1qC corresponding to amino acid residues 225-240 (YDMVGI
QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:11); (c) amino acid residues
225-232 of SEQ ID NO:3 (SEQ ID NO:12) or amino acid residues of a
C1qC corresponding to amino acid residues 225-232 (YDMVGIQG) of SEQ
ID NO:3 (SEQ ID NO:12); (d) amino acid residue Tyr 225 of SEQ ID
NO:3 or an amino acid residue of a C1qC 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:13) or amino acid residues of a C1qC
corresponding to amino acid residues 174-196
(HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3 (SEQ ID NO:13); (f) amino
acid residues 184-192 of SEQ ID NO:3 (SEQ ID NO:14) or amino acid
residues of a C1qC corresponding to amino acid residues 184-192
(RSGVKVVTF) of SEQ ID NO:3 (SEQ ID NO:14); (g) amino acid residues
185-187 of SEQ ID NO:3 or amino acid residues of a C1qC
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 C1qC corresponding to amino acid residue Ser 185 of
SEQ ID NO:3.
[0125] In certain embodiments, the anti-C1q antibody binds to amino
acid residue Lys 219 and Ser 202 of the human C1qA as shown in SEQ
ID NO:1 or amino acids of a human C1qA corresponding to Lys 219 and
Ser 202 as shown in SEQ ID NO:1, and amino acid residue Tyr 225 of
the human C1qC as shown in SEQ ID NO:3 or an amino acid residue of
a human C1qC corresponding to Tyr 225 as shown in SEQ ID NO:3. In
certain embodiments, the anti-C1q antibody binds to amino acid
residue Lys 219 of the human C1qA as shown in SEQ ID NO:1 or an
amino acid residue of a human C1qA corresponding to Lys 219 as
shown in SEQ ID NO:1, and amino acid residue Ser 185 of the human
C1qC as shown in SEQ ID NO:3 or an amino acid residue of a human
C1qC corresponding to Ser 185 as shown in SEQ ID NO:3.
[0126] In some embodiments, the anti-C1q antibody binds to a C1q
protein and binds to one or more amino acids of the C1q protein
within amino acid residues selected from: (a) amino acid residues
218-240 of SEQ ID NO:3 (SEQ ID NO:10) or amino acid residues of a
C1qC corresponding to amino acid residues 218-240 (WLAVNDYYDMVGI
QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:10); (b) amino acid residues
225-240 of SEQ ID NO:3 (SEQ ID NO:11) or amino acid residues of a
C1qC corresponding to amino acid residues 225-240 (YDMVGI
QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:11); (c) amino acid residues
225-232 of SEQ ID NO:3 (SEQ ID NO:12) or amino acid residues of a
C1qC corresponding to amino acid residues 225-232 (YDMVGIQG) of SEQ
ID NO:3 (SEQ ID NO:12); (d) amino acid residue Tyr 225 of SEQ ID
NO:3 or an amino acid residue of a C1qC 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:13) or amino acid residues of a C1qC
corresponding to amino acid residues 174-196
(HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3 (SEQ ID NO:13); (f) amino
acid residues 184-192 of SEQ ID NO:3 (SEQ ID NO:14) or amino acid
residues of a C1qC corresponding to amino acid residues 184-192
(RSGVKVVTF) of SEQ ID NO:3 (SEQ ID NO:14); (g) amino acid residues
185-187 of SEQ ID NO:3 or amino acid residues of a C1qC
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 C1qC corresponding to amino acid residue Ser 185 of
SEQ ID NO:3.
[0127] In some embodiments, the anti-C1q antibody of this
disclosure inhibits the interaction between C1q and C1s. In some
embodiments, the anti-C1q antibody inhibits the interaction between
C1q and C1r. In some embodiments the anti-C1q antibody inhibits the
interaction between C1q and C1s and between C1q and C1r. In some
embodiments, the anti-C1q antibody inhibits the interaction between
C1q and another antibody, such as an autoantibody. In some
embodiments, the anti-C1q 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 C1q antibody inhibits an
interaction, such as the C1q-C1s interaction, at approximately
equimolar concentrations of C1q and the anti-C1q antibody. In other
embodiments, the anti-C1q antibody binds to C1q 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-C1q antibody binds C1q with
a binding stoichiometry that ranges from 20:1 to 1.0:1 or less
than1.0:1. In certain embodiments, the anti-C1q antibody binds C1q
with a binding stoichiometry that ranges from 6:1 to 1.0:1 or less
than1.0:1. In certain embodiments, the anti-C1q antibody binds C1q
with a binding stoichiometry that ranges from 2.5:1 to 1.0:1 or
less than1.0:1. In some embodiments, the anti-C1q antibody inhibits
the interaction between C1q and C1r, or between C1q and C1s, or
between C1q and both C1r and C1s. In some embodiments, the anti-C1q
antibody inhibits the interaction between C1q and C1r , between C1q
and C1s, and/or between C1q and both C1r and C1s. In some
embodiments, the anti-C1q antibody binds to the C1q A-chain. In
other embodiments, the anti-C1q antibody binds to the C1q B-chain.
In other embodiments, the anti-C1q antibody binds to the C1q
C-chain. In some embodiments, the anti-C1q antibody binds to the
C1q A-chain, the C1q B-chain and/or the C1q C-chain. In some
embodiments, the anti-C1q antibody binds to the globular domain of
the C1q A-chain, B-chain, and/or C-chain. In other embodiments, the
anti-C1q antibody binds to the collagen-like domain of the C1q
A-chain, the C1q B-chain, and/or the C1q C-chain.
[0128] Where antibodies of this disclosure inhibit the interaction
between two or more complement factors, such as the interaction of
C1q and C1s, or the interaction between C1q and C1r , 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.
[0129] In some embodiments, the antibodies of this disclosure
inhibit 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
C4-cleavage are well known in the art. The EC.sub.50 values for
antibodies of this disclosure with respect C4-cleavage may be less
than 3 .mu.g/ml; 2.5 .mu.g/ml; 2.0 .mu.g/ml; 1.5 .mu.g/ml; 1.0
.mu.g/ml; 0.5 .mu.g/ml; 0.25 .mu.g/ml; 0.1 .mu.g/ml; 0.05 .mu.g/ml.
In some embodiments, the antibodies of this disclosure inhibit
C4-cleavage at approximately equimolar concentrations of C1q and
the respective anti-C1q antibody.
[0130] 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 EC.sub.50 values for
antibodies of this disclosure with respect to inhibition of
autoantibody-dependent and complement-dependent cytotoxicity may be
less than 3 .mu.g/ml; 2.5 .mu.g/ml; 2.0 .mu.g/ml; 1.5 .mu.g/ml; 1.0
.mu.g/ml; 0.5 .mu.g/ml; 0.25 .mu.g/ml; 0.1 .mu.g/ml; 0.05
.mu.g/ml.
[0131] 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 EC.sub.50 values for antibodies of this disclosure
with respect CDCC inhibition may be 1 less than 3 .mu.g/ml; 2.5
.mu.g/ml; 2.0 .mu.g/ml; 1.5 g/ml; 1.0 .mu.g/ml; 0.5 .mu.g/ml;0.25
.mu.g/ml; 0.1 .mu.g/ml; 0.05 .mu.g/ml. In some embodiments, the
antibodies of this disclosure inhibit CDCC but not
antibody-dependent cellular cytotoxicity (ADCC).
[0132] In some embodiments, the antibodies of this disclosure
inhibit C1F hemolysis (also referred to as CH50 hemolysis) 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 or wherein control antibodies are used that do not bind
to a complement factor or another antibody such as an autoantibody
(see, e.g., Example 3). Methods for measuring C1F hemolysis are
well known in the art (see, e.g., Example 3). The EC.sub.50 values
for antibodies of this disclosure with respect to C1F hemolysis may
be less than 3 .mu.g/ml; 2.5 .mu.g/ml; 2.0 .mu.g/ml; 1.5 .mu.g/ml;
1.0 .mu.g/ml; 0.5 .mu.g/ml; 0.25 .mu.g/ml; 0.1 .mu.g/ml; 0.05
.mu.g/ml. In some embodiments, the anti-C1q antibodies of this
disclosure neutralize at least 50% of C1F hemolysis at a dose of
less than 200 ng/ml, less than100 ng/ml, less than 50 ng/ml, or
less than 20 ng/ml. In some embodiments, the antibodies of this
disclosure neutralize C1F hemolysis at approximately equimolar
concentrations of C1q and the anti-C1q antibody. In some
embodiments, the anti-C1q antibodies of this disclosure neutralize
hemolysis in a human C1F hemolysis assay. In some embodiments, the
antibodies of this disclosure neutralize hemolysis in a human,
mouse, and rat C1F hemolysis assay (see, e.g., Example 3).
[0133] In some embodiments, the alternative pathway may amplify CDC
initiated by C1q binding and subsequent C1s activation; in at least
some of these embodiments, the antibodies of this disclosure
inhibit the alternative pathway by 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 were absent.
[0134] In some embodiments, the antibodies of this disclosure
prevent synaptic loss in a cellular in vitro model or an in vivo
model of synaptic loss, such as an in vivo mouse model. In vivo
mouse models may include Tg2576, a mouse amyloid precursor protein
(APP) transgenic model of Alzheimer's disease, R6/2 NT-CAG150, a
transgenic model for Huntington's disease, or SMA.DELTA.7, a mouse
model for Spinal Muscular Atrophy, or DBA/2J, a genetic mouse model
of glaucoma. In general, any neurodegenerative disease model, such
as an Alzheimer's disease and/or Huntington's disease model, may be
used that displays synapse loss.
[0135] Methods for measuring synaptic loss in vitro or in vivo are
well known in the art. In vitro lesion formation may be reduced by
at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, or at least 95%, or by an amount
that ranges from at least 30% to at least 95%, relative to a
control experiment in which antibodies of this disclosure are
absent. The EC.sub.50 values for antibodies of this disclosure with
respect to the prevention of in vitro lesion formation may be less
than 3 .mu.g/ml; 2.5 .mu.g/ml; 2.0 .mu.g/ml; 1.5 .mu.g/ml; 1.0
.mu.g/ml; 0.5 .mu.g/ml; 0.25 .mu.g/ml; 0.1 .mu.g/ml; 0.05 .mu.g/ml.
In vivo synaptic loss may be reduced by at least 5%, at least 10%,
at least 15%, at least 20%, at least 35%, at least 40%, or at least
50%, or by an amount that ranges from at least 5% to at least 50%,
relative to a control experiment in which antibodies of this
disclosure are absent.
[0136] The present disclosure provides anti-C1q antibodies. The
antibodies of this disclosure may have one or more of the following
characteristics. The antibodies of this disclosure may be
polyclonal antibodies, monoclonal antibodies, humanized antibodies,
human antibodies, antibody fragments, bispecific and polyspecific
antibodies, multivalent antibodies, or heteroconjugate antibodies.
Antibody fragments of this disclosure may be functional fragments
that bind the same epitope as any of the anti-C1q antibodies of
this disclosure. In some embodiments, the antibody fragments of
this disclosure specifically bind to and neutralize a biological
activity of C1q. In some embodiments, the antibody fragments are
miniaturized versions of the anti-C1q antibodies or antibody
fragments of this disclosure that have the same epitope of the
corresponding full-length antibody, but have much smaller molecule
weight. Such miniaturized anti-C1q antibody fragments may have
better brain penetration ability and a shorter half-life, which is
advantageous for imaging and diagnostic utilities (see e.g., Lutje
S et al., Bioconjug Chem. 2014 Feb 19; 25(2):335-41; Tavare R et
al., Proc Natl Acad Sci USA. 2014 Jan. 21; 111(3):1108-13; and
Wiehr S et al., Prostate. 2014 May; 74(7):743-55). Accordingly, in
some embodiments, anti-C1q antibody fragments of this disclosure
have better brain penetration as compared to their corresponding
full-length antibodies and/or have a shorter half-life as compared
to their corresponding full-length antibodies. In some embodiments,
anti-C1q antibodies of the present disclosure are bispecific
antibodies recognizing a first antigen and a second antigen. In
some embodiments, the first antigen is a C1q antigen. In some
embodiments, the second antigen is an antigen facilitating
transport across the blood-brain-barrier, including without
limitation, 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, and ANG1005. The
antibodies of this disclosure may further contain engineered
effector functions, amino acid sequence modifications or other
antibody modifications known in the art; e.g., the constant region
of the anti-C1q antibodies described herein may be modified to
impair complement activation.
[0137] In some embodiments, the anti-C1q antibodies of this
disclosure prevent Alzheimer's disease and/or Huntington's disease,
or one or more symptoms of such neurodegenerative diseases. In
certain embodiments, prevention of Alzheimer's disease and/or
Huntington's disease, or one or more symptoms of such
neurodegenerative diseases by the anti-C1q antibodies of the
present disclosure may be measured by inhibition of A.beta.-induced
C3 deposition and/or inhibit synapse loss in the hippocampus in an
in vivo mouse model of Alzheimer's disease and/or Huntington's
disease. In some embodiments, the anti-C1q antibodies of this
disclosure inhibit A.beta.-induced C3 deposition and/or inhibit
synapse loss in the hippocampus in an in vivo mouse model of
Alzheimer's disease and/or Huntington's disease. Methods for
measuring A.beta.-induced C3 deposition and/or inhibit synapse loss
in the hippocampus in vivo are well known in the art (see also
Examples 5-9 for exemplary methods)
[0138] Additional anti-C1q antibodies, e.g., antibodies that
specifically bind to a C1q protein of the present disclosure, may
be identified, screened, and/or characterized for their
physical/chemical properties and/or biological activities by
various assays known in the art.
Antibody Preparation
[0139] Anti-C1q antibodies of the present disclosure can encompass
polyclonal antibodies, monoclonal antibodies, humanized antibodies,
chimeric antibodies, human antibodies, antibody fragments (e.g.,
Fab, Fab'-SH, Fv, scFv, and F(ab').sub.2 fragments), bispecific and
polyspecific antibodies, multivalent antibodies, heteroconjugate
antibodies, library derived antibodies, antibodies having modified
effector functions, fusion proteins containing an antibody portion,
and any other modified configuration of the immunoglobulin molecule
that includes an antigen recognition site, such as an epitope
having amino acid residues of a C1q protein of the present
disclosure, including glycosylation variants of antibodies, amino
acid sequence variants of antibodies, and covalently modified
antibodies. The anti-C1q antibodies may be human, murine, rat, or
of any other origin (including chimeric or humanized
antibodies).
[0140] (1) Polyclonal Antibodies
[0141] Polyclonal antibodies, such as polyclonal anti-C1q
antibodies, are generally raised in animals by multiple
subcutaneous (sc) or intraperitoneal (ip) injections of the
relevant antigen and an adjuvant. It may be useful to conjugate the
relevant antigen (e.g., a purified or recombinant C1q protein of
the present disclosure) to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin (KLH),
serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor,
using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are independently lower alkyl groups. Examples of
adjuvants which may be employed include Freund's complete adjuvant
and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate). The immunization protocol may be selected by one
skilled in the art without undue experimentation.
[0142] The animals are immunized against the desired antigen,
immunogenic conjugates, or derivatives by combining, e.g., 100
.mu.g (for rabbits) or 5 .mu.g (for mice) of the protein or
conjugate with 3 volumes of Freund's complete adjuvant and
injecting the solution intradermally at multiple sites. One month
later, the animals are boosted with 1/5 to 1/10 the original amount
of peptide or conjugate in Freund's complete adjuvant by
subcutaneous injection at multiple sites. Seven to fourteen days
later, the animals are bled and the serum is assayed for antibody
titer. Animals are boosted until the titer plateaus. Conjugates
also can be made in recombinant-cell culture as protein fusions.
Also, aggregating agents such as alum are suitable to enhance the
immune response.
[0143] (2) Monoclonal Antibodies
[0144] Monoclonal antibodies, such as monoclonal anti-C1q
antibodies, are obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible naturally
occurring mutations and/or post-translational modifications (e.g.,
isomerizations, amidations) that may be present in minor amounts.
Thus, the modifier "monoclonal" indicates the character of the
antibody as not being a mixture of discrete antibodies.
[0145] For example, the monoclonal anti-C1q antibodies may be made
using the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), or may be made by recombinant DNA methods
(U.S. Pat. No. 4,816,567).
[0146] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as hereinabove described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization (e.g., a purified or recombinant C1q protein of the
present disclosure). Alternatively, lymphocytes may be immunized in
vitro. Lymphocytes then are fused with myeloma cells using a
suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)).
[0147] The immunizing agent will typically include the antigenic
protein (e.g., a purified or recombinant C1q protein of the present
disclosure) or a fusion variant thereof. Generally peripheral blood
lymphocytes ("PBLs") are used if cells of human origin are desired,
while spleen or lymph node cells are used if non-human mammalian
sources are desired. The lymphoctyes are then fused with an
immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal
Antibodies: Principles and Practice, Academic Press (1986), pp.
59-103.
[0148] Immortalized cell lines are usually transformed mammalian
cells, particularly myeloma cells of rodent, bovine or human
origin. Usually, rat or mouse myeloma cell lines are employed. The
hybridoma cells thus prepared are seeded and grown in a suitable
culture medium that may contain one or more substances that inhibit
the growth or survival of the unfused, parental myeloma cells. For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine (HAT medium), which are substances that
prevent the growth of HGPRT-deficient-cells.
[0149] In some embodiments, immortalized myeloma cells are those
that fuse efficiently, support stable high-level production of
antibody by the selected antibody-producing cells, and are
sensitive to a medium such as HAT medium. Among these, are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors (available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA), as well as SP-2 cells and derivatives
thereof (e.g., X63-Ag8-653) (available from the American Type
Culture Collection, Manassas, Va. USA). Human myeloma and
mouse-human heteromyeloma cell lines have also been described for
the production of human monoclonal antibodies (Kozbor, J. Immunol.
, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987)).
[0150] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen (e.g., a C1q protein of the present disclosure). In
some embodiments, the binding specificity of monoclonal antibodies
produced by hybridoma cells is determined by immunoprecipitation or
by an in vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunosorbent assay (ELISA).
[0151] The culture medium in which the hybridoma cells are cultured
can be assayed for the presence of monoclonal antibodies directed
against the desired antigen (e.g., a C1q protein of the present
disclosure). In some embodiments, the binding affinity and
specificity of the monoclonal antibody can be determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such
techniques and assays are known in the in art. For example, binding
affinity may be determined by the Scatchard analysis of Munson et
al., Anal. Biochem., 107:220 (1980).
[0152] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, supra). Suitable culture media for this
purpose include, for example, D-MEM or RPMI-1640 medium. In
addition, the hybridoma cells may be grown in vivo as tumors in a
mammal.
[0153] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose chromatography, hydroxylapatite
chromatography, gel electrophoresis, dialysis, affinity
chromatography, and other methods as described above.
[0154] Anti-C1q monoclonal antibodies may also be made by
recombinant DNA methods, such as those disclosed in U.S. Pat. No.
4,816,567, and as described above. DNA encoding the monoclonal
antibodies is readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that specifically
bind to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells serve as a source of such DNA.
Once isolated, the DNA may be placed into expression vectors, which
are then transfected into host-cells such as E. coli cells, simian
COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that
do not otherwise produce immunoglobulin protein, in order to
synthesize monoclonal antibodies in such recombinant host-cells.
Review articles on recombinant expression in bacteria of DNA
encoding the antibody include Skerra et al., Curr. Opin. Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Rev. 130:151-188
(1992).
[0155] In certain embodiments, anti-C1q antibodies can be isolated
from antibody phage libraries generated using the techniques
described in McCafferty et al., Nature, 348:552-554 (1990).
Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J.
Mol. Biol., 222:581-597 (1991) described the isolation of murine
and human antibodies, respectively, from phage libraries.
Subsequent publications describe the production of high affinity
(nanomolar ("nM") range) human antibodies by chain shuffling (Marks
et al., Bio/Technology, 10:779-783 (1992)), as well as
combinatorial infection and in vivo recombination as a strategy for
constructing very large phage libraries (Waterhouse et al., Nucl.
Acids Res., 21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies of desired
specificity (e.g., those that bind a C1q protein of the present
disclosure).
[0156] The DNA encoding antibodies or fragments thereof may also be
modified, for example, by substituting the coding sequence for
human heavy- and light-chain constant domains in place of the
homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et
al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Typically
such non-immunoglobulin polypeptides are substituted for the
constant domains of an antibody, or they are substituted for the
variable domains of one antigen-combining site of an antibody to
create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0157] The monoclonal antibodies described herein (e.g., anti-C1q
antibodies of the present disclosure or fragments thereof) may by
monovalent, the preparation of which is well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and a modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues may be substituted with another amino acid
residue or are deleted so as to prevent crosslinking. In vitro
methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce fragments thereof, particularly
Fab fragments, can be accomplished using routine techniques known
in the art.
[0158] Chimeric or hybrid anti-C1q antibodies also may be prepared
in vitro using known methods in synthetic protein chemistry,
including those involving crosslinking agents. For example,
immunotoxins may be constructed using a disulfide-exchange reaction
or by forming a thioether bond. Examples of suitable reagents for
this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate.
[0159] (3) Humanized Antibodies
[0160] Anti-C1q antibodies of the present disclosure or antibody
fragments thereof may further include humanized or human
antibodies. Humanized forms of non-human (e.g., murine) antibodies
are chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fab, Fab'-SH, Fv, scFv, F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementarity determining region (CDR) of
the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann
et al., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct.
Biol. 2: 593-596 (1992). In some embodiments, the anti-C1q antibody
is a chimeric antibody comprising the heavy and light chain
variable domains of any of the anti-C1q antibody described herein
(e.g., antibody M1 and 4A4B 11) and constant regions from a human
immunoglobulin.
[0161] Methods for humanizing non-human anti-C1q antibodies are
well known in the art. Generally, a humanized antibody has one or
more amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers, Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);
Verhoeyen et al., Science 239:1534-1536 (1988), or through
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0162] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody. Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies. Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285
(1992); Presta et al., J. Immunol. 151:2623 (1993).
[0163] Furthermore, it is important that antibodies be humanized
with retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, humanized antibodies
are prepared by a process of analyzing the parental sequences and
various conceptual humanized products using three-dimensional
models of the parental and humanized sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to
those skilled in the art. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the recipient and
import sequences so that the desired antibody characteristic, such
as increased affinity for the target antigen or antigens (e.g., C1q
proteins of the present disclosure), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0164] Various forms of the humanized anti-C1q antibody are
contemplated. For example, the humanized anti-C1q antibody may be
an antibody fragment, such as an Fab, which is optionally
conjugated with one or more cytotoxic agent(s) in order to generate
an immunoconjugate. Alternatively, the humanized anti-C1q antibody
may be an intact antibody, such as an intact IgG1 antibody.
[0165] (4) Human Antibodies
[0166] Alternatively, human anti-C1q antibodies can be generated.
For example, it is now possible to produce transgenic animals
(e.g., mice) that are capable, upon immunization, of producing a
full repertoire of human antibodies in the absence of endogenous
immunoglobulin production. The homozygous deletion of the antibody
heavy-chain joining region (J.sub.H) gene in chimeric and germ-line
mutant mice results in complete inhibition of endogenous antibody
production. Transfer of the human germ-line immunoglobulin gene
array in such germ-line mutant mice will result in the production
of human antibodies upon antigen challenge. See, e.g., Jakobovits
et al., Proc. Nat'l Acad. Sci. USA, 90:2551 (1993); Jakobovits et
al., Nature, 362:255-258 (1993); Bruggermann et al., Year in
Immunol., 7:33 (1993); U.S. Pat. No. 5,591,669 and WO 97/17852.
[0167] Alternatively, phage display technology can be used to
produce human anti-C1q antibodies and antibody fragments in vitro,
from immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. McCafferty et al., Nature 348:552-553 (1990);
Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991). According to
this technique, antibody V domain genes are cloned in-frame into
either a major or minor coat protein gene of a filamentous
bacteriophage, such as M13 or fd, and displayed as functional
antibody fragments on the surface of the phage particle. Because
the filamentous particle contains a single-stranded DNA copy of the
phage genome, selections based on the functional properties of the
antibody also result in selection of the gene encoding the antibody
exhibiting those properties. Thus, the phage mimics some of the
properties of the B-cell. Phage display can be performed in a
variety of formats, reviewed in, e.g., Johnson, Kevin S. and
Chiswell, David J., Curr. Opin Struct. Biol. 3:564-571 (1993).
Several sources of V-gene segments can be used for phage display.
Clackson et al., Nature 352:624-628 (1991) isolated a diverse array
of anti-oxazolone antibodies from a small random combinatorial
library of V genes derived from the spleens of immunized mice. A
repertoire of V genes from unimmunized human donors can be
constructed and antibodies to a diverse array of antigens
(including self-antigens) can be isolated essentially following the
techniques described by Marks et al., J. Mol. Biol. 222:581-597
(1991), or Griffith et al., EMBO J. 12:725-734 (1993). See also
U.S. Pat. Nos. 5,565,332 and 5,573,905. Additionally, yeast display
technology can be used to produce human anti-C1q antibodies and
antibody fragments in vitro (e.g., WO 2009/036379; WO 2010/105256;
WO 2012/009568; US 2009/0181855; US 2010/0056386; and Feldhaus and
Siegel (2004) J. Immunological Methods 290:69-80). In other
embodiments, ribosome display technology can be used to produce
human anti-C1q antibodies and antibody fragments in vitro (e.g.,
Roberts and Szostak (1997) Proc Natl Acad Sci 94:12297-12302;
Schaffitzel et al. (1999) J. Immunolical Methods 231:119-135;
Lipovsek and Pl uckthun (2004) J. Immunological Methods
290:51-67).
[0168] The techniques of Cole et al., and Boerner et al., are also
available for the preparation of human anti-C1q monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147(1):
86-95 (1991). Similarly, human anti-C1q antibodies can be made by
introducing human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly and
antibody repertoire. This approach is described, for example, in
U.S. Pat. Nos. 5,545,807; 5,545,806, 5,569,825, 5,625,126,
5,633,425, 5,661,016 and in the following scientific publications:
Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al.,
Nature 368: 856-859 (1994); Morrison, Nature 368: 812-13 (1994),
Fishwild et al., Nature Biotechnology 14: 845-51 (1996), Neuberger,
Nature Biotechnology 14: 826 (1996) and Lonberg and Huszar, Intern.
Rev. Immunol. 13: 65-93 (1995).
[0169] Finally, human anti-C1q antibodies may also be generated in
vitro by activated B-cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275).
[0170] (5) Antibody Fragments
[0171] In certain embodiments there are advantages to using
anti-C1q antibody fragments, rather than whole anti-C1q antibodies.
Smaller fragment sizes allow for rapid clearance.
[0172] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., J. Biochem. Biophys. Method. 24:107-117 (1992); and Brennan et
al., Science 229:81 (1985)). However, these fragments can now be
produced directly by recombinant host-cells, for example, using
nucleic acids encoding anti-C1q antibodies of the present
disclosure. Fab, Fv and scFv antibody fragments can all be
expressed in and secreted from E. coli, thus allowing the
straightforward production of large amounts of these fragments. A
anti-C1q antibody fragments can also be isolated from the antibody
phage libraries as discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host-cell culture. Production of Fab and F(ab').sub.2 antibody
fragments with increased in vivo half-lives are described in U.S.
Pat. No. 5,869,046. In other embodiments, the antibody of choice is
a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No.
5,571,894 and U.S. Pat. No. 5,587,458. The anti-C1q, anti-C1r , or
anti-C1q antibody fragment may also be a "linear antibody," e.g.,
as described in U.S. Pat. No. 5,641,870. Such linear antibody
fragments may be monospecific or bispecific.
[0173] (6) Bispecific and Polyspecific Antibodies
[0174] Bispecific antibodies (BsAbs) are antibodies that have
binding specificities for at least two different epitopes,
including those on the same or another protein (e.g., one or more
C1q proteins of the present disclosure). Alternatively, one part of
a BsAb can be armed to bind to the target C1q antigen, and another
can be combined with an arm that binds to a second protein. Such
antibodies can be derived from full length antibodies or antibody
fragments (e.g., F(ab').sub.2 bispecific antibodies).
[0175] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy-chain/light
chain pairs, where the two chains have different specificities.
Millstein et al., Nature, 305:537-539 (1983). Because of the random
assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829 and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0176] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion may be with an immunoglobulin heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. In some embodiments, the first heavy-chain constant region
(C.sub.H1) containing the site necessary for light chain binding is
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0177] In some embodiments of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only half of the
bispecific molecules provides for an easy way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies, see, for example, Suresh et al.,
Methods in Enzymology 121: 210 (1986).
[0178] According to another approach described in WO 96/27011 or
U.S. Pat. No. 5,731,168, the interface between a pair of antibody
molecules can be engineered to maximize the percentage of
heterodimers which are recovered from recombinant-cell culture. The
interface may comprise at least a part of the C.sub.H3 region of an
antibody constant domain. In this method, one or more small amino
acid side chains from the interface of the first antibody molecule
are replaced with larger side chains (e.g., tyrosine or
tryptophan). Compensatory "cavities" of identical or similar size
to the large side chains(s) are created on the interface of the
second antibody molecule by replacing large amino acid side chains
with smaller ones (e.g., alanine or threonine). This provides a
mechanism for increasing the yield of the heterodimer over other
unwanted end-products such as homodimers.
[0179] Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent sodium arsenite to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-TNB derivative to form
the bispecific antibody. The bispecific antibodies produced can be
used as agents for the selective immobilization of enzymes.
[0180] Fab' fragments may be directly recovered from E. coli and
chemically coupled to form bispecific antibodies. Shalaby et al.,
J. Exp. Med. 175: 217-225 (1992) describes the production of fully
humanized bispecific antibody F(ab').sub.2 molecules. Each Fab'
fragment was separately secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific
antibody. The bispecific antibody thus formed was able to bind to
cells overexpressing the ErbB2 receptor and normal human T-cells,
as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0181] Various techniques for making and isolating bivalent
antibody fragments directly from recombinant-cell culture have also
been described. For example, bivalent heterodimers have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. The "diabody" technology described by
Hollinger et al., Proc. Nat'l Acad. Sci. USA, 90: 6444-6448 (1993)
has provided an alternative mechanism for making
bispecific/bivalent antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L, domains of one fragment are forced to pair
with the complementary V.sub.L, and V.sub.H domains of another
fragment, thereby forming two antigen-binding sites. Another
strategy for making bispecific/bivalent antibody fragments by the
use of single-chain Fv (sFv) dimers has also been reported. See
Gruber et al., J. Immunol., 152:5368 (1994).
[0182] Antibodies with more than two valencies are also
contemplated. For example, trispecific antibodies can be prepared.
Tutt et al., J. Immunol. 147:60 (1991).
[0183] Exemplary bispecific antibodies may bind to two different
antigens. In some embodiments a bispecific antibody binds to a
first antigen, C1q, and a second antigen facilitating transport
across the blood-brain barrier. Numerous antigens are known in the
art that facilitate transport across the blood-brain barrier (see,
e.g., Gabathuler R., Approaches to transport therapeutic drugs
across the blood-brain barrier to treat brain diseases, Neurobiol.
Dis. 37 (2010) 48-57). Such second antigens include, without
limitation, 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, including CRM197 (a non-toxic mutant of diphtheria
toxin), llama single domain antibodies such as TMEM 30(A)
(Flippase), protein transduction domains such as TAT, Syn-B, or
penetratin, poly-arginine or generally positively charged peptides,
and Angiopep peptides such as ANG1005 (see, e.g., Gabathuler,
2010).
[0184] (7) Multivalent Antibodies
[0185] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The anti-C1q antibodies of
the present disclosure or antibody fragments thereof can be
multivalent antibodies (which are other than of the IgM class) with
three or more antigen binding sites (e.g., tetravalent antibodies),
which can be readily produced by recombinant expression of nucleic
acid encoding the polypeptide chains of the antibody. The
multivalent antibody can comprise a dimerization domain and three
or more antigen binding sites. In some embodiments, the
dimerization domain comprises an Fc region or a hinge region. In
this scenario, the antibody will comprise an Fc region and three or
more antigen binding sites amino-terminal to the Fc region. In some
embodiments, the multivalent antibody herein contains three to
about eight, and in some embodiments four, antigen binding sites.
The multivalent antibody contains at least one polypeptide chain
(and in some embodiments two polypeptide chains), wherein the
polypeptide chain or chains comprise two or more variable domains.
For instance, the polypeptide chain or chains may comprise
VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2
is a second variable domain, Fc is one polypeptide chain of an Fc
region, X1 and X2 represent an amino acid or polypeptide, and n is
0 or 1. Similarly, the polypeptide chain or chains may comprise
V.sub.H-C.sub.H1-flexible linker-V.sub.H-C.sub.H1-Fc region chain;
or V.sub.H-C.sub.H1-V.sub.H-C.sub.H1-Fc region chain. The
multivalent antibody herein may further comprise at least two (and
in some embodiments four) light chain variable domain polypeptides.
The multivalent antibody herein may, for instance, comprise from
about two to about eight light chain variable domain polypeptides.
The light chain variable domain polypeptides contemplated here
comprise a light chain variable domain and, optionally, further
comprise a CL domain.
[0186] (8) Heteroconjugate Antibodies
[0187] Heteroconjugate antibodies are also within the scope of the
present disclosure. Heteroconjugate antibodies are composed of two
covalently joined antibodies (e.g., anti-C1q antibodies of the
present disclosure or antibody fragments thereof). For example, one
of the antibodies in the heteroconjugate can be coupled to avidin,
the other to biotin. Such antibodies have, for example, been
proposed to target immune system cells to unwanted cells, U.S. Pat.
No. 4,676,980, and have been used to treat HIV infection.
International Publication Nos. WO 91/00360, WO 92/200373 and EP
0308936. It is contemplated that the antibodies may be prepared in
vitro using known methods in synthetic protein chemistry, including
those involving crosslinking agents. For example, immunotoxins may
be constructed using a disulfide exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980. Heteroconjugate
antibodies may be made using any convenient cross-linking methods.
Suitable cross-linking agents are well known in the art, and are
disclosed in U.S. Pat. No. 4,676,980, along with a number of
cross-linking techniques.
[0188] (9) Effector Function Engineering
[0189] It may also be desirable to modify an anti-C1q antibody of
the present disclosure to modify effector function and/or to
increase serum half-life of the antibody. For example, the Fc
receptor binding site on the constant region may be modified or
mutated to remove or reduce binding affinity to certain Fc
receptors, such as Fc.gamma.RI, Fc.gamma.RII, and/or Fc.gamma.RIII.
In some embodiments, the effector function is impaired by removing
N-glycosylation of the Fc region (e.g., in the CH 2 domain of IgG)
of the antibody. In some embodiments, the effector function is
impaired by modifying regions such as 233-236, 297, and/or 327-331
of human IgG as described in PCT WO 99/58572 and Armour et al.,
Molecular Immunology 40: 585-593 (2003); Reddy et al., J.
Immunology 164:1925-1933 (2000).
[0190] The constant region of the anti-complement antibodies
described herein may also be modified to impair complement
activation. For example, complement activation of IgG antibodies
following binding of the C1 component of complement may be reduced
by mutating amino acid residues in the constant region in a C1
binding motif (e.g., C1q binding motif). It has been reported that
Ala mutation for each of D270, K322, P329, P331 of human IgG1
significantly reduced the ability of the antibody to bind to C1q
and activating complement. For murine IgG2b, C1q binding motif
constitutes residues E318, K320, and K322. Idusogie et al. (2000)
J. Immunology 164:4178-4184; Duncan et al. (1988) Nature 322:
738-740. As the C1s binding motif E318, K320, and K322 identified
for murine IgG2b is believed to be common for other antibody
isotypes (Duncan et al. (1988) Nature 322:738-740), C1q binding
activity for IgG2b can be abolished by replacing any one of the
three specified residues with a residue having an inappropriate
functionality on its side chain. It is not necessary to replace the
ionic residues only with Ala to abolish C1q binding. It is also
possible to use other alkyl-substituted non-ionic residues, such as
Gly, Ile, Leu, or Val, or such aromatic non-polar residues as Phe,
Tyr, Trp and Pro in place of any one of the three residues in order
to abolish C1q binding. In addition, it is also possible to use
such polar non-ionic residues as Ser, Thr, Cys, and Met in place of
residues 320 and 322, but not 318, in order to abolish C1s binding
activity. In addition, removal of carbohydrate modifications of the
Fc region necessary for complement binding can prevent complement
activation Glycosylation of a conserved asparagine (Asn-297) on the
CH2 domain of IgG heavy chains is essential for antibody effector
functions (Jefferis et al. (1998) Immunol Rev 163:59-76).
Modification of the Fc glycan alters IgG conformation and reduces
the Fc affinity for binding of complement protein C1q and effector
cell receptor FcR (Alhorn et al. (2008) nos ONE 2008; 3:e1413).
Complete removal of the Fc glycan abolishes CDC and ADCC.
Deglycosylation can be performed using glycosidase enzymes for
example Endoglycosidase S (EndoS), a 108 kDa enzyme encoded by the
gene endoS of Streptococcus pyogenes that selectively digests
asparagine-linked glycans on the heavy chain of all IgG subclasses,
without action on other immunoglobulin classes or other
glycoproteins (Collin et al. (2001) EMBO J 2001,20:3046-3055).
[0191] To increase the serum half-life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that
is responsible for increasing the in vivo serum half-life of the
IgG molecule.
[0192] (10) Other Amino Acid Sequence Modifications
[0193] Amino acid sequence modifications of anti-C1q antibodies of
the present disclosure, or antibody fragments thereof, are also
contemplated. For example, it may be desirable to improve the
binding affinity and/or other biological properties of the
antibodies or antibody fragments. Amino acid sequence variants of
the antibodies or antibody fragments are prepared by introducing
appropriate nucleotide changes into the nucleic acid encoding the
antibodies or antibody fragments, or by peptide synthesis. Such
modifications include, for example, deletions from, and/or
insertions into and/or substitutions of, residues within the amino
acid sequences of the antibody. Any combination of deletion,
insertion, and substitution is made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics (i.e., the ability to bind or physically interact
with a C1q protein of the present disclosure). The amino acid
changes also may alter post-translational processes of the
antibody, such as changing the number or position of glycosylation
sites.
[0194] A useful method for identification of certain residues or
regions of the anti-C1q antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells in Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
the target antigen. Those amino acid locations demonstrating
functional sensitivity to the substitutions then are refined by
introducing further or other variants at, or for, the sites of
substitution. Thus, while the site for introducing an amino acid
sequence variation is predetermined, the nature of the mutation per
se need not be predetermined. For example, to analyze the
performance of a mutation at a given site, alanine scanning or
random mutagenesis is conducted at the target codon or region and
the expressed antibody variants are screened for the desired
activity.
[0195] Amino acid sequence insertions include amino-("N") and/or
carboxy-("C") terminal fusions ranging in length from one residue
to polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody to an
enzyme or a polypeptide which increases the serum half-life of the
antibody.
[0196] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also contemplated.
Conservative substitutions are shown in the Table A below under the
heading of "preferred substitutions". If such substitutions result
in a change in biological activity, then more substantial changes,
denominated "exemplary substitutions" in Table A, or as further
described below in reference to amino acid classes, may be
introduced and the products screened.
TABLE-US-00003 TABLE A Amino Acid Substitutions Original Exemplary
Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile
val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; arg gln
Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu
(E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile
(I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine;
ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu;
phe; ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala
Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp;
phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine
leu
[0197] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0198] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0199] (2) neutral hydrophilic: cys, ser, thr;
[0200] (3) acidic: asp, glu;
[0201] (4) basic: asn, gln, his, lys, arg;
[0202] (5) residues that influence chain orientation: gly, pro;
and
[0203] (6) aromatic: trp, tyr, phe.
[0204] Non-conservative substitutions entail exchanging a member of
one of these classes for another class.
[0205] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability (particularly where
the antibody is an antibody fragment, such as an Fv fragment).
[0206] In some embodiments, the substitutional variant involves
substituting one or more hypervariable region residues of a parent
antibody (e.g. a humanized or human anti-C1q antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and the antigen (e.g.,
a C1q protein of the present disclosure). Such contact residues and
neighboring residues are candidates for substitution according to
the techniques elaborated herein. Once such variants are generated,
the panel of variants is subjected to screening as described herein
and antibodies with superior properties in one or more relevant
assays may be selected for further development.
[0207] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0208] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
0-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0209] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0210] Nucleic acid molecules encoding amino acid sequence variants
of the anti-IgE antibody are prepared by a variety of methods known
in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the antibodies (e.g., anti-C1q
antibody of the present disclosure) or antibody fragments.
[0211] (11) Other Antibody Modifications
[0212] Anti-C1q antibodies of the present disclosure, or antibody
fragments thereof, can be further modified to contain additional
non-proteinaceous moieties that are known in the art and readily
available. In some embodiments, the moieties suitable for
derivatization of the antibody are water-soluble polymers.
Non-limiting examples of water-soluble polymers include, but are
not limited to, polyethylene glycol (PEG), copolymers of ethylene
glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl
alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
polypropylene glycol homopolymers, polypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and
may be branched or unbranched. The number of polymers attached to
the antibody may vary, and if more than one polymer is attached,
they can be the same or different molecules. In general, the number
and/or type of polymers used for derivatization can be determined
based on considerations including, but not limited to, the
particular properties or functions of the antibody to be improved,
whether the antibody derivative will be used in a therapy under
defined conditions, etc. Such techniques and other suitable
formulations are disclosed in Remington: The Science and Practice
of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., Philadelphia College
of Pharmacy and Science (2000).
Nucleic Acids, Vectors, and Host Cells
[0213] Anti-C1q antibodies of the present disclosure may be
produced using recombinant methods and compositions, e.g., as
described in U.S. Pat. No. 4,816,567. In some embodiments, isolated
nucleic acids having a nucleotide sequence encoding any of the
anti-C1q antibodies of the present disclosure are provided. Such
nucleic acids may encode an amino acid sequence containing the VL
and/or an amino acid sequence containing the VH of the anti-C1q
antibody (e.g., the light and/or heavy chains of the antibody). In
some embodiments, one or more vectors (e.g., expression vectors)
containing such nucleic acids are provided. In some embodiments, a
host cell containing such nucleic acid is also provided. In some
embodiments, the host cell contains (e.g., has been transduced
with): (1) a vector containing a nucleic acid that encodes an amino
acid sequence containing the VL of the antibody and an amino acid
sequence containing the VH of the antibody, or (2) a first vector
containing a nucleic acid that encodes an amino acid sequence
containing the VL of the antibody and a second vector containing a
nucleic acid that encodes an amino acid sequence containing the VH
of the antibody. In some embodiments, the host cell is eukaryotic,
e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g.,
Y0, NS0, Sp20 cell).
[0214] Methods of making an anti-C1q antibody of the present
disclosure are provided. In some embodiments, the method includes
culturing a host cell of the present disclosure containing a
nucleic acid encoding the anti-C1q 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). See also Example 1.
[0215] For recombinant production of an anti-C1q antibody of the
present disclosure, a nucleic acid encoding the anti-C1q 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).
[0216] Suitable vectors containing a nucleic acid sequence encoding
any of the anti-C1q 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, Strategene, and
Invitrogen.
[0217] Expression vectors generally are replicable polynucleotide
constructs that contain a nucleic acid of the present disclosure.
The expression vector may replicable in the host cells either as
episomes or as an integral part of the chromosomal DNA. Suitable
expression vectors include but are not limited to plasmids, viral
vectors, including adenoviruses, adeno-associated viruses,
retroviruses, cosmids, and expression vector(s) disclosed in PCT
Publication No. WO 87/04462. Vector components may generally
include, but are not limited to, one or more of the following: a
signal sequence; an origin of replication; one or more marker
genes; suitable transcriptional controlling elements (such as
promoters, enhancers and terminator). For expression (i.e.,
translation), one or more translational controlling elements are
also usually required, such as ribosome binding sites, translation
initiation sites, and stop codons.
[0218] 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-C1q antibody of the
present disclosure.
[0219] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells.
For example, anti-C1q antibodies of the present disclosure may be
produced in bacteria, in particular when glycosylation and Fc
effector function are not needed. For expression of antibody
fragments and polypeptides in bacteria (e.g., U.S. Pat. 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,
N.J., 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.
[0220] In addition to prokaryotes, eukaryotic microorganisms, such
as filamentous fungi or yeast, are also suitable cloning or
expression hosts for antibody-encoding vectors, including fungi and
yeast strains whose glycosylation pathways have been "humanized,"
resulting in the production of an antibody with a partially or
fully human glycosylation pattern (e.g., Gerngross, Nat. Biotech.
22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215
(2006)).
[0221] Suitable host cells for the expression of glycosylated
antibody can also be derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frupperda cells. Plant
cell cultures can also be utilized as hosts (e.g., U.S. Pat. Nos.
5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429,
describing PLANTIBODIES.TM. technology for producing antibodies in
transgenic plants.).
[0222] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells
(Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and
myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
Pharmaceutical Compositions
[0223] Anti-C1q antibodies of the present disclosure can be
incorporated into a variety of formulations for therapeutic use
(e.g., by administration) or in the manufacture of a medicament
(e.g., for treating or preventing a neurodegenerative disease, such
as Alzheimer's disease or Huntington's disease) by combining the
antibodies with appropriate pharmaceutically acceptable carriers or
diluents, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms. Examples of such formulations
include, without limitation, tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants, gels,
microspheres, and aerosols. Pharmaceutical compositions can
include, depending on the formulation desired,
pharmaceutically-acceptable, non-toxic carriers of diluents, which
are 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 include, without limitation, distilled water,
buffered water, physiological saline, PBS, Ringer's solution,
dextrose solution, and Hank's solution. A pharmaceutical
composition or formulation of the present disclosure can further
include other carriers, adjuvants, or non-toxic, nontherapeutic,
nonimmunogenic stabilizers, excipients and the like. The
compositions can also include additional substances to approximate
physiological conditions, such as pH adjusting and buffering
agents, toxicity adjusting agents, wetting agents and
detergents.
[0224] A pharmaceutical composition of the present disclosure can
also include any of a variety of stabilizing agents, such as an
antioxidant for example. When the pharmaceutical composition
includes a polypeptide, the polypeptide can 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, and enhance solubility or uptake). Examples of such
modifications or complexing agents include, without limitation,
sulfate, gluconate, citrate and phosphate. The polypeptides of a
composition can also be complexed with molecules that enhance their
in vivo attributes. Such molecules include, without limitation,
carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,
sodium, potassium, calcium, magnesium, manganese), and lipids.
[0225] Further examples of formulations that are suitable for
various types of administration can 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).
[0226] For oral administration, the active ingredient can 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) can 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
can be used to make compressed tablets. Both tablets and capsules
can be manufactured as sustained release products to provide for
continuous release of medication over a period of hours. Compressed
tablets can 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 can contain coloring and
flavoring to increase patient acceptance.
[0227] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can 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 can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives.
[0228] 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 in vivo 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 sterile, substantially isotonic and made
under GMP conditions.
[0229] Formulations may be optimized for retention and
stabilization in the brain or central nervous system. 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.
[0230] Other strategies for increasing retention include the
entrapment of the antibody, such as an anti-C1q antibody of the
present disclosure, 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.
[0231] 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.
[0232] 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 will 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 individual invention. 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.
Pharmaceutical Dosages
[0233] Pharmaceutical compositions of the present disclosure
containing an anti-C1q antibody of the present disclosure may be
used (e.g., administered to an individual in need of treatment with
anti-C1q antibody, such as a human individual) in accord with known
methods, such as intravenous administration as a bolus or by
continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, intracranial, intraspinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral,
topical, or inhalation routes.
[0234] Dosages and desired drug concentration of pharmaceutical
compositions of the present disclosure may vary depending on the
particular use envisioned. The determination of the appropriate
dosage or route of administration is well within the skill of an
ordinary artisan. Animal experiments provide reliable guidance for
the determination of effective doses for human therapy.
Interspecies scaling of effective doses can be performed following
the principles described in Mordenti, J. and Chappell, W. "The Use
of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and
New Drug Development, Yacobi et al., Eds, Pergamon Press, New York
1989, pp. 42-46.
[0235] For in vivo administration of any of the anti-C1q antibodies
of the present disclosure, normal dosage amounts may vary from
about 10 ng/kg up to about 100 mg/kg of an individual's and/or
subject's body weight or more per day, depending upon the route of
administration. In some embodiments, the dose amount is about 1
mg/kg/day to 10 mg/kg/day. For repeated administrations over
several days or longer, depending on the severity of the disease,
disorder, or condition to be treated, the treatment is sustained
until a desired suppression of symptoms is achieved.
[0236] An exemplary dosing regimen may include administering an
initial dose of an anti-C1q antibody, of about 2 mg/kg, followed by
a weekly maintenance dose of about 1 mg/kg every other week. Other
dosage regimens may be useful, depending on the pattern of
pharmacokinetic decay that the physician wishes to achieve. For
example, dosing an individual from one to twenty-one times a week
is contemplated herein. In certain embodiments, dosing ranging from
about 3 .mu.g/kg to about 2 mg/kg (such as about 3 .mu.g/kg, about
10 .mu.g/kg, about 30 .mu.g/kg, about 100 .mu.g/kg, about 300
.mu.g/kg, about 1 mg/kg, or about 2 mg/kg) may be used. In certain
embodiments, dosing frequency is three times per day, twice per
day, once per day, once every other day, once weekly, once every
two weeks, once every four weeks, once every five weeks, once every
six weeks, once every seven weeks, once every eight weeks, once
every nine weeks, once every ten weeks, or once monthly, once every
two months, once every three months, or longer. Progress of the
therapy is easily monitored by conventional techniques and assays.
The dosing regimen, including the anti-C1q antibody administered,
can vary over time independently of the dose used.
[0237] Dosages for a particular anti-C1q antibody may be determined
empirically in individuals who have been given one or more
administrations of the anti-C1q antibody. Individuals are given
incremental doses of an anti-C1q antibody. To assess efficacy of an
anti-C1q antibody, any clinical symptom of a neurodegenerative
disorder (e.g., Alzheimer's disease and Huntington's disease),
inflammatory disorder, or autoimmune disorder can be monitored.
[0238] Administration of an anti-C1q antibody of the present
disclosure can be continuous or intermittent, depending, for
example, on the recipient's physiological condition, whether the
purpose of the administration is therapeutic or prophylactic, and
other factors known to skilled practitioners. The administration of
an anti-C1q antibody may be essentially continuous over a
preselected period of time or may be in a series of spaced
doses.
[0239] Guidance regarding particular dosages and methods of
delivery is provided in the literature; see, for example, U.S. Pat.
Nos. 4,657,760; 5,206,344; or 5,225,212. It is within the scope of
the invention that different formulations will be effective for
different treatments and different disorders, and that
administration intended to treat a specific organ or tissue may
necessitate delivery in a manner different from that to another
organ or tissue. Moreover, dosages may be administered by one or
more separate administrations, or by continuous infusion. For
repeated administrations over several days or longer, depending on
the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs. However, other dosage
regimens may be useful. The progress of this therapy is easily
monitored by conventional techniques and assays.
Therapeutic Uses
[0240] The present disclosure provides anti-C1q antibodies, and
antigen-binding fragments thereof, which can bind to and neutralize
a biologic activity of C1q. These anti-C1 q antibodies are useful
for preventing, reducing risk, or treating Alzheimer's disease
and/or Huntington's disease. Accordingly, as disclosed herein,
anti-C1q antibodies of the present disclosure may be used for
treating, preventing, or reducing risk of Alzheimer's disease
and/or Huntington's disease in an individual. In some embodiments,
the individual has Alzheimer's disease or Huntington's disease. In
some embodiments, the individual is a human.
[0241] In some embodiments, the Alzheimer's disease and/or
Huntington's disease is associated with loss of nerve connections
or synapses, including CF1-dependent synapse loss. In some
embodiments, the synapse loss is dependent on the complement
receptor 3 (CR3)C3 or complement receptor CR1 in some embodiments,
the synapse loss is associated with pathological activity-dependent
synaptic pruning. In some embodiments, synapses are phagocytosed by
microglia.
Alzheimer 's Disease
[0242] As disclosed herein, "Alzheimer's disease, " "AD," or
"Alzheimer disease" may be used interchangeably and refer to a
common form of dementia. Alzheimer s disease worsens as it
progresses, and eventually leads to death. The cause and
progression of Alzheimer's disease are not well understood.
Alzheimer's disease is generally characterized by two types of
brain lesions: senile plaques and neurofibrillary tangles.
[0243] Symptoms of Alzheimer's disease may include, without
limitation, loss of cognitive function, short term memory loss,
problems with the executive functions of attentiveness, planning,
flexibility, and abstract thinking, impairments in semantic memory,
confusion, irritability, aggression, mood swings, apathy,
depression, impairment with learning and memory, difficulty with
language, difficulty with executive functions, difficulty with
perception (agnosia), difficulty with execution of movements
(apraxia), long-term memory loss, loss of reading and writing
skills, loss of complex motor functions, and loss of bodily
functions. The anti-C1q antibody may also be administered ex vivo
in brain slice models of Alzheimer's disease.
[0244] Accordingly, the anti-C1q antibodies of the present
disclosure may be used to treat, prevent, or improve one or more
symptoms of Alzheimer's disease. In some embodiments, the present
disclosure provides methods of treating, preventing, or improving
one or more symptoms in subjects having Alzheimer's disease by
administering an anti-C1q antibody of the present disclosure to,
for example, inhibit the interaction between C1q and an
autoantibody, such as an anti-ganglioside autoantibody, the
interaction of C1q and C1r , and/or the interaction of C1q and C1s.
The anti-C1q antibody may also be administered ex vivo in brain
slice models of Alzheimer's disease.
Huntington's Disease
[0245] As disclosed herein, "Huntington's disease" and "HD" may be
used interchangeably and refer to an inherited neurodegenerative
genetic disorder that causes the progressive degeneration of nerve
cells in the brain, affects muscle coordination, and leads to
cognitive decline and psychiatric problems. Huntington's disease is
the most common genetic cause of abnormal involuntary writhing
movements called chorea. Huntington's disease worsens as it
progresses. Complications such as pneumonia, heart disease, and
physical injury from falls reduce life expectancy to around twenty
years from the point at which symptoms begin. Huntington's disease
is caused by inherited defect in a single gene (the Huntingtin
gene). Huntington's disease is an autosomal dominant disorder,
which means that a person needs only one copy of the defective gene
to develop Huntington's disease.
[0246] Huntington's disease usually causes movement, cognitive, and
psychiatric disorders with a wide spectrum of signs and symptoms.
Symptoms of Huntington's disease may include, without limitation,
problems with mood, problems with cognition, lack of coordination,
involuntary jerking or writhing movements (chorea), involuntary,
sustained contracture of muscles (dystonia), muscle rigidity, slow,
uncoordinated fine movements, slow or abnormal eye movements,
impaired gait, posture and balance, difficulty with the physical
production of speech, difficulty swallowing, difficulty planning,
organizing and prioritizing tasks, inability to start a task or
conversation, lack of flexibility, the tendency to get stuck on a
thought, behavior or action (perseveration), lack of impulse
control that can result in outbursts, acting without thinking and
sexual promiscuity, problems with spatial perception that can
result in falls, clumsiness or accidents, lack of awareness of
one's own behaviors and abilities, difficulty focusing on a task
for long periods, slowness in processing thoughts or "finding"
words, difficulty in learning new information, depression, loss of
interest in normal activities, social withdrawal, insomnia or
excessive sleeping, fatigue, tiredness and loss of energy, feelings
of worthlessness or guilt, indecisiveness, distractibility and
decreased concentration, frequent thoughts of death, dying or
suicide, changes in appetite, reduced sex drive,
obsessive-compulsive disorder, mania, bipolar disorder,
irritability, apathy, and anxiety.
[0247] Accordingly, the anti-C1q antibodies of the present
disclosure may be used to treat, prevent, or improve one or more
symptoms of Huntington's disease. In some embodiments, the present
disclosure provides methods of treating, preventing, or improving
one or more symptoms in subjects having Huntington's disease by
administering an anti-C1q antibody of the present disclosure to,
for example, inhibit the interaction between C1q and an
autoantibody, such as an anti-ganglioside autoantibody, the
interaction of C1q and C1 r, and/or the interaction of C1q and C1s.
The anti-C1q antibody may also be administered ex vivo in brain
slice models of Huntington's disease.
Combination Treatments
[0248] The antibodies of the present disclosure may be used,
without limitation, in combination with any additional treatment
for Alzheimer's disease and/or Huntington's disease.
[0249] In some embodiments, an anti-C1q antibody of this disclosure
is administered in therapeutically effective amounts in combination
with a second anti-complement factor antibody (e.g., a neutralizing
anti-complement factor antibody), such as an anti-C1s or anti-C1r
antibody, or a second anti-C1q antibody. In some embodiments, an
anti-C1q antibody of this disclosure is administered in
therapeutically effective amounts with a second and a third
neutralizing anti-complement factor antibody, such as a second
anti-C1q antibody, an anti-C1s antibody, and/or an anti-C1r
antibody.
[0250] In some embodiments, the anti-C1q antibodies of this
disclosure are administered in combination with an inhibitor of
antibody-dependent cellular cytotoxicity (ADCC). ADCC inhibitors
may include, without limitation, soluble NK cell inhibitory
receptors such as the killer cell Ig-like receptors (KIRs), which
recognize HLA-A, HLA-B, or HLA-C and C-type lectin CD94/NKG2A
heterodimers, which recognize HLA-E (see, e.g., Lopez-Botet M., T.
Bellon, M. Llano, F. Navarro, P. Garcia & M. de Miguel. (2000),
Paired inhibitory and triggering NK cell receptors for HLA class I
molecules. Hum. Immunol. 61: 7-17; Lanier L.L. (1998) Follow the
leader: NK cell receptors for classical and nonclassical MHC class
I. Cell 92: 705-707.), and cadmium (see, e.g., Immunopharmacology
1990; Volume 20, Pages 73-8).
[0251] In some embodiments, the antibodies of this disclosure are
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 Comstatin-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
C1q binding sites in their Fc domains (e.g., Fab fragments or
antibody otherwise engineered not to bind C1q).
Diagnostic Uses
[0252] The anti-C1q antibodies of this disclosure also have
diagnostic utility. This disclosure therefore provides for methods
of using the anti-C1q antibodies of this disclosure, for diagnostic
purposes, such as the detection of C1q in an individual or in
tissue samples derived from an individual. In some embodiments, the
individual is a human. In some embodiments, the individual is a
human patient suffering from Alzheimer's disease or Huntington's
disease. In some embodiments, the anti-C1q antibodies of this
disclosure are used to detect synapses and synapse loss. For
example, synapse loss may be measured in an individual suffering
from Alzheimer's disease or Huntington's disease. The phenomenon of
synapse loss in neurodegeneration is well understood in the art.
See, e.g., U.S. Patent Publication Nos. 2012/0195880 and
2012/0328601.
[0253] In some embodiments, the diagnostic methods involve the
steps of administering an anti-C1q antibody of this disclosure to
an individual and detecting the antibody bound to a synapse of the
individual. Antibody-binding to synapses may be quantified, for
example, by non-invasive techniques such as positron emission
tomography (PET), X-ray computed tomography, single-photon emission
computed tomography (SPECT), computed tomography (CT), and computed
axial tomography (CAT).
[0254] In some embodiments, the diagnostic methods involve
detecting synapses in a biological sample, such as a biopsy
specimen, a tissue, or a cell. An anti-C1q antibody is contacted
with the biological sample and synapse-bound antibody is detected.
The detection method may involve quantification of the
synapse-bound antibody. Antibody detection in biological samples
may occur with any method known in the art, including
immunofluorescence microscopy, immunocytochemistry,
immunohistochemistry, ELISA, FACS analysis or
immunoprecipitation.
[0255] The quantification of synapse-bound antibodies provides a
relative measure for the number of synapses present in the
individual. Typically, synapses are quantified repeatedly over a
period of time. The exact periodicity of synapse quantification
depends on many factors, including the nature of the
neurodegenerative disease (e.g., Alzheimer's disease and/or
Huntington's disease), the stage of disease progression, treatment
modalities and many other factors. Repeat measurements commonly
reveal progressive synapse loss in individuals having a
neurodegenerative disease (e.g., Alzheimer's disease and/or
Huntington's disease). Alternatively, relative synapse counts may
be compared in populations of diseased individuals, and healthy
control individuals at a single time point. In diseased individuals
undergoing treatment, the treatment's efficacy can be assessed by
comparing the rates of synapse loss in the treated individuals with
the rates of synapse loss in a control group. Control group members
have received either no treatment or a control treatment, such as a
placebo control.
Kits
[0256] The invention also provides kits containing anti-C1q
antibodies of this disclosure for use in the methods of the present
disclosure. Kits of the invention may include one or more
containers comprising a purified anti-C1q antibody of this
disclosure. In some embodiments, the kits further include
instructions for use in accordance with the methods of this
disclosure. In some embodiments, these instructions comprise a
description of administration of the anti-C1q antibody to treat or
diagnose, e.g., Alzheimer's disease and/or Huntington's disease,
according to any of the methods of this disclosure. In some
embodiments, the instructions comprise a description of how to
detect C1q, for example in an individual, in a tissue sample, or in
a cell. The kit may further comprise a description of selecting an
individual suitable for treatment based on identifying whether that
subject has the disease and the stage of the disease.
[0257] 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 invention 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.
[0258] The label or package insert indicates that the composition
is used for treating, e.g., Alzheimer's disease and Huntington's
disease. Instructions may be provided for practicing any of the
methods described herein.
[0259] The kits of this invention are 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.
[0260] 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.
[0261] The invention will be more fully understood by reference to
the following Examples. They should not, however, be construed as
limiting the scope of the invention. All citations throughout the
disclosure are hereby expressly incorporated by reference.
EXAMPLES
Example 1
Production of Anti-C1q Antibodies
[0262] The anti-C1q antibody M1 was generated by Antibody Solutions
Inc. (Sunnyvale Calif.) by immunizing C1q knockout mice with human
C1q using standard mouse immunization and hybridoma screening
technologies (Milstein, C (1999). Bioessays 21: 966-73; Mark Page,
Robin Thorpe, The Protein Protocols Handbook 2002, Editors: John M.
Walker. pp 1111-1113).
[0263] The anti-C1q antibodies 1C7, 2A1, 3A2, and 5A3 were
generated at ImmunoPrecise Ltd (Victoria, BC Canada) by immunizing
mice with human C1q protein purified from human plasma (Complement
Technology Inc. Tyler Tex., Cat #A-099). In brief, female BALB/c
mice were injected intraperitoneal with 25 .mu.g of protein in
complete Freund's adjuvant (CFA) on Day 0 and boosts were done with
25 .mu.g of C1q enzyme in incomplete Freund's adjuvant (IFA) on
days 21, 42, 52, and a final intravenous boost on Day 63. Four days
following the final boost the mice were euthanized, spleens removed
and splenocytes were fused with the myeloma cell line SP2/0. Fused
cells were grown on hypoxanthine-aminopterin-thymidine (HAT)
selective semisolid media for 10-12 days and the resulting
hybridomas clones were transferred to 96-well tissue culture plates
and grown in HAT medium until the antibody titer was high. The
antibody-rich supernatants of the clones were isolated and tested
in an ELISA assay for reactivity with C1q. Positive clones were
isotyped and cultured for 32 days (post HAT selection) to identify
stable expressing clones.
[0264] A hybridoma cell line producing the anti-C1q antibody M1 and
referred to as mouse hybridoma C1q-M1 7788-1(M) 051613 was
deposited with ATCC on 6/6/2013 having ATCC Accession Number
PTA-120399. M1 was shown to bind specifically to human and mouse
C1q and to neutralize biological functions of C1q, such as
complement mediated hemolysis (see, e.g., Example 3).
Example 2
Anti-C1q Antibodies Specifically Bind to C1q
ELISA Screening
[0265] Anti-C1q antibodies 1C7, 2A1, 3A2, and 5A3 were screened for
C1q-binding using standard ELISA protocols.
[0266] Briefly, the day before the assay was performed, 96-well
microtiter plates were coated at 0.2 .mu.g/well of C1q-enzyme
antigen in 100 .mu.L/well carbonate coating buffer pH 9.6 overnight
at 4.degree. C. Control wells were coated with human transferrin.
Next, the plates were blocked with 3% milk powder in PBS for 1 hour
at room temperature. Then, hybridoma tissue culture supernatants
were plated at 100 .mu.L/well for 1 hour at 37.degree. C. with
shaking. The secondary antibody (1:10,000 goat anti-mouse
IgG/IgM(H+L)-HRP) was applied at 100 .mu.L/well for 1.5 hours at
room temperature with shaking. TMB substrate was added at 50
.mu.L/well for 5 minutes at room temperature in the dark. The
reaction was stopped with 50 .mu.L/well 1M HCl and absorbance
readings were taken at a wavelength of 450 nm.
[0267] Four hybridoma supernatants containing the anti-C1q
antibodies 1C7, 2A1, 3A2, and 5A3 were tested for binding to human
C1q (FIG. 1). By ELISA, all four supernatants showed strong binding
signals in the presence of human C1q, whereas only background
signals were observed in control wells containing human
transferrin. This experiment demonstrated that the anti-C1q
antibodies 1C7, 2A1, 3A2, and 5A3 specifically bind to human
C1q.
Kinetic Analyses
[0268] The interactions of the full length anti-C1q antibody M1
with human and mouse C1q proteins were first measured in a kinetic
mode and thermodynamic dissociation constants were subsequently
calculated. Additionally, M1 binding data was compared with
corresponding data obtained using the reference antibody 4A4B11.
4A4B11 is described in U.S. Pat. No. 4,595,654. The 4A4B 11
producing hybridoma cell line is available from ATCC (ATCC
HB-8327TM).
[0269] C1q-antibody interactions were measured using an OCTE.TM.
System according to standard protocols and manufacturer's
instructions. Briefly, human and mouse C1q proteins were
immobilized separately on a biosensor at three concentrations (3
nM, 1.0 nM, and 0.33 nM). Next, the anti-C1q antibody M1 was
injected onto the C1q-coated biosensor at a concentration of 2.0
.mu.g/ml and the association constants (k.sub.on) and dissociation
constants (k.sub.off) for anti-C1q antibodies M1 and 4A4B11 were
measured. The data were fit by non-linear regression analysis and
using the Octet Data Analysis software to yield affinity (K.sub.D)
and kinetic parameters (k.sub.on/off ) for the interactions of M1
and 4A4B11 with human and mouse C1q respectively (see Table B).
TABLE-US-00004 TABLE B Kinetic Analysis of M1 and 4A4B11 Antibody
Antigen K.sub.D (M) k.sub.on (1/Ms) k.sub.off (1/s) M1 human C1q
1.28*10.sup.-11 5.18*10.sup.6 6.31*10.sup.-5 M1 mouse C1q
3.23*10.sup.-11 1.81*10.sup.6 5.84*10.sup.-5 4A4B11 human C1q
2.29*10.sup.-11 4.49*10.sup.6 1.03*10.sup.-4 4A4B11 mouse C1q
undetectable undetectable undetectable
[0270] In this experimental series, anti-C1q antibody M1 was shown
to bind both human and mouse C1q proteins with very high affinities
(K.sub.D<10.sup.-10M). By comparison, the reference antibody
4A4B11 was found to bind to human C1q, whereas binding to mouse C1q
was undetectable. Whereas the affinities of M1 and 4A4B11 for human
C1q were on the same order of magnitude (i.e., in the double-digit
picomolar range; K.sub.D.about.10-30 pM), the affinity of M1 for
mouse C1q was determined to be about four orders of magnitude
higher (K.sub.D.about.30 pM) than that the affinity of 4A4B 11 for
mouse C1q (K.sub.D.about.40 nM).
Anti-C1q Antibodies M1 and 4A4B11 Do Not Compete for
C1q-Binding
[0271] Blocking experiments were performed to determine whether the
anti-C1q antibodies M1 and 4A4B11 bind to the same or overlapping
epitopes of human C1q or whether M1 and 4A4B11 bind to separate C1q
epitopes.
[0272] To this end, M1 was coated on a biosensor chip (BIACORE.TM.)
and subsequently contacted with a combination of human C1q and M1,
a combination of human C1q and 4A4B11, or human C1q alone.
C1q-binding to M1 was followed for 10 min and dissociation of
M1-C1q complexes was subsequently followed for 20 min. Relative
binding signals were recorded at the end of the association and
dissociation periods. Table C shows the results of these
experiments.
TABLE-US-00005 TABLE C Analysis of Simultaneous Interactions of M1
and 4A4B11 with human C1q Associa- Dissocia- Sensor Antigen
Solution tion Response tionResponse Ab ID: ID: Ab ID: (nm) @600 s
(nm) @1200 s M1 hC1q M1 -0.0119 -0.00945 M1 hC1q 4A4B11 0.8213
0.82139 M1 hC1q None (Ag 0.4715 0.45137 Only)
[0273] It was found that C1q alone bound effectively to immobilized
M1 antibody on the biosensor chip. Preincubation of C1q with
soluble M1 antibody prevented all binding of the resulting M1-C1q
complex to immobilized Ml. By contrast, preincubation of C1q with
4A4B11 did not prevent the interaction of the resulting 4A4B11-C1q
complex with immobilized M1. The larger relative binding signals
observed in the binding experiment involving the 4A4B11-C1q complex
relative to the binding experiment involving C1q alone is due to
the fact that the relative binding signals correlate with the
molecular weight of the soluble binding partners and that the
4A4B11-C1q complex has a higher molecular weight than C1q
alone.
[0274] These results demonstrate that 4A4B11 does not compete with
M1 for C1q binding. Therefore, 4A4B11 and M1 may recognize separate
epitopes on C1q.
Example 3
Anti-C1q Antibodies Inhibit Complement-Mediated Hemolysis
[0275] Anti-C1q antibodies were tested in human and rodent
hemolytic assays (CH50) for their ability to neutralize C1q and
block its activation of the downstream complement cascade. CH50
assays were conducted essentially as described in Current Protocols
in Immunology (1994) Supplement 9 Unit 13.1. In brief, 5
microliters (.mu.l) of human serum (Cedarlane, Burlington, N.C.),
0.625 .mu.l of Wistar rat serum, or 2.5 .mu.l of C57B1/6 mouse
serum was diluted to 50 .mu.l of GVB buffer (Cedarlane, Burlington,
N.C.) and added to 50 .mu.l of the monoclonal antibodies (1 .mu.g)
diluted in GVB buffer. The antibody:serum mixture was pre-incubated
for 30 minutes on ice and then added to 100 .mu.l of EA cells
(2.times.10.sup.8/ml) for rat and human assays, and
4.times.10.sup.7/ml for mouse assays. The EA cells were generated
exactly as specified in Current Protocols using Sheeps blood in
Alsever's (Cedarlane Cat #CL2581) and hemolysin (Cedalane Cat
#CL9000). The EA cells, serum and antibody mixture was incubated
for 30 minutes at 37.degree. C. and then placed on ice. Next 1.2 ml
of 0.15 M NaCl was added to the mixture and the OD.sub.412 of the
sample was read in a spectrophotometer to determine the amount of
cell lysis. The percent inhibition of the test antibodies was
determined relative to a control mouse IgG1 antibody (Abcam ab
18447).
[0276] A modified CH50 assay (also referred to as C1F hemolysis
assay) was performed that provided limiting quantities of the C1
complex from human serum to provide greater sensitivity for
assessing C1 activity and potential C1 inhibition. In brief, the
assay was conducted as follows. First, 3.times.10.sup.7 sheep red
blood cells (RBC) were incubated with anti-sheep RBC IgM antibody
to generate activated erythrocytes (EA cells). The EA cells were
then incubated with purified C4b protein to create EAC4b cells.
EAC4b cells were subsequently incubated with diluted
(1:1000-1:10000) normal human serum (NHS) that was pre-incubated
with or without anti-C1q and control mouse IgG antibodies, to
provide a limiting quantity of human C1. Next, the resulting EAC14
cells were incubated with purified human C2 protein to generate
EAC14b2a cells. Finally, guinea pig serum was added in an EDTA
buffer and incubated at 37.degree. C. for 30 minutes. Cell lysis
was measured in a spectrophotometer at a wavelength of 450 nm.
[0277] First, four C1q-binding antibodies (1C7, 2A1, 3A2, and 5A3)
were tested in the human CH50 assay at a single concentration (1
.mu.g) (FIG. 2). All four antibodies were found to inhibit
hemolysis. The anti-C1q antibody 1C7 inhibited hemolysis at greater
than 90%, 2A1 inhibited hemolysis at greater than 40%, 3A2
inhibited hemolysis at greater than 60%, and 5A3 inhibited
hemolysis at greater than 50%.
[0278] Next, anti-C1q antibodies 1C7 and 3A2 were tested in the
human CH50 hemolysis assay in a dose-response format (FIG. 3).
Anti-C1q antibody 4A4B11 was used as a reference. Both 1C7 and 3A2
antibodies inhibited CH50 hemolysis in a dose-dependent manner.
Approximately 100 ng of the 1C7 antibody and approximately 200 ng
of the 3A2 were required to inhibit 50% of the hemolysis observed
(FIG. 3).
[0279] Anti-C1q antibody M1 was tested for its C1q neutralizing
activity in human, mouse, and rat CH50 assays (FIG. 4A-C). Testing
was conducted in dose-response formats. Anti-C1q antibody 4A4B11
was used as a reference. M1 was demonstrated to neutralize C1q
activity in human, mouse, and rat CH50 hemolysis assays in a
dose-dependent manner (FIG. 4A-4C). By contrast, 4A4B11 was found
to neutralize C1q activity only in the human CH50 assay, whereas
the reference antibody was inactive in the mouse and rat CH50
hemolysis assays (up to 2 .mu.g). In the human and rat CH50
hemolysis assays M1 inhibited greater than 90% and up to 100% of
hemolysis (FIGS. 4A and 4C); in the mouse assay M1 inhibited
greater than 50% of hemolysis (FIG. 4B). In the human CH50 assay,
less than 125 ng of M1 were required to achieve 50% inhibition of
hemolysis. In the mouse CH50 assay, approximately 500 ng of M1 were
required to achieve 50% inhibition of hemolysis. In the rat CH50
assay, less than 16 ng were required to achieve 50% inhibition of
hemolysis.
Example 4
Epitope Mapping for Antibody 4A4B11 and M1
[0280] In order to determine the nature of the epitope (i.e.,
linear or conformational), the inhibition of the interaction
between the C1Q protein and the antibodies 4A4B11 (ANN-001) and M1
(ANN-005) by unstructured peptides generated by proteolysis of the
C1q antigen was evaluated. If the peptides generated by complete
proteolysis of the antigen are able to inhibit the binding of the
antigen on the antibody, the interaction is not based on
conformation, and the epitope is linear. If the peptides generated
by complete proteolysis of the antigen are unable to inhibit the
binding of the antigen on the antibodies 4A4B11 and Ml, the
conformation is necessary for interaction. Based on the data
described in detail below, unstructured peptides generated by
digestion of native C1q did not compete with intact C1q for binding
to the 4A4B11 (ANN-001) and M1 (ANN-005) antibodies (see FIG. 2),
suggesting that the C1q epitope for these antibodies is a complex
conformational epitope.
[0281] In order to determine the key residues of the conformational
C1q epitope that binds of ANN-001 and ANN-005 on C1Q antigen with
high resolution the antibody/antigen complexes were incubated with
deuterated cross-linkers and subjected to multi-enzymatic
proteolytic cleavage. After enrichment of the cross-linked
peptides, the samples were analyzed by high resolution mass
spectrometry (nLC-Orbitrap MS) and the data generated analyzed
using XQuest software. The analysis described below indicates that
antibody 4A4B11 (ANN-001) binds to an epitope that includes amino
acids S202 and K219 of human C1QA and Y225 of human C1QC, and
antibody M1 (ANN-005) binds to an epitope that includes amino acid
K219 of human C1QA and S185 of human C1QC. See the amino acid
sequence alignment of human and mouse C1qA and C1qC as shown
below.
TABLE-US-00006 Amino acid sequence alignment of human and mouse
ClqA MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIR
human METSQGWLVACVLTMTLVWTVAEDVCRAPNGKDGAPGNPGRPGRPGLKGERGEPGAAGIR
mouse ** .:**** ***:::*. *:**:****:**.* .*.*** ********:*****.***
TGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAI human
TGIRGFKGDPGESGPPGKPGNVGLPGPSGPLGDSGPQGLKGVKGNPGNIRDQPRPAFSAI mouse
***.*.*** ** ** *.**.** ******** * *.** ** ****.**********
RRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSR human
RQNPMTLGNVVIFDKVLTNQESPYQNHTGRFICAVPGFYYFNFQVISKWDLCLFIKSSSG mouse
*.** *******.*:****.*****:***:*:***:***.***:*:*::** * ***
GQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFL human
GQPRDSLSFSNTNNKGLFQVLAGGTVLQLRRGDEVWIEKDPAKGRIYQGTEADSIFSGFL mouse
** * **.*.:*.*******::** ****::**:**:**** **:****:****:***** IFPSA
human (SEQ ID NO: 1) IFPSA mouse (SEQ ID NO: 4) ***** Amino acid
sequence alignment of human and mouse ClqC
MDVGPSSLPHLGLKLLLLLLLLP-LRGQANTGCYGIPGMPGLPGAPGKDGYDGLPGPKGE human
MVVGPSCQPPCGLCLLLLFLLALPLRSQASAGCYGIPGMPGMPGAPGKDGHDGLQGPKGE mouse
* ****. ** ** ****:** **.**.:**********:********:*** *****
PGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQ human
PGIPAVPGTRGPKGQKGEPGMPGHRGKNGPRGTSGLPGDPGPRGPPGEPGVEGRYKQKHQ mouse
*****:** ***********:*** ***** * *:** *** * ***** ******* *
SVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLC human
SVFTVTRQTTQYPEANALVRFNSVVTNPQGHYNPSTGKFTCEVPGLYYFVYYTSHTANLC mouse
********* * * .*:*:***:*:*****.*:.*******:*********::*******
VLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSG human
VHLNLNLARVASFCDHMFNSKQVSSGGVLLRLQRGDEVWLSVNDYNGMVGIEGSNSVFSG mouse
* * . .:*.:**.* :::**.********* *;****;**** .****:**:***** FLLFPD
human (SEQ ID NO: 3) FLLFPD mouse (SEQ ID NO: 5) ******
1. Identification of the C1q/Antibody Complexes by Mass
Spectrometry
[0282] The C1q/antibody complexes were generated by mixing
equimolar solutions of C1q antigen and antibody (4 .mu.M in 5 .mu.l
each). One .mu.l of the mixture obtained was mixed with 1 .mu.l of
a matrix composed of a re-crystallized sinapinic acid matrix (10
mg/ml) in acetonitrile/water (1:1, v/v), TFA 0.1% (K200 MALDI Kit).
After mixing, 1 .mu.l of each sample was spotted on the MALDI plate
(SCOUT 384). After crystallization at room temperature, the plate
was introduced in the MALDI mass spectrometer and analyzed
immediately. The analysis has been repeated in triplicate. FIG. 5
shows the presence of the antigen, antibody and antigen/antibody
complexes for C1q/4A4B 11 (FIG. 5A) and C1q/M1 (FIG. 5B). Peaks are
present at the predicted molecular weights of monomeric antibody
(.about.150 kDa) and C1q monomer (.about.460 kDa) and there is a
1:1 complex of antibody:antigen present at .about.615 kDa.
2. Unstructured C1Q Peptides Generated by Proteolysis do not
Compete for Binding of C1Q to Antibody
[0283] To determine if the C1q/antibody complexes could be competed
with peptides the C1q antigen was digested with immobilized pepsin.
25 .mu.l of the antigen with a concentration of 10 .mu.M were mixed
with immobilized pepsin 5 .mu.M and incubate at room temperature
for 30 minutes. After the incubation time the sample was
centrifuged and the supernatant was pipetted. The completion of the
proteolysis was controlled by High-Mass MALDI mass spectrometry in
linear mode. The pepsin proteolysis was optimized in order to
obtain a large amount of peptide in the 1000-3500 Da range. Next, 5
.mu.l of the antigen peptides generated by proteolysis were mixed
with 5 .mu.l of ANN-001 or ANN-005 (8 .mu.M) and incubated at
37.degree. C. for 6 hours. After incubation of ANN-001 or ANN-005
with the C1Q antigen peptides, 5 .mu.l of the mixture was mixed
with 5 .mu.l of the C1Q antigen (4 .mu.M) so the final mix
contained 2 .mu.M/2 .mu.M/2.5 .mu.M of C1Q antigen/4A4B 11 or
M1/C1Q antigen Peptides.
[0284] The MALDI ToF MS analysis was performed using CovalX's HM3
interaction module with a standard nitrogen laser and focusing on
different mass ranges from 0 to 2000 kDa. For the analysis, the
following parameters have been applied for Mass Spectrometer:
Linear and Positive mode; Ion Source 1: 20 kV; Ion Source 2: 17 kV;
Pulse Ion Extraction: 400 ns; for HM3: Gain Voltage: 3.14 kV; Gain
Voltage: 3.14 kV; Acceleration Voltage: 20 kV.
[0285] To calibrate the instrument, an external calibration with
clusters of Insulin, B SA and IgG has been applied. For each
sample, 3 spots were analyzed (300 laser shots per spots). The
presented spectrum corresponds to the sum of 300 laser shots. The
MS data were analyzed using the Complex Tracker analysis software
version 2.0 (CovalX Inc).
[0286] The results are shown in FIG. 6, and demonstrate that C1q
peptides do not compete with intact C1q for binding to monoclonal
antibody ANN-005 (M1).
3. Identification of the Conformational Epitopes for C1q Binding to
ANN-001 and ANN-005
[0287] Using chemical cross-linking, High-Mass MALDI mass
spectrometry and nLC-Orbitrap mass spectrometry the interaction
interface between the antigen C1Q and two monoclonal antibodies
ANN-001 and ANN-005 was characterized. 50 of the sample C1Q antigen
(concentration 4.mu.M) was mixed with 50 of the sample ANN-001
(Concentration 4 .mu.M) or ANN-005 (Concentration 4 .mu.M) in order
to obtain an antibody/antigen mix with final concentration 2 .mu.M.
The mixture was incubated at 37.degree. C. for 180 minutes. In a
first step, 1 mg of DiSuccinimidylSuberate H12 (DSS-H12)
cross-linker was mixed with 1 mg of DiSuccinimidylSuberate D12
(DSS-D12) cross-linker. The 2 mg prepared were mixed with 1 ml of
DMF in order to obtain a 2 mg/ml solution of DSS H12/D12. 10 .mu.l
of the antibody/antigen mix prepared previously were mixed with 1
.mu.l of the solution of cross-linker d0/d12 prepared (2 mg/ml).
The solution was incubated 180 minutes at room temperature in order
to achieve the cross-linking reaction. In order to facilitate the
proteolysis, it was necessary to reduce the disulfide bound present
in this protein. The cross-linked sample was mixed with 20 .mu.l of
ammonium bicarbonate (25 mM, pH 8.3). After mixing 2.5 .mu.l of DTT
(500 mM) is added to the solution. The mixture was then incubated 1
hour at 55.degree. C. After incubation, 2.5 .mu.l of iodioacetamide
(1 M) was added before 1 hour of incubation at room temperature in
a dark room. After incubation, the solution was diluted 1/5 by
adding 120 .mu.l of the buffer used for the proteolysis. 145 .mu.l
of the reduced/alkyled cross-linked sample was mixed with 2 .mu.l
of trypsin (Sigma, T6567). The proteolytic mixture was incubated
overnight at 37.degree. C. For a-chymotrypsin proteolysis, the
buffer of proteolysis was Tris-HCL 100 mM, CaCl.sub.2 10 mM, pH7.8.
The 145 .mu.l of the reduced/alkyled cross-linked complex was mixed
with 2 .mu.l of .alpha.-chymotrypsin 200 .mu.M and incubated
overnight at 30.degree. C. For this analysis, an nLC in combination
with Orbitrap mass spectrometry were used. The cross-linker
peptides were analyzed using Xquest version 2.0 and stavrox
software. The peptides identified and cross-linked amino acids are
indicated in Table D below.
TABLE-US-00007 TABLE D Clq cross-linked peptides and contact
residues necessary for ANN-001 and ANN-005 binding Protease Clq
Contact Digest X-linked Peptide Subunit Residue Antibody Trypsin
GLFQVVSGGMVLQLQQGDQVWVEK ClqA K219 ANN-001 (SEQ ID NO: 15, residues
196-219 of SEQ ID NO: 1) Trypsin FQVVSGGMVLQL ClqA S202 ANN-001
(SEQ ID NO: 16, residues 198-209 of SEQ ID NO: 1) Chymotrypsin
YDMVGIQGSDSVFSGF ClqC Y225 ANN-001 (SEQ ID NO: 11, residues 225-240
of SEQ ID NO: 3) Trypsin GLFQVVSGGMVLQLQQGDQVWVEK ClqA K219 ANN-005
(SEQ ID NO: 15, residues 196-219 of SEQ ID NO: 1) Chymotrypsin
RSGVKVVTF ClqC S185 ANN-005 (SEQ ID NO: 14, residues 184-192 of SEQ
ID NO: 3)
[0288] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Example 5
Materials and Methods
Tissue Preparation
[0289] Mice were perfused with 4% PFA and their brains extracted
and processed for immunohistochemistry (IHC). Briefly, the tissue
was postfixed for 2 hr in PFA before washing in PBS and incubating
for 48 hr in 30% sucrose in PBS. The brains were subsequently
frozen in OCT and 14 mm cryosections were cut.
[0290] Immunohistochemistry
Brain sections were dried at 37.degree. C. for 30 minutes, washed
in PBS, and then incubated with blocking solution for 1 hour (hr)
at room temperature (150 mM NaCl 50 mM Tris base, 5% BSA, 100 mM
L-lysine, 0.2% triton, 0.04% sodium azide). Sections were
subsequently incubated with relevant primary antibody diluted in
blocking solution overnight at 4.degree. C.
[0291] For C1q staining, undiluted rabbit anti-C1q antibody
(Stephen AH et al., J Neurosci. 2013 Aug. 14; 33(33):13460-74) was
used. For C3 staining, goat anti-mouse C3 (Cappel 55730) was used
at a dilution of 1/200. Synapses were labeled with rat anti-mouse
PSD-95 (Millipore MAB1596) used at a dilution of 1/100 and with
rabbit anti-mouse synapsin (Synaptic systems 106103) used at a
dilution of 1/500.
[0292] Sections were then washed in PBS and incubated with
fluorescently tagged secondary antibodies diluted in blocking
buffer for 2 hr at room temperature. For C3 staining, donkey
anti-goat Alexa Fluor 488 (Life Technologies A11055) and donkey
anti-goat Alexa Fluor 594 (Life Technologies A11058) secondary
antibodies were used; for PSD-95 staining, donkey anti-mouse Alexa
Fluor 488 (Life Technologies A21202) secondary antibody was used;
for synapsin staining, donkey anti-rabbit Alexa Fluor 594 (Life
Technologies A21207) secondary antibody was used; and for C1q
staining, donkey anti-rabbit Alexa Fluor 488 (Life Technologies
A21206) secondary antibody was used. After washing in PBS, sections
were mounted in vectashield (Vector Labs H-1400).
Imaging
[0293] Sections were imaged on a Zeiss LSM 700 confocal microscope
using a 63.times. oil objective and a 1AU pinhole. Single plane
images from each channel were captured in relevant brain regions.
Image J software was then used to threshold the images and quantify
the number of co-localized puncta.
A.beta. Oligomer Production
[0294] As a source of Abeta (A.beta.) oligomers, dimeric forms of
disulfide-crosslinked (A.beta.1-40 S26C)2 (21st Century Bio) was
separated using a Superdex 75 SEC column (GE Healthcare) (Shankar
et al., Nature Medicine 2008).
Acute in vivo Model of Alzheimer's Disease
[0295] An acute in vivo model of A.beta. synaptotoxicity in
Alzheimer's disease was generated by injecting 5 ng A.beta.
oligomers or saline (1 .mu.l vol.) at 0.5 .mu.l/min via
intracerebroventricular (ICV) injection using a Hamilton syringe
into anesthetized wild-type (WT) mice following the coordinates for
left ventral placement (bregma-0.4 mm, 1.0 mm lateral to midline,
and 2.5 mm below dura at 0.degree. angle).
[0296] For C1q neutralizing antibody trial, 5 .mu.g C1q
neutralizing antibody or control IgG (1 .mu.l vol.) was injected to
the left ventricle of C57BL/6J mice, followed by 5 ng A .beta.
oligomers or saline (1 .mu.l vol.). Adult (2-3 mo) wild-type (WT)
mice (C57BL/6J; JAX 000664) or C1qA knockout (KO) mice (Botto et
al., 1998) were then given an intraperitoneal (IP) injection of C1q
neutralizing antibody or control IgG (20 mg/kg), and were allowed
to wake and resume normal activities.
[0297] For C3 levels, mice were sacrificed 18 hours post injection.
For synapse quantification, mice received additional
intraperitoneal (IP) injections of either C1q neutralizing antibody
or control IgG (20 mg/kg) at t=24 hr and 48 hr, then sacrificed at
t=72 hr.
In vivo Model of Huntington's Disease
[0298] zQ175 is a knock-in model of Huntington's disease in which
188 CAG repeats have been inserted into the mouse Huntingtin gene
alongside the human polyproline region. It was generated from a
spontaneous expansion of the CAG repeat region in a litter of CAG
140 mice. These mice phenocopy many aspects of the human disease
with robust motor and cognitive deficits starting around 30 weeks
of age, as shown by a reduced performance on the rotarod and in a
procedural two choice-swim test. They also show specific striatal
atrophy, an early event in human Huntington's disease pathology
with a 21% loss of volume at 30 weeks. Striatal medium spiny
neurons (MSNs) are particularly vulnerable to mHTT insult and
dramatic loss of this neuronal population in Huntington's disease
patients is a hallmark of the disease. In line with this, MSNs in
zQ175 mice show hyper-excitability from as early as 12 weeks,
followed by progressive loss of corticostriatal transmission
(Heikkinen et al., 2012; Menalled et al., 2012).
Production and Characterization of Anti-C1q Antibodies
[0299] The C1q blocking antibody M1 was generated and characterized
as described in Examples 1-4 above.
Example 6
A.beta. Oligomer Injection Induces C1q Deposition at Synapses in
Wild-Type Mice
[0300] In order to determine whether A.beta. oligomer-induced
synaptotoxicity is associated with C1q deposition at synapses,
wild-type mice were injected by intracerebroventricular (ICV)
injection with A.beta. monomers and A.beta. oligomers, and 18 hours
post-injection brains were harvested and analyzed for C1q
deposition in the hippocampus using an anti-C1q specific antibody
(FIG. 8A). Localization of C1q to synapses was evaluated by
staining sections simultaneously for C1q and the synaptic marker
PSD-95 (FIG. 8B). FIG. 8A shows that A.beta. oligomers induce much
higher levels of C1q deposition in the brain, as compared to the
A.beta. monomers. FIG. 8B shows that C1q is co-localized at
synapses with PSD-95. Moreover, consistent with the results in FIG.
8A, A.beta. oligomers induced a greater amount of C1q
co-localization with PSD-95 at synapses (FIG. 8B).
Example 7
C1q Deficiency Suppresses A.beta. Oligomer-Induced C3 Deposition
and Synapse Loss in the Hippocampus of Wild-Type Mice
[0301] In order to determine whether A.beta. oligomer-induced
synaptotoxicity is dependent on C1q function, synapse number was
evaluated in wild-type (WT) mice and C1q knockout (KO) mice
injected with A.beta. oligomers. Intracerebroventricular (ICV)
injection of either soluble A.beta. oligomers or A.beta. monomers
was performed in healthy adult (2-3 mo) WT and C1q KO mice. After
72 hours, brains were harvested for immunohistochemistry (IHC)
using synapsin as pre-synaptic marker and PSD95 as a post-synaptic
marker. Synapse number was quantified by measuring the number of
co-localized synapsin and PSD-95 puncta. The results in FIG. 9
shows that a significant loss of structural synapses in the CA1
region of the hippocampus occurs A.beta. oligomer-treated WT mice
(FIGS. 9A and 9B), but does not occur in C1q KO mice (FIG. 9C).
These results demonstrate that C1q deficiency prevents the loss of
synaptic puncta, which is typically seen in wild-type mice after
A.beta. oligomer treatment.
[0302] In order to determine whether the protective effect of C1q
deficiency is associated with the prevention of complement C3
deposition, the brain sections were also stained for C3. FIG. 10
shows that the deposition of C3 induction by A.beta. oligomer
treatment in wild-type (WT) mice is absent in C1q KO mice treated
A.beta. oligomers. These results indicate that A.beta. oligomers
are unable to induce C3 deposition in the absence of C1q, and
suggests that C1q blocking antibodies, such as the M1 antibody, may
prevent A.beta. oligomer-induced C3 deposition.
Example 8
C1q Antibody M1 Prevents Complement Deposition and Synapse Loss in
an In Vivo Mouse Model of Alzheimer's Disease
[0303] The ability of the anti-C1q antibody M1 to suppress synapse
loss in an in vivo mouse model of Alzheimer's disease (AD) was
tested using the acute A.beta. oligomer model of AD in which
A.beta. oligomers are injected directly into the ventricles of the
brain. Two sets of mice were given an initial IP injection and ICV
injection of a blocking anti-C1q antibody and a mouse IgG1 control
alongside an ICV injection of A.beta. oligomers. One set of mice
was sacrificed after 18 hr to determine whether the antibody was
capable of reducing A.beta. oligomer-induced C3 levels (a
downstream complement component). The other set of mice received a
further IP injection of the blocking anti-C1q antibody 48 hr after
the initial surgery, and sacrificed at 72 hr to assess levels of
synaptic markers.
[0304] FIG. 11 shows that in wild-type (WT) mice co-injected with
the C1q blocking antibody and A.beta. oligomers there is a
significant reduction in C3 staining (FIG. 11A) in the brain and
more synaptic puncta (FIG. 11B), as compared to mice co-injected
with the IgG1 control and A.beta. oligomers.
Example 9
C1q Antibody M1 Prevents Complement Deposition in an In Vivo Mouse
Model of Huntington's Disease
[0305] The efficacy of the C1q antibody M1 was also tested in an in
vivo mouse model of Huntington's disease (zQ125 transgenic mice).
zQ125 transgenic mice received two IP injections over a 48 hr
period of 20 mg/kg of the M1 antibody or of a mouse IgG control.
Consistent with the results of Example 8 above using a mouse model
of Alzheimer's disease, there was less C3 deposition in the
disease-affected regions of the zQ175 mice injected with the C1q
blocking antibody, as compared to mice injected with the IgG
control or control mice that did not receive an injection (FIG.
12).
DEPOSIT OF MATERIAL
[0306] The following materials have been deposited according to the
Budapest Treaty in the American Type Culture Collection, ATCC
Patent Depository, 10801 University Blvd., Manassas, Va.
20110-2209, USA (ATCC):
TABLE-US-00008 Deposit ATCC Sample ID Isotype Date Accession No.
Mouse hybridoma C1qM1 IgG1, Jun. 6, PTA-120399 7788-1(M) 051613
producing kappa 2013 anti-C1q antibody M1
[0307] The hybridoma cell line producing the M1 antibody (mouse
hybridoma C1qM1 7788-1(M) 051613) has been deposited with ATCC
under conditions that assure that access to the culture will be
available during pendency of the patent application and for a
period of 30 years, or 5 years after the most recent request, or
for the effective life of the patent, whichever is longer. A
deposit will be replaced if the deposit becomes nonviable during
that period. The deposit is available as required by foreign patent
laws in countries wherein counterparts of the subject application,
or its progeny are filed. However, it should be understood that the
availability of the deposit does not constitute a license to
practice the subject invention in derogation of patent rights
granted by governmental action.
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Sequence CWU 1
1
161245PRTHomo sapiens 1Met Glu Gly Pro Arg Gly Trp Leu Val Leu Cys
Val Leu Ala Ile Ser1 5 10 15 Leu Ala Ser Met Val Thr Glu Asp Leu
Cys Arg Ala Pro Asp Gly Lys 20 25 30 Lys Gly Glu Ala Gly Arg Pro
Gly Arg Arg Gly Arg Pro Gly Leu Lys 35 40 45 Gly Glu Gln Gly Glu
Pro Gly Ala Pro Gly Ile Arg Thr Gly Ile Gln 50 55 60 Gly Leu Lys
Gly Asp Gln Gly Glu Pro Gly Pro Ser Gly Asn Pro Gly65 70 75 80 Lys
Val Gly Tyr Pro Gly Pro Ser Gly Pro Leu Gly Ala Arg Gly Ile 85 90
95 Pro Gly Ile Lys Gly Thr Lys Gly Ser Pro Gly Asn Ile Lys Asp Gln
100 105 110 Pro Arg Pro Ala Phe Ser Ala Ile Arg Arg Asn Pro Pro Met
Gly Gly 115 120 125 Asn Val Val Ile Phe Asp Thr Val Ile Thr Asn Gln
Glu Glu Pro Tyr 130 135 140 Gln Asn His Ser Gly Arg Phe Val Cys Thr
Val Pro Gly Tyr Tyr Tyr145 150 155 160 Phe Thr Phe Gln Val Leu Ser
Gln Trp Glu Ile Cys Leu Ser Ile Val 165 170 175 Ser Ser Ser Arg Gly
Gln Val Arg Arg Ser Leu Gly Phe Cys Asp Thr 180 185 190 Thr Asn Lys
Gly Leu Phe Gln Val Val Ser Gly Gly Met Val Leu Gln 195 200 205 Leu
Gln Gln Gly Asp Gln Val Trp Val Glu Lys Asp Pro Lys Lys Gly 210 215
220 His Ile Tyr Gln Gly Ser Glu Ala Asp Ser Val Phe Ser Gly Phe
Leu225 230 235 240 Ile Phe Pro Ser Ala 245 2253PRTHomo sapiens 2Met
Met Met Lys Ile Pro Trp Gly Ser Ile Pro Val Leu Met Leu Leu1 5 10
15 Leu Leu Leu Gly Leu Ile Asp Ile Ser Gln Ala Gln Leu Ser Cys Thr
20 25 30 Gly Pro Pro Ala Ile Pro Gly Ile Pro Gly Ile Pro Gly Thr
Pro Gly 35 40 45 Pro Asp Gly Gln Pro Gly Thr Pro Gly Ile Lys Gly
Glu Lys Gly Leu 50 55 60 Pro Gly Leu Ala Gly Asp His Gly Glu Phe
Gly Glu Lys Gly Asp Pro65 70 75 80 Gly Ile Pro Gly Asn Pro Gly Lys
Val Gly Pro Lys Gly Pro Met Gly 85 90 95 Pro Lys Gly Gly Pro Gly
Ala Pro Gly Ala Pro Gly Pro Lys Gly Glu 100 105 110 Ser Gly Asp Tyr
Lys Ala Thr Gln Lys Ile Ala Phe Ser Ala Thr Arg 115 120 125 Thr Ile
Asn Val Pro Leu Arg Arg Asp Gln Thr Ile Arg Phe Asp His 130 135 140
Val Ile Thr Asn Met Asn Asn Asn Tyr Glu Pro Arg Ser Gly Lys Phe145
150 155 160 Thr Cys Lys Val Pro Gly Leu Tyr Tyr Phe Thr Tyr His Ala
Ser Ser 165 170 175 Arg Gly Asn Leu Cys Val Asn Leu Met Arg Gly Arg
Glu Arg Ala Gln 180 185 190 Lys Val Val Thr Phe Cys Asp Tyr Ala Tyr
Asn Thr Phe Gln Val Thr 195 200 205 Thr Gly Gly Met Val Leu Lys Leu
Glu Gln Gly Glu Asn Val Phe Leu 210 215 220 Gln Ala Thr Asp Lys Asn
Ser Leu Leu Gly Met Glu Gly Ala Asn Ser225 230 235 240 Ile Phe Ser
Gly Phe Leu Leu Phe Pro Asp Met Glu Ala 245 250 3245PRTHomo sapiens
3Met Asp Val Gly Pro Ser Ser Leu Pro His Leu Gly Leu Lys Leu Leu1 5
10 15 Leu Leu Leu Leu Leu Leu Pro Leu Arg Gly Gln Ala Asn Thr Gly
Cys 20 25 30 Tyr Gly Ile Pro Gly Met Pro Gly Leu Pro Gly Ala Pro
Gly Lys Asp 35 40 45 Gly Tyr Asp Gly Leu Pro Gly Pro Lys Gly Glu
Pro Gly Ile Pro Ala 50 55 60 Ile Pro Gly Ile Arg Gly Pro Lys Gly
Gln Lys Gly Glu Pro Gly Leu65 70 75 80 Pro Gly His Pro Gly Lys Asn
Gly Pro Met Gly Pro Pro Gly Met Pro 85 90 95 Gly Val Pro Gly Pro
Met Gly Ile Pro Gly Glu Pro Gly Glu Glu Gly 100 105 110 Arg Tyr Lys
Gln Lys Phe Gln Ser Val Phe Thr Val Thr Arg Gln Thr 115 120 125 His
Gln Pro Pro Ala Pro Asn Ser Leu Ile Arg Phe Asn Ala Val Leu 130 135
140 Thr Asn Pro Gln Gly Asp Tyr Asp Thr Ser Thr Gly Lys Phe Thr
Cys145 150 155 160 Lys Val Pro Gly Leu Tyr Tyr Phe Val Tyr His Ala
Ser His Thr Ala 165 170 175 Asn Leu Cys Val Leu Leu Tyr Arg Ser Gly
Val Lys Val Val Thr Phe 180 185 190 Cys Gly His Thr Ser Lys Thr Asn
Gln Val Asn Ser Gly Gly Val Leu 195 200 205 Leu Arg Leu Gln Val Gly
Glu Glu Val Trp Leu Ala Val Asn Asp Tyr 210 215 220 Tyr Asp Met Val
Gly Ile Gln Gly Ser Asp Ser Val Phe Ser Gly Phe225 230 235 240 Leu
Leu Phe Pro Asp 245 4245PRTMus musculus 4Met Glu Thr Ser Gln Gly
Trp Leu Val Ala Cys Val Leu Thr Met Thr1 5 10 15 Leu Val Trp Thr
Val Ala Glu Asp Val Cys Arg Ala Pro Asn Gly Lys 20 25 30 Asp Gly
Ala Pro Gly Asn Pro Gly Arg Pro Gly Arg Pro Gly Leu Lys 35 40 45
Gly Glu Arg Gly Glu Pro Gly Ala Ala Gly Ile Arg Thr Gly Ile Arg 50
55 60 Gly Phe Lys Gly Asp Pro Gly Glu Ser Gly Pro Pro Gly Lys Pro
Gly65 70 75 80 Asn Val Gly Leu Pro Gly Pro Ser Gly Pro Leu Gly Asp
Ser Gly Pro 85 90 95 Gln Gly Leu Lys Gly Val Lys Gly Asn Pro Gly
Asn Ile Arg Asp Gln 100 105 110 Pro Arg Pro Ala Phe Ser Ala Ile Arg
Gln Asn Pro Met Thr Leu Gly 115 120 125 Asn Val Val Ile Phe Asp Lys
Val Leu Thr Asn Gln Glu Ser Pro Tyr 130 135 140 Gln Asn His Thr Gly
Arg Phe Ile Cys Ala Val Pro Gly Phe Tyr Tyr145 150 155 160 Phe Asn
Phe Gln Val Ile Ser Lys Trp Asp Leu Cys Leu Phe Ile Lys 165 170 175
Ser Ser Ser Gly Gly Gln Pro Arg Asp Ser Leu Ser Phe Ser Asn Thr 180
185 190 Asn Asn Lys Gly Leu Phe Gln Val Leu Ala Gly Gly Thr Val Leu
Gln 195 200 205 Leu Arg Arg Gly Asp Glu Val Trp Ile Glu Lys Asp Pro
Ala Lys Gly 210 215 220 Arg Ile Tyr Gln Gly Thr Glu Ala Asp Ser Ile
Phe Ser Gly Phe Leu225 230 235 240 Ile Phe Pro Ser Ala 245
5246PRTMus musculus 5Met Val Val Gly Pro Ser Cys Gln Pro Pro Cys
Gly Leu Cys Leu Leu1 5 10 15 Leu Leu Phe Leu Leu Ala Leu Pro Leu
Arg Ser Gln Ala Ser Ala Gly 20 25 30 Cys Tyr Gly Ile Pro Gly Met
Pro Gly Met Pro Gly Ala Pro Gly Lys 35 40 45 Asp Gly His Asp Gly
Leu Gln Gly Pro Lys Gly Glu Pro Gly Ile Pro 50 55 60 Ala Val Pro
Gly Thr Arg Gly Pro Lys Gly Gln Lys Gly Glu Pro Gly65 70 75 80 Met
Pro Gly His Arg Gly Lys Asn Gly Pro Arg Gly Thr Ser Gly Leu 85 90
95 Pro Gly Asp Pro Gly Pro Arg Gly Pro Pro Gly Glu Pro Gly Val Glu
100 105 110 Gly Arg Tyr Lys Gln Lys His Gln Ser Val Phe Thr Val Thr
Arg Gln 115 120 125 Thr Thr Gln Tyr Pro Glu Ala Asn Ala Leu Val Arg
Phe Asn Ser Val 130 135 140 Val Thr Asn Pro Gln Gly His Tyr Asn Pro
Ser Thr Gly Lys Phe Thr145 150 155 160 Cys Glu Val Pro Gly Leu Tyr
Tyr Phe Val Tyr Tyr Thr Ser His Thr 165 170 175 Ala Asn Leu Cys Val
His Leu Asn Leu Asn Leu Ala Arg Val Ala Ser 180 185 190 Phe Cys Asp
His Met Phe Asn Ser Lys Gln Val Ser Ser Gly Gly Val 195 200 205 Leu
Leu Arg Leu Gln Arg Gly Asp Glu Val Trp Leu Ser Val Asn Asp 210 215
220 Tyr Asn Gly Met Val Gly Ile Glu Gly Ser Asn Ser Val Phe Ser
Gly225 230 235 240 Phe Leu Leu Phe Pro Asp 245 631PRTHomo sapiens
6Gly Leu Phe Gln Val Val Ser Gly Gly Met Val Leu Gln Leu Gln Gln1 5
10 15 Gly Asp Gln Val Trp Val Glu Lys Asp Pro Lys Lys Gly His Ile
20 25 30 726PRTHomo sapiens 7Gly Leu Phe Gln Val Val Ser Gly Gly
Met Val Leu Gln Leu Gln Gln1 5 10 15 Gly Asp Gln Val Trp Val Glu
Lys Asp Pro 20 25 820PRTHomo sapiens 8Ser Gly Gly Met Val Leu Gln
Leu Gln Gln Gly Asp Gln Val Trp Val1 5 10 15 Glu Lys Asp Pro 20
918PRTHomo sapiens 9Ser Gly Gly Met Val Leu Gln Leu Gln Gln Gly Asp
Gln Val Trp Val1 5 10 15 Glu Lys1023PRTHomo sapiens 10Trp Leu Ala
Val Asn Asp Tyr Tyr Asp Met Val Gly Ile Gln Gly Ser1 5 10 15 Asp
Ser Val Phe Ser Gly Phe 20 1116PRTHomo sapiens 11Tyr Asp Met Val
Gly Ile Gln Gly Ser Asp Ser Val Phe Ser Gly Phe1 5 10 15 128PRTHomo
sapiens 12Tyr Asp Met Val Gly Ile Gln Gly1 5 1323PRTHomo sapiens
13His Thr Ala Asn Leu Cys Val Leu Leu Tyr Arg Ser Gly Val Lys Val1
5 10 15 Val Thr Phe Cys Gly His Thr 20 149PRTHomo sapiens 14Arg Ser
Gly Val Lys Val Val Thr Phe1 5 1524PRTHomo sapiens 15Gly Leu Phe
Gln Val Val Ser Gly Gly Met Val Leu Gln Leu Gln Gln1 5 10 15 Gly
Asp Gln Val Trp Val Glu Lys 20 1612PRTHomo sapiens 16Phe Gln Val
Val Ser Gly Gly Met Val Leu Gln Leu1 5 10
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