U.S. patent application number 15/511597 was filed with the patent office on 2019-08-08 for light-emitting versions of the monoclonal antibody to c3d (mab 3d29) for imaging.
The applicant listed for this patent is The Johns Hopkins University, The Regents of the University of Colorado, a body corporate. Invention is credited to Catherine A. Foss, V. Michael Holers, Martin G. Pomper, Joshua M. Thurman.
Application Number | 20190240355 15/511597 |
Document ID | / |
Family ID | 55534005 |
Filed Date | 2019-08-08 |
![](/patent/app/20190240355/US20190240355A9-20190808-D00001.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00002.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00003.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00004.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00005.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00006.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00007.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00008.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00009.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00010.png)
![](/patent/app/20190240355/US20190240355A9-20190808-D00011.png)
View All Diagrams
United States Patent
Application |
20190240355 |
Kind Code |
A9 |
Pomper; Martin G. ; et
al. |
August 8, 2019 |
Light-Emitting Versions of the Monoclonal Antibody to C3D (MAB
3D29) for Imaging
Abstract
The presently disclosed subject matter provides compositions and
kits comprising light-emitting versions of the monoclonal antibody
to C3d (mAB 3d29) for imaging and methods of use thereof for
detecting infectious and inflammatory cells in vivo. The presently
disclosed subject matter also provides methods for detecting and/or
monitoring a Mycobacterium tuberculosis (M. tuberculosis) infection
in a subject, as well as methods of treating a M. tuberculosis
infection in a subject.
Inventors: |
Pomper; Martin G.;
(Baltimore, MD) ; Foss; Catherine A.; (Baltimore,
MD) ; Thurman; Joshua M.; (Aurora, CO) ;
Holers; V. Michael; (Aurora, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of Colorado, a body corporate
The Johns Hopkins University |
Denver
Baltimore |
CO
MD |
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170290930 A1 |
October 12, 2017 |
|
|
Family ID: |
55534005 |
Appl. No.: |
15/511597 |
Filed: |
September 15, 2015 |
PCT Filed: |
September 15, 2015 |
PCT NO: |
PCT/US2015/050232 PCKC 00 |
371 Date: |
March 15, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14624347 |
Feb 17, 2015 |
9259488 |
|
|
15511597 |
|
|
|
|
PCT/US2013/055400 |
Aug 16, 2013 |
|
|
|
14624347 |
|
|
|
|
62050568 |
Sep 15, 2014 |
|
|
|
61684691 |
Aug 17, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5695 20130101;
G01N 33/534 20130101; A61P 31/04 20180101; A61K 49/0043 20130101;
C07K 16/18 20130101; C07K 2317/92 20130101; G01N 2469/10 20130101;
A61K 51/1009 20130101; A61K 49/0058 20130101; A61K 51/1018
20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C07K 16/18 20060101 C07K016/18; A61K 51/10 20060101
A61K051/10 |
Claims
1. A method for detecting and/or monitoring a Mycobacterium
tuberculosis (M. tuberculosis) infection in a subject, the method
comprising: (a) administering to a subject an effective amount of a
monoclonal antibody or antibody derivative which binds to C3d in
the subject, wherein the monoclonal antibody or antibody derivative
is conjugated to an imaging tag; and (b) detecting a signal
generated by the imaging tag to detect and/or monitor the location
of the M. tuberculosis infection in the subject.
2. The method of claim 1 wherein the antibody or antibody
derivative comprises 3d29 or a derivative thereof.
3. The method of claim 1, wherein the antibody or antibody
derivative binds to infected tissue in the subject.
4. The method of claim 3 wherein the infected tissue comprises
inflamed tissue.
5. The method of claim 4 wherein the infected tissue is selected
from the group consisting of lung, spleen, and any other
extrapulmonary infected tissue.
6. The method of claim 5 wherein the antibody or antibody
derivative co-localizes with alveolar and peripheral phagocytes in
M. tuberculosis infected lung sections in the subject and/or
co-localizes with aggregates of macrophages in the lungs of
infected subjects.
7. The method of claim 1 wherein the imaging tag is a fluorescent
tag and/or a radiolabel.
8. The method of claim 1 wherein the imaging tag comprises any
radioiodine nuclide.
9. The method of claim 1 wherein the imaging tag comprises
.sup.125I, .sup.123I, .sup.124I or .sup.131I.
10. The method of claim 1 wherein the imaging tag comprises
LISSAMINE, IRDye680RD or IRDye800CW.
11. The method of claim 1 wherein the step of detecting the signal
comprises performing an imaging method selected from the group
consisting of computed tomography (CT), fluorescence imaging, and
single-photon emission computed tomography (SPECT), positron
emission tomography (PET) and combinations thereof.
12. The method of claim 1 wherein the step of administering
comprises injecting the antibody or antibody derivative into the
subject.
13. The method of claim 12, wherein injecting comprises intravenous
injection or intraperitoneal injection.
14. The method of claim 1, further comprising treating the subject
for M. tuberculosis infection.
15. The method of claim 14, wherein treating comprises
administering to the subject an effective amount of an antibiotic
agent, an anti-inflammatory agent, or a combination thereof.
16. The method of claim 1, wherein the subject is human.
17. A method of treating a M. tuberculosis infection in a subject
in need thereof, the method comprising: (a) administering to a
subject an effective amount of a monoclonal antibody or antibody
derivative which binds to C3d, wherein the monoclonal antibody or
antibody derivative is conjugated to an imaging tag, and wherein
the antibody or antibody derivative binds to infected tissue in the
subject; and (b) detecting a signal generated by the imaging tag to
detect and/or monitor the location of the M. tuberculosis infection
in the subject; and (c) administering to the subject an effective
amount of an antibiotic agent, an anti-inflammatory agent, or a
combination thereof.
18. The method of claim 17, wherein the infected tissue comprises
inflamed tissue.
19. The method of claim 18, wherein the antibiotic agent and/or
anti-inflammatory agent are administered to the location of the M.
tuberculosis infection in the subject.
20. The method of claim 17, wherein the subject is human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/050,568, filed Sep. 15, 2014, which is
incorporated herein by reference in its entirety.
BACKGROUND
[0002] Mycobacterium tuberculosis is a pathogen that evades the
host immune system by living within alveolar and peripheral
macrophages. Host evasion is partially accomplished by M. tb
coating itself with complement fragment 3d (C3d), which directs it
for phagocytosis by the host macrophage and inhibits the full
Complement response. Because C3d is generated only during specific
types of inflammatory events and binds its target rapidly, C3d
serves as an excellent biomarker for imaging infections and other
specific inflammatory events.
[0003] The complement system is an important arm of the innate
immune system, providing critical protection against invasive
pathogens (Ricklin et al., 2001)) and contributing to the
pathogenesis of numerous autoimmune and inflammatory diseases
(Walport, 2001). During the course of complement activation, the C3
protein undergoes proteolytic cleavage at several different sites
(FIG. 1). The cleavage fragments are fixed to nearby tissues
through a covalent linkage originating from the thioester site on
C3 with hydroxyl or primary amine groups on acceptor surfaces
(3-5). Thus, the deposition of C3 fragments on tissue surfaces
constitutes a durable signal of tissue inflammation. For this
reason, tissue-bound C3 fragments are commonly used clinically and
experimentally as biomarkers of immune activation. Renal biopsies
from patients with glomerulonephritis, for example, are routinely
immunostained for C3 fragments, and the detection of glomerular C3
fragments serves as a sensitive and robust indicator of disease
activity (Schulze et al., 1993). C3 deposition has also been
recognized to occur in all stages of age-related macular
degeneration (Hageman et al., 2001).
[0004] Because tissue-bound C3 fragments are associated with local
inflammation, they also have been exploited as addressable binding
ligands for targeted therapeutics and diagnostic agents in several
tissues, including the kidneys, the heart, the brain, and the eyes
(Atkinson et al., 2005; Serkova et al., 2010; Sargsyan et al.,
2012; Rohrer et al., 2009; Rohrer et al., 2012). These targeted
agents have employed recombinant forms of complement receptor 2
(CR2), a protein that can discriminate between intact C3 in the
plasma and tissue-bound C3 fragments. The rationale for this
approach is that systemically administered agents can be delivered
to sites of inflammation through their affinity with the iC3b and
C3d fragments. By directing therapeutic agents to molecular
targets, one can achieve a high degree of local activity with the
drug while minimizing its systemic side effects (Webb, 2011).
Previous studies also have used a CR2-targeted contrast agent to
detect tissue-bound C3 fragments and renal disease activity by MRI
(Serkova et al., 2010; Sargsyan et al., 2012). Although specific
for the cleaved forms of C3, CR2-targeted agents probably bind
these fragments with a relatively low affinity (reported values
range from 1 to 10 .mu.M at physiologic ionic strength) (Guthridge
et al., 2001; Isenman et al., 2010; Dempsey et al., 1996).
Higher-affinity targeting vectors for epitopes on the cleaved forms
of C3 could potentially deliver therapeutic and diagnostic agents
to sites of inflammation with even greater efficiency, durability,
and specificity.
[0005] Informative monoclonal antibodies (mAbs) against
tissue-bound C3 fragments have many biomedical applications. They
could be used as in vivo delivery vehicles for new therapeutic and
diagnostic agents. They also could potentially modulate the
biologic functions of the C3 fragments. Such antibodies also could
be useful for identifying specific C3 fragments (e.g., C3b, iC3b,
C3dg, and C3d) and quantifying their relative abundance. There are,
however, several barriers to the generation of such antibodies by
standard methods. Like CR2, the antibodies must recognize epitopes
of cleaved C3 that are not exposed on intact C3 (which circulates
at a concentration of 1 to 2 mg/ml). This is feasible, however,
since internal regions of C3d (and also iC3b and C3dg) are exposed
by conformational changes in C3 during its activation and
subsequent proteolytic processing of its fragments (Janssen et al.,
2006). Another difficulty is that standard methods for generating
and cloning hybridomas may expose the hybridoma cells to C3 and C3
fragments in serum-containing media, or to C3 synthesized by cells,
such as macrophages, that are used in the cultures. C3 and C3
fragments in the media could mask positive hybridoma clones or
affect the growth of such clones through engagement of the B cell
receptors.
SUMMARY
[0006] In an aspect, the presently disclosed subject matter
provides a method for detecting and/or monitoring a Mycobacterium
tuberculosis (M. tuberculosis) infection in a subject, the method
comprising: (a) administering to a subject an effective amount of a
monoclonal antibody or antibody derivative which binds to C3d in
the subject, wherein the monoclonal antibody or antibody derivative
is conjugated to an imaging tag; and (b) detecting a signal
generated by the imaging tag to detect and/or monitor the location
of the M. tuberculosis infection in the subject.
[0007] In another aspect, the presently disclosed subject matter
provides for the use of a monoclonal antibody or antibody
derivative which binds to C3d for detecting and/or monitoring a M.
tuberculosis infection in a subject, wherein the antibody or
antibody derivative is conjugated to an imaging tag.
[0008] In yet another aspect, the presently disclosed subject
matter provides for the use of antibody 3d29 or a derivative
thereof for detecting and/or monitoring a M. tuberculosis infection
in a subject, wherein the antibody or antibody derivative is
conjugated to an imaging tag.
[0009] In some embodiments, the antibody or antibody derivative
comprises 3d29 or a derivative thereof. In some embodiments, the
antibody or antibody derivative (e.g., 3d29 or a derivative
thereof) binds to infected tissue in the subject. In some
embodiments, the infected tissue comprises inflamed tissue. In some
embodiments, the infected tissue is selected from the group
consisting of lung, spleen, and any other extrapulmonary infected
tissue. In some embodiments, the antibody or antibody derivative
co-localizes with alveolar and peripheral phagocytes in M.
tuberculosis infected lung sections in the subject and/or
co-localizes with aggregates of macrophages in the lungs of
infected subjects. In some embodiments, the imaging tag is a
fluorescent tag and/or a radiolabel. In some embodiments, the
imaging tag comprises any radioiodine nuclide. In some embodiments,
the imaging tag comprises .sup.125I, .sup.123I, .sup.124I, or
.sup.131I. In some embodiments, the imaging tag comprises
LISSAMINE, IRDye680RD or IRDye800CW.
[0010] In some embodiments, the step of detecting the signal
comprises performing an imaging method selected from the group
consisting of computed tomography (CT), fluorescence imaging, and
single-photon emission computed tomography (SPECT), positron
emission tomography (PET) and combinations thereof.
[0011] In some embodiments, the step of administering comprises
injecting the antibody or antibody derivative into the subject. In
some embodiments, injecting comprises intravenous or
intraperitoneal injection.
[0012] In some embodiments, the method further comprises treating
the subject for M. tuberculosis infection. In some embodiments,
treating comprises administering to the subject an effective amount
of an antibiotic agent, an anti-inflammatory agent, or a
combination thereof. In some embodiments, the subject is human.
[0013] In another aspect, the presently disclosed subject matter
provides a method of treating a M. tuberculosis infection in a
subject in need thereof, the method comprising: (a) administering
to a subject an effective amount of a monoclonal antibody or
antibody derivative which binds to C3d, wherein the monoclonal
antibody or antibody derivative is conjugated to an imaging tag,
and wherein the antibody or antibody derivative binds to infected
tissue in the subject; (b) detecting a signal generated by the
imaging tag to detect and/or monitor the location of the M.
tuberculosis infection in the subject; and (c) administering to the
subject an effective amount of an antibiotic agent, an
anti-inflammatory agent, or a combination thereof. In some
embodiments, the infected tissue comprises inflamed tissue. In some
embodiments, the antibiotic agent and/or anti-inflammatory agent
are administered to the location of the M. tuberculosis infection
in the subject. In some embodiments, the antibiotic agent and/or
anti-inflammatory agent are administered to the location of the
inflammation in the subject. In some embodiments, the subject is
human.
[0014] In one aspect, the presently disclosed subject matter
provides a purified monoclonal antibody or antibody derivative
which binds to a complement C3 activation fragment and is capable
of imaging the complement C3 activation fragment in vivo when bound
to an imaging tag. In a particular embodiment, the imaging tag is a
fluorescent tag and/or a radiolabel.
[0015] In certain aspects, the presently disclosed subject matter
provides an imaging kit for visualizing a complement C3 activation
fragment comprising the antibody or antibody derivative.
[0016] In other aspects, the presently disclosed subject matter
provides a method for detecting infection or inflammation in a
subject, the method comprising administering to the subject an
antibody or antibody derivative linked to a labeling substance,
wherein the antibody or antibody derivative binds to a complement
C3 activation fragment, and wherein binding to the complement C3
activation fragment means that the subject has an infection or
inflammation.
[0017] Certain aspects of the presently disclosed subject matter
having been stated hereinabove, which are addressed in whole or in
part by the presently disclosed subject matter, other aspects will
become evident as the description proceeds when taken in connection
with the accompanying Examples and Figures as best described herein
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Having thus described the presently disclosed subject matter
in general terms, reference will now be made to the accompanying
Figures, which are not necessarily drawn to scale, and wherein:
[0019] FIG. 1 shows metabolism of C3 to iC3b and C3d during
complement activation. During complement activation, the C3 protein
undergoes proteolytic cleavage at several locations. The C3d domain
is present within the C3, C3b, and iC3b molecules. However,
conformational changes in the 3D structure of C3 expose C3d
epitopes during cleavage of the C3 molecule;
[0020] FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D show the generation
of mAbs that recognize C3 activation fragments. Anti-human C3d
hybridomas were generated: FIG. 2A shows the hybridomas were
screened against recombinant human C3d by ELISA, and 9 of the
clones bound to the protein (clone 7C10 was used as a positive
control, and the remaining clones were newly identified); FIG. 2B
shows reactivity of the clones against reduced intact human C3 and
recombinant human C3d by Western blot analysis was tested. Three
patterns of reactivity were seen: Group 1 clones bound strongly to
reduced C3d; Group 2 clones bound to the .alpha. chain of reduced
intact C3; and Group 3 clones did not bind well to either moiety.
The asterisk denotes the mAb whose results are shown. The rightmost
blot shows the result using a polyclonal antibody against mouse C3.
The lower molecular weight bands detected by the mAbs in the C3
samples are likely contaminants; FIG. 2C shows clone 3d11
recognized all of the human C3 .alpha. chain fragments by Western
blot analysis. The appearance of the .alpha., .alpha.', .alpha.'1,
C3dg, and C3d fragments from purified human proteins are shown. The
lower molecular weight bands detected in the C3 and iC3b samples
are likely contaminants; and FIG. 2D shows immunoprecipitation of
C3 fragments in mouse serum demonstrated that the Group 1 clones
recognize the iC3b form (.alpha.'1 chain) and C3dg, but do not bind
to the C3 and C3b (.alpha. and .alpha.' chains). Clone 3d16
demonstrated some binding to the iC3b and C3dg fragments. The
results using 3d8b were from a separate gel. The immunoprecipitated
proteins were visualized by Western blot analysis with mAb 3d11
under reducing conditions;
[0021] FIG. 3 shows that proteins in mouse serum do not reduce the
binding of 3 d29 to platebound C3d. The anti-C3d mAbs were tested
in a C3d ELISA in which increasing concentrations of serum from
wild-type and C3-deficient (C3-/-) mice were added to the
reactions. Binding of the anti-C3d mAbs was not reduced by
wild-type or C3-/- serum in any of the dilutions tested. The
results for mAb 3d29 are shown;
[0022] FIG. 4 shows surface plasmon resonance of clones 3d8b, 3d9a,
and 3d29 against recombinant human C3d demonstrate high-affinity
binding. Surface plasmon resonance was performed using recombinant
human C3d. The protein was immobilized on a CM5 chip (100 RU), and
samples containing variable concentrations of the antibodies (90,
30, or 10 nM) were added. The data were fitted using a 1:1 Langmuir
binding model and equilibrium dissociation constants (KD) were
calculated. mAb 171 was used as a negative control, and the results
of binding with mAb 171 (blue line) were compared with the results
using mAb and mAb 3d8b, both at 90 nM. The anti-C3d mAbs
demonstrated high-affinity binding, and the KDs are shown for each
mAb studied;
[0023] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, and FIG. 5E show that
clones 3d3, 3d15, and 3d16 stabilize C3 convertase on sheep
erythrocytes. Sheep erythrocytes were sensitized with antibody and
opsonized with human C3b. They were then treated with factor B,
factor D, and properdin to generate AP C3 convertases (C3bBbP) on
the cell surfaces. One microgram of antibody was added to a
150-.mu.l reaction mix, and the cells were used immediately as
shown in FIG. 5A and FIG. 5C, or incubated for 2 hours as shown in
FIG. 5B and FIG. 5D. n=4-6 for each condition: FIG. 5A shows when
guinea pig serum was added to the erythrocytes as a source of MAC
and the average number of lytic sites was calculated (Z value),
cells treated with clones 3d3, 3d15, and 3d16 demonstrated a
greater MAC formation than control-treated cells; FIG. 5B shows
when the cells were incubated 2 hours prior to addition of the
guinea pig serum, the same 3 clones showed greater Z values,
indicating that these clones stabilize the C3 convertase on the
cell surface; FIG. 5C and FIG. 5D show the experiment was repeated
for clones 3d3, 3d15, and 3d16 in the presence or absence of factor
B. In the absence of factor B, MAC formation was eliminated,
demonstrating that the reaction required formation of the
alternative pathway C3 convertase; and FIG. 5E shows the same
reaction was repeated but with the addition of 400 ng of factor H.
None of the antibodies tested interfered with the ability of factor
H to dissociate the C3 convertase and prevent MAC formation. This
experiment was performed in duplicate, and the mean of these
results is shown;
[0024] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, and FIG. 6E show
inhibition of the CR2-C3d interaction by anti-C3d mAbs: FIG. 6A
shows a competition ELISA was performed to test whether the
anti-C3d mAbs interfere with the binding of a recombinant construct
of the 2 N-terminal domains of CR2 (MBP-CR2) and plate-bound C3d.
The percentage of MBP-CR2 binding (y-axis) (kept at a constant
concentration of 10 .mu.g/ml) to C3d was determined in the presence
of individual anti-C3d mAbs (x-axis) at a concentration of 26
.mu.g/ml. Values are normalized to a positive control in which
C3d-coated wells were incubated with MBP-CR2 in the absence of
anti-C3d mAbs (not shown). Also shown for each sample is a negative
control in which the wells were coated with BSA instead of C3d;
FIG. 6B, FIG. 6C, and FIG. 6D show capacity of the Group 1 mAbs
(3d8b, 3d9a, and 3d29) to block MBP-CR2 binding to plate-bound C3d
at mAb concentrations ranging from 1.625 to 26 .mu.g/ml; and FIG.
6E shows that 3d10 did not block the binding of CR2 to plate-bound
C3d over the same concentration range;
[0025] FIG. 7A, FIG. 7B, and FIG. 7C show clones 3d8b, 3d9a, and
3d29 bind to mouse C3 fragments generated in vitro: FIG. 7A shows
normal mouse serum was activated on zymosan particles, and binding
of the antibodies to the C3-opsonized particles was tested. The
opsonized particles were incubated with 1 .mu.g of each antibody,
and bound antibody was detected by flow cytometry. Polyclonal
antimouse C3 was used as a positive control. Clones 3d8b, 3d9, and
3d29 bound to the opsonized particles. This assay was repeated on
separate occasions, and a representative result is shown; FIG. 7B
shows zymosan particles were opsonized with C3 using normal mouse
serum and were then incubated with biotinylated 3d29. Incubating
3d29 with the particles in the presence of the activated serum
failed to reduce binding of 3d29 to the particle surface and
actually increased binding. ***P<0.001; and FIG. 7C shows the
addition of fresh mouse serum to the supernatant when the antibody
was incubated with the particles did not reduce binding of
biotinylated 3d29 to the particle surface;
[0026] FIG. 8A and FIG. 8B show that clones 3d8b, 3d9a, and 3d29
bind to mouse C3 fragments generated in vivo: FIG. 8A shows kidney
tissue sections from factor H-deficient mice (fH-/-) were used to
test binding of the antibodies to C3 tissue deposits. Factor H mice
are known to have abundant deposition of C3 fragments along the
glomerular capillaries without IgG at this location. This was
confirmed by immunostaining with a polyclonal antibody against
mouse C3. Kidney tissue sections were then incubated with 5
.mu.g/ml of each clone. Clones 3d8b, 3d9, and 3d29 bound to the
capillaries in a pattern identical to that of polyclonal anti-C3.
The remaining 6 clones did not demonstrate substantive binding (the
result for clone 3d31 is shown); and FIG. 8B shows kidneys from
factor I-deficient (fI-/-) mice were immunostained with a
polyclonal antibody against C3 and with mAb 3d29. The fI-/- mice
cannot generate iC3b. The absence of glomerular staining in fI-/-
mice by mAb 3d29 confirms that the mAb does not recognize C3b.
Glomeruli are indicated with arrowheads. Original magnification,
.times.400 for all panels, including the inset;
[0027] FIG. 9A and FIG. 9B show that clones 3d8b, 3d9a, and 3d29
target tissue-bound C3 fragments after systemic in vivo injection:
FIG. 9A shows factor H- deficient mice were injected with 0.5 mg of
each antibody. After 24 hours the mice were sacrificed, and
immunofluorescence microscopy was performed to detect glomerular
IgG. Mice injected with clones 3d8b, 3d9, and 3d29 demonstrated IgG
deposition along the capillary walls in a pattern indistinguishable
from that of C3 deposition (as shown by control staining of a
section with a polyclonal anti-C3 antibody). These mice do not have
detectable C3 deposits along the tubules, and no IgG was seen in
the tubulointerstitium. To confirm that the detection antibody was
not binding to endogenous IgG, clone 3d29 was biotinylated and the
experiment was repeated. Streptavidin-FITC was used to detect the
injected antibody, and again, it could be seen along the capillary
loops; and FIG. 9B shows wild-type C57BL/6 mice demonstrate C3
deposits along the basolateral aspect of the tubules. Unmanipulated
C57BL/6 mice were injected with biotinylated 3d29 or with a
biotinylated control antibody. The mice were sacrificed after 24
hours, and 3d29 was detected in the kidneys using strepatavidin-PE.
The antibody was detected along the tubules in a pattern
indistinguishable from the C3 deposits. Original magnification,
.times.400;
[0028] FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E, FIG. 10F,
and FIG. 10G show clones 3d29 target tissue-bound C3 fragments in
the retina in a model of CNV. Four laser spots in each eye were
created by Argon laser photocoagulation: FIG. 10A shows FITC-3d29
strongly bound to CNV lesions in flat mounts made from wild-type
mice; FIG. 10B shows low-intensity staining was observed for HB5, a
control antibody, to the edge of the CNV lesions in flat mounts
made from wild-type mice; FIG. 10C shows low-intensity staining of
FITC-3d29 was observed in CNV lesions in flat mounts made from
fB-/- mice; FIG. 10D shows bright-field image revealing 4
depigmented CNV lesions in a wild-type mouse; FIG. 10E shows
fluorescence image of the same fundus demonstrating that no
fluorescence is detectable in live CNV mice injected with 0.2 mg
FITC-HB5; FIG. 10F shows bright-field image revealing 4 depigmented
CNV lesions in a wild-type mouse injected with FITC-3d29; and FIG.
10G shows fluorescence image of the same fundus demonstrating that
fluorescence is clearly detectable in live CNV mice injected with
0.2 mg FITC-3d29. Original magnification, .times.630 for FIG. 10A,
FIG. 10B, and FIG. 10C and resolution element ("resel") of
approximately 4 .mu.m for FIG. 10D, and FIG. 10E; and
[0029] FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, and FIG.
11F show: FIG. 11A shows coronal and sagittal views of
[.sup.125I]3d29 SPECT-CT after 24 h of uptake showing abundant
focal pulmonary uptake as well as in spleen (Sp) and metabolized
radioiodine in thyroid; FIG. 11B shows sagittal view of
[.sup.125I]isotype control uptake in an infected mouse after 24
hours of uptake. Only stomach (Sto) and thyroid uptake are visible;
FIG. 11C shows ex vivo biodistribution of carrier-free
[.sup.125I]3d29 and [.sup.125I]isotype in infected mice showing
three-fold higher total lung uptake of 3d29 in infected mice; FIG.
11D shows coronal and sagittal [.sup.125I]3d29 SPECT-CT in healthy
mice after a 24 h uptake; FIG. 11E shows sagittal view of
[.sup.125I]isotype control uptake in an infected mouse after 24
hours of uptake; and FIG. 11F shows ex vivo microscopy showing
co-localization of injected fluorescent 3d29 with CD68+ phagocytes
in infected lungs (top panel) with only trace binding to luminal
alveolar macrophages in a healthy mouse.
[0030] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
DETAILED DESCRIPTION
[0031] The presently disclosed subject matter now will be described
more fully hereinafter with reference to the accompanying Figures,
in which some, but not all embodiments of the presently disclosed
subject matter are shown. Like numbers refer to like elements
throughout. The presently disclosed subject matter may be embodied
in many different forms and should not be construed as limited to
the embodiments set forth herein; rather, these embodiments are
provided so that this disclosure will satisfy applicable legal
requirements. Indeed, many modifications and other embodiments of
the presently disclosed subject matter set forth herein will come
to mind to one skilled in the art to which the presently disclosed
subject matter pertains having the benefit of the teachings
presented in the foregoing descriptions and the associated Figures.
Therefore, it is to be understood that the presently disclosed
subject matter is not to be limited to the specific embodiments
disclosed and that modifications and other embodiments are intended
to be included within the scope of the appended claims.
[0032] The practice of the present invention will typically employ,
unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant nucleic acid (e.g., DNA) technology,
immunology, and RNA interference (RNAi) which are within the skill
of the art. Non-limiting descriptions of certain of these
techniques are found in the following publications: Ausubel, F., et
al., (eds.), Current Protocols in Molecular Biology, Current
Protocols in Immunology, Current Protocols in Protein Science, and
Current Protocols in Cell Biology, all John Wiley & Sons, N.Y.,
edition as of December 2008; Sambrook, Russell, and Sambrook,
Molecular Cloning. A Laboratory Manual, 3.sup.rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and
Lane, D., Antibodies--A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 1988; Freshney, R. I.,
"Culture of Animal Cells, A Manual of Basic Technique", 5th ed.,
John Wiley & Sons, Hoboken, N.J., 2005. Non-limiting
information regarding therapeutic agents and human diseases is
found in Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic
and Clinical Pharmacology, McGraw-Hill/Appleton & Lange
10.sup.th ed. (2006) or 11th edition (July 2009). Non-limiting
information regarding genes and genetic disorders is found in
McKusick, V. A.: Mendelian Inheritance in Man. A Catalog of Human
Genes and Genetic Disorders. Baltimore: Johns Hopkins University
Press, 1998 (12th edition) or the more recent online database:
Online Mendelian Inheritance in Man, OMIM.TM.. McKusick-Nathans
Institute of Genetic Medicine, Johns Hopkins University (Baltimore,
Md.) and National Center for Biotechnology Information, National
Library of Medicine (Bethesda, Md.), as of May 1, 2010, World Wide
Web URL: http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian
Inheritance in Animals (OMIA), a database of genes, inherited
disorders and traits in animal species (other than human and
mouse), at http://omia.angis.org.au/contact.shtml.
[0033] In some embodiments, the presently disclosed subject matter
provides compositions, methods and kits for imaging and therapy of
infectious disease and inflammation. In other embodiments, the
presently disclosed subject matter provides antibody and antibody
derivatives (e g, minibodies, diabodies) for imaging a variety of
infectious and inflammatory entities, such as experimental models
of chronic bacterial infection, disseminated tuberculosis and
rheumatoid arthritis. In still other embodiments, fluorescent or
radiolabeled versions of antibody 3d29 are used in the presently
disclosed methods. In further embodiments, fluorescent antibody is
used for imaging in vivo or in cellulo or for
fluorescence-activated cell sorting.
[0034] In some embodiments, the agents are capable of detecting
infectious or inflammatory cells in vivo (the radioactive or
near-infrared emitting versions) or in cellulo (the optical
version). In other embodiments, this is the first time that
complement has been imaged specifically in vivo for the purpose of
studying infection or inflammation.
[0035] During complement activation the C3 protein is cleaved, and
C3 activation fragments are covalently fixed to tissues.
Tissue-bound C3 fragments are a durable biomarker of tissue
inflammation, and these fragments have been exploited as
addressable binding ligands for targeted therapeutics and
diagnostic agents. Cross-reactive murine monoclonal antibodies
against human and mouse C3d have been generated, the final C3
degradation fragment generated during complement activation. Three
monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially
bind to the iC3b, C3dg, and C3d fragments in solution, but do not
bind to intact C3 or C3b were generated. The same three clones also
bind to tissue-bound C3 activation fragments when injected
systemically. Using mouse models of renal and ocular disease, it
was confirmed that, following systemic injection, the antibodies
accumulated at sites of C3 fragment deposition within the
glomerulus, the renal tubulointerstitium, and the posterior pole of
the eye. To detect antibodies bound within the eye, optical imaging
was used and accumulation of the antibodies within retinal lesions
in a model of choroidal neovascularization (CNV) was observed.
[0036] The results demonstrate that imaging methods that use these
antibodies provide a sensitive means of detecting and monitoring
complement activation-associated tissue inflammation. It was found
that [.sup.125I]3d29 but not [.sup.125I]isotype control SPECT-CT
sensitively detects granulomas and inflamed spleen in infected
mice. Healthy mice display minimal spleen uptake of [.sup.125I]3d29
while [.sup.125I]isotype signal is restricted to stomach and
thyroid due to radioiodine metabolite (FIG. 11A, FIG. 11B, FIG.
11D, and FIG. 11E). Ex vivo biodistribution of low dose
[.sup.125I]3d29 and [.sup.125I]isotype control in M. tb. infected
mice showed a 3:1 elevation of 3d29 uptake in infected lungs over
isotype (FIG. 11C). The focal nature of [.sup.125I]3d29 binding to
granulomas and inflamed spleen allow them to be clearly observed
over blood pool and other less inflamed tissues. 3d29-LISSAMINE
conjugate co-localizes with alveolar and peripheral phagocytes in
M. tb infected lung sections while only trace uptake is detected in
uninfected luminal alveoolar phagocytes (FIG. 11F). No binding of
isotype conjugate was observed.
[0037] Novel methods have been used herein to develop 9 murine
monoclonal antibodies against human C3d that cross-react with both
mouse and cynomolgus C3d. Three of these high-affinity antibodies
discriminate the cleaved forms of C3 from the intact C3 protein.
Furthermore, the presently disclosed studies demonstrate that these
antibodies can be used to target tissue sites of complement
activation in vivo despite high levels of intact C3 in the
circulation. Methods are reported herein that were used to develop
these monoclonal antibodies against C3d and evidence is presented
that these reagents target tissue-bound C3d in vivo.
[0038] The optimal treatment of chronic infections and autoimmune
diseases requires methods for accurately detecting and localizing
tissue inflammation. Complement C3 activation fragments are fixed
to pathogens and to host cells during the immune response, and thus
can serve as biomarkers of ongoing inflammation. Several probes
have been developed that detect tissue-bound C3 deposits, including
a monoclonal antibody to C3d (mAb 3d29) that does not recognize
native C3 or C3b. To determine whether this antibody can be used to
noninvasively monitor Mycobacterium tuberculosis (M. tb) infection,
female C3HeB/FeJ mice were infected with aerosolized M. tb. 3d29
was covalently labeled with Iodine-125. Infected and non-infected
control mice were then injected with the radiolabeled probe.
Single-photon emission computed tomography (SPECT)/CT imaging at 24
and 48 hours post-radiotracer injection was performed. Results
showed that [.sup.125I]3d29 was detected by SPECT and co-registered
with CT images in order to localize [.sup.125I]3d29 in injected
mice. Lung tissue from similar animals was also immunostained for
3d29 and macrophages. Strong signal was detected by SPECT imaging
in the lungs and spleens of infected mice, consistent with the
location of granulomas in the infected animals. Low level signal
was seen in the spleens of uninfected mice and no signal was seen
in the lungs of healthy mice Immunofluorescence microscopy revealed
that 3d29 in the lungs of infected mice co-localized with
aggregates of macrophages (detected with anti-CD68 antibodies and
DPA-713-IRDye680LT). 3d29 was detected in the cytoplasm of
macrophages, consistent with the location of internalized M. tb.
3d29 was also seen in alveolar epithelial cells, indicating that it
detects M. tb. phagocytosed by other CD68-positive cells. In
conclusion, the results demonstrated that radiolabeled 3d29 can be
used to detect and localize areas of infection with M. tb.
Infection with M. tb is one of the leading causes of mortality
worldwide, and incomplete treatment has led to multidrug
resistant-strains of the disease. The presently disclosed imaging
method is useful to ensure the effective and complete treatment of
infected patients and is useful for monitoring disease activity in
a wide range of other infectious and autoimmune diseases.
[0039] Accordingly, in an aspect the presently disclosed subject
matter provides a method for detecting and/or monitoring a
Mycobacterium tuberculosis (M. tuberculosis) infection in a
subject, the method comprising: (a) administering to a subject an
effective amount of a monoclonal antibody or antibody derivative
which binds to C3d in the subject, wherein the monoclonal antibody
or antibody derivative is conjugated to an imaging tag; and (b)
detecting a signal generated by the imaging tag to detect and/or
monitor the location of the M. tuberculosis infection in the
subject.
[0040] In another aspect, the presently disclosed subject matter
provides for the use of a monoclonal antibody or antibody
derivative which binds to C3d for detecting and/or monitoring a M.
tuberculosis infection in a subject, wherein the antibody or
antibody derivative is conjugated to an imaging tag.
[0041] In yet another aspect, the presently disclosed subject
matter provides for the use of antibody 3d29 or a derivative
thereof for detecting and/or monitoring a M. tuberculosis infection
in a subject, wherein the antibody or antibody derivative is
conjugated to an imaging tag.
[0042] In some embodiments, the antibody or antibody derivative
comprises 3d29 or a derivative thereof. Suitable 3d29 antibodies
and derivatives of 3d29 antibodies and their sequences can be found
in international PCT application publication no. WO 2014/028865,
which is incorporated herein by reference in its entirety. In some
embodiments, the antibody or antibody derivative binds to infected
tissue in the subject. In some embodiments, the infected tissue
comprises inflamed tissue. In some embodiments, the infected tissue
is selected from the group consisting of lung, spleen and any other
extrapulmonary infected tissue. In some embodiments, the antibody
or antibody derivative co-localizes with alveolar and peripheral
phagocytes in M. tuberculosis infected lung sections in the subject
and/or co-localizes with aggregates of macrophages in the lungs of
infected subjects. In some embodiments, the imaging tag is a
fluorescent tag and/or a radiolabel. In some embodiments, the
imaging tag comprises any radioiodine nuclide. In some embodiments,
the imaging tag comprises .sup.125I, .sup.123I, .sup.124I, or
.sup.131I. In some embodiments, the imaging tag comprises
LISSAMINE, IRDye608RD or IRDye800CW.
[0043] In some embodiments, the step of detecting the signal
comprises performing an imaging method selected from the group
consisting of computed tomography (CT), fluorescence imaging, and
single-photon emission computed tomography (SPECT), positron
emission tomography (PET), and combinations thereof. In some
embodiments, the step of detecting the signal comprises performing
SPECT/CT imaging. In some embodiments, the step of detecting the
signal comprises performing PET/CT.
[0044] In some embodiments, the step of administering comprises
injecting the antibody or antibody derivative into the subject. In
some embodiments, injecting comprises intravenous or
intraperitoneal injection.
[0045] In some embodiments, the method further comprises treating
the subject for M. tuberculosis infection. In some embodiments,
treating comprises administering to the subject an effective amount
of an antibiotic agent, an anti-inflammatory agent, or a
combination thereof.
[0046] As used herein, "anti-inflammatory agent" refers to an agent
that may be used to prevent or reduce an inflammatory response or
inflammation in a cell, tissue, organ, or subject. Exemplary
anti-inflammatory agents include, without limitation, steroidal
anti-inflammatory agents, a nonsteroidal anti-inflammatory agent,
or a combination thereof. In some embodiments, anti-inflammatory
agents include clobetasol, alclofenac, alclometasone dipropionate,
algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenac
sodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen,
apazone, balsalazide disodium, bendazac, benoxaprofen, benzydamine
hydrochloride, bromelains, broperamole, budesonide, carprofen,
cicloprofen, cintazone, cliprofen, clobetasol propionate,
clobetasone butyrate, clopirac, cloticasone propionate,
cormethasone acetate, cortodoxone, deflazacort, desonide,
desoximetasone, dexamethasone, dexamethasone acetate, dexamethasone
dipropionate, diclofenac potassium, diclofenac sodium, diflorasone
diacetate, diflumidone sodium, diflunisal, difluprednate,
diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab,
enolicam sodium, epirizole, etodolac, etofenamate, felbinac,
fenamole, fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone,
fentiazac, flazalone, fluazacort, flufenamic acid, flumizole,
flunisolide acetate, flunixin, flunixin meglumine, fluocortin
butyl, fluorometholone acetate, fluquazone, flurbiprofen,
fluretofen, fluticasone propionate, furaprofen, furobufen,
halcinonide, halobetasol propionate, halopredone acetate, ibufenac,
ibuprofen, ibuprofen aluminum, ibuprofen piconol, ilonidap,
indomethacin, indomethacin sodium, indoprofen, indoxole, intrazole,
isoflupredone acetate, isoxepac, isoxicam, ketoprofen, lofemizole
hydrochloride, lomoxicam, loteprednol etabonate, meclofenamate
sodium, meclofenamic acid, meclorisone dibutyrate, mefenamic acid,
mesalamine, meseclazone, methylprednisolone suleptanate,
momiflumate, nabumetone, naproxen, naproxen sodium, naproxol,
nimazone, olsalazine sodium, orgotein, orpanoxin, oxaprozin,
oxyphenbutazone, paranyline hydrochloride, pentosan polysulfate
sodium, phenbutazone sodium glycerate, pirfenidone, piroxicam,
piroxicam cinnamate, piroxicam olamine, pirprofen, prednazate,
prifelone, prodolic acid, proquazone, proxazole, proxazole citrate,
rimexolone, romazarit, salcolex, salnacedin, salsalate,
sanguinarium chloride, seclazone, sermetacin, sudoxicam, sulindac,
suprofen, talmetacin, talniflumate, talosalate, tebufelone,
tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, tetrydamine,
tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium,
triclonide, triflumidate, zidometacin, zomepirac sodium, aspirin
(acetylsalicylic acid), salicylic acid, corticosteroids,
glucocorticoids, tacrolimus, pimecorlimus, prodrugs thereof,
co-drugs thereof, and combinations thereof. The anti-inflammatory
agent may also be a biological inhibitor of proinflammatory
signaling molecules including antibodies to such biological
inflammatory signaling molecules. The anti-inflammatory agent may
be included in a pharmaceutical composition comprising the antibody
or antibody derivative which binds C3d (e.g., 3d29 or a derivative
thereof), optionally together with an antibiotic agent. In some
embodiments, the anti-inflammatory agent is conjugated directly or
indirectly to the antibody or antibody derivative (e.g., antibody
or antibody derivative which binds to C3d, e.g., 3d29 or a
derivative thereof), for example, to target the anti-inflammatory
agent to the location of the M. tuberculosis infection and/or
inflammation in the subject (e.g., infected and/or inflamed tissue,
e.g., lungs and/or spleen).
[0047] Antibiotic agents include without limitation those that
affect the bacterial cell wall, such as penicillins and
cephalosporins, the cell membrane, such as polymyxins, interfere
with essential bacterial enzymes, such as rifamycins, lipiarmycins,
quinolones, and sulfonamides, target protein synthesis, such as
macrolides, lincosamides and tetracyclines, cyclic lipopeptides,
such as daptomycin, glycylcyclines, such as tigecycline,
oxazolidinones, such as linezolid, lipiarmycins, such as
fidaxomicin, fluoroquinolones, such as gemifloxacin,
lipoglycopeptides, such as telavancin, and macrocyclics, such as
fidaxomicin. Exemplary antibiotic agents include, without
limitation, rifampicin, pyrazinamide, ethambutol, streptomycin,
isoniazid, amoxicillin, ampicillin, bacampicillin, carbenicillin,
cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin,
oxacillin, penicillin G, penicillin V, piperacillin, pivampicillin,
pivmecillinam, ticarcillin, cefacetrile (cephacetrile), cefadroxil
(cefadroxyl), cefalexin (cephalexin), cefaloglycin (cephaloglycin),
cefalonium (cephalonium), cefaclor, cefamandole, cefmetazole,
cefcapene, cefdaloxime, aztreonam, imipenem, doripenem, meropenem,
ertapenem, azithromycin, erythromycin, clarithromycin,
dirithromycin, roxithromycin, ketolides, telithromycin,
clindamycin, lincomycin, pristinamycin, amikacin, gentamicin,
kanamycin, neomycin, flumequine, nalidixic acid, oxolinic acid,
piromidic acid, ciprofloxacin, enoxacin, lomefloxacin,
balofloxacin, gatifloxacin, grepafloxacin, levofloxacin,
sulfamethizole, sulfamethoxazole, sulfisoxazole, demeclocycline,
doxycycline, minocycline, oxytetracycline, tetracycline,
chloramphenicol, metronidazole, tinidazole, nitrofurantoin,
vancomycin, telavancin, linezolid, bacitracin, polymyxin B, and
viomycin.
[0048] The antibiotic agent may be included in a pharmaceutical
composition comprising the antibody or antibody derivative which
binds C3d (e.g., 3d29 or a derivative thereof), optionally together
with an anti-inflammatory agent. In some embodiments, the
antibiotic agent is conjugated directly or indirectly to the
antibody or antibody derivative (e.g., antibody or antibody
derivative which binds to C3d, e.g., 3d29 or a derivative thereof),
for example, to target the antibiotic agent to the location of the
M. tuberculosis infection and/or inflammation in the subject (e.g.,
infected and/or inflamed tissue, e.g., lungs and/or spleen).
[0049] In some embodiments, the subject is human.
[0050] In another aspect, the presently disclosed subject matter
provides a method of treating a M. tuberculosis infection in a
subject in need thereof, the method comprising: (a) administering
to a subject an effective amount of a monoclonal antibody or
antibody derivative which binds to C3d, wherein the monoclonal
antibody or antibody derivative is conjugated to an imaging tag,
and wherein the antibody or antibody derivative binds to infected
tissue in the subject; (b) detecting a signal generated by the
imaging tag to detect and/or monitor the location of the M.
tuberculosis infection in the subject; and (c) administering to the
subject an effective amount of an antibiotic agent, an
anti-inflammatory agent, or a combination thereof. In some
embodiments, the infected tissue comprises inflamed tissue. In some
embodiments, the antibiotic agent and/or anti-inflammatory agent
are administered to the location of the M. tuberculosis infection
in the subject. In some embodiments, the antibiotic agent and/or
anti-inflammatory agent are administered to the location of the
inflammation in the subject. In some embodiments, the subject is
human.
[0051] "Sequence identity" or "identity" in the context of proteins
or polypeptides refers to the amino acid residues in two amino acid
sequences that are the same when aligned for maximum correspondence
over a specified comparison window.
[0052] Thus, "percentage of sequence identity" refers to the value
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the amino acid sequence
in the comparison window may comprise additions or deletions (i.e.,
gaps) as compared to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the two
sequences. The percentage is calculated by determining the number
of positions at which the identical amino acid residue occurs in
both sequences to yield the number of matched positions, dividing
the number of matched positions by the total number of positions in
the window of comparison and multiplying the results by 100 to
yield the percentage of sequence identity. Useful examples of
percent sequence identities include, but are not limited to, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer
percentage from 50% to 100%. These identities can be determined
using any of the programs described herein.
[0053] Sequence alignments and percent identity or similarity
calculations may be determined using a variety of comparison
methods designed to detect homologous sequences including, but not
limited to, the MegAlign.TM. program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Within the context of this application it will be understood that
where sequence analysis software is used for analysis, that the
results of the analysis will be based on the "default values" of
the program referenced, unless otherwise specified. As used herein
"default values" will mean any set of values or parameters that
originally load with the software when first initialized. The
"Clustal V method of alignment" corresponds to the alignment method
labeled Clustal V (described by Higgins and Sharp (1989) CABIOS
5:151-153; Higgins et al. (1992) Comput. Appl. Biosci. 8:189-191)
and found in the MegAlign.TM. program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
[0054] It is well understood by one skilled in the art that many
levels of sequence identity are useful in identifying proteins or
polypeptides (e.g., from other species) wherein the proteins or
polypeptides have the same or similar function or activity. Useful
examples of percent identities include, but are not limited to,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer
percentage from 50% to 100%. Indeed, any integer amino acid
identity from 50% to 100% may be useful in describing the present
presently disclosed subject matter, such as 51%, 52%, 53%, 54%,
55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99%.
[0055] The term "antibody," also known as an immunoglobulin (Ig),
is a large Y-shaped protein produced by B cells that is used by the
immune system to identify and neutralize foreign objects such as
bacteria and viruses by recognizing a unique portion (epitope) of
the foreign target, called an antigen. As used herein, the term
"antibody" also includes an "antigen-binding portion" of an
antibody (or simply "antibody portion"). The term "antigen-binding
portion," as used herein, refers to one or more fragments of an
antibody that retain the ability to specifically bind to an
antigen. It has been shown that the antigen-binding function of an
antibody can be performed by fragments of a full-length antibody.
Examples of binding fragments encompassed within the term
"antigen-binding portion" of an antibody include: (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989)
Nature 341:544-546), which consists of a VH domain; and (vi) an
isolated complementarity determining region (CDR). Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded
for by separate genes, they can be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a
single protein chain in which the VL and VH regions pair to form
monovalent polypeptides (known as single chain Fv (scFv); e.g.,
Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.
Natl. Acad Sci. USA 85:5879-5883; and Osbourn et al. (1998) Nature
Biotechnology 16:778). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. Any VH and VL sequences of specific scFv
can be linked to human immunoglobulin constant region cDNA or
genomic sequences, in order to generate expression vectors encoding
complete IgG polypeptides or other isotypes. VH and V1 can also be
used in the generation of Fab, Fv or other fragments of
immunoglobulins using either protein chemistry or recombinant DNA
technology. Other forms of single chain antibodies, such as
diabodies are also encompassed. Diabodies are bivalent, bispecific
antibodies in which VH and VL domains are expressed on a single
polypeptide chain, but using a linker that is too short to allow
for pairing between the two domains on the same chain, thereby
forcing the domains to pair with complementary domains of another
chain and creating two antigen binding sites (e.g., Holliger et al.
(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al.
(1994) Structure 2:1121-1123).
[0056] Still further, an antibody or antigen-binding portion
thereof may be part of larger immunoadhesion polypeptides, formed
by covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion polypeptides include use of the streptavidin
core region to make a tetrameric scFv polypeptide (Kipriyanov et
al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a
cysteine residue, a marker peptide and a C-terminal polyhistidine
tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov
et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such
as Fab and F(ab').sub.2 fragments, can be prepared from whole
antibodies using conventional techniques, such as papain or pepsin
digestion, respectively, of whole antibodies. Moreover, antibodies,
antibody portions and immunoadhesion polypeptides can be obtained
using standard recombinant DNA techniques, as described herein.
[0057] Antibodies may be polyclonal or monoclonal; xenogeneic,
allogeneic, or syngeneic; or modified forms thereof (e.g.
humanized, chimeric, etc.). Antibodies may also be fully human. The
terms "monoclonal antibodies" and "monoclonal antibody
composition," as used herein, refer to a population of antibody
polypeptides that contain only one species of an antigen binding
site capable of immunoreacting with a particular epitope of an
antigen, whereas the term "polyclonal antibodies" and "polyclonal
antibody composition" refer to a population of antibody
polypeptides that contain multiple species of antigen binding sites
capable of interacting with a particular antigen. A monoclonal
antibody composition typically displays a single binding affinity
for a particular antigen with which it immunoreacts.
[0058] The term "humanized antibody", as used herein, is intended
to include antibodies made by a non-human cell having variable and
constant regions which have been altered to more closely resemble
antibodies that would be made by a human cell. For example, by
altering the non-human antibody amino acid sequence to incorporate
amino acids found in human germline immunoglobulin sequences. The
humanized antibodies of the presently disclosed subject matter may
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo),
for example in the CDRs. The term "humanized antibody", as used
herein, also includes antibodies in which CDR sequences derived
from the germline of another mammalian species, such as a mouse,
have been grafted onto human framework sequences.
[0059] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities. Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals.
[0060] The subject treated by the presently disclosed methods in
their many embodiments is desirably a human subject, although it is
to be understood that the methods described herein are effective
with respect to all vertebrate species, which are intended to be
included in the term "subject." Accordingly, a "subject" can
include a human subject for medical purposes, such as for the
treatment of an existing condition or disease or the prophylactic
treatment for preventing the onset of a condition or disease, or an
animal subject for medical, veterinary purposes, or developmental
purposes. Suitable animal subjects include mammals including, but
not limited to, primates, e.g., humans, monkeys, apes, and the
like; bovines, e.g., cattle, oxen, and the like; ovines, e.g.,
sheep and the like; caprines, e.g., goats and the like; porcines,
e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys,
zebras, and the like; felines, including wild and domestic cats;
canines, including dogs; lagomorphs, including rabbits, hares, and
the like; and rodents, including mice, rats, and the like. An
animal may be a transgenic animal. In some embodiments, the subject
is a human including, but not limited to, fetal, neonatal, infant,
juvenile, and adult subjects. Further, a "subject" can include a
patient afflicted with or suspected of being afflicted with a
condition or disease. Thus, the terms "subject" and "patient" are
used interchangeably herein.
[0061] More particularly, as described herein, the presently
disclosed compositions can be administered to a subject for therapy
by any suitable route of administration, including orally, nasally,
transmucosally, ocularly, rectally, intravaginally, parenterally,
including intramuscular, subcutaneous, intramedullary injections,
as well as intrathecal, direct intraventricular, intravenous,
intra-articular, intra-sternal, intra-synovial, intra-hepatic,
intralesional, intracranial, intraperitoneal, intranasal, or
intraocular injections, intracisternally, topically, as by powders,
ointments or drops (including eyedrops), including buccally and
sublingually, transdermally, through an inhalation spray, or other
modes of delivery known in the art. The presently disclosed
compositions can also be administered intratumorally, such that the
compositions are directly administered into a solid tumor, such as
by injection or other means.
[0062] In general, the "effective amount" or "therapeutically
effective amount" of an active agent or drug delivery device refers
to the amount necessary to elicit the desired biological response.
As will be appreciated by those of ordinary skill in this art, the
effective amount of an agent or device may vary depending on such
factors as the desired biological endpoint, the agent to be
delivered, the composition of the encapsulating matrix, the target
tissue, and the like.
[0063] As used herein, the active agents may be combined and
administered in a single dosage form, may be administered as
separate dosage forms at the same time, or may be administered as
separate dosage forms that are administered alternately or
sequentially on the same or separate days. In one embodiment of the
presently disclosed subject matter, the active agents are combined
and administered in a single dosage form. In another embodiment,
the active agents are administered in separate dosage forms (e.g.,
wherein it is desirable to vary the amount of one but not the
other). The single dosage form may include additional active agents
for the treatment of the disease state.
[0064] Further, the presently disclosed compositions can be
administered alone or in combination with adjuvants that enhance
stability of the agents, facilitate administration of
pharmaceutical compositions containing them in certain embodiments,
provide increased dissolution or dispersion, increase activity,
provide adjuvant therapy, and the like, including other active
ingredients. Advantageously, such combination therapies utilize
lower dosages of the conventional therapeutics, thus avoiding
possible toxicity and adverse side effects incurred when those
agents are used as monotherapies.
[0065] When administered sequentially, the agents can be
administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or
longer of one another. In other embodiments, agents administered
sequentially, can be administered within 1, 5, 10, 15, 20 or more
days of one another. When administered in combination, the
effective concentration of each of the agents to elicit a
particular biological response may be less than the effective
concentration of each agent when administered alone, thereby
allowing a reduction in the dose of one or more of the agents
relative to the dose that would be needed if the agent was
administered as a single agent. The effects of multiple agents may,
but need not be, additive or synergistic. The agents may be
administered multiple times.
[0066] In some embodiments, when administered in combination, the
two or more agents can have a synergistic effect. As used herein,
the terms "synergy," "synergistic," "synergistically" and
derivations thereof, such as in a "synergistic effect" or a
"synergistic combination" or a "synergistic composition" refer to
circumstances under which the biological activity of a combination
of an agent and at least one additional therapeutic agent is
greater than the sum of the biological activities of the respective
agents when administered individually.
[0067] Synergy can be expressed in terms of a "Synergy Index (SI),"
which generally can be determined by the method described by F. C.
Kull et al. Applied Microbiology 9, 538 (1961), from the ratio
determined by:
Q.sub.a/Q.sub.A+Q.sub.b/Q.sub.B=Synergy Index (SI)
wherein:
[0068] Q.sub.A is the concentration of a component A, acting alone,
which produced an end point in relation to component A;
[0069] Q.sub.a is the concentration of component A, in a mixture,
which produced an end point;
[0070] Q.sub.B is the concentration of a component B, acting alone,
which produced an end point in relation to component B; and
[0071] Q.sub.b is the concentration of component B, in a mixture,
which produced an end point.
[0072] Generally, when the sum of Q.sub.a/Q.sub.A and
Q.sub.b/Q.sub.B is greater than one, antagonism is indicated. When
the sum is equal to one, additivity is indicated. When the sum is
less than one, synergism is demonstrated. The lower the SI, the
greater the synergy shown by that particular mixture. Thus, a
"synergistic combination" has an activity higher that what can be
expected based on the observed activities of the individual
components when used alone. Further, a "synergistically effective
amount" of a component refers to the amount of the component
necessary to elicit a synergistic effect in, for example, another
therapeutic agent present in the composition.
[0073] As used herein, the term "reduce" or "inhibit," and
grammatical derivations thereof, refers to the ability of an agent
to block, partially block, interfere, decrease, reduce or
deactivate a pathway or mechanism of action. Thus, one of ordinary
skill in the art would appreciate that the term "reduce"
encompasses a complete and/or partial loss of activity, e.g., a
loss in activity by at least 10%, in some embodiments, a loss in
activity by at least 20%, 30%, 50%, 75%, 95%, 98%, and up to and
including 100%.
[0074] In another aspect, the presently disclosed subject matter
provides a pharmaceutical composition alone or in combination with
one or more additional therapeutic agents in admixture with a
pharmaceutically acceptable excipient. In some embodiments, the
pharmaceutical composition comprises an antibody or antibody
derivative which binds to C3d in combination with an antibiotic
agent, an anti-inflammatory agent, or both an antibiotic agent and
anti-inflammatory agent, optionally with a pharmaceutically
acceptable carrier, diluent, or excipient, for example, for
detecting and/or diagnosing and/or monitoring a M. tuberculosis
infection in a subject.
[0075] One of skill in the art will recognize that the
pharmaceutical compositions include the pharmaceutically acceptable
salts of the compounds described above. Pharmaceutically acceptable
salts are generally well known to those of ordinary skill in the
art, and include salts of active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituent moieties found on the compounds described herein. When
compounds of the present disclosure contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent.
[0076] Examples of pharmaceutically acceptable base addition salts
include sodium, potassium, calcium, ammonium, organic amino, or
magnesium salt, or a similar salt. When compounds of the present
disclosure contain relatively basic functionalities, acid addition
salts can be obtained by contacting the neutral form of such
compounds with a sufficient amount of the desired acid, either neat
or in a suitable inert solvent. Examples of pharmaceutically
acceptable acid addition salts include those derived from inorganic
acids like hydrochloric, hydrobromic, nitric, carbonic,
monohydrogencarbonic, phosphoric, monohydrogenphosphoric,
dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or
phosphorous acids and the like, as well as the salts derived from
relatively nontoxic organic acids like acetic, propionic,
isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,
lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,
citric, tartaric, methanesulfonic, and the like. Also included are
salts of amino acids such as arginate and the like, and salts of
organic acids like glucuronic or galactunoric acids and the like
(see, for example, Berge et al. (1977) "Pharmaceutical Salts", J.
of Pharm. Sci. 66, 1-19). Certain specific compounds of the present
disclosure contain both basic and acidic functionalities that allow
the compounds to be converted into either base or acid addition
salts.
[0077] Accordingly, pharmaceutically acceptable salts suitable for
use with the presently disclosed subject matter include, by way of
example but not limitation, acetate, benzenesulfonate, besylate,
benzoate, bicarbonate, bitartrate, bromide, calcium edetate,
carnsylate, carbonate, citrate, edetate, edisylate, estolate,
esylate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,
lactobionate, malate, maleate, mandelate, mesylate, mucate,
napsylate, nitrate, pamoate (embonate), pantothenate,
phosphate/diphosphate, polygalacturonate, salicylate, stearate,
subacetate, succinate, sulfate, tannate, tartrate, or teoclate.
Other pharmaceutically acceptable salts may be found in, for
example, Remington: The Science and Practice of Pharmacy (20.sup.th
ed.) Lippincott, Williams and Wilkins (2000).
[0078] In therapeutic and/or diagnostic applications, the compounds
of the disclosure can be formulated for a variety of modes of
administration, including systemic and topical or localized
administration. Techniques and formulations generally may be found
in Remington: The Science and Practice of Pharmacy (20.sup.th ed.)
Lippincott, Williams and Wilkins (2000).
[0079] Use of pharmaceutically acceptable inert carriers to
formulate the compounds herein disclosed for the practice of the
disclosure into dosages suitable for systemic administration is
within the scope of the disclosure. With proper choice of carrier
and suitable manufacturing practice, the compositions of the
present disclosure, in particular, those formulated as solutions,
may be administered parenterally, such as by intravenous injection.
The compounds can be formulated readily using pharmaceutically
acceptable carriers well known in the art into dosages suitable for
oral administration. Such carriers enable the compounds of the
disclosure to be formulated as tablets, pills, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral
ingestion by a subject (e.g., patient) to be treated.
[0080] For nasal or inhalation delivery, the agents of the
disclosure also may be formulated by methods known to those of
skill in the art, and may include, for example, but not limited to,
examples of solubilizing, diluting, or dispersing substances, such
as saline; preservatives, such as benzyl alcohol; absorption
promoters; and fluorocarbons.
[0081] Pharmaceutical compositions suitable for use in the present
disclosure include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. Generally, the compounds
according to the disclosure are effective over a wide dosage range.
For example, in the treatment of adult humans, dosages from 0.01 to
1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to
40 mg per day are examples of dosages that may be used. A
non-limiting dosage is 10 to 30 mg per day. The exact dosage will
depend upon the route of administration, the form in which the
compound is administered, the subject to be treated, the body
weight of the subject to be treated, and the preference and
experience of the attending physician.
[0082] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions.
[0083] Following long-standing patent law convention, the terms
"a," "an," and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a subject" includes a plurality of subjects, unless the context
clearly is to the contrary (e.g., a plurality of subjects), and so
forth.
[0084] Throughout this specification and the claims, the terms
"comprise," "comprises," and "comprising" are used in a
non-exclusive sense, except where the context requires otherwise.
Likewise, the term "include" and its grammatical variants are
intended to be non-limiting, such that recitation of items in a
list is not to the exclusion of other like items that can be
substituted or added to the listed items.
[0085] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing amounts, sizes,
dimensions, proportions, shapes, formulations, parameters,
percentages, parameters, quantities, characteristics, and other
numerical values used in the specification and claims, are to be
understood as being modified in all instances by the term "about"
even though the term "about" may not expressly appear with the
value, amount or range. Accordingly, unless indicated to the
contrary, the numerical parameters set forth in the following
specification and attached claims are not and need not be exact,
but may be approximate and/or larger or smaller as desired,
reflecting tolerances, conversion factors, rounding off,
measurement error and the like, and other factors known to those of
skill in the art depending on the desired properties sought to be
obtained by the presently disclosed subject matter. For example,
the term "about," when referring to a value can be meant to
encompass variations of, in some embodiments, .+-.100% in some
embodiments .+-.50%, in some embodiments .+-.20%, in some
embodiments .+-.10%, in some embodiments .+-.5%, in some
embodiments .+-.1%, in some embodiments .+-.0.5%, and in some
embodiments .+-.0.1% from the specified amount, as such variations
are appropriate to perform the disclosed methods or employ the
disclosed compositions.
[0086] Further, the term "about" when used in connection with one
or more numbers or numerical ranges, should be understood to refer
to all such numbers, including all numbers in a range and modifies
that range by extending the boundaries above and below the
numerical values set forth. The recitation of numerical ranges by
endpoints includes all numbers, e.g., whole integers, including
fractions thereof, subsumed within that range (for example, the
recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as
fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and
any range within that range.
EXAMPLES
[0087] The following Examples have been included to provide
guidance to one of ordinary skill in the art for practicing
representative embodiments of the presently disclosed subject
matter. In light of the present disclosure and the general level of
skill in the art, those of skill can appreciate that the following
Examples are intended to be exemplary only and that numerous
changes, modifications, and alterations can be employed without
departing from the scope of the presently disclosed subject matter.
The synthetic descriptions and specific examples that follow are
only intended for the purposes of illustration, and are not to be
construed as limiting in any manner to make compounds of the
disclosure by other methods.
Example 1
Methods
Reagents
[0088] Recombinant Human C3d:
[0089] Recombinant human C3d was used as an immunogen for antibody
generation. It was also used as a target antigen in ELISA binding
studies and Western blot analysis. C3d was generated using the pGEX
expression system (GE Healthcare) in E. coli as previously
described (Li et al., 2008). The C3d construct comprised amino
acids 996-1303 of the precursor Pro-C3 protein. Briefly,
ampicillin-resistant colonies were expanded to 1 liter in
Luria-Bertani (LB) broth. The cultures were grown at 37.degree. C.
until an A600 of 0.3 was achieved. Cultures were induced with 0.3
mM isopropyl-.beta.-D-thiogalactoside at 30.degree. C. overnight
before harvesting by centrifugation. Harvested pellets were
resuspended in glutathione S-transferase column buffer (50 mM
Tris-HCl, pH 8.0, 250 mM NaCl, 1 mM EDTA) and lysed by sonication.
Lysate was clarified by centrifugation and applied to a GSTrap HP
column (GE Biosciences). C3d was cleaved from the column by
digesting with 50 units of thrombin overnight at 4.degree. C. and
subsequently purified by size-exclusion chromatography. The purity
of C3d was verified using SDS-PAGE. A second form of recombinant
human C3d encompassing the same region was also produced as
previously described (Kulik et al., 2007). Binding of the
antibodies to this construct by ELISA was performed to ensure that
the antibodies bound a C3d epitope that was present on protein
generated through independent methods.
[0090] Recombinant Murine C3d:
[0091] Murine C3d was cloned from murine cDNA using a forward
primer containing a BamH I restriction site (5' CGC GGA TCC GCG GCT
GTG GAC GGG GAG 3') and a reverse primer containing an EcoRI
restriction site (5' CCG GAA TTC CGG TCA TCA ACG GCT GGG GAG GTG
3'). The amplified fragment was inserted into pGEX vector and
generated by the same methods used for the human C3d. This
recombinant murine C3d was used as a target antigen in ELISA
binding studies.
[0092] Recombinant CR2 SCR1-2:
[0093] Recombinant maltose-binding protein-tagged (MBP-tagged) CR2
SCR1-2 (MBP-CR2) comprising residues 1-133 of wild-type CR2 and
encompassing the first 2 SCR modules were expressed in E. coli as
previously described (Szakonyi et al., 206; Young et al., 2007;
Young et al., 2008). Briefly, MBP-CR2 SCR1-2-transformed colonies
of E. coli BL21 were expanded to 4 liters in LB media and grown at
37.degree. C. until an A600 of 0.3 was obtained. Cultures were then
induced with 0.3 mM IPTG at 20.degree. C. overnight before
harvesting by centrifugation. Resulting cell pellets were
resuspended in a column buffer containing 20 mM Tris-HCl (pH 7.4),
0.2 M NaCl, and 1 mM EDTA prior to lysis by sonication. The
resulting lysate was clarified by centrifugation and recombinant
MBP-CR2, which was initially purified by successive
amylose-affinity and size-exclusion chromatographic steps. Finally,
the recombinant MBP-CR2 was applied to a C3d-affinity column
generated by binding GST-tagged C3d to a GSTrap column (GE
Biosciences) and eluted with a linear NaCl gradient. The resulting
protein was then concentrated, buffer-exchanged into PBS (1.6 mM
MgCl2, 0.9 mM KCl, 0.5 mM KH2PO4, 45.6 mM NaCl, 2.7 mM Na2HPO4, pH
7.4), and its purity tested by SDS-PAGE.
[0094] Purified Complement Proteins:
[0095] Binding studies were also performed using commercially
available purified complement proteins (C3, C3b, iC3b, and C3d; all
from CompTech).
Mice and Animal Models
[0096] To generate monoclonal antibodies against C3d, mice with a
targeted deletion of the C3 gene were immunized with recombinant
human C3d. These mice were generated as previously described
(Wessels et al., 1995). C57BL/6 wild-type mice were used for some
in vivo experiments, and serum was collected from these mice for in
vitro assays that required murine complement proteins. Mice with
targeted deletion of the gene for factor H were generated as
previously described (Pickering et al., 2002). Kidney sections from
these mice were used to test binding of the anti-C3d antibodies to
tissue-bound C3 fragments in vitro, and fH-/- mice were injected
with purified anti-C3d antibodies to test binding of the antibodies
to tissue-bound C3 fragments in vivo. Kidney sections from mice
that have targeted deletion of the gene for factor I, and thus do
not generate iC3b, were used to test whether the antibodies bind to
the C3b fragment (Rose et al., 2008). Mice with targeted deletion
of the gene for complement factor B gene were used as a negative
control for binding of the FITC-labeled anti-C3d antibodies against
CNV lesions (Matsumoto et al., 1997). To induce CNV lesions,
3-month-old mice were anesthetized (xylazine and ketamine, 20 and
80 mg/kg, respectively) and their pupils were dilated (2.5%
phenylephrine HCl and 1% atropine sulfate). Argon laser
photocoagulation (532 nm, 100 nm spot size, 0.1-second duration,
100 mW) was used to generate 4 laser spots in each eye surrounding
the optic nerve, using a hand-held coverslip as a contact lens
(Rohrer et al., 2009). For tail vein injections, the vein was
vasodilated by heat, a 25-G needle was inserted, and a volume of
100 .mu.l was injected. The dosing and treatment schedule is
outlined in the Results section.
Immunization Protocol and Hybridoma Generation
[0097] The humoral immune response to the immunizations was
assessed by ELISA using C3d as the target. The mice developed high
titers of anti-C3d antibodies after 3 injections of 60 to 100 .mu.g
of protein (the first injection using complete Freund's adjuvant
and the second and third injections using incomplete Freund's
adjuvant). The mice were then injected intraperitoneally with 100
.mu.g of C3d, and after 72 hours the spleen was harvested for
fusion to Sp2/0 hybridoma cells (Kulik et al., 2009). To prevent
exposure of the anti-C3d hybridomas to C3d during the cloning
process, the cells were grown in serum-free media supplemented with
hypoxanthine-aminopterin-thymidine (HAT) (Sigma-Aldrich), and
peritoneal macrophages from C3-/- mice were used as the feeder
cells during this process. Single-cell clones were generated and
specificity of the clones to C3d was confirmed by ELISA, as
described below.
ELISAs
[0098] C3d ELISAs:
[0099] To assess the reactivity of antibodies against C3d, ELISAs
were performed using purified forms of C3 activation fragments from
several different sources (see Reagents section above). Direct
ELISAs were performed by affixing 30-50 .mu.g of the C3 fragment to
the ELISA plate overnight at 4.degree. C. and pH 7.4. The plates
were blocked with 1% BSA in PBS for 2 hours at room temperature.
Antibody was added to the wells at 5 .mu.g/ml, incubated, and the
plates were washed 4 times. Bound antibodies were then detected
with HRP-conjugated anti-mouse IgG (MP Biomedicals). Sandwich
ELISAs were performed by incubating polyclonal anti-human C3d
antibody (Dako) to the ELISA plates in order to capture the C3d.
Binding of the antibodies to the captured C3d was then detected as
above.
[0100] C3d-CR2/Anti-C3d Monoclonal Antibody Competition Assay:
[0101] Plates were incubated overnight at 4.degree. C. with
wild-type C3d at a concentration of 5 .mu.g/ml in a 50 mM sodium
bicarbonate buffer (pH 8.8). After coating, plates were blocked
using 1% BSA in PBS (pH 7.4), for 1 hour at room temperature.
Plates were then washed 3 times using PBS-Tween 20 (0.05%).
Recombinant wild-type MBP-CR2 (10 .mu.g/ml) was added to half of
the C3d-coated wells to act as a positive control. To the other
half of the C3d-coated wells, 10 .mu.g/ml of recombinant wild-type
MBP-CR2 containing 1 of the following anti-C3d monoclonal
antibodies: 3d8B; 3d31; 3d15; 3d9a; 3d11; 3d16; 3d10; 3d3; or 3d29
at concentrations ranging from 1.625 to 26 .mu.g/ml in PBS was
added. After a 1-hour incubation period, the plates were washed and
then incubated with commercially available HRP-conjugated anti-MBP
antibody (New England BioLabs). After a further 1-hour incubation
period, binding of MBP-CR2 to the plate-bound C3d was detected with
2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS).
Western Blot Analysis and Pull-Down Studies
[0102] Western blot analysis was performed by resolving 1 .mu.g of
purified complement protein on a 10% Bis-Tris polyacrylamide gel
(Invitrogen) under denaturing conditions. The protein was then
transferred to a nitrocellulose membrane. C3 fragments were then
detected by incubating the membrane with 25 .mu.g of each antibody
(0.5 mg/ml) for 1 hour at room temperature, and bound antibody was
detected with HRP-conjugated anti-mouse IgG Immunoprecipitation of
complement fragments was performed by adding 100 .mu.g of antibody
to 75 .mu.l of protein G sepharose (GE Healthcare) preblocked with
1% BSA for 1 hour. The antibodies were incubated with the protein G
sepharose for 2 hours, excess antibody was removed by washing the
sepharose with PBS, 150 .mu.l of serum from RAG-1 knockout mice was
added, and the mixture was incubated overnight at 4.degree. C. The
protein G sepharose was then washed 3 times in PBS, resuspended in
loading buffer, and separated by SDS-PAGE. The isolated C3
fragments were detected by Western blot analysis using mAb
3d11.
Measurement of Antibody Affinities by Surface Plasmon Resonance
[0103] The binding of clones 3d8b, 3d9a, and 3d29 to recombinant
human C3d was examined using a BIAcore 3000 (Biacore) at the
University of Colorado Biophysics Core. C3d was immobilized on a
carboxymethyl-dextran (CM5) chip using random amine coupling with
1-ethyl-3-[3-dimethylaminopropyl] carbodiimide
hydrochloride/N-hydroxysulfosuccinimide as the activating reagent.
Recombinant human C3d was immobilized at a concentration of 50
mg/ml in 100 mM sodium acetate, pH 5.0. The remaining activated
groups on the surface of the chip were blocked with a 1-M
ethanolamine solution (pH 8.5). Experiments were conducted in 10 mM
HEPES, 150 mM NaCl, and 0.005% P20 (pH 7.4), and the chip was
regenerated between runs with two 10-.mu.l injections of 10 mM
NaOH. Each antibody was injected at concentrations of 90, 30, and
10 nM for 1 minute at a flow rate of 50 .mu.l per minute, and
dissociation of the resulting antibody-C3d complexes was monitored
for 10 minutes. All injections were performed in triplicate to
verify reproducibility and all data were double referenced using a
blank flow cell and a blank injection of buffer to account for
nonspecific binding and baseline drift, respectively. Data were fit
using a 1:1 Langmuir binding model and data analysis was performed
using SCRUBBER-2 software (distributed by David Myszka of the
University of Utah Center for Biomolecular Interactions).
Complement Assays
[0104] Zymosan Activation Assay:
[0105] Zymosan particles were opsonized with murine C3 fragments by
incubating the particles with complement-sufficient mouse serum as
previously described (Thurman et al., 2005). The particles were
washed and then incubated with 2 .mu.g of purified anti-C3d
antibody, and bound antibody was detected with FITC-conjugated
anti-mouse IgG (MP Biotech). The samples were analyzed by flow
cytometry and compared with a positive control (C3 deposition
detected with a polyclonal anti-mouse C3; MP Biomedicals) or with a
negative control (no serum added). In some experiments,
biotinylated antibodies were incubated with the particles in the
presence of the activated serum, or fresh mouse serum was added to
the particles at the incubation step. Bound antibody was then
detected with streptavidin-FITC in order to test whether C3 and C3
fragments in the serum would compete with C3 on the zymosan surface
for the antibody.
[0106] Alternative Pathway Hemolytic Assay:
[0107] This assay was performed as previously described (Thurman et
al., 2005). Briefly, rabbit erythrocytes (Colorado Serum Company)
were washed and then resuspended in a solution of 1.1% NaCl,
0.0025% Na-5,5 diethyl barbiturate (pH 7.35), 8 mM EGTA, and 2 mM
MgCl2 (GVB/Mg/EGTA). Fifty microliters of this suspension was added
to human serum (5-100 .mu.l), and buffer solution was added to
bring the final volume up to 150 .mu.l. Erythrocytes in buffer
without serum were used as a negative control, and erythrocytes
added to 100 .mu.l of distilled water were used as a positive
control (complete lysis). Samples were incubated at 37.degree. C.
for 30 minutes, with occasional shaking to keep the cells in
suspension. The reactions were stopped by adding 1.5 ml of cold PBS
and the samples were spun at 1,000 g for 5 minutes. The optical
density of each supernatant was read at 415 nm using a
spectrophotometer (Bio-Rad). The concentration of serum that caused
approximately 50% lysis of the erythrocytes was determined. The
reactions were then repeated with the addition of 0 to 40 .mu.g of
each antibody. The percent lysis for each reaction was compared
with serum alone, and the change in lysis was reported as a
percentage.
[0108] Buffers:
[0109] DGVB2+ buffer: 1 mM MgCl2, 0.15 mM CaCl2, 71 mM NaCl, 0.1%
(w/v) gelatin, 2.5% (w/v) dextrose, and 2.47 mM sodium
5',5''-diethyl barbiturate (pH 7.35); Mg2+ EGTA buffer: 10 mM
Na2EGTA, 7 mM MgCl2, 59 mM NaCl, 0.083% (w/v) gelatin, 2.075% (w/v)
dextrose, and 2.05 mM sodium 5',5''-diethyl barbiturate (pH
7.3-7.6); 10 mM EDTA buffer: 10 mM Na2EDTA, 128 mM NaCl, 0.1% (w/v)
gelatin, and 4.45 mM sodium 5',5''-diethyl barbiturate (pH 7.35);
40 mM EDTA buffer: 40 mM Na2EDTA, 85 mM NaCl, 0.1% (w/v) gelatin,
and 2.96 mM sodium 5',5''-diethyl barbiturate (pH 7.35).
[0110] Preparation of Cell-Bound C3b:
[0111] Ab-sensitized sheep erythrocytes (EA cells, 5 ml,
5.times.108/ml) obtained from CompTech were washed twice and
resuspended in 5 ml of DGVB2+ buffer, mixed with 37.5 .mu.g of
human C1 in 5 ml of DGVB2+, and incubated for 15 minutes at
30.degree. C. The resulting cells (EAC1) were washed twice and
resuspended in 5 ml of DGVB2+, mixed with 50 .mu.g of human C4
suspended in 5 ml of DGVB2+, and incubated for 15 minutes at
30.degree. C. These cells (EAC1, 4) were washed twice and suspended
in 5 ml of DGVB2+, mixed with 250 .mu.g of human C3 and 5 .mu.g of
human C2 suspended in 5 ml of DGVB2+, and incubated for 30 minutes
at 30.degree. C. The resulting cells (EAC1, 4, 2, 3) were washed
and resuspended in 5 ml of 10 mM EDTA buffer and incubated at
37.degree. C. for 2 hours to allow for dissociation of the active
classical pathway convertases. The resulting C3b-coated cells were
washed twice in 5 ml 10 mM EDTA buffer, twice in 5 ml of 10 mM Mg2+
EGTA buffer, and resuspended in 10 mM Mg2+ EGTA buffer to a final
concentration of 1.times.108 per milliliter. They were stored at
4.degree. C. and used within 1 week.
[0112] Effects of Anti-C3 mAbs on the Activity of Cell-Bound C3bBbP
Complexes:
[0113] C3b-coated sheep erythrocytes were prepared as described
(Hourcade et al., 1995; Whaley et al., 1985). C3b-coated sheep
erythrocytes (100 .mu.l), 50 .mu.l of purified factor D (5 ng in
Mg2+ EGTA buffer), 50 .mu.l of properdin (45 .mu.g in Mg2+ EGTA
buffer), and 50 .mu.l of factor B (3-5 .mu.g in Mg2+ EGTA buffer)
were mixed together and incubated at 30.degree. C. for 30 minutes.
In some cases, the factor B was replaced by 50 .mu.l of Mg2+ EGTA
buffer. Samples were chilled to 4.degree. C. and treated with 150
.mu.l 40 mM EDTA buffer (40 mM Na2EDTA, 85 mM NaCl, 0.1% [w/v]
gelatin, and 2.96 mM sodium 5',5''-diethyl barbiturate, pH 7.35),
containing in some cases 1 .mu.g of mouse anti-human C3d mAb.
Samples were then incubated for 0 to 3 hours at 30.degree. C. to
permit spontaneous C3bBbP dissociation. In some cases, this
incubation was undertaken with or without 400 .mu.g of factor H for
30 minutes to assess factor H-dependent convertase decay
acceleration. Functional convertases were then quantified by adding
150 .mu.l of a 1:20 dilution of guinea pig serum (Colorado Serum)
in 40 mM EDTA buffer to all samples, followed by incubation at
37.degree. C. for 60 minutes. Additional samples included cell
lysis controls in which cells were treated with 450 .mu.l of
distilled water alone and a negative control in which cells were
treated with 450 .mu.l of DGVB2+ buffer alone. All samples were
then centrifuged and the OD414 of the supernatants was determined.
Hemolytic activity levels were expressed as Z values, the average
number of lytic sites or MAC pores formed per red blood cell, and
were calculated using the expression: Z=-ln (1-y), where y is the
proportion of lysed cells. Each determination was the average of
duplicate points. All complement proteins were of human origin and
were purchased from CompTech.
Immunofluorescence Microscopy
[0114] For immunofluorescence microscopy, sagittal sections of the
kidneys were snap-frozen in OCT compound (Sakura Finetek USA).
Five-micrometer sections were cut with a cryostat and stored at
-80.degree. C. until used. The slides were later fixed with acetone
and stained with antibody against mouse C3 or mouse IgG. The slides
were then counterstained with hematoxylin (Vector Laboratories) and
viewed using an Olympus BX51 microscope. The anti-C3d antibodies
were used at a concentration of 2 .mu.g/ml for tissue staining. For
immunofluorescence microscopy of RPE/choroid, flat-mount
preparations were incubated with FITC-labeled antibodies. In brief,
eyes were collected and immersion fixed in 4% paraformaldehyde for
30 minutes at 4.degree. C. after which the anterior chamber, lens,
and retina were removed. The eyecups were incubated in blocking
solution (3% BSA, 10% normal goat serum, and 0.4% Triton-X in
tris-buffered saline) for 1 hour followed by anti-C3d antibodies
(1:100 of 1 mg/ml solution) overnight at 4.degree. C. in blocking
solution. Following extensive washing, eyecups were flattened using
4 relaxing cuts, coverslips were applied with Fluoromount (Southern
Biotechnology Associates), and slides were examined by confocal
microscopy (Leica TCS SP2 AOBS; Leica).
Fundus Imaging
[0115] Fundus imaging was performed using the Micron III retinal
imaging microscope (Phoenix Research Laboratories), which is based
on a custom optical system with a 300-W xenon light source and a
3-chip CCD camera, operating at 30 frames per second in
linear/diagnostic mode. For imaging, mice were anesthetized, their
pupils were dilated as described above and secured in the imaging
cradle. Optical contact between the cornea of the mouse and the
lens of the optical system was established through a drop of
methylcellulose. A fundus photograph was obtained using
bright-field imaging to focus on the CNV lesions, after which the
mode was switched to FITC fluorescence imaging (excitation at 490
nm). JPEG images were exported to Adobe Photoshop to assemble the
photos and to extract images of individual lesions. Images obtained
with this system have a resolution element of approximately 4
.mu.m. To improve visualization of individual lesions, contrast
enhancement using identical parameters for control and experimental
images was applied.
Statistics
[0116] Data were analyzed using GraphPad Prism software (GraphPad)
and the results for groups are presented as the mean.+-.SEM.
Comparison between groups was performed using unpaired 2-tailed t
tests. A P value of less than 0.05 was considered significant.
Study Approval
[0117] The mice were housed and maintained in the University of
Colorado Center for Laboratory Animal Care in accordance with the
NIH Guidelines for the Care and Use of Laboratory Animals. The CNV
model procedures and fundus imaging were performed in accordance
with the ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research and were approved by the IACUC of the Medical
University of South Carolina. All other animal experiments and
procedures were approved by the IACUC of the University of
Colorado.
FIG. 11 Methods
[0118] Aerosol-infect C3HeB/FeJ (granulomatous TB) with live M. tb
and wait .about.8 weeks before imaging. Uninfected and infected
mice were injected with .about.3 mCi of [.sup.125I]3d29 for
SPECT-CT scans including an infected an uninfected mouse injected
with 3 mCi each of [.sup.125I]isotype control for a specific
binding study. Uptake times included 24 and 48 h post-injection and
were optimized at 24 hours post-injection for granuloma
visualization. Infected mice were also subjected to ex vivo
bisdistribution studies by injecting .about.7 Ci of [125I]3d29 or
[125I]isotype followed by a 24 hour uptake. All major tissues were
collected to measure the % injected dose/gram of tissue. Lung
sections were probed in vitro with 3d29-LISSAMINE or
isotype-LISSAMINE conjugates and co-stained with anti-CD68 and
anti-TSPO antibody to localize the cellular distribution of 3d29 in
this mouse model of pulmonary TB.
Example 2
Development of Murine mAbs Against Recombinant Human C3 d
[0119] During complement activation, C3 undergoes a conformational
change that exposes a reactive thioester bond (Serkova et al.,
2010; Janssen et al., 2006). The thioester domain (TED) of C3
rotates during the conversion of C3 into C3b, altering the
orientation of the TED on the surface of the molecule (Janssen et
al., 2006). Domains within this region of C3b become increasingly
exposed during the subsequent cleavages that generate iC3b, C3dg,
and finally C3d (FIG. 1). In order to generate mAbs against
epitopes on this region of C3, recombinant human C3d was produced
using an E. coli expression system (Shaw et al., 2010) and
immunized mice bearing a targeted deletion of the C3 gene (C3-/-
mice) (Wessels et al., 1995). Although C3-/- mice have impaired
humoral immunity (Wessels et al., 1995), immunization of these mice
has previously been used to generate antibodies against human C3
(Li et al., 2007). It was found that C3-/- mice immunized with
recombinant C3d immunogen in adjuvant developed a strong humoral
response to C3d (data not shown).
[0120] Two fusions were performed using splenocytes of mice with
high antibody titers against C3d, but both fusions failed to yield
any reactive clones. Because the desired hybridomas would produce
monoclonal antibodies specific to C3d, it was hypothesized that the
failure to generate any clones was because C3 fragments generated
by serum in the tissue culture media or from macrophages used in
the cloning process bound to the B cell receptors of the reactive
cells. This could potentially lead to apoptosis of the cells or
interfere with the screening ELISA assay. Therefore, a third fusion
was performed in which hybridomas were grown in serum-free media
formulations. Because macrophages also have the capacity to
synthesize all of the proteins of the alternative complement
pathway and generate C3 fragments (Strunk et al., 1983), the feeder
cells used during cloning were obtained by peritoneal lavage of
C3-/- mice. Single-cell clones were generated and screened against
C3d by ELISA, and 9 hybridomas producing monoclonal antibodies with
strong ELISA reactivity against human C3d were identified (FIG.
2A). This ELISA was repeated multiple times during the cloning and
purification process, and a representative experiment is shown.
[0121] To confirm that the monoclonal antibodies reacted with C3d
and not with a contaminant in the immunogen, the antibodies were
tested against C3d using a sandwich ELISA in which recombinant C3d
was captured with a polyclonal anti-C3d capture antibody. To test
the reactivity of the clones against murine C3d (which has 84%
sequence identity with human C3d), indirect and sandwich ELISAs
using recombinant murine C3d were performed. Direct ELISAs were
also performed to test binding of the antibodies to recombinant
human C3d from a second construct as well as to commercially
available purified human C3d, and to recombinant cynomolgus C3d.
The 9 clones all showed strong reactivity against each of these
targets (data not shown). To test the antibodies for
cross-reactivity against other plasma proteins, the C3d ELISA was
repeated in the presence of wild-type and C3-/- mouse serum (FIG.
3). Serum was added across a range of dilutions (1:5 to 1:6,400),
and no effect on the binding of the antibodies to C3d was
observed.
Example 3
Specificity of mAbs 3d8b, 3d9a, and 3d29 to C3 Activation
Fragments
[0122] Western blot analysis of human C3 and C3d was performed
following separation of C3d and C3 fragments by SDS-PAGE, and the
blots were probed with the 9 anti-C3d clones. Although one might
expect epitopes recognized in SDS-denatured C3d to also be exposed
on the intact C3 .alpha. chain in its denatured form, the
antibodies demonstrated differential recognition of C3 and C3d in
this assay (FIG. 2B). The antibodies displayed 3 distinct binding
patterns by Western blot analysis: strong binding to C3d without
substantial binding to C3 (Group 1); strong binding to the C3
.alpha. chain and C3d (Group 2); or weak binding to both proteins
(Group 3). A commercially available polyclonal antibody against
mouse C3 (MP Biomedicals; product 55557) bound fragments that
approximate the sizes of the a and 13 chains of C3 and is shown on
the right-most blot for comparison. The lower molecular weight
bands may represent contaminants or degraded C3. Antibodies from
each group displayed nearly identical patterns when run side by
side (data not shown). Clone 3d11 recognized all of the C3 .alpha.
chain fragments by Western blot analysis (FIG. 2C). To evaluate the
binding of the antibodies to the different C3 fragments in their
native form, immunoprecipitation reactions were performed using
activated murine serum that contained a mixture of the various C3
fragments (FIG. 2D). The immunoprecipitated proteins were then
detected by Western blot analysis with mAb 3d11. Antibodies 3d8b
and 3d29 (Group 1) pulled down murine iC3b, C3dg, and C3d
fragments; 3d11 (Group 2) did not pull down any murine C3
fragments; and 3d16 (Group 3) pulled down iC3b and C3dg fragments.
The affinities of mAbs 3d8b, 3d9a, and 3d29 for human C3d were
tested by surface plasmon resonance (FIG. 4). The measured
affinities were: 3d8b: KD=0.47 nM; 3d9a: KD=0.37 nM; and 3d29:
KD=1.06 nM.
Example 4
Effects of Anti-C3d mAbs on Surface-Bound C3 Convertase
Activity
[0123] The alternative pathway C3 convertase is composed of C3b in
complex with the factor B fragment Bb and the fluid-phase protein
properdin (P). While C3bBbP dissociation occurs spontaneously
(T1/2.about.5-10 minutes), this process is greatly accelerated by
the fluid-phase complement regulator factor H. This latter reaction
plays a critical role in protecting cells and tissues from
complement-mediated damage and in preserving C3 homeostasis.
Certain anti-C3 autoantibodies, referred to as C3 nephritic factors
(C3Nef), stabilize the alternative pathway C3 convertase and confer
to it resistance to factor H, thus permitting uncontrolled
complement activation (Daha et al., 1976). To assess whether the
anti-C3d antibodies have C3Nef-like activity, human C3bBbP
complexes preassembled on sheep erythrocytes were first incubated
with the anti-C3d antibodies or with buffer alone for various
durations. The hemolytic activity of the remaining convertases was
then quantified (FIG. 5A). The Group 1 mAbs (3d8b, 3d9a, and 3d29)
did not have any effect on erythrocyte lysis, nor did the Group 2
clone 3d31. The loss of hemolytic activity due to spontaneous
convertase dissociation during the incubation period in these
samples was comparable to that of the control cells. In contrast,
the Group 3 clones (3d3, 3d15, and 3d16) stabilized the convertase,
causing greater erythrocyte lysis immediately (FIG. 5A), and after
a 2-hour incubation period (FIG. 5B). In all cases, hemolysis was
absolutely dependent on the presence of factor B in the preassembly
step (FIGS. 5, C and D), thus confirming that the alternative
pathway C3 convertase mediated the Group 3 effects. EGTA was
included as a calcium chelator, thus precluding the involvement of
the other complement activation pathways in the process. The impact
of the anti-C3d antibodies on factor H activity was also examined.
Factor H is an alternative pathway regulatory protein that limits
alternative pathway activation by accelerating the decay of C3
convertase (Weiler et al., 1976) or by serving as a cofactor for
factor I-mediated cleavage (inactivation) of C3b (Pangburn et al.,
1977). The addition of factor H inhibited lysis of the erythrocytes
in reactions containing each of the anti-C3d antibodies, indicating
that none of the antibodies blocked the factor H binding site on
the surface of C3b (FIG. 5E). This is consistent with recent data
indicating that the binding site on C3b for the amino-terminal 4
short consensus repeats (SCRs) of factor H (CFH1-4), which harbor
the factor I cofactor and C3bBb decay acceleration activities of
factor H, lies outside the TED domain (which approximates to the
C3d cleavage product) (Wu et al., 2009). Finally, the antibodies
were tested in an alternative pathway hemolysis assay using normal
human serum and rabbit erythrocytes. This is a standard assay for
measuring alternative pathway activity on activator surfaces. Even
when clones 3d8b, 3d9a, and 3d29 were added to the reaction mix at
high concentrations, they did not increase erythrocyte lysis (data
not shown).
Example 5
Effect of Anti-C3d mAbs on Binding of C3d by CR2
[0124] C3d is a ligand for CR2, which is expressed on B cells and
follicular dendritic cells. Recognition of C3d by CR2 on B cells
lowers the threshold for B cell activation by the B cell receptor
(Lyubchenko et al., 2005). CR2 also binds to the iC3b, C3dg, and
C3d fragments of C3, similar to the Group 1 monoclonal antibodies.
Consequently, signaling by CR2 is important in the development of
the humoral immune response and autoimmunity (Dempsey et al.,
1996). It was tested whether the mAbs against C3d block this
interaction (FIG. 6A). Using an in vitro CR2-C3d binding assay, it
was found that clones 3d8b, 3d9a, 3d11, 3d29, and 3d31 blocked CR2
from binding C3d, whereas the other antibodies did not. Dose
response curves for the Group 1 antibodies demonstrated nearly
complete inhibition of CR2 binding by 3d8b (FIG. 6B). Clones 3d9a
and 3d29 achieved approximately 80% inhibition of binding by CR2
when added at high concentrations (FIG. 6C and FIG. 6D). The
inability of 3d10 to block CR2 binding at any of the concentrations
tested is shown in FIG. 6E. These results raise the possibility
that mAbs 3d8b, 3d9a, 3d11, 3d29, and 3d31 may have
immunomodulatory function.
Example 6
Binding of Anti-C3d mAbs to Surface-Bound C3 Activation Fragments
In Vitro
[0125] To assess the ability of the mAbs to bind native C3
fragments bound to activating surfaces, zymosan particles were
opsonized with C3 fragments by incubation with mouse serum (Thurman
et al., 2005). The particles were then incubated with the
antibodies, and bound antibodies were detected by flow cytometry
(FIG. 7A). mAbs 3d8b, 3d9a, and 3d29 bound to the opsonized zymosan
particles, whereas the other mAbs did not. To test whether intact
C3 or C3 activation fragments in serum could compete with the C3
fragments on the zymosan surface, this assay was repeated and the
particles were incubated with antibody in the presence of activated
or fresh serum (FIGS. 7, B and C). The addition of serum to the
reactions did not reduce binding of the antibody to the particles.
To test the binding of these antibodies to C3 deposits in tissues,
sections were made from the kidneys of factor H-deficient (fH-/-)
mice. The glomeruli of these mice are characterized by
glomerulonephritis and have abundant deposits of the C3 activation
fragments iC3b and C3dg/C3d (28, 29). Clones 3d8b, 3d9a, and 3d29
bound to sites in the acetone-fixed sections in a pattern
indistinguishable from that obtained using a polyclonal antibody
against C3 (FIG. 8A and FIG. 8B). 3d8b, 3d9a, and 3d29 did not bind
to the glomeruli of factor I-deficient mice (FIG. 8B; results not
shown for 3d8b and 3d9a), confirming that the antibodies are
specific to downstream cleavage fragments (iC3b, C3dg, and C3
d).
Example 7
In Vivo Targeting of Anti-C3d mAbs to Tissue Sites of Complement
Activation
[0126] Next, it was sought to determine whether the antibodies
would bind to tissue-bound C3 fragments when injected in vivo. The
antibodies were injected intravenously into fH-/- mice, which do
not have glomerular deposits of endogenous IgG (29). After 24
hours, the kidneys were harvested and immunostained for IgG (FIG.
9A). mAbs 3d8b, 3d9a, and 3d29 were readily detected along the
glomerular basement membrane in a pattern indistinguishable from
that of the C3 fragments, demonstrating that they bound to C3
deposits in the glomerular capillary wall after intravenous
injection. To confirm that endogenous deposits of IgG were not
being detected, mAb 3d29 was biotinylated and injected into fH-/-
mice. Glomerular binding of the antibody was detected using
streptavidin-FITC (FIG. 9A). C3 fragments are ordinarily deposited
along the tubular basement membrane of wild-type mice (Thurman et
al., 2003). Tubular C3 deposits are not seen in fH-/- mice, likely
because most C3 is consumed in the fluid phase in these mice
(Pickering et al., 2002; Renner et al., 2011). No IgG was detected
along the tubular basement membrane of fH-/- mice injected with the
anti-C3d antibodies. However, when biotinylated 3d29 was injected
into wild-type mice, it was detected along the tubular basement
membrane and colocalized with the C3 deposits (FIG. 9B). These
results indicate that mAbs 3d8b, 3d9a, and 3d29 target and bind to
tissue deposits of C3 activation fragments in the glomeruli of
nephritic mice and in the tubulointerstitium of unmanipulated
wild-type mice.
Example 8
In Vivo Imaging of Anti-C3d mAbs Targeted to Ocular Sites of
Complement Activation
[0127] To test whether the targeted antibodies could be visualized
in vivo, a system amenable to optical imaging, the eye, was
employed. Complement activation is involved in the pathology of
age-related macular degeneration (AMD). Complement components,
including C3 (Hageman et al., 2001), anaphlatoxins C3a and C5a
(Nozaki et al., 2006), as well as components of the membrane attack
complex (MAC) (Hageman et al., 2001) have been identified within
pathological structures in AMD (e.g., drusen, Bruch's membrane),
and single nucleotide polymorphisms in complement genes are risk
factors for AMD (Leveziel et al., 2011). AMD results in vision loss
from either atrophy of the retinal pigmented epithelium (RPE)
followed by loss of photoreceptors, or choroidal neovascularization
(CNV) followed by loss of photoreceptors. The latter process can be
mimicked in mice by damaging the blood-retina barrier using laser
photocoagulation, which triggers ingrowth of choroidal blood
vessels into the subretinal space in a complement-dependent fashion
(Rohrer et al., 2009). Likewise, complement deposition has been
shown to occur at the site of injury (Rohrer et al., 2009; Nozaki
et al., 2006). Using the systemic CR2 targeting strategy, it was
shown that complement inhibition delivered in this fashion (CR2-fH)
can ameliorate CNV (Rohrer et al., 2009; Rohrer et al., 2012). It
was evaluated whether complement activation in the RPE/choroid of
laser-damaged mice using the anti-C3d mAbs could be directly
imaged. First, the antibodies were tested to determine which of
them recognize C3d epitopes in the CNV lesion sites in flat-mounted
RPE/choroid. Since fluorescently labeled antibodies are required
for in vivo imaging, only FITC-labeled antibodies were tested. Of
the FITC-labeled mAbs, clone 3d29 demonstrated the best binding to
the CNV lesion in lightly fixed tissues (4% paraformaldehyde for 30
minutes) (FIG. 10A). An isotype control antibody (HB5) was also
tested in order to confirm the specificity of binding by 3d29 (FIG.
10B). Since complement factor B knockout mice (fB-/-) show no
increase in C3 in the RPE/choroid in response to the lesion and
fail to develop significant CNV (11), fB-/- mice were used as
negative controls for FITC-labeled mAb binding (FIG. 10C). For in
vivo imaging, CNV lesions were generated and 200 .mu.g (200 .mu.l
of antibody at a concentration of 1 mg/ml) of FITC-labeled 3d29 or
HB5 was injected intravenously on day 3 after CNV induction, a time
point previously shown to correspond to the peak of C3 deposition
within the lesion (Rohrer et al., 2009). Fundus imaging of the
animals 6, 24, and 48 hours after the injection was used. The CNV
lesions are readily apparent in bright-field images as depigmented
areas (FIGS. 10, D and F). At the 6-hour time point, unbound
FITC-labeled antibody was still visible in the retinal and
choroidal vasculature, obscuring positive staining. Twenty-four
hours after antibody injection, a strong fluorescent signal was
detected in the CNV lesions of 3d29-injected mice (FIG. 10G).
Little signal was detected in the lesions of HB5-injected mice
(FIG. 10E). At 48 hours, while the positive signal for
3d29-injected mice was still present, the intensity was less
pronounced. These results indicate that 3d29 is retained in
RPE/choroidal tissue deposits of C3 activation fragments at the
posterior pole of CNV-lesioned mice at a high enough concentration
that it can be visualized in the living eye using conventional
imaging techniques.
Example 9
Discussion
[0128] This report describes the development of 3 mAbs (the Group 1
antibodies 3d8b, 3d9a, and 3d29) against the C3 activation fragment
C3d that do not bind to intact C3 in its native conformation. These
3 antibodies recognize an epitope on iC3b, C3dg, and C3d that is
either generated or exposed during complement activation. This
epitope is probably closely related to the CR2 binding site that is
buried within the native C3 structure (van den Elsen et al., 2011).
To successfully create these antibodies, several modifications were
made to standard methods of hybridoma fusion: the hybridoma cells
were grown under serum-free conditions, and macrophages from C3-/-
mice were used as feeder cells during the cloning process. This
approach allowed the generation of 9 mAbs against human C3d that
also reacted with murine and cynomolgus C3d.
[0129] mAbs 3d8b, 3d9a, and 3d29 demonstrated strong binding to
SDS-denatured C3d by Western blot analysis, but no detectable
binding to SDS-denatured C3 or C3b (FIG. 2B). The same mAbs
specifically pulled down iC3b, C3dg, and C3d from a mixture that
also contained intact C3 and C3b (FIG. 2D). These 3 mAbs also bound
to C3 fragments on the surface of opsonized zymosan particles in
vitro (FIG. 7A), demonstrating the ability to bind sur-face-bound
C3 fragments. Certain anti-C3 antibodies are known to stabilize C3
convertases, effectively amplifying complement activation. The 3
clones that target tissue-bound C3 fragments did not show any
activating activity with the use of several different in vitro
assays (FIG. 5). Based on their ability to compete for CR2 binding
to C3d (FIG. 6), an overlapping or closely associated binding site
is assumed. The Group 3 antibodies (3d3, 3d15, and 3d16) stabilized
C3 convertases that were preassembled on sheep red blood cells
(FIG. 5). None of the antibodies described here prevented factor
H-mediated dissociation of the C3 convertase. When mice with
glomerulonephritis were injected with mAbs 3d8b, 3d9a, or 3d29, the
antibodies accumulated at the site of C3 fragment deposits within
the glomeruli, demonstrating that the antibodies can be used to
target tissue-bound iC3b and C3d at this location (FIG. 9A). When
injected into wild-type mice, these antibodies bound to C3
fragments deposited along the tubular basement membrane (which have
deposition of C3 fragments at baseline; FIG. 9B). Because C3
fragments are present in the plasma of fH-/- mice, and wild-type
mice have high circulating levels of intact C3, these experiments
verified that mAbs 3d8b, 3d9a, and 3d29 preferentially bind to the
tissue-bound iC3b and C3d activation fragments, even in the
presence of circulating C3 and C3 fragments.
[0130] A major obstacle to the development of a high-affinity
targeting protein for C3 activation fragments is that the protein
must distinguish the cleavage fragments from intact C3. The high
affinity of these antibodies for C3d and the ability to deliver
agents to sites of C3d deposition in vivo, make them invaluable
tools for the development of diagnostic and therapeutic agents. The
ability to block the C3d-CR2 interaction further raises the
possibility that these antibodies will have immunomodulatory
effects. In addition, when used for tissue analysis ex vivo, these
antibodies can also be used to specifically detect deposits of
iC3b, C3dg, and C3d fragments in tissues (FIG. 8). These antibodies
cross-react with murine, human, and cynomolgus C3d, making them
suitable for both preclinical and clinical studies. 41 commercially
available mAbs against cleavage fragments of human C3 have been
identified (not including antibodies against C3a), 11 of which are
described by the vendors as reacting with iC3b and/or C3d. None of
the available antibodies had been tested for species
cross-reactivity, CR2 inhibition, or in vivo targeting, and only 1
of the antibodies is reported as functional in ELISAs, Western blot
analysis, flow cytometry, and immunohistochemistry (Quidel antibody
A209). Unlike the antibodies described (FIG. 5), however, that
antibody stabilized the C3 convertase on sheep erythrocytes (data
not shown). Similarly, 3 other commercial antibodies specific to
epitopes in C3d have been tested in this assay and were all found
to stabilize the C3 convertase (Dennis Hourcade, unpublished
observations). Thus, although a wide range of antibodies against
human C3 are available, based on data available from the vendors
and based on our own experiments, none of the commercial antibodies
against iC3b or C3d are comparable to mAbs 3d8b, 3d9a, or 3
d29.
[0131] The detection of glomerular C3 deposition is critical for
the accurate diagnosis of glomerulonephritis, and renal biopsy
tissue is routinely stained for C3 fragments. The antibodies and
methods described herein may advance our ability to detect and
monitor tissue C3 deposition. An MRI-based method for the
noninvasive detection of glomerular C3 has been developed, and
these high-affinity antibodies may improve the sensitivity of this
method. In the current study, it has been demonstrated that
FITC-labeled 3d29 was visualized in live animals using conventional
fluorescence imaging. This enabled noninvasive detection of C3d
deposits within the RPE/choroid of mice with CNV. Finally, targeted
complement inhibitors have also demonstrated great promise for the
treatment of inflammatory diseases (Atkinson et al., 2005; Sekine
et al., 2011; Song et al., 2003). These antibodies may provide a
high-affinity targeting vector for the delivery of novel
therapeutic agents to sites of tissue inflammation.
[0132] The exact epitope of these antibodies has not yet been
identified. The antibodies were screened against a panel of C3d
mutants, but identification of the exact epitope on C3d was not
successful (data not shown). This suggests that the antibodies may
recognize a complex epitope not evaluated by that assay. Epitope
mapping studies of these antibodies are underway. However, subtle
differences between the antibodies, such as the superior ability of
3d8b to block CR2 binding compared with that of 3d9a and 3d29,
suggest that they recognize distinct epitopes. Identification of
the binding site for each antibody may help predict biologic
functions of the antibodies, as one may then predict interactions
of the C3 molecules that will be interrupted by the antibodies.
[0133] Autoimmune diseases are frequently life long and are
characterized by flares and remissions. The immunomodulatory drugs
used to treat these diseases are effective, but can cause serious
side effects. Thus, as with cancer, the treatment of autoimmune
diseases hinges upon the accurate assessment of disease activity.
Unfortunately, current molecular imaging methods for detecting
tissue inflammation, such as white blood cell scans, lack the
senbodies sitivity and specificity necessary for monitoring
autoimmune disease activity (Sargsyan et al., 2012). Because C3
fragments are abundant and durable markers of inflammation, they
represent a powerful biomarker of tissue inflammation. Quantitative
methods of detecting tissue C3 fragment deposits would improve our
ability to monitor a patient's disease activity and response to
therapy and would advance the application of "personalized
medicine" to the autoimmune diseases. Our studies demonstrate that
mAbs 3d8b, 3d9a, and 3d29 can be employed as molecular imaging
probes for the detection of complement activation.
[0134] In conclusion, mAbs against C3 activation fragments have
been successfully generated. Three of the antibodies recognize
breakdown products of C3 (iC3b, C3dg, and C3d) but do not bind to
intact C3 in its native state. It has been demonstrated that these
antibodies target tissue-bound C3 fragments in vivo, despite high
circulating levels of intact C3. Antibodies specific to
tissue-bound C3 activation fragments may be employed for targeted
delivery of therapeutic and diagnostic agents to sites of tissue
inflammation. Radiologic methods of detecting these antibodies
could provide an important new tool for detecting and monitoring
tissue inflammation. It has been demonstrated that fluorescently
labeled antibody was detected in live animals with CNV. Now that
therapeutic complement inhibitors have been approved for clinical
use (Rother et al., 2007), noninvasive methods of detecting
complement activation within tissues will be increasingly important
in therapeutic decision making.
REFERENCES
[0135] All publications, patent applications, patents, and other
references mentioned in the specification are indicative of the
level of those skilled in the art to which the presently disclosed
subject matter pertains. All publications, patent applications,
patents, and other references (e.g., websites, databases, etc.)
mentioned in the specification are herein incorporated by reference
in their entirety to the same extent as if each individual
publication, patent application, patent, and other reference was
specifically and individually indicated to be incorporated by
reference. It will be understood that, although a number of patent
applications, patents, and other references are referred to herein,
such reference does not constitute an admission that any of these
documents forms part of the common general knowledge in the art. In
case of a conflict between the specification and any of the
incorporated references, the specification (including any
amendments thereof, which may be based on an incorporated
reference), shall control. Standard art-accepted meanings of terms
are used herein unless indicated otherwise. Standard abbreviations
for various terms are used herein.
[0136] Although the foregoing subject matter has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be understood by those skilled in
the art that certain changes and modifications can be practiced
within the scope of the appended claims. [0137] Ricklin D,
Hajishengallis G, Yang K, Lambris J D. Complement: a key system for
immune surveillance and homeostasis. Nat Immunol. 2010;
11(9):785-797. [0138] Walport M J. Complement. Second of two parts.
N Engl J Med. 2001; 344:1140-1144. [0139] Law S K, Dodds A W. The
internal thioester and the covalent binding properties of the
complement proteins C3 and C4. Protein Sci. 1997; 6(2):263-274.
[0140] Sahu A, Kozel T R, Pangburn M K. Specificity of the
thioester-containing reactive site of human C3 and its significance
to complement activation. Biochem J. 1994; 2:429-436. [0141] Sahu
A, Pangburn M K. Covalent attachment of human complement C3 to IgG.
Identification of the amino acid residue involved in ester linkage
formation. J Biol Chem. 1994; 269(46):28997-29002. [0142] Schulze
M, Pruchno C J, Burns M, Baker P J, Johnson R J, Couser W G.
Glomerular C3c localization indicates ongoing immune deposit
formation and complement activation in experimental
glomerulonephritis. Am J Pathol. 1993; 142(1):179-187. [0143]
Hageman G S, Luthert P J, Victor Chong N H, Johnson L V, Anderson D
H, Mullins R F. An integrated hypothesis that considers drusen as
biomarkers of immunemediated processes at the RPE-Bruch's membrane
interface in aging and age-related macular degeneration. Prog Retin
Eye Res. 2001; 20(6):705-732. [0144] Atkinson C, et al. Targeted
complement inhibition by C3d recognition ameliorates tissue injury
without apparent increase in susceptibility to infection. J Clin
Invest. 2005; 115(9):2444-2453. [0145] Serkova N J, et al. Renal
inflammation: targeted iron oxide nanoparticles for molecular MR
imaging in mice. Radiology. 2010; 255(2):517-526. [0146] Sargsyan S
A, et al. Detection of glomerular complement C3 fragments by
magnetic resonance imaging in murine lupus nephritis. Kidney Int.
2012; 81(2):152-159. [0147] Rohrer B, et al. A targeted inhibitor
of the alternative complement pathway reduces angiogenesis in a
mouse model of age-related macular degeneration. Invest Ophthalmol
Vis Sci. 2009; 50(7):3056-3064. [0148] Rohrer B, Coughlin B,
Bandyopadhyay M, Holers V M. Systemic human CR2-targeted complement
alternative pathway inhibitor ameliorates mouse laser-induced
choroidal neovascularization. J Ocul Pharmacol Ther. 2012;
28(4):402-409. [0149] Webb S. Pharma interest surges in antibody
drug conjugates. Nat Biotechnol. 2011; 29(4):297-298. [0150]
Guthridge J M, et al. Structural studies in solution of the
recombinant N-terminal pair of short consensus/complement repeat
domains of complement receptor type 2 (CR2/CD21) and interactions
with its ligand C3dg. Biochemistry. 2001; 40(20):5931-5941. [0151]
Isenman D E, Leung E, Mackay J D, Bagby S, van den Elsen J M.
Mutational analyses reveal that the staphylococcal immune evasion
molecule Sbi and complement receptor 2 (CR2) share overlapping
contact residues on C3d: implications for the controversy regarding
the CR2/C3d cocrystal structure. J Immunol. 2010; 184(4):1946-1955.
[0152] Dempsey P W, Allison M E, Akkaraju S, Goodnow C C, Fearon D
T. C3d of complement as a molecular adjuvant: bridging innate and
acquired immunity. Science. 1996; 271(5247):348-350. [0153] Janssen
B J, Christodoulidou A, McCarthy A, Lambris J D, Gros P. Structure
of C3b reveals conformational changes that underlie complement
activity. Nature. 2006; 444(7116):213-216. [0154] Shaw C D, et al.
Delineation of the complement receptor type 2-C3d complex by
site-directed mutagenesis and molecular docking. J Mol Biol. 2010;
404(4):697-710. [0155] Wessels M R, Butko P, Ma M, Warren H B, Lage
A L, Carroll M C. Studies of group B streptococcal infection in
mice deficient in complement component C3 or C4 demonstrate an
essential role for complement in both innate and acquired immunity.
Proc Natl Acad Sci USA. 1995; 92(25):11490-11494. [0156] Li Y,
Williams M E, Cousar J B, Pawluczkowycz A W, Lindorfer M A, Taylor
R P. Rituximab-CD20 complexes are shaved from Z138 mantle cell
lymphoma cells in intravenous and subcutaneous SCID mouse models. J
Immunol. 2007; 179(6):4263-4271. [0157] Strunk R C, Kunke K S,
Giclas P C. Human peripheral blood monocyte-derived macrophages
produce haemolytically active C3 in vitro. Immunology. 1983;
49(1):169-174. [0158] Daha M R, Fearon D T, Austen K F. C3
nephritic factor (C3NeF): stabilization of fluid phase and
cellbound alternative pathway convertase. J Immunol. 1976;
116(1):1-7. [0159] Weiler J M, Daha M R, Austen K F, Fearon D T.
Control of the amplification convertase of complement by the plasma
protein beta1H. Proc Natl Acad Sci USA. 1976; 73(9):3268-3272.
[0160] Pangburn M K, Schreiber R D, Muller-Eberhard H J. Human
complement C3b inactivator: isolation, characterization, and
demonstration of an absolute requirement for the serum protein
betalH for cleavage of C3b and C4b in solution. J Exp Med. 1977;
146(1):257-270. [0161] Wu J, Wu Y Q, Ricklin D, Janssen B J,
Lambris J D, Gros P. Structure of complement fragment C3b-factor H
and implications for host protection by complement regulators. Nat
Immunol. 2009; 10(7):728-733. [0162] Lyubchenko T, dal Porto J,
Cambier J C, Holers V M. Coligation of the B cell receptor with
complement receptor type 2 (CR2/CD21) using its natural ligand
C3dg: activation without engagement of an inhibitory signaling
pathway. J Immunol. 2005; 174(6):3264-3272. [0163] Thurman J M, et
al. A novel inhibitor of the alternative complement pathway
prevents antiphospholipid antibody-induced pregnancy loss in mice.
Mol Immunol. 2005; 42(1):87-97. [0164] Paixao-Cavalcante D, Hanson
S, Botto M, Cook H T. Pickering M C. Factor H facilitates the
clearance of GBM bound iC3b by controlling C3 activation in fluid
phase. Mol Immunol. 2009; 46(10):1942-1950. [0165] Pickering M C,
et al. Uncontrolled C3 activation causes membranoproliferative
glomerulonephritis in mice deficient in complement factor H. Nat
Genet. 2002; 31(4):424-428. [0166] Thurman J M, Ljubanovic D,
Edelstein C L, Gilkeson G S, Holers V M. Lack of a functional
alternative complement pathway ameliorates ischemic acute renal
failure in mice. J Immunol. 2003; 170(3):1517-1523. [0167] Renner
B, et al. Binding of factor H to tubular epithelial cells limits
interstitial complement activation in ischemic injury. Kidney Int.
2011; 80(2):165-173. [0168] Nozaki M, et al. Drusen complement
components C3a and C5a promote choroidal neovascularization. Proc
Natl Acad Sci USA. 2006; 103(7):2328-2333. [0169] Leveziel N, et
al. Genetic factors associated with age-related macular
degeneration. Ophthalmologica. 2011; 226(3):87-102. [0170] Van den
Elsen J M, Isenman D E. A crystal structure of the complex between
human complement receptor 2 and its ligand C3d. Science. 2011;
332(6029):608-611. [0171] Sekine H, et al. The benefit of targeted
and selective inhibition of the alternative complement pathway for
modulating autoimmunity and renal disease in MRL/lpr mice.
Arthritis Rheum. 2011; 63(4):1076-1085. [0172] Song H, He C, Knaak
C, Guthridge J M, Holers V M, Tomlinson S. Complement receptor
2-mediated targeting of complement inhibitors to sites of
complement activation. J Clin Invest. 2003; 111(12):1875-1885.
[0173] Sargsyan S A, Thurman J M. Molecular imaging of autoimmune
diseases and inflammation. Mol Imaging. 2012; 11(3):251-264. [0174]
Rother R P, Rollins S A, Mojcik C F, Brodsky R A, Bell L. Discovery
and development of the complement inhibitor eculizumab for the
treatment of paroxysmal nocturnal hemoglobinuria. Nat Biotechnol.
2007; 25(11):1256-1264. [0175] Li K, et al. Solution structure of
the complex formed between human complement C3d and full-length
complement receptor type 2. J Mol Biol. 2008; 384(1):137-150.
[0176] Kulik L, et al. Intrinsic B cell hypo-responsiveness in mice
prematurely expressing human CR2/CD21 during B cell development.
Eur J Immunol. 2007; 37(3):623-633. [0177] Szakonyi G, et al.
Structure of the Epstein-Barr virus major envelope glycoprotein.
Nat Struct Mol Biol. 2006; 13 (11): 996-1001. [0178] Young K A,
Chen X S, Holers V M, Hannan J P. Isolating the Epstein-Barr virus
gp350/220 binding site on complement receptor type 2 (CR2/CD21). J
Biol Chem. 2007; 282(50):36614-36625. [0179] Young K A, Herbert A
P, Barlow P N, Holers V M, Hannan J P. Molecular basis of the
interaction between complement receptor type 2 (CR2/CD21) and
Epstein-Barr virus glycoprotein gp350. J Virol. 2008;
82(22):11217-11227. [0180] Rose K L, et al. Factor I is required
for the development of membranoproliferative glomerulonephritis in
factor H-deficient mice. J Clin Invest. 2008; 118(2):608-618.
[0181] Matsumoto M, et al. Abrogation of the alternative complement
pathway by targeted deletion of murine factor B. Proc Natl Acad Sci
USA. 1997; 94(16):8720-8725. [0182] Kulik L, et al. Pathogenic
natural antibodies recognizing annexin IV are required to develop
intestinal ischemia-reperfusion injury. J Immunol. 2009;
182(9):5363-5373. [0183] Hourcade D E, Wagner L M, Oglesby T J.
Analysis of the short consensus repeats of human complement factor
B by site-directed mutagenesis. J Biol Chem. 1995;
270(34):19716-19722. [0184] Whaley K. Measurement of complement.
In: Whaley K, ed. Methods in Complement for Clinical Immunologists.
New York, N.Y., USA: Churchill Livingstone; 1985:77-139.
[0185] Although the foregoing subject matter has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be understood by those skilled in
the art that certain changes and modifications can be practiced
within the scope of the appended claims.
* * * * *
References