U.S. patent application number 16/000710 was filed with the patent office on 2020-11-12 for methods of treating hemolytic disorders comprising administering an anti-c3b antibody.
The applicant listed for this patent is NOVELMED THERAPEUTICS, INC.. Invention is credited to Rekha Bansal.
Application Number | 20200354440 16/000710 |
Document ID | / |
Family ID | 1000005178505 |
Filed Date | 2020-11-12 |
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United States Patent
Application |
20200354440 |
Kind Code |
A9 |
Bansal; Rekha |
November 12, 2020 |
METHODS OF TREATING HEMOLYTIC DISORDERS COMPRISING ADMINISTERING AN
ANTI-C3b ANTIBODY
Abstract
A method of treating a hemolytic disorder in a subject in need
thereof includes administering to the subject a therapeutically
effective amount of an antibody that binds to a component of
alternative pathway C3 convertase and selectively inhibits C3a,
C5a, C3b, C5b, and C5b-9 produced exclusively by the alternative
pathway, without inhibiting any of the classical pathway's ability
to produce C3a, C5a, C3b, C5b, and C5b-9.
Inventors: |
Bansal; Rekha; (Cleveland,
OH) |
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Applicant: |
Name |
City |
State |
Country |
Type |
NOVELMED THERAPEUTICS, INC. |
Cleveland |
OH |
US |
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20190031747 A1 |
January 31, 2019 |
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Family ID: |
1000005178505 |
Appl. No.: |
16/000710 |
Filed: |
June 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14679713 |
Apr 6, 2015 |
9988441 |
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16000710 |
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PCT/US2013/063401 |
Oct 4, 2013 |
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14679713 |
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61709796 |
Oct 4, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 2317/76 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1-57. (canceled)
58. A method of treating a hemolytic disorder in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of an anti-C3b antibody, wherein
the anti-C3b antibody or antigen binding fragment thereof
comprises: a light chain variable region that includes three CDRs
having the amino acid sequences of SEQ ID NOs: 73-75 and a heavy
chain variable domain that includes three CDRs having an amino acid
sequence of SEQ ID NOs: 40-42.
59. The method of claim 58, the anti-C3b antibody or antigen
binding fragment thereof being administered at an amount effective
to prevent C3b formation responsible for extravascular hemolysis
and C5b-9 responsible for intravascular hemolysis.
60. The method of claim 58, the hemolytic disorder being selected
from the group consisting of Paroxysmal Nocturnal Hemoglobinuria
(PNH), Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic
Thrombocytopenic Purpura (TTP), Hemolytic-Uremic Syndrome (HUS),
Disseminated Intravascular Coagulation (DIC), Antiphospholipid
Syndrome (APS), Post-Transfusion Purpura, and Immune
Thrombocytopenia (NAITP).
61. The method of claim 58, wherein the hemolytic disorder is
associated with C3b induced activation of blood cells and the
anti-C3b antibody or antigen binding fragment thereof is
administered at amount effective to inhibit C3b induced activation
of blood cells.
62. The method of claim 61, wherein the activation of blood cells
includes neutrophil activation, monocyte activation, platelet
activation and T-lymphocyte activation.
63. The method of claim 58, wherein the anti-C3b antibody or
antigen binding fragment thereof is administered to the subject
with one or more symptoms selected from the group consisting of:
(a) the subject has red blood cells opsonized with C3b; (b) the
subject has leukocytes opsonized with C3b; (c) the subject has
platelets opsonized with C3b; (d) the subject has anemia; (e) the
subject has higher than normal levels of LDH; (f) the subject has
higher than normal levels of free hemoglobin; (g) the subject has
lower than normal levels of platelets; (h) the subject has higher
than normal levels of reticulocyte counts; and (i) the subject has
higher than normal levels of bilirubin.
64. The method of claim 63, wherein the anti-C3b antibody or
antigen binding fragment thereof reduces all or one of listed
symptoms (a)-(i) to normal levels.
65. The method of claim 58, wherein the subject is being treated
for extravascular hemolysis.
66. A method of treating cellular and/or tissue damage caused by
alternative complement pathway induced inflammation in a subject,
the method comprising administering to the subject a
therapeutically effective amount of an anti-C3b antibody or antigen
binding fragment thereof, wherein the anti-C3b antibody or antigen
binding fragment thereof comprises: a light chain variable region
that includes three CDRs having the amino acid sequences of SEQ ID
NOs: 73-75 and a heavy chain variable domain that includes three
CDRs having an amino acid sequence of SEQ ID NOs: 40-42.
67. The method of claim 66, wherein the anti-C3b antibody or
antigen binding fragment thereof is administered at amount
effective to inhibit C3b induced activation of blood cells.
68. The method of claim 67, wherein the activation of blood cells
includes neutrophil activation, monocyte activation, platelet
activation and T-lymphocyte activation.
69. The method of claim 66, wherein the tissue damage is associated
with cellular damage.
70. The method of claim 66, wherein the tissue damage is associated
with organ damage.
Description
RELATED APPLICATION
[0001] This application claims priority benefit of U.S. Provisional
Patent Application Ser. No. 61/709,796, filed on Oct. 4, 2012, the
content of which is incorporated herein by reference in its
entirety.
BACKGROUND
[0002] The complement system can be activated through three
distinct enzymatic cascades, referred to as the "classical
pathway", "Lectin/MBL", and "alternative" pathway" (CP, MBL, and AP
respectively). MBL is not discussed here. The classical pathway is
responsible for aiding in host defense against antigens to prevent
infection of cells. The lectin pathway is a variation of the
classical pathway. The alternative pathway is currently thought to
be responsible for 80-95% of total complement activity in cases
where trigger of complement activation is the classical pathway
("AP amplification loop"). The alternative pathway by itself is
activated in a number of disease indications where complement
components have been found in elevated state.
[0003] There are three "alternative pathway specific proteins";
Factors B, D, and P, which play a major role in the; a) initiation
and propagation of the alternative pathway and b) classical pathway
propagation via the alternative pathway amplification loop.
Proteins C3 and C3b, the key players in complement system, are
common to all classical and alternative complement pathways. While
there may be a one type of C3, there are three different types of
C3b produced as each C3 convertase is different. AP C3 convertase
is composed of PC3bBb, the classical C3 convertase is made up of
different proteins. Therefore it is hard to believe that the cut
would be all identical to produce similar C3b molecules. As a
result, C3b produced by the alternative pathway is different
compared to C3b produced via the classical pathway.
[0004] The classical pathway (CP) is initiated by antigen-antibody
complex. The CP progression involves proteins such as C1Q, C1r/C1s,
C4, and C2. The CP C3 convertase consists of C3bC4b2a. This complex
can cleave the C3 into C3b and C3a. This C3b is derived from
classical pathway convertase and is usually required for
opsonization of various pathogens and bacteria. Inhibition of this
C3b is undesirable. C3b coated cells are removed via complement
receptors present on various cells.
[0005] Both complement pathways independently produce C3a, C3b,
C5a, C5b, C5b-9, and sC5b-9 as complement activation
byproducts.
[0006] During classical pathway triggered activation of the
alternative pathway, Classical pathway C3 convertase also cleaves
C3 into C3b which can work independent of the alternative pathway
with full amplification of the classical pathway in 1% normal human
serum in the presence of Ca.sup.2+/Mg.sup.2+ ions. Classical
pathway C5 convertase can cleave C5 to generate C5a and C5b. The
C5b molecule then inserts into the lipid bilayer of the cell to
initiate the formation of C5b-9 or sC5b-9.
[0007] In alternative pathway activation, C3b produced by the
complement system can bind properdin and Factor B to form the
complex "PC3bB". Factor D then cleaves Factor B, within the
complex, into Bb and Ba. This cleavage results in the release of Ba
from the complex and the forma.sup.tion .sup.of the AP convertase
PC3bBb. PC3bBb cleaves C3 into C3a and C3b, thereby perpetuating
the amplification loop of the alternative pathway for the benefit
of the alternative pathway. PC3bBb can then cleave C5 to make C5b
and C5a. The C5b molecule then inserts into a lipid bilayer of a
cell and forms the nucleus for MAC deposition.
[0008] The classical pathway can also initiate the propagation of a
part of the alternative pathway known as the amplification loop.
Within the amplification loop, C3b binds properdin and Factor B to
form the complex "PC3bB". Factor D then cleaves Factor B, within
the complex, into Bb and Ba. This cleavage results in the release
of Ba from the complex and the formation of the AP convertase
PC3bBb. PC3bBb cleaves C3 into C3a and C3b, thereby perpetuating
the amplification loop.
[0009] C3b is therefore both a component and a byproduct of the
complement system irrespective of the type of complement pathway
activation. During the amplification of the AP, as the PC3bBb (AP
C3 Convertase) generates increasing amounts of C3b, an
amplification loop is established so that activation of the
alternative pathway can continue. Furthermore, the classical
pathway can also generate C3b, which can bind factor B and thereby
engage the alternative pathway, even though the trigger is CP
mediated. This allows more C3b to deposit on a target, which leads
to enhanced amplification of AP activation.
[0010] Addition of newly formed C3b to the existing AP C3
convertase PC3bBb generates the AP C5 convertase. Addition of newly
formed C3b to the existing CP C3 convertase generates CP C5
convertase. Both C5 convertases have the ability to cleave C5 to
produce C5b and C5a. The terminal complex produced as a result of
complement activation is known as the MAC complex (also known as
C5b-9 or sC5b-9), which is responsible for lysis of cells in a
subject. Both C3a and C5a are potent anaphylatoxins that are
responsible for activating platelets, neutrophils, and monocytes.
As a result, inflammatory molecules such as elastase, TNF-.alpha.,
IL-1, VEGF, and peroxides are released. Formation of C5b-9/sC5b-9
is responsible for tissue damage and tissue injury/tissue damage
seen in "other diseases"
[0011] Classical complement pathway activation provides a valuable
first-line defense against potential pathogens and can generate
C3a/C3b, C5a/C5b, and C5b-9/sC5b-9. Therefore, exacerbation of the
classical pathway can produce large amounts of complement
byproducts. As described elsewhere, both C3a and C5a are potent
anaphylatoxins, C3b mediates opsonization, and C5b is responsible
for wanted killing of the pathogens. Here, both C3a and C5a would
generate beneficial responses and are produced to kill the
invaders. This pathway is required for host defense and therefore
must not be inhibited.
[0012] Alternative pathway activation in Mg++ ions, without the
calcium ions, guarantees only the AP activation. In disease state,
this pathway is activated independent of the classical pathway.
This pathway is not required for host defense and therefore can be
inhibited in its entirety.
SUMMARY
[0013] Embodiments described herein relate to antibodies that
prevent C3b formation responsible for extravascular hemolysis and
C5b-9 responsible for intravascular hemolysis. The invention
further relates to methods for treatment of subjects suffering from
disorders that involve lysis of red blood cells and platelets via
intravascular and extravascular route. The invention also covers
protection of neutrophils, monocytes, platelets, and T-lymphocytes
against complement attack. This is accomplished by antibodies of
the claimed genus that block the formation and deposition of C3b on
cells and C5b-9 on cells that are deficient in GPI linked
proteins.
[0014] This application summarizes a group of complement inhibitor
monoclonal antibodies that prevent the formation of alternative
pathway derived C3b and C5b-9 formation. These antibodies are being
claimed as a genus in this particular application. Although these
antibodies bind different targets within the alternative pathway,
they have unique feature as they all inhibit alternative pathway
generated C3b called 'C3b'' but not the classical pathway generated
C3b.
[0015] It is Removal of cells causes cytopenia depending upon the
cell type under attack--neutropenia, monocytopenia,
thrombocytopenia, lymphocytopenia, and leukopenia. Thus, inhibition
of AP activation by a claimed genus of monoclonal antibodies can
prevent cytopenia in a subject (human) Cytopenia is commonly
observed in hematological disorders such as Paroxysmal Nocturnal
Hemoglobinuria (PNH), Idiopathic Thrombocytopenic Purpura (ITP),
Thrombotic Thrombocytopenic Purpura (TTP), Hemolytic-Uremic
Syndrome (HUS), Disseminated Intravascular Coagulation (DIC),
Antiphospholipid Syndrome (APS), Post-Transfusion Purpura, Neonatal
Allo-Immune Thrombocytopenia (NAITP). The antibodies of the claimed
genus are capable of preventing cytopenia, cellular activation,
cell dysfunction, inflammation, extravascular hemolysis,
intravascular hemolysis and tissue injury.
[0016] Both the classical and the alternative pathways upon
activation produce C3b molecules. The two C3b although called the
same but are different. C3b molecules produced by alternative
pathway but not the classical pathway in PNH bind erythrocytes,
neutrophils, monocytes, platelets, and T lymphocytes. This binding
results in clearance of such cells via extravascular hemolysis.
Removal via extravascular hemolysis causes cytopenia and increased
levels of bilirubin and LDH. C5b-9 (also known as MAC) deposits
onto the cell membrane and results in lysis of anucleated cells
such as erythrocytes and platelets. Clear evidence of C3b
deposition and C5b-9 deposition have not been reported previously.
Erythrocyte lysis results in increased LDH levels, increased
reticulocyte counts and decreased levels of hemoglobin in
erythrocytes.
[0017] In PNH, we found that C3b and C5b-9 have been associated
with both anucleated and nucleated cells. These patterns of C3b and
C5b-9 binding to a variety of cells deficient in GPI linked
proteins suggests destruction/partial destruction, activation, or
dysfunction of such cells. Current invention is to prevent the
formation and deposition of such molecules on a variety of
anucleated and nucleated cells that are responsible for
pathological outcomes in diseases where the absence of GPI liked
proteins is associated with pathology. Neutralizing antibodies that
prevent the formation of C3b and C5b-9 via the alternative pathway
are covered under this invention.
[0018] As an example of hematological disorder where cytopenia
occurs is PNH. Cytopenia covers leukopenia, neutropenia,
monocytopenia, thrombocytopenia, lymphocytopenia. Nearly all types
of cells appear to be deficient in GPI linked proteins in PNH. Such
cells are subject to complement attack via C3b deposition and
extravascular removal and/or destruction via extravascular route.
All antibodies that are selective blocker of only alternative
pathway-derived C3b and C5b-9 are covered under this invention. A
set of such antibodies that perform such function are covered under
this invention.
[0019] T-lymphocytes, monocytes, and neutrophils are all deficient
in GPI linked proteins and therefore are subject to complement
attack and deposition of C3b and C5b-9. C3b coated cells would be
deprived of the proper function and C5b-9 desposition would cause
cell death resulting in loss of the cells. As an example, the
neutrophil is the key cell fighting bacterial and fungal infection
in the body. These neutrophils in PNH patients may not ingest germs
effectively and are therefore less able to fight infection. These
patients, whose white blood cells don't work properly, are much
more likely to develop a second infection. It is known via the
laboratory testing that by adding GM-CSF in the laboratory testing,
it is possible to restore the ability of the white blood cells to
ingest bacteria and fight infection. Thus addition of GM-CSF is
proposed as a potential use in patients to increase the ability of
neutrophils to behave normally The main function of GM-CSF is
known. The protein is a cytokine that functions as a white blood
cell growth factor in general. GM-CSF stimulates stem cells to
produce granulocytes (neutrophils, eosinophils, and basophils) and
monocytes. Thus increased amount of GM-CSF seen in the PNH blood
samples indicates that PNH cells are dying. Thus adding GM-CSF with
and without the claimed genus of antibodies could help improve the
cell quality in general and increase the ability of cells to fight
infections.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a schematic showing the complement cascade of the
classical pathway and the alternative pathway. Lectin pathway is
not shown as it is not the part of the invention. The FIG. 1 shows
a schematic representation of the CP and the AP. In this figure, we
show that both CP and AP are distinct and not connected. The
schematic only shows how the antibodies of the current genus work
and not the way antibodies of the other invention work. AP
amplification is shown in the upper right hand side and consists of
PC3b, PC3bB, PC3bBb. As can be seen in the schematic, PC3bBb then
acts to perpetuate the cycle by cleaving C3 into more C3b which
binds to P to again form PC3b. Application of the invention
completely inhibits the alternative pathway without affecting the
classical pathway by specifically targeting the components of this
amplification loop. These antibodies prevent the amplification loop
of the alternative complement pathway without affecting the
classical pathway (as shown on the left side of the schematic in
FIG. 1).
[0021] Based on the old convergence theory describing C3 being the
convergence point, those with ordinary skill in the art would
expect any activation of the classical pathway to invariably have
the effect of alternative pathway activation. This is because the
two pathways are believed to "overlap" at the starting point of the
C3. According this theory, C3b produced via the classical pathway
participates in the AP amplification loop. The invention that is
the subject of this patent is the development of a new and unique
genus of complement inhibiting antibodies which challenge that
assumption. The claimed invention, this new genus of antibodies,
specific targets components of the alternative pathway
amplification loop in such a way as to inhibit the alternative
pathway regardless of whether or not the AP amplification loop has
been otherwise triggered by the classical pathway. Thus anti-C3b
antibodies of the current invention only inhibit the AP and not the
CP amplification loop or the CP propagation. The uniqueness of the
invention is not only which components these antibodies target, but
how they target those components. Similarly, we describe the
anti-Ba, anti-Bb, and-P and anti-C3b for the invention.
[0022] FIG. 2 illustrates three assay figure tracings from real
data generated from one of the invented antibodies as a
representative FIG. 4. One line represents untreated sample whereas
the second line represents the antibody treated sample. The Panel A
is a CP assay conducted in 1% NHS in CP buffer. The second panel is
a CP assay in 10% NHS that allows CP amplification loop to
contribute into the AP. The third panel (Panel C) shows inhibition
by the invented antibodies of the genus that inhibit the AP without
affecting the CP (Panel B). All antibodies showing this pattern
would belong to the invented genus.
[0023] FIG. 3 is plots showing the binding affinities of the
invented antibodies to their respective targets (C3b, Bb, and
P).
[0024] FIG. 4 is a graph showing that the invented antibodies
inhibit alternative pathway dependent hemolysis of rabbit
erythrocytes (rRBC) in Human Serum (NHS). There exist a multitude
of antibodies which inhibit the activities of Properdin (Factor P),
Factor Bb, and C3b. All such antibodies inhibit the alternative
pathway and not the CP (FIG. 7). However, these antibodies will act
on their targets in such a way as to inhibit the alternative
pathway without inhibiting the classical pathway.
[0025] FIG. 5 is a graph showing that the invented antibodies do
not inhibit classical pathway dependent lysis of Antibody
Sensitized Sheep Erythrocytes (sRBC). The current state of the act
teaches that activation of the classical pathway invariably results
in activation of the alternative pathway at the amplification loop,
which begins with cleavage of C3 by CP produced C3 convertase. The
claimed invention makes possible the therapeutic inhibition of the
alternative pathway, despite classical pathway activity. As shown,
the Anti-C3b, Anti-Ba, Anti-Bb, and Anti-P antibodies of the
invented genus do not inhibit the classical pathway and are
specific to the alternative complement pathway (FIGS. 2-3).
Therefore, the invention could have potential application in any
disease characterized or mediated by a pathological over-activation
of the alternative complement pathway.
[0026] FIG. 6 illustrates plots showing that the invented
antibodies inhibit the formation of C3b in serum, a marker for
extravascular hemolysis.
[0027] FIG. 7 illustrates plots showing that the invented
antibodies inhibit the formation of C5b-9 in serum, a marker for
intravascular hemolysis.
[0028] FIG. 8 is a graph showing that the invented antibodies
inhibit the formation of C3a in Whole Blood Inflammation. Both C3a
(cleaved from C3) and C5a (cleaved from C5) are potent
anaphylatoxins (triggers of local inflammation) that are produced
upon complement activation. Both the classical pathway and the
alternative pathway produce these molecules. The Figure shows the
inhibition of C3a derived from the alternative complement pathway.
Classical pathway trigger does not exist in this model.
[0029] FIG. 9 is a graph showing that the invented antibodies
inhibit the formation of C5a in Whole Blood Inflammation. The
claimed invention selectively inhibits C3a (FIGS. 8) and C5a (FIG.
9) produced from the alternative pathway.
[0030] FIGS. 10A-D illustrate graphs showing that the invented
antibodies Inhibit formation of sC5b-9 in Whole Blood.
[0031] FIG. 11 is a graph showing that the invented antibodies
inhibit neutrophil activation. The neutrophils activation occurs
due to the activation of the AP and not CP or CP-induced AP.
[0032] FIG. 12 is a graph showing that the invented antibodies
inhibit monocyte activation. The monocyte activation occurs due to
the activation of the AP and not CP or CP-induced AP.
[0033] FIG. 13 is a graph showing that the invented antibodies
inhibit platelet activation. The platelet activation occurs due to
the activation of the AP and not CP or CP-induced AP.
[0034] FIG. 14 is a graph showing that the invented antibodies
inhibit monocyte-platelet aggregates. The monocyte-platelet
aggregation occurs due to the activation of the AP and not CP or
CP-induced AP.
[0035] FIG. 15 is a graph showing that the invented antibodies
inhibit elastase release from neutrophils. The neutrophil elastase
is produced from neutrophils that are activated via the C3a/C5a
produced from the alternative pathway.
[0036] FIG. 16 is a graph showing antibodies of the invention
prevent LDH release in an in vivo model of PNH.
[0037] FIG. 17 is a graph showing antibodies of the invention
prevent HgB release in vivo in a model of PNH.
[0038] FIG. 18 illustrates a graph showing cells in blood from PNH
patients.
DETAILED DESCRIPTION
Definitions
[0039] Unless specifically defined herein, all terms used in this
document have the same meaning as would be understood by those of
ordinary skill in the art of the present invention. The following
definitions are provided for clarity, and to define their intended
meaning as used in the specification and claims to describe the
present invention.
Definitions--Complement Pathways
[0040] "CLASSICAL PATHWAY" refers to complement which is triggered
by antigen-antibody complexes for activation and may or may not
also trigger the alternative pathway amplification loop for its
propagation.
[0041] "ALTERNATIVE PATHWAY" refers to complement activation which
is triggered by a cell surface (or cell-surface like material)
looking like a foreign surface. The absence of GPI linked protein
makes the surface of the PNH cell foreign enough to activate the
alternative pathway. The alternative pathway may also begin with
spontaneous proteolytic generation of C3b from complement factor
C3, where C3b has the ability to bind B and P both.
[0042] "ALTERNATIVE PATHWAY SPECIFIC PROTEIN" refers to C3b, factor
B, factor Bb, factor D, and/or properdin. Here C3b refers to C3b as
a part of the AP and not CP.
[0043] "AP AMPLIFICATION LOOP" refers to a looping series of
reactions in which C3b formed makes AP C3 convertase. This
convertase cleaves C3 and generates more C3b, which feeds back into
the loop. This self-perpetuating cycle of reactions generates large
amounts of C3b.
[0044] "C3b" is term used for both C3b derived from AP and CP
pathways.
[0045] "ALTERNATIVE PATHWAY-DEPENDENT C5a" describes the formation
of C5a produced from activity of the alternative pathway of the
complement system in whole blood. For example, "AP-dependent C5a
formation" refers to the formation of C5a via activation of the
alternative pathway, which is independent of the classical
pathway.
[0046] "ALTERNATIVE PATHWAY-DEPENDENT sC5b-9" describes the
formation of sC5b-9 produced from activity of the alternative
pathway of the complement system. For example, "AP-dependent sC5b-9
(soluble MAC) formation" refers to the formation of sC5b-9 via
activation of the alternative pathway, which is independent of the
classical pathway.
[0047] "ALTERNATIVE PATHWAY-DEPENDENT C5b-9" describes the
formation of C5b-9 produced from activity of the alternative
pathway of the complement system. For example, "AP-dependent C5b-9
formation (Deposited MAC)" refers to the formation of C5b-9 via
activation of the alternative pathway, which is independent of the
classical pathway.
[0048] "C3a DEPENDENT CELLULAR ACTIVATION" describes the activation
of neutrophils, monocytes, platelets, T lymphocytes, endothelial
cells, mast cells, and platelets which occurs when Alternative
Pathway-Dependent C3a binds to C3a receptors, which are present on
these cells. These cells are found, in their C3a activated state,
in various different diseases (see OTHER DISEASES).
[0049] "C5a DEPENDENT CELLULAR ACTIVATION" describes the activation
of neutrophils, monocytes, platelets, T lymphocytes, endothelial
cells, mast cells, and platelets which occurs when Alternative
Pathway-Dependent C5a binds to C5a receptors, which are present on
these cells. These cells are found, in their C5a activated state,
in various different diseases (see "OTHER DISEASES").
[0050] "C5b-9 and sC5b-9 DEPENDENT TISSUE INJURY/CELLULAR DAMAGE"
describes the cellular damage caused by the formation of sC5b-9
and/or C5b-9. These molecules either bind to the cellular surface
and/or insert themselves into the cell's plasma membrane resulting
in pathological conditions also described as "TISSUE INJURY".
Tissue injury occurs in various diseases and can result in the
damage to various organs.
[0051] "MEMBRANE ATTACK COMPLEX" ("MAC") refers to a complex of the
terminal five complement components (C5b-C9) that inserts into and
disrupts cell membranes. This complex is also referred to as C5b-9.
MAC complex is produced by both the alternative pathway and by the
classical complement pathway. The complex that is associated with
"S protein" is called sC5b-9, a soluble form of MAC. The invented
antibodies inhibit alternative pathway associated C5b-9 and
sC5b-9.
[0052] "C3a, C5a, C5b-9, sC5b-9 AND INFLAMMATION" describes
inflammation caused by the products of AP activation and activity;
and in particular, the AP products C3a, C5a, C5b-9, and sC5b-9
generating from AP activity. These molecules cause C3a DEPENDENT
CELLULAR ACTIVATION, C5a DEPENDENT CELLULAR ACTIVATION, C5b-9 and
sC5b-9 DEPENDENT CELLULAR DAMAGE, and result in the prevalence of
CYTOKINE ACTIVATED CELLS, PROTEASE ACTIVATED CELLS, and PEROXIDE
ACTIVATED CELLS, all of which can be implemented in various
different diseases and disease pathologies.
Definitions--Whole Blood & Inflammation
[0053] "WHOLE BLOOD" describes complete blood with the same
composition of cells, chemicals, proteins, etc. as blood found in
human blood vessels. The isolated blood contains all components of
the complement system including inflammatory cells that are
responsible for inflammatory responses.
[0054] "INFLAMMATION IN WHOLE BLOOD" describes the cascade of
reactions beginning with alternative pathway activation in whole
blood, the resulting production of C3a, C5a, and C5b-9 and sC5b-9
in whole blood, the resulting activation of neutrophils monocytes
and platelets in whole blood, and ultimately, the production of
inflammatory cytokines in whole blood (in vivo or ex vivo). Several
inflammatory mediators are found to be secreted into the plasma.
These inflammatory mediators are TNF, IL-1, IL-6, IL-8 and several
others. Not included in the list.
[0055] "ALTERNATIVE PATHWAY (AP)-DEPENDENT INFLAMMATION IN
HEMOLYTIC DISEASES" refers to an increase in alternative complement
pathway activity, as measured by continued or increased formation,
and/or release, of one or more of the following components C3a,
C3b, C5a, C5b-9, or sC5b-9, and all the anticipated consequences
thereof, in PNH and other hemolytic diseases. Such anticipated
consequences include; continued or increased AP-dependent
MAC-mediated deposition and/or lysis of cells, continued or
increased AP-dependent activation of platelets, monocytes,
neutrophils, mast cells, or basophils; and/or continued or
increased AP-dependent formation or release of TNF-.alpha., IL-1,
or neutrophil elastase.
[0056] "OTHER ORPHAN AND NON_ORPHAN HEMATOLOGICAL AND NON
HEMATOLOGICAL ACUTE AND CHRONIC DISEASES" describes a list of
diseases where one of the elevated components measured is derived
from the activation of the alternative pathway system. These
components include but not limited to; C3a/C3b, P, Ba/Bb, C5a/C5a,
and C5b-9 /sC5b-9. Elevated levels of these components have been
found associated with one or more diseases. These components are
responsible for cellular activation and release of inflammatory
mediators. These, in turn, ultimately cause tissue damage, defining
the disease in both hematological and non-hematological
diseases.
[0057] "ALTERNATIVE PATHWAY (AP)-DEPENDENT INFLAMMATION IN PNH"
refers to an increase in alternative complement pathway activity,
as measured by continued or increased formation, and/or release, of
C3a, C3b, C5a, C5b, C5b-9, and/or sC5b-9, and all the anticipated
consequences thereof. Such anticipated consequences include;
continued or increased AP-dependent C3b and MAC-mediated deposition
or lysis of cells, continued or increased AP-dependent activation
of platelets, monocytes, neutrophils, mast cells, or basophils;
and/or continued or increased AP-dependent formation or release of
TNF-.alpha., IL-1, or neutrophil elastase.
[0058] "AUTOIMMUNE DISEASE" refers to a condition where the immune
response of a subject is inappropriately directed against
substances and tissues normally present in the body.
[0059] "CELLULAR LYSIS" indicates tissue injury in part. Cellular
lysis occurs as a result of C5b-9 formation of the cell surface.
Such deposition of C5b-9 leads to cellular injury and in case of
tissues the cell injury is a tissue injury.
Definitions--Inhibitory Antibodies and Agents
[0060] "AGENT" "COMPOUND" refers to any substance, molecule,
element, compound, entity, or any combination thereof. An agent can
be, among other things, a protein, oligopeptide, small organic
molecule, polysaccharide, polynucleotide, or other biochemical
substance. It can be a natural product, a synthetic compound, a
chemical compound, or a combination of two or more substances of
different origins. Unless otherwise specified, the terms "agent",
"substance", and "compound" can be used interchangeably.
[0061] "Alternative pathway specific antibody" refers to an
antibody or fragment thereof that can bind to an alternative
pathway protein to inhibit activation and/or progression of the
alternative pathway in a subject.
[0062] "ANTIBODIES TO AP PROTEINS" describe anti-P, anti-Ba,
anti-Bb, anti-C3b antibodies that neutralize the activity of the
alternative pathway without inhibiting the classical pathway.
Definitions--Pharmacology
[0063] "PHARMACOKINETIC ACTIVITY" or "PHARMACOKINETICS" refers to
the mechanisms of absorption and distribution of an administered
drug, the rate at which a drug action begins and the duration of
the effect, the chemical changes of the substance in the body, and
the effects and routes of excretion of the metabolites of the
drug
[0064] "THERAPEUTICALLY EFFECTIVE AMOUNT" is defined as an amount
sufficient to completely inhibit AP activity in vivo
[0065] As used herein, a "prophylactically effective amount" is
defined as an amount sufficient to prevent the onset of a disease
or disorder in a subject.
[0066] As used herein, the terms "administering," "administration,"
and like refer to ways in which the antibody or antigen binding
fragment thereof can be given to the subject, including, but not
limited to, oral administration, intravenous administration,
intraperitoneal, intramuscular, subcutaneous administration, aural
administration, or rectal administration.
Definitions--Antibodies
[0067] "ANTIBODY" used in the broadest sense includes monoclonal
antibodies, including full length or partial length monoclonal
antibodies, and polyclonal antibodies from mouse, rabbit or human
species. The antibodies can also be egenrated in other mammalas. In
its most widely recognized form, an antibody contains two heavy (H)
chains and two light (L) chains inter-connected by disulfide bonds.
Each heavy chain is comprised of a heavy chain variable region
(abbreviated herein as VH) and a heavy chain constant region. The
heavy chain constant region is comprised of three domains, CH1, CH2
and CH3. Each light chain is comprised of a light chain variable
region (abbreviated herein as VL) and a light chain constant
region. The light chain constant region is comprised of one domain,
CL. The VH and VL regions can be further subdivided into regions of
hyper-variability, termed complementarity determining regions
(CDRs), interspersed with regions that are more conserved, termed
framework regions (FR). Each VH and VL is composed of three CDRs
and four Frameworks arranged from amino-terminus to
carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2,
1-R3, CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The term
"antibody" encompasses whole antibodies and antibody fragments
thereof, derived from any antibody-producing mammal (e.g., mouse,
rat, rabbit, and primate including human), that specifically bind
to proteins such as properdin, C3b, Ba, and Bb or portions thereof.
Exemplary antibodies include polyclonal, monoclonal and recombinant
antibodies; multi-specific antibodies (e.g., bispecific
antibodies); humanized antibodies; murine antibodies; chimeric,
mouse-human, mouse-primate, primate-human monoclonal antibodies;
and anti-idiotype antibodies, and may be any intact molecule or
fragment thereof.
[0068] "OTHER ANTIBODIES" refer to antibodies developed in living
organism including and not limited to animals and humans for
therapeutic use in humans and animals. Any antibodies raised in a
living organism is capable of inhibiting AP mediated lysis
(Assay-3) but not the CP mediated lysis or the CP amplification
loop.
[0069] "ANTIBODY FRAGMENT" refers to a portion derived from or
related to a full-length antibody, particularly an anti-C3b,
anti-P, and anti-Ba, or anti-Bb antibody, generally including the
antigen binding or variable region thereof (see "ANTIGEN BINDING
FRAGMENT"). The term "antibody fragment" refers to a portion
derived from a full-length alternative pathway inhibitory antibody,
generally including the antigen binding and variable region
thereof. Other antibodies include nano bodies, diabodies, linear
antibodies, single-chain antibody molecules and multispecific
antibodies formed from antibody fragments. Examples of antibody
fragments include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, or
scFv fragments (and any PEGylated variations of any of the
forgoing).
[0070] "ANTIGEN BINDING FRAGMENT" of an antibody refers to the one
or more fragments of an intact antibody that retain the ability to
specifically bind to a given antigen.
[0071] Antigen binding functions of an antibody can be performed by
fragments of an intact antibody containing the Complementarity
Determining Regions (CDRs). Examples of antigen binding
fragments:
[0072] "Fab" fragments (single chain variable regions with VH and
VL);
[0073] "Monovalent Fragments" (antibody fragments consisting of the
VL, VH, CL and CH1 domains);
[0074] "F(ab')2" fragments (bivalent fragments comprising two Fab
fragments linked by a disulfide bridge at the hinge region);
[0075] "Fd" fragments (which consist of the VH and CH1 domains of
an antibody);
[0076] "Fv" fragment (which consist of the VL and VH domains of a
single arm of an antibody);
[0077] single domain antibody ("dAb"), which consist of a VH domain
or a VL domain;
[0078] an isolated Complementarity Determining Region ("CDR").
[0079] A "FUNCTIONAL DERIVATIVE" of an antibody is any compound
which is either taken from, or incorporates within itself, the
functional region of the antibody. Functional derivatives of
antibodies include, but are not limited to, antigen binding
fragments, CDRs, humanized antibodies, "Fab" fragments, "Fd"
fragments, chimeric antibodies, monoclonal antibodies, recombinant
antibodies, and single chain antibodies.
[0080] CDRs, as antigen binding fragments, can also be incorporated
into single domain antibodies, maxi bodies, mini bodies,
intrabodies, diabodies, triabodies, tetra bodies, v-NAR and
bis-scFv. Antigen binding fragments of antibodies can be grafted
into scaffolds based on polypeptides such as Fibronectin type III
(Fn3). Antigen binding fragments can be incorporated into single
chain molecules comprising a pair of tandem Fv segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions.
[0081] As used herein, the term "Fc region" refers to the region of
the antibody that induces effector functions.
[0082] "AFFINITY" refers to the chemical strength of the
interaction between an antibody and an antigen at single antigenic
sites.
[0083] "BINDING SPECIFICITY" refers to the ability of an individual
antibody or antigen binding fragment to bind to a particular
target, e.g., the binding specificity of an antibody to bind only
to its target.
[0084] "COMPOUNDS," "BLOCKER", "INHIBITOR", or "ANTAGONIST" refers
to a chemical substance, or force, that retards or prevents a
chemical or physiological reaction or response. Common blockers or
inhibitors include, but are not limited to, antisense molecules,
antibodies, antagonists and their derivatives. For example, an
antibody that binds to a component of an AP specific interaction
between that component and another component of the AP. Such an
antibody would be an inhibitor or blocker of that interaction and,
by extension, the AP.
[0085] "CHIMERIC ANTIBODY" is a recombinant protein that contains
the variable domains and CDRs derived from an antibody of from a
non-human species of animal, while the remainder of the antibody
molecule is derived from a human antibody. The replacement of the
non-binding region of the antibody with a human constant region
enables the chimeric antibody to retain its specificity in
recognizing and binding the targeted antigen while having reduced
antigenicity in humans (compared to the original mouse
antibody).
[0086] "HUMANIZED ANTIBODY" is an antibody that consists of
non-human CDRs and humanized framework regions. Humanized
antibodies are typically recombinant proteins in which only the
antibody complementarity-determining regions are of non-human
origin.
[0087] As used herein, a "single-chain Fv" or "scFv" antibody
fragment comprises the VH and VL domains of an antibody, wherein
these domains are present in a single polypeptide chain.
[0088] As used herein, the term immunogenicity refers to the
ability of an antigen to initiate an immune response in a
subject.
[0089] "COMPLEMENTARITY DETERMINING REGIONS (CDRs)" are the key
binding regions of the antibody. There are typically three CDRs
found within the variable regions of each of the two heavy and
light chain variable regions. CDRs can be shuffled around, in terms
of location, to create a particular binding affinity. See also
"ANTIGEN BINDING FRAGMENTS."
[0090] "EFFECTOR FUNCTIONS" refer to those biological activities
attributable to the native Fc region of an antibody, and vary with
the antibody isotype. Examples of antibody effector functions
include: C1q binding and complement dependent cytotoxicity; Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g., B cell receptor); lack of activation of platelets that
express Fc receptor; and B cell activation. In order to minimize or
eliminate side effects of a therapeutic antibody, it may be
preferable to minimize or eliminate effector functions.
[0091] As used herein, the term "reduced Fc effector function(s)"
refers to the function(s) of an antibody wherein the antibody does
not act against an antigen that recognizes the Fc region of the
antibody. Examples of reduced Fc effector functions can include,
but are not limited to, reduced Fc binding to the antigen, lack of
Fc activation against an antigen, an Fc region that contains
mutations to prevent normal Fc effector functions, or prevention of
the activation of platelets and other cells that have Fc
receptors.
[0092] "HUMAN ANTIBODY" is an antibody in which all components of
the antibody are of human origin, including the framework, CDRs,
and constant regions. The term "humanized" antibody is an antibody
of non-human origin that retains the binding specificity of the
non-human antibody while being less immunogenic in humans See
CHIMERIC ANTIBODY and HUMANIZED ANTIBODY.
[0093] "PURIFIED ANTIBODY" refers to antibodies which have been
isolated from contaminants. In preferred embodiments, the antibody
will be purified (1) to greater than 95% by weight of antibody as
determined by the Lowry method, and most preferably more than 99%
by weight, (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under
reducing or non-reducing conditions using Coomassie blue, or
preferably, silver stain.
[0094] "ISOTYPE" refers to the antibody class (e.g., IgM, IgE, IgG
such as IgG1 or IgG4) that is provided by the heavy chain constant
region genes. Isotype also includes modified versions of one of
these classes, where modifications have been made to alter the Fc
function, for example, to enhance or reduce effector functions or
binding to Fc receptors.
[0095] "MONOCLONAL ANTIBODY" refers to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical. A
monoclonal antibody is directed against a single determinant on the
antigen. For example, the monoclonal antibodies useful in the
present invention may be prepared by the hybridoma methodology or
they may be made using recombinant DNA methods in bacterial or
eukaryotic animal or plant cells. The "monoclonal antibodies" may
also be isolated from phage antibody libraries, or generated using
in vitro, in vivo, and cell culture methods. Monoclonal antibodies
include those that bind to a unique sequence of amino acids and
have a single specific epitope on its target antigen.
[0096] "POLYCLONAL ANTIBODY PREPARATIONS," unlike monoclonal
antibody preparations, include different antibodies directed
against different determinants (epitopes). As used herein, the term
"polyclonal" refers to an antibody that recognizes multiple epitope
sites on a single antigen.
[0097] "RECOMBINANT ANTIBODY" includes all antibodies that are
prepared, expressed, created or isolated by recombinant means and
methods.
[0098] "SINGLE CHAIN ANTIBODY" refers to an antibody in which the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes. These genes can be joined, using recombinant
methods, by an artificial peptide linker. Joining the genes results
in the production of a single protein chain in which the VL and VH
regions pair to form monovalent molecules (known as single chain
Fv, "scFv"). Such single chain antibodies include one or more
"antigen binding fragments" of an antibody. See ANTIGEN BINDING
FRAGMENT.
[0099] "THERAPEUTIC ANTIBODY" refers to an antibody that may be
considered effective in a therapeutic or prophylactic context with
regard to a disease or condition of interest.
Definitions--Amino Acids and Amino Acid Sequence
[0100] "AMINO ACID," in the broadest sense, refers to the naturally
occurring amino acids which can be divided into groups based upon
the chemical characteristic of the side chain of the respective
amino acids. "Hydrophobic" amino acids are Ile, Leu, Met, Phe, Trp,
Tyr, Val, Ala, Cys and Pro. "Hydrophilic" amino acids are, Asn,
Gln, Ser, Thr, Asp, Glu, Lys, Arg and His. The "uncharged
hydrophilic" amino acids are Ser, Thr, Asn and Gln. The "acidic"
amino acids are Glu and Asp. The "basic" amino acids are Lys, Arg
and His. As used herein, the amino acid residues are abbreviated as
follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp;
D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E),
glutamine (Gln; Q), glycine (Gly; G), histidine (His; H),
isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine
(Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser;
S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and
valine (Val; V).
[0101] "CONSERVATIVE AMINO ACID SUBSTITUTION" is illustrated by a
substitution among amino acids within each of the following groups:
(1) glycine, alanine, valine, leucine, and isoleucine, (2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,
(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)
lysine, arginine and histidine.
[0102] "IDENTICAL," in the context of two or more nucleic acids or
polypeptide sequences, refer to two or more sequences or
subsequences that are the same. Two sequences are "substantially
identical" if two sequences have a specified percentage of amino
acid residues or nucleotides that are the same (i.e., 60% identity,
optionally 65%, 70%, 75%,80%, 85%, 90%, 95%, or 99% identity over a
specified region, or, when not specified, over the entire
sequence), when compared and aligned for maximum correspondence
over a comparison window, or designated region as measured using
one of the following sequence comparison algorithms or by manual
alignment and visual inspection. Optionally, the identity exists
over a region that is at least about 50 nucleotides (or 10 amino
acids) in length, or more preferably over a region that is 100 to
500 or 1000 or more nucleotides (or 20, 50, 200 or more amino
acids) in length. The percent identity between two amino acid
sequences can also be determined using the algorithm of Meyers and
Miller.
Definitions--PNH and Hemolytic Diseases
[0103] As used herein, the term "HEMOLYTIC DISEASES" refers to any
disorder or disease in which cellular lysis, cellular damage and
inflammation play a role in the pathology of the disease. Hemolytic
disease is also an inflammatory disorder or disease wherein AP
activation causes cellular lysis, cellular damage, and
inflammation. Hemolytic diseases include diseases characterized by
pathologic lysis of erythrocytes and/or platelets. Anucleated cells
such as erythrocytes and platelets are subject to full lysis. Lysis
of erythrocytes releases hemoglobin which has pathological outcome
for blood and organs. Nucleated cells such as neutrophils,
monocytes, T lymphocytes can be attacked by the MAC but do not
undergo full lysis.
[0104] "INTRAVASCULAR HEMOLYSIS" refers to the lysis of anucleated
and nucleated cells which is caused by AP activation and the
associated production and deposition of C5b-9 on cell surfaces.
[0105] "EXTRAVASCULAR HEMOLYSIS" refers to lysis of cells due to
C3b deposition and removal via complement receptors. C3b is
produced via the activation of the classical and the alternative
pathway. This invention is focused on C3b produced via the
alternative complement pathway.
[0106] "TRAP ANTAGONIST" is a receptor-Fc fusion protein consisting
of the antibody Fab fused to the Fc portion of human IgG1. In a
preferred embodiment, an expression plasmid encoding the target
protein is transfected into CHO cells, which secrete the trap
antagonist into the culture medium. The resulting antagonist trap
binds its ligands using the binding domains of high-affinity
receptors, having greater affinity for properdin.
[0107] "SUBCUTANEOUS ADMINISTRATION" refers to introduction of a
drug under the skin of an animal or human patient, preferable
within a pocket between the skin and underlying tissue, by
relatively slow, sustained delivery from a drug receptacle. The
pocket may be created by pinching or drawing the skin up and away
from underlying tissue. There are various formulations available
specially those skilled in the art are well aware of such
formulations.
[0108] "TISSUE INJURY" refers to the tissue where C5b-9 (MAC) is
found to injure the tissue. Tissue injury is caused by the MAC and
can be inhibited by the antibodies that prevent MAC formation. One
example shown in the application is the quantifiable death of
erythrocytes in a time dependent manner in the presence of normal
human serum that contains physiological levels of complement
components. This demonstration of lysis of cells is quantifiable by
the loss of scattering at OD700. Nucleated cells present in tissues
are also injured by complement similar to erythrocytes. Inhibition
of erythrocyte lysis and therefore tissue injury can be prevented
by the use of antibodies of this invention. Tissue injury can occur
in any part of the body/organs and can lead to pathological outcome
such as arthritis. In hematological disorder where all cells that
lack the GPI are subject to MAC attack, tissue injury and damage
can be prevented by the use of such antibodies. This definition can
be extended to many diseases where tissue injury occurs as a result
of AP activation but not CP activation.
[0109] Embodiments described herein relate to methods for treating
a subject suffering from hemolytic disease, hemolytic related, or
PNH-like, condition by administering to an afflicted subject an
effective amount of one (or several) of a specific genus of
inhibitory antibodies that inhibit intravascular and extravascular
lysis mediated only the alternative complement pathway without
affecting the classical complement pathway. The antibodies of this
genus have been identified and selected, from a variety of
antibodies inhibiting the complement system, for their specific and
unique effect on specific components of the alternative pathway.
The inhibitory antibodies of the claimed genus are selective for
the alternative complement pathway. The antibodies produced from
this combination of selection criteria are useful for a multitude
of hemolytic conditions.
[0110] Both, the classical and the alternative pathways are
independent. Lectin or the MBL pathway is part of the classical
pathway. Both pathways independently generate C3b, C3a, C5b, C5a,
and C5b-9. Antibodies of the present invention inhibit C3b and
C5b-9 formation, molecules produced via both pathways. These
monoclonals do not inhibit classical pathway derived C3b and C5b-9
whether the amplication loop is a part of the process or not. This
invention leaves the C3b produced via the classical pathway intact
for host defense such as opsonization. This invention leaves the
C5b-9 produced via the classical pathway intact for host
defense.
[0111] Prior art uses inhibitors do not appear to be selective
because, the classical pathway feeds into the alternative pathway
and also work in co-ordinance with the alternative pathway.
Classical pathway uses the amplification loop of the alternative
pathway. Inhibitors of AP developed in such a setting would inhibit
the amplified activity of the classical pathway.
[0112] Uniquely, complement attack does not damage normal cells,
abnormal cells are those that lack the important regulators of the
complement system such as CD55 and CD59. These abnormal cells are
found in PNH. PNH cells lack CD55 and CD59, our invention shows
that both CD55 and CD59 are absent in nearly all types of cells
including erythrocytes, platelets, T-lymphocytes, neutrophils, and
monocytes--but the total population of each type of cells may be
different--for example, the % of abnormal cells can vary from less
than 1% to 10% or 10% to 100%.
[0113] In PNH, abnormal erythrocytes undergo lysis and release
hemoglobin as a result of AP activation. The released hemoglobin
can be damaging to kidneys. Breakthrough due to lysis of
erythrocytes is considered important and drugs have been discovered
and currently being used to control intravascular hemolysis. This
drug due to its downstream action does not prevent extravascular
hemolysis and therefore patients continue to remain anemic.
[0114] To prevent extravascular hemolysis from taking place;
several major categories of complement inhibitors can be developed;
a) Classical pathway inhibitors that prevent C3b deposition
produced via the classical pathway onto the cell surface, b)
Classical pathway inhibitors that prevent C3b formation produced
via the amplification loop, c) AP inhibitors that prevent the
formation of CP derived C3b formation, and d) AP inhibitors that
prevent C3b produced via the alternative pathway without affecting
the classical pathway. The inventor of the current application
claims those inhibitors that selectively target the alternative
pathway derived C3b formation without affecting the classical
pathway derived C3b formation. The rationale for such an approach
is that such inhibitors would leave the C3b produced via the
Classical pathway for host defense. The present inventors claim a
genus of monoclonal antibodies that prevent the formation of C3b
only via the alternative pathway without affecting the classical
pathway derived C3b.
[0115] To prevent intravascular hemolysis; several major categories
of complement inhibitors can be developed; a) Classical pathway
inhibitors that prevent C3b deposition produced via the classical
pathway onto the cell surface, b) Classical pathway inhibitors that
prevent C5b-9 formation produced via the amplification loop, c) AP
inhibitors that prevent the formation of CP derived C5b-9
formation, and d) AP inhibitors that prevent C5b-9 produced via the
alternative pathway without affecting the classical pathway. The
inventor of the current application claims those inhibitors that
selectively target the alternative pathway derived C5b-9 formation
without affecting the classical pathway derived C5b-9 formation.
The rationale for such an approach is that such inhibitors would
leave the C5b-9 produced via the Classical pathway for host
defense. The present inventors claim a genus of monoclonal
antibodies that prevent the formation of C5b-9 only via the
alternative pathway without affecting the classical pathway derived
C5b-9.
[0116] C3b and C5b-9 are produced via both the classical and the
alternative pathways. The two C3 convertases (CP C3 convertase and
AP C3 convertase) with different molecular structure have been
identified; (C4b2a) and (PC3bBb). These C3 convertases cleave C3
and generate two different types of C3b molecules. Since both
complement pathways are independent, this invention only targets
C3b production via the alternative pathway without affecting the
C3b produced via the classical pathway or from CP amplification
loop. A genus of monoclonal antibodies that selectively targets the
alternative pathway derived C3b are the focus of the current
invention.
[0117] Antibodies of the present invention would control
extravascular hemolysis in vivo and its associated clinical
outcomes such as increased reticulocite counts, hemoglobin (HgB)
and LDH in clinical trials. In certain embodiments, the present
invention comprises a method of treating a subject having
hematological disorder wherein erythrocytes, neutrophils,
monocytes, platelets and T lymphocytes are deficient in GPI linked
proteins, the method comprising administering an effective amount
of an inhibitor that inhibits the alternative complement pathway to
prevent the formation and deposition of C3b, PC3b, PC3bBb and
P(C3b)n(Bb)n. Such an action is important for preventing extra- and
intra-vascular hemolysis and episodes of hemolytic crisis. In other
embodiments, the invention comprises a method of treating a subject
previously treated with Eculizumab or a comparable drug wherein the
subject already is exhibiting extravascular hemolysis, the present
invention is expected to dis-assemble to convertase and halt the
progression of extravascular hemolysis.
[0118] In certain embodiments, the methods of the present invention
comprise treating a subject having complement-mediated hemolytic
disorder affecting blood cells, wherein the subject exhibits at
least one of the following characteristics; a) the subject exhibits
signs or symptoms continued loss of red blood cells by ongoing or
intermittent intravascular hemolysis and/or extravascular
hemolysis; b) the subject has red blood cells opsonized by
fragments of C3; c) the subject requires periodic blood
transfusions; c) the subject has low normal or below normal levels
of hemoglobin;e) the subject has low normal or below normal levels
of platelets; f) the subject has high normal or above normal
reticulocytes; g) the subject has high normal or above normal
bilirubin; h) the subject has iron overload or is at risk of iron
overload.
[0119] As preferred embodiments useful to accomplish the above
methods, the present invention provides agents and compositions
that inhibit the activity of the complement alternative pathway.
Such agents and compositions comprise fusion proteins carrying the
binding regions of the antibodies from the claimed genus and or
antibodies themselves. These agents are expected to prevent the
initiation of C3 convertase formation and formation of C3b, prevent
deposition of C3b onto cells that lack the GPI linked proteins. As
a result, extravascular hemolysis is down-regulated, number of
transfusions are reduced, cytopenia is reduced, and intravascular
hemolysis is reduced. In another aspect of the present invention
where reduction in cytopenia is claimed, cytopenia includes
leukocytopenia, thrombocytopenia, erythrocytopenia, leukocytopenia,
lymphocytopenia, and neutropenia. These processes can occur as a
result of cellular aggregate formation and removal of such
aggregates from subject's circulation. Reduction in cell number can
also occur due to extravascular hemolysis.
[0120] In preferred embodiments, the inhibitor of the complement
alternative pathway may comprise a fusion of the "Fab", or a
fragment comprising at least the variable region of the antibody or
a biologically active fragment thereof to the Fc region of the
antibody. In another aspect of the current invention, the inhibitor
of the complement alternative pathway may comprise only the
blocking Anti-C3b antibody, Anti-Factor Bb antibody, Anti-Properdin
antibody, and Anti-Factor D antibodies specially those that block
both the formation of C3b and C5b-9. If such antibodies block the
formation of C3b and not the formation of C5b-9, then such
antibodies are excluded from the current invention. The selected
antibodies of the genus should only inhibit the alternative pathway
but not the classical pathway.
[0121] In a particular preferred embodiment, the inhibitor of the
complement alternative pathway is a genus of neutralizing
monoclonal antibodies that have the following characteristics:
[0122] Inhibit the alternative pathway derived C3b and do not
inhibit the classical pathway derived C3b. The classical pathway
derived C3b is required for opsonization and for host defense. Thus
the selected genus of the antibodies perform specific function.
[0123] These antibodies doe not inhibit the classical pathway and
therefore do not inhibit the formation of C3b via the classical
pathway.
[0124] The present invention provides in one aspect a method of
treating a subject having a complement-mediated hemolytic disorder
affecting blood cells, the method comprising administering an
effective amount of the antibody and its antigen binding fragments
that inhibit activation of the complement alternative pathway,
wherein the antibody inhibits the formation of both the C3b and
C5b-9 responsible for extravascular and intravascular hemolysis
repectively.
[0125] In certain embodiments of any of the methods described
herein, the subject has one or more of the following
characteristics: a) the subject exhibits signs or symptoms of
cytopenia by ongoing or intermittent intravascular hemolysis and/or
extravascular hemolysis; b) the subject has cell bound C3b wherein
the cell is selected from the group comprising leukocytes,
lymphocytes, erythrocytes, platelets, and monocytes, basophils; c)
the subject requires periodic blood transfusions; the subject has
low normal or below normal levels of hemoglobin; the subject has
low normal or below normal levels of platelets; the subject has
high normal or above normal reticulocytes; the subject has high
normal or above normal bilirubin; or the subject has iron overload
or is at risk of iron overload; or the subject has high number of
dead cells.
[0126] In some embodiments, the method includes administering an
effective amount of a monoclonal antibody selected from the
collection of antibodies of this invention which inhibit the
activity of the complement alternative pathway and therefore;
[0127] Increase in the total number of cells to normal levels.
[0128] Increase the total number of surviving red blood cells
increase to normal levels.
[0129] Increase the total number of neutrophils to normal
levels.
[0130] Increase the total number of monocytes to normal levels.
[0131] Increase the total number of T-lymphocytes to normal
levels.
[0132] Increase the total number of platelets to normal levels.
[0133] Decrease the total number of dead cells, increase the total
number of healthy cells.
[0134] Decrease the total number of cellular aggregates.
[0135] Decrease in the total LDH to normal levels.
[0136] Decrease bilirubin to normal levels.
[0137] Decrease hemoglobin in plasma.
[0138] Decrease in C3a, C3b, C5a, C5b, C5b-9, and sC5b-9
levels.
[0139] Decrease in activated cells.
[0140] Decrease in inflammatory cytokine levels.
[0141] Decrease in cellular activation.
[0142] In some embodiments, the antibodies of the current invention
dampen and/or inhibit the activation of AP without inhibiting CP.
They inhibit alternative pathway-dependent lysis and activation of
cells involved in inflammation, inhibit production of inflammatory
molecules and ultimately inhibit a myriad of pathologies associated
with various hemolytic diseases. These antibodies also inhibit the
formation of C3b responsible for extravascular hemolysis and
intravascular hemolysis mediated via C5b-9.
[0143] C3b of alternative complement pathway (C3b)--This key
molecule is important in the amplification of the alternative
pathway. When associated with a disease state, AP activation causes
C3b to be produced and deposited on various cells, including blood
cells. This, in turn, causes extravascular lysis of erythrocytes
and platelets, the root cause of erythrocytopenia and
thrombocytopenia respectively. For this reason, pathological
overproduction of C3b is detrimental and must be inhibited or
controlled. C3b is also deposited on neutrophils, monocytes, and
T-lymphocytes. Deposition of C3b on these nucleated cells does not
cause lysis of these cells but causes cell damage and equivalent to
tissue injury. C3b receptors are found on these nucleated cells and
the density of such receptors is increased during complement
activation and diseases. As a result, such cells become
dysfunctional. It is the invention of this application to
demonstrate that not only erythrocytes would be subject to lysis
but also the platelets would be subject to lysis. Both erythrocytes
and platelets are a nucleated cells. The nucleated cells such as
neutrophils, monocytes, and platelets--they all would bind C3b and
are removed via extravascular lysis.
[0144] We believe that if C3b formation is inhibited only via the
alternative pathway, no C3b produced via the alternative pathway
will be available for deposition on cells and therefore would
inhibit cell loss via extravascular lysis and/or removal by the
host liver/or spleen. Inhibition of removal would mean cytopenia
which includes all cells will be inhibited. In hematological
disorders such as PNH, not only erythrocytopenia is observed, but
also thrombocytopenia, neutropenia, monocytopenia, lymphocytopenia
is observed. It is important to address removal of leukocytes and
platelets as these are new findings.
[0145] Neutrophils and other cells bear C3b receptors and therefore
bound C3b could be detected with the anti-C3b antibody. Neutrophils
coated with C3b are incapable of fighting infections therefore the
neutralizing monoclonals of the claimed invention would prevent
infection.
[0146] Similar to erythrocytes, platelets are also anucleated and
therefore have the ability to lyse.
[0147] C3b produced via the classical pathway is designated as
(C3b) to differentiate this C3b from those produced via the
alternative pathway. C3b would remain intact for opsonization and
removal of bacteria and therefore must not be inhibited. The
antibodies of the current genus do not inhibit the classical
pathway and therefore do not inhibit associated side products such
as C3b, C3a, C5b, C5a, and C5b-9 produced via the classical
pathway. The process of opsonophagocytosis begins with deposition
of C3b on the surface of cells and the subsequent uptake by
phagocytic cells. Inappropriate and/or uncontrolled production of
C3b leads to inappropriate and/or uncontrolled
opsonophagocytosis.
[0148] C3a--The C3a molecule is a peptide with a molecular weight
of 9,000 Da and a high affinity for C3a receptors (C3aR). C3aRs are
present on neutrophils, monocytes, platelets, mast cells, and T
lymphocytes. Binding of C3a to C3aR activates the release of
inflammatory molecules from the triggered/activated cells. Upon
activation, these cells: a) form intra- and inter- cellular
aggregates, b) invade the normal tissue and host themselves causing
pathology, and c) release inflammatory mediators such as
TNF-.alpha., IL_1, 11-18, IL-27, peroxides and proteases that can
degrade the matrix and initiate inflammation and tissue
destruction. For example activated/triggered monocytes express CD1
1b and release Tumor Necrosis Factor alpha (TNF-.alpha.). Activated
monocytes can form aggregates with platelets. Activated neutrophils
also express CD1 1b and release peroxides and neutrophil elastase.
Activated platelets express a higher concentration of CD62P and
form aggregates with neutrophils and monocytes. Both mast cells and
T lymphocytes are also activated by C3a. C3a initiates the release
of TNF-.alpha. from monocytes. TNF-.alpha. is known to play a key
role in the pathological outcomes and conditions. Platelets also
bear C3a receptors. Upon activation by C3a, platelets express
CD62P, an activation marker. CD62P is responsible for inter
cellular aggregate formation. These aggregates are removed from
circulation, which ultimately leads to thrombocytopenia.
[0149] C5a also plays a role in activation of platelets. Regardless
of the method of platelet activation, activated platelets express
CD62P, which is also called P-selectin. P-selectin also mediates
platelet-monocyte conjugation. This binding triggers the release of
tissue factor from monocytes.
[0150] C5a/C5b--AP C5 convertase (P(C3b)n(Bb)n) cleaves C5 and
produces C5a and C5b. C5a is known to activate neutrophils and
monocytes as C3aR and C5aR receptors have been found on these
cells. Upon activation, neutrophils and monocytes produce inter and
intracellular aggregates and release inflammatory markers such as
neutrophils elastase, peroxides and a variety of matrix proteases
that degrade the tissue matrices. Similar to C3a, C5a also causes
the release of inflammatory mediators relevant to several
pathologies and associated hemolytic diseases.
[0151] C5b-9 and sC5b-9--These complexes are also called "MAC", the
C5b-9 is a complex that forms on the cell surface and causes tissue
injury. As demonstrated in FIG. 6, rabbit erythrocytes (rRBC)
activate the AP in whole blood. In response, C5b-9 is integrated in
the cell membrane, causing lysis of these cells by human
complement. This assay represents a way of demonstrating tissue
injury using an erythrocyte hemolysis assay. The sC5b-9 is a MAC
complex that is formed by the association of protein S to C5b. C5b
binds S instead of depositing on a cell surface. Protein S enables
the formation of "soluble MAC," abbreviated as sC5b-9. Soluble MAC
also activates platelets and other cell types.
[0152] C3a and C5a activates cells, activated cells express markers
such as CD62P and CD1 1b. These activated cells form aggregates.
Aggregates are removed from circulation leaving patient
cytopenic.
[0153] The antibodies of the present invention can prevent AP
derived formation of C3a, C3b, C5a, C5b, and C5b-9. As a result,
cellular activation is prevented. If there is no activation, there
is no release of inflammatory markers. Thus, the antibodies of this
invention are capable of blocking, preventing the progression of
the disease.
Role of Alternative Pathway in Hemolytic Diseases
[0154] Based on the available literature and associated data, it
appears that in chronic hemolysis, complement activation is
mediated predominantly via the formation of C5b-9 on cell surfaces.
It does not differentiate between the classical pathway derived or
the alternative pathway derived. This invention targets the C5b-9
formed via the alternative complement pathway, but not the
classical complement pathway. This invention would leave the
classical pathway intact for host defense against infection.
[0155] Hemolytic diseases include those in which lysis of
erytrhocytes results in a release of hemolglobin. Such actions
reduce the total concentration of erythrocytes in the blood.
Paroxysmal nocturnal hemoglobinuria ("PNH") is a rare hemolytic
disease. It is an autoimmune disorder of the blood wherein
erythrocytes are destroyed by activities of the body's own
complement pathways. PNH results from somatic mutations which
render cells unable to synthesize the glycosyl-phosphatidylinositol
("GPI") anchor. The GPI anchor protects cells against complement
attack. PNH cells are deficient in complement-regulating surface
proteins that include the decay-accelerating factor ("DAF"), or
CD55, and membrane inhibitor of reactive lysis ("MIRL"), or
CD59.
[0156] In PNH, lysis of erythrocytes causes a pathologic reduction
in the total erythrocyte count (i.e., hemolytic anemia). The
presence of hemoglobin in the urine (hemoglobinuria) is
particularly evident after sleeping and usually causes the urine to
appear dark in color. Subjects with PNH will also have free
hemoglobin in their bloodstream (hemoglobinemia). Hemolytic anemia
is due to intravascular lysis of red blood cells by complement
component C5b-9 (MAC). Reduced numbers of erytrhocytes and
platelets cause dysphagia, fatigue, erectile dysfunction,
thrombosis and recurrent abdominal pain.
[0157] Erythrocyte Lysis--Erythrocytes are anucleated cells and are
responsible for maintaining the hemoglobins. These cells are known
to be subject to complement attack in PNH due to the absence of
CD55 and CD59 from the cell surface. These cells are therefore
subject to C3b deposition and removal via extravascular lysis.
These CD59 deficient cells also allow deposition of C5b-9 and
erythrocyte removal via intravascular lysis. Lysis results in
hemoglobin release from these erythrocytes causing hemolytic anemia
and therefore decrease in the number of erythrocytes in general
causing erythrocytopenia. Thus erytrhocytes are subject to removal
via both extra- and intra- vascular lysis. Additionally, excessive
free hemoglobin can cause kidney damage and system loss of iron.
Haptoglobin helps ameliorate the situation by binding free
hemoglobin and facilitating enzymatic degradation of the bound
hemoglobin.
[0158] Pathologic intravascular hemolysis, such as that associated
with PNH and other hemolytic diseases, often results in
concentrations of free hemoglobin high enough to completely deplete
haptoglobin. Once haptoglobin has been depleted, the burden is then
on the kidneys to re-absorb the free hemoglobin. Once the kidneys
reach their capacity for hemoglobin re-absorption, hemoglobinuria
begins. The release of free hemoglobin during intravascular
hemolysis results in excessive oxidation of nitric oxide (NO) to
nitrate (NO.sub.3.sup.-) The depletion of NO causes enhanced smooth
muscle contraction, vasoconstriction and platelet activation and
aggregation. The systemic consequences of excess free hemolglobin
in blood also effect abdominal pain, erectile dysfunction,
esophageal spasm, and thrombosis.
[0159] As a routine laboratory test, blood smears are, generally,
evaluated to identify morphologic abnormalities of RBCs (Red Blood
Cells), reticulocyte count (to determine bone marrow compensation
for RBC loss), lactate dehydrogenase (LDH), and levels of free
hemoglobin (from hemolysis). Concentrations of bilirubin,
haptoglobin, hemosiderin, and free hemoglobin can measure the
extent of hemolysis and help differentiate between intravascular
vs. extravascular hemolysis. RBC numbers, levels of RBC (i.e.,
cell-bound) hemoglobin, and hematocrit are often evaluated to
determine the extent of any anemia and/or any other associated
symptom of hemolytic disease. Levels of Lactate Dehydrogenase (LDH)
can also provide some information with regards to the extent of
ongoing cell death.
[0160] Lysis of erythrocytes sometimes could give erroneous and
inconsistent results due to persistent extravascular and
intravascular hemolysis. Therefore cells that do not undergo lysis
would be better for determining the clone size in PNH patients.Such
examples are neutrophils and other mononuclear cells.
[0161] Convertase Laden Erythrocytes--In PNH, erythrocytes that
lack the CD55, would be prone to C3b deposition. Such cells not
only have the C3b but also have the entire C3 convertase.
Antibodies of the current genus, prevent the formation of C3b,
C3bBb, PC3bBb formation and therefore would prevent the lysis of
erythrocytes via extra- and intra-vascular hemolysis. Thus these
antibodies would prevent the formation of both the C3 and C5
convertases.
[0162] Antibodies of the current invention, those that selectively
prevent the formation of C3b and C5b-9 produced via the alternative
pathway would inhibit extra- and intra-vascular hemolysis with
resultant benefit in total anemia. Platelet Lysis--Platelets are
anucleated and therefore subject to complement attack via the
alternative pathway. Similar to erythrocytes, platelets are also
destroyed via the similar mechanism. Lysis of platelets would occur
in PNH patients where platelets lack the CD55 and CD59 on its cell
surface. Platelet lysis means reduction in platelet number and
therefore blood clotting ability of blood in PNH patients. The
reduction in platelet number results in increased levels of
platelet contents including but not limited to platelet factor 4
(PF4), platelet derived growth factor (PDGF), beta thromboglobulin,
P-selectin. This includes all contents that are reported now or in
future are covered under this invention. Thus antibodies of the
current invention would decrease thrombocytopenia associated with
patients with hematological disorders.
[0163] Lysis of Nucleated Cells--Under this category fall cells
such as Neutrophils, monocytes, and lymphocytes. These cells are
known to be CD55/CD59 positive and have recently been considered
reliable cells for establishing PNH clone. Often higher percentage
of leukocytes are detected with CD59 than shown with erythrocytes.
Erythrocytes generally have a lower life span compared to
leukocytes. Nucleated cells do not lyse and therefore are present
in blood for longer duration compared to erythrocytes and therefore
are more confirmed markers of PNH.
[0164] Neutrophils bear C3b receptors and therefore would bind such
molecules. We show increased staining of C3b, properdin, and Bb on
neutrophils indicating that the convertase forms on such cells.
Antibodies of the current invented genus of antibodies is capable
to preventing the formation of alternative pathway derived C3
convertase but not classical pathway derived C3b. Similar finds
have been noted on all nucleated cells. It was surprising to note
that nearly all nucleated cells showed heavy staining with both C3b
and C5b-9. Both of these molecules deposit on cell surface as a
result of AP activation.
[0165] It is the intent of the proposed invention to prevent the
formation and deposition of C3b and C5b-9 on the nucleated and
non-nucleated cells. AP specific selected antibodies inhibit the
pathway upsteam and prevent the deposition of both molecules that
cause extra and intravascular lysis and damage. Nucleated cells
when laden with C3b and C5b-9 are likely to become dysfunctional
and recognized lend themselves to death. Dead cells are recognized
by the stain specific for cell death.
[0166] Role of C3a and C5a in Inflammation: Elevated levels of C3a
and C5a are predicted in PNH due to the continuous activation of
the alternative pathway leading to lysis of erythrocytes. C3a and
C5a have potent pro-inflammatory and immuno-regulatory functions.
They increase vascular permeability and serve as chemo attractants,
which promote soft tissue swelling. The anaphylatoxins activate
neutrophils and monocytes, which results in the production of
pro-inflammatory mediators such as TNF-.alpha. IL-1, IL-6, IL-8,
and IL-17 [47-50]. C5a is a potent chemotactic protein that induces
neutrophil chemotaxis, de-granulation, neutrophil elastase release,
and superoxide generation. Neutrophils contain a potent arsenal of
vasoactive, proteolytic and cytotoxic substances, which are
produced to mediate many of the manifestations of inflammation and
cellular lysis in hemolytic diseases such as PNH. Compounds of the
current invention inhibit the detrimental inflammation, tissue
injury, and cellular lysis.
[0167] BLOOD TRANSFUSION AND ANTI COMPLEMENT ANTIBODIES--Blood cell
transfusion is given when the patient has too few red blood cells
(anemia). Blood tests in PNH show changes consistent with
intravascular hemolytic anemia: low hemoglobin, raised lactate
dehydrogenase, raised reticulocytes (immature red cells released by
the bone marrow to replace the destroyed cells), raised bilirubin
(a breakdown product of hemoglobin), and decreased levels of
haptoglobin. Anemia causes weakness and tiredness. In severe cases,
it can cause shortness of breath or a rapid heartbeat. Transfusions
are usually used when the hemoglobin level is less than 8 grams per
deciliter. Sometimes instead of a transfusion, you may get a red
blood cell growth factor--a drug that helps your body make more red
blood cells. This growth factor is called erythropoietin
(Procrit.RTM., Epogen.RTM., Aransep.RTM.). Platelet, another
anucleated cell would also decrease in number based on
complement-mediated lysis. Doctors prescribe platelet transfusions
to keep the platelet count above 10,000 to 20,000 (per cubic
millimeter). Transfused platelets last only two to three days. The
antibodies of the current invention are expected to preserve the
added platalets and prevent the destruction of platalets made by
the patient's body.
[0168] White blood cell (granulocyte) transfusions are rare. This
is because the granulocytes last only a few hours in the
bloodstream. Donated white blood cells must be used right away and
do not last long. A common example is filgrastim (Neupogen.RTM.)
for increasing the number of neutrophils/leukocytes. The antibodies
of the claimed genus can help prevent the damage and lysis of cells
that are increased by the additives.
[0169] To prevent cytopenia in general, the antibody of the claimed
genus could prevent C3b formation and deposition and C5b-9
formation and deposition, the two main functions of the AP derived
moieties are important for hemolytic disorder whether it is with or
wthoiut the additives.
Role of Complement System Activation in PNH and Other Hemolytic
Diseases and Conditions
[0170] Elevated levels of C3a, C5a, C3b, C5b, and C5b-9 can gauge
the level of activation, inflammation and hemolysis in disease
conditions. Examples of complement-associated disorders involving
hematologic disorders include, but are not limited to: Catastrophic
anti-phospholipid syndrome (CAPS), Cold Agglutinin Disease (CAD),
which increases c3b, Thrombotic thrombocytopenic purpura (TTP),
which increases CD46, factor H, and factor I, Idiopathic
thrombocytopenic purpura, where C3 and C4 detected are on
platelets, Serum sickness, where abnormal factor H leads to
increased glomerular C3 deposition, Endotoxemia, Sepsis, Atypical
hemolytic uremic syndrome (ahus), where there is enhanced formation
of c3bbb convertase and resistance to complement regulators,
Paroxysmal Nocturnal Hemoglobinuria (PNH), where it has been shown
a C5 antibody treatment reduced thromboembolism risk, Septic shock,
sickle cell anemia, which elevates c3b, Hypereosinophilic syndrome,
which increases c5a, anti-phospholipid, Autoimmune
lymphoproliferative syndrome, Dego's disease, where c5b-9 is
activated, Evan's syndrome, essential mixed cryoglobulinemia, and
pure red cell aplasia. Antibodies of the invention genus of
antibodies, selected with The Screening Method (see Page 23), can
prevent local damage and have shown benefit in whole blood models
of the disease. Antibodies of the current invention prevent blood
inflammation and cellular lysis and the associated maladies.
Classical Pathway Versus Alternative Pathway C3 Convertases and
PNH
[0171] Both the classical and the alternative pathway C3
convertases are responsible for the cleavage of central C3.
Eculizumab inhibits both the CP and the AP at the C5 level and does
not inhibit the formation of C3a and C3b. This is a severe
disadvantage of Eculizumab since both excess C3a and excess C3b
have been implemented in several disease conditions. C3b produced
by both, or either, pathways can coat cells. These cells can then
be removed via the opsonization process.
[0172] Conceptually, inhibition of the AP, but not the CP, will
allow for CP dependent production of C3b which may be required, in
case of infection, for opsonization via the CP. The antibodies of
the invention genus of antibodies only inhibit the AP. They do not
inhibit the CP or any amplification loop of the CP. Inhibition of
C3b is essential to prevent extravascular lysis (and effective
removal) of erythrocytes, while CP dependent C3b is essential to
host defense. In diseases such as PNH, it is the alternative
pathway that is problematic, not the classical pathway. Thus, to
combat overproduction of these proteins, the best treatment is to
shut down the alternative pathway alone. Present thinking on the
subject is that the two pathways and inextricably connected, and
that it may not be possible to shut down the AP without inhibiting
the CP. The present invention presents a challenge to the present
thinking and offers a method for shutting down the AP without, to
any degree, inhibiting the CP. Any treatment that shuts down or
significantly inhibits the classical pathway will jeopardize the
body's ability to fight infection. C3b production is needed for the
removal of unwanted cells, such as infectious bacteria. Therefore,
it is desirable and advantageous to preserve production of CP
dependent C3b while inhibiting AP dependent C3b.
Selection and Identification of the Claimed Genus of Antibodies
[0173] While both the classical and alternative pathways produce
C3a and C5a, the present invention selectively inhibits AP produced
C3a and C5a. Equimolar concentrations of C3a and C3b are produced
as a result of the C3 cleavage. Thus inhibition of C3b formation in
vitro can be demonstrated by the assays described herein. The
formation of C3b evidences the concurrent formation of C3a (and
vice versa). Any antibody, targeting a component of the C3
convertase complex, which acts to inhibit the cleavage of C3 into
C3b and C3a, will inhibit C3b and C3a in equal measure.
[0174] Inhibition of C3a production will also inhibit all of the
activities of C3a. Such activities include: subsequent activation
of neutrophils, monocytes, platelets, basophils, and T lymphocytes,
as well as production of inflammatory markers. (See FIGS. 13
through 18.) C3b deposits on the cell surfaces via C3b receptors.
C3b deposition is required for opsonization/removal of erythrocytes
and other cells which cause pathological outcomes in other
hemolytic diseases
[0175] U.S. Pat. Nos. 6,333,034 & 7,423,128 claims antibodies
that inhibit both CP and AP mediated complement activation and
therefore host defense is compromised. These antibodies play in
role how antibodies prevent the formation of properdin oligomer.
Properdin is a thrombospondin type 1 repeat and consists of six
repeats of thrombospondin typel. These antibodies inhibit the
binding of properdin to C3b and prevent the formation of C3c. C3b
cleavage results in the formation of C3c. Thus these antibodies
prevent the cleavage of C3b.
[0176] In another aspect, the alternative pathway specific antibody
of the present invention can bind to the alternative pathway
protein without reducing the levels of that protein in the
human.
Therapeutic Antibodies
[0177] Referring to FIG. 1, we show that both CP and AP are
distinct and not connected. It is known that CP has an
amplification loop and that connects the CP and the AP. The
schematic only shows how the antibodies of the current genus work
and not the way antibodies of other inventions work. AP
amplification is shown in the upper right hand side and consists of
PC3b, PC3bB, PC3bBb. As can be seen in the schematic, PC3bBb then
acts to perpetuate the cycle by cleaving C3 into more C3b which
binds to P to again form PC3b. Application of the antibodies
selected using the screening method described herein completely
inhibits the alternative pathway without affecting the classical
pathway by specifically targeting the components of this
amplification loop. These antibodies prevent the amplification loop
of the alternative complement pathway without affecting the
classical pathway (as shown on the left side of the schematic in
FIG. 1).
[0178] Based on the old conversion of pathway at C3 theory, those
with ordinary skill in the art would expect any activation of the
classical pathway to invariably have the effect of triggering and
propagating the alternative pathway. This is because the two
pathways are believed to "overlap" at the starting point of the C3.
According to this theory, C3b produced via the classical pathway
participates in the AP amplification loop. The genus of antibodies
selected using the method described herein specifically targets
components of the alternative pathway amplification loop in such a
way as to inhibit the alternative pathway regardless of whether or
not the AP amplification loop has been otherwise triggered by the
classical pathway. Thus, for example, anti-C3b antibodies described
herein only inhibit the AP and not the CP amplification loop or the
CP propagation.
The "Screening Method": Selection of Antibodies that Inhibit the
Alternative Complement Pathway, Do Not Inhibit the Classical
Complement Pathway, and Are Specific for Components of the AP C3
Convertase
[0179] Antibodies specific for complement proteins belonging to the
alternative pathway (whether part of the CP amplification loop or
an alternative pathway by itself), such as C3b, P, Ba, and Bb can
be screened using a "Screening Method" described herein to select
antibodies to inhibit alternative complement pathway without
affecting the CP or the amplification loop of the CP. C3b, P, Ba,
and Bb are large proteins of, respectively, 210K, 50K, 33K, and 66K
molecular weight. One skilled in the art can generate millions of
antibodies to each of these proteins. Production of antibodies to a
target is, by itself, meaningless without further selection of
those antibodies as having a specific therapeutic function.
[0180] In some embodiments, the Screening Method can include a
two-stage screening process. The first stage utilizes three
successive screening assays to identify Type AP antibodies
(antibodies which specifically inhibit the AP). The selection
process leads to identification of alternative pathway specific
antibodies which are similar in functionality but targeted to a
wide variety of antigens. These selected antibodies cannot be
differentiated based on the targets they bind to or the species of
animals in which they were raised. Upon sequencing, it is clear
that such antibodies have widely different CDRs (the regions
involved in binding to antigens). The functionality, and ultimate
therapeutic value, of these antibodies can't be defined by their
sequences alone. The Screening Method described herein can identify
and define a genus of antibodies against Properdin (P), Factor C3b
(C3b), and/or Factor B (Ba, or Bb) which have the desired
functionality and effect.
A1) STEP 1: Selection Based on Function
[0181] Three distinct types of antibodies can be identified using
specific assays. The antibodies can be referred to as Type CP, Type
CP/AP, and Type AP, and are defined as follows.
[0182] Type CP: These inhibit the classical pathway but not the
alternative pathway.
[0183] Type CP/AP: These inhibit both the classical pathway and
alternative pathway.
[0184] Type AP: These inhibit the alternative pathway but not the
classical pathway.
[0185] Identification of these three different types of complement
inhibiting antibodies is accomplished using three different assays;
a CP only assay, a combined CP and AP assay, and an AP only
assay.
Type CP
[0186] For classical pathway activation, antibody sensitized sheep
cells are used as an activator in 1% normal human serum in the
presence of Ca2+/Mg2+. The calcium ions are required for the
activation of the classical pathway for the initial trigger of the
C1q/C1r/s complexes. CP will not occur in the absence of the
Calcium ions. Mg2+ is required for alternative pathway activation.
In 1% normal human serum containing Ca2+/Mg2+, only the CP proceeds
to completion. Without the requisite levels of NHS which is 10%,
the alternative pathway will not have a significant presence. FIG.
2, Assay-1 shows that shows that CP activation leads to the lysis
of antibody sensitized sheep red blood cells. Antibody bound sRBCs
act as a trigger for the classical pathway. The observed CP
activity is isolated from AP activity by using a 1% buffer solution
containing Ca2+ and Mg2+. FIG. 6 also shows that none of the
selected antibodies materially inhibit CP mediated hemolysis of the
sRBCs in 1% human serum in classical pathway condition.
Type CP/AP
[0187] For classical pathway activation, along with the
amplification loop of the alternative pathway, antibody sensitized
sheep red blood cells are used as an activator in 10% Ca2+/Mg2+ in
normal human serum. The difference between the assays used to
identify Type CP antibodies and those used to identify Type AP/CP
antibodies is the concentration of normal human serum which is 10%
in Type CP/AP. The concentration of Ca2+/Mg2+ used in the
identification of Type AP/CP antibodies, by providing the level of
Mg2+ required for AP activation, allows for both the CP and the AP
to be active. Antibody sensitized sRBCs only activate the CP. They
do not, by themselves, activate the Alternative Pathway. However,
in the presence of sufficient NHS, activated CP will utilize the
amplification loop of the AP. Thus, the assay system is designed to
evaluate the performance of complement inhibitors under conditions
in which both pathways are active (FIG. 3, Assay 2 and FIG. 4 Panel
B). Under these conditions, the C3b produced via the classical
pathway can feed into the alternative pathway causing
"amplification of the alternative pathway loop" of the alternative
pathway and can serve as a trigger indirectly. In other words, the
AP has been activated by the CP. Antibodies that prevent CP
initiated activation of the AP have been described in (R
Gupta-Bansal, J B Parent, K R Brunden, Molecular immunology.
37(5):191-201). These antibodies reduced hemolysis of the sheep red
blood cells in these assays. However, at this stage in The
"Screening Method", the antibodies have yet to be differentiated
according to how (and where) they inhibit the process. Antibodies
which inhibit the classical pathway's activation of the AP by
inhibiting any stage of the CP are not included in the selected
genus of antibodies. Accordingly, the antibodies that inhibit the
classical pathway initiated amplification of the alternative
pathway have been excluded from the selected genus of
antibodies.
Type AP
[0188] Rabbit RBCs (rRBC) are used to activate the AP in 10% normal
human serum in the presence of Mg2+ and in the absence of Ca2+.
Because the CP requires the presence of Ca2+, the classical pathway
will not be active under these conditions. Thus, in 10% NHS in
Mg2+, only the AP proceeds to completion. As shown in FIG. 2,
Assay-3, AP activation leads to cellular lysis of the rRBCs. It
should be noted that this assay demonstrates that the alternative
pathway can be activated, and progress to completion, in the
absence of active classical pathway function. The AP does not
require initiation by the classical pathway in order to proceed.
FIG. 4 clearly shows that the invention genus of antibodies
inhibits AP dependent hemolysis of rRBCs in 10% normal human
serum.
[0189] When an antibody's effect on AP activation and progression
is observed in isolation, with the AP as a stand-alone process, the
information obtained is different than the information obtained
from observation of the antibody's effects in conditions where both
the CP and the AP are active. The information obtained here is also
different than that obtained from observation of the antibody's
effect in conditions where only the CP is active.
Analysis of the Three Assays
[0190] If the presence of a particular antibody(s) in one of these
three assays was found to reduce the rate of hemolysis, it was
concluded that that antibody inhibits the pathway, or pathways,
which were active in that assay. Thus, for example, if an antibody
was found to reduce hemolysis in all three assays, it was concluded
that the antibody inhibited both the AP and the CP (Type CP/AP). If
an antibody was found to inhibit hemolysis only in assays
containing 1% human serum (with Ca2+/Mg2+ buffer) it was concluded
that that antibody inhibited the CP but not the AP (Type CP). If an
antibody was found to inhibit hemolysis only in the assays
containing 10% human serum and Mg2+ (but not Ca2+), it was
concluded that the antibody inhibited the AP but not the CP.
[0191] This is the first stage of The Screening Method. Antibodies
passing these selection criteria have been shown to: 1) inhibit the
alternative pathway under conditions in which the alternative
pathway is active in isolation (i.e., without concurrent activation
of the classical pathway), and 2) have no effect on CP activity
(either in isolation or when concurrent with AP activity).
[0192] The invention uses this combination of assays to first
identify Type AP antibodies. However, additional screening steps
are needed in order to identify the selected genus of antibodies.
Additional screening is necessary because these assays will
identify antibodies in both the upstream and the downstream portion
of the AP and the CP.
A2) STEP 2: Selection of those Type AP Antibodies which Act on C3
Convertase Formation
[0193] The second step of the Screening Method is to identify which
Type AP antibodies inhibit only the functional activity of AP C3
convertase. In other words, this step identifies those Type AP
antibodies which act "up-stream" of the alternative complement
pathway system, at the amplification loop of the AP, rather than
"down-stream."
[0194] This step is accomplished by first establishing a solid
phase ELISA based binding assay. This assay allows for the direct
detection of C3b and C5b-9 produced via the alternative pathway.
Detections of these proteins represent an early component (C3b) and
a late component (C5b-9) of the alternative complement pathway. If
an antibody inhibits production of C5b-9 but not C3b, it is likely
to be acting on the C5 convertase of the AP. By contrast, C3b
production will be inhibited by antibodies that inhibit the
activity of the C3 convertase. Inhibition of C3b production will
also inhibit production of C5b-9 (because C5b-9 is produced
downstream of C3b). Thus, Stage 2 of The Screening Method separates
antibodies inhibiting C3 convertase (up-stream) from those
inhibiting C5 convertase (down-stream).
[0195] The Screening Method identified antibodies that selectively
inhibit the AP C3 convertase. This stage of selection utilizes an
assay in which human serum at 10% in the presence of Mg++ is
allowed to incubate over an LPS coating. LPS is a specific
activator of the alternative pathway and can allow formation of AP
derived C3 convertase and C5 convertase. As shown in FIG. 6, the
selected genus of antibodies prevents the formation of C3b, a
central component of the alternative pathway amplification loop.
FIG. 7 shows that they also inhibit formation of C5b-9.
[0196] At the conclusion of the this stage selection process,
antibodies that prevent the AP dependent cellular lysis and C3b
formation (FIG. 6) are selected as being members of the selected
genus of antibodies. These antibodies are defined by the fact that
they all: 1) selectively inhibit the alternative pathway without
inhibiting the classical pathway (FIGS. 2, 4, and 5), and 2)
inhibit the alternative pathway dependent C3b formation, by acting
on C3 convertase formation, an upstream component of the AP.
[0197] FIGS. 10 and 11 show that the selected genus of antibodies
inhibits formation of complement proteins C3a and C5a,
respectively.
Selected Genus of Antibodies
[0198] Application of the Screening Method has thus far produced
several antibodies from the selected genus of antibodies.
C3b As Target Protein
Mouse Anti Human C3b (Anti-C3b)
[0199] C3b is a large protein and therefore multiple antibodies can
be produced against various segments of this protein. There exist
multiple sites where-on an antibody might bind and inhibit the
protein's activity in any variety of ways. Depending on how and
where an antibody binds to C3b, the effect of that antibody could
range from inconsequential to complete inhibition. Injecting a
mouse with Human C3b will result in the production of a myriad of
mouse antibodies against the Human C3b protein.
[0200] The selected genus of antibodies include those that bind to
C3b in such a way as to prevent the interaction of C3b with Factor
B. The effect of these antibodies is necessarily isolated to the
alternative pathway since no such interaction exists within the
classical pathway. These antibodies prevent the formation of
C3a/C3b, C5a/C5b, and C5b-9/sC5b-9 critical for pathological
outcome causing disease initiation and progression. Inhibition of
the formation of each of these molecules, by the alternative
pathway, has significant physiological consequences. Inhibition of
alternative pathway produced C3b (herein referred to as "aC3b")
impacts extravascular hemolysis of erythrocytes. The C3b produced
by the classical pathway is not inhibited by these antibodies and
therefore is required for opsonization of foreign
particles/bacteria that are coated with CP produced C3b (herein
referred to as "cC3b"). Thus, the selected genus of antibodies
prevents the formation of aC3b and not cC3b by such antibodies that
have this as a common function. The inhibition of C3a formation has
direct effect on monocytes activation and production of TNF-a which
is a validated marker of inflammation.
Properdin as Target Protein
[0201] As is the case with C3b, Properdin is a large protein with
many potential sites where antibodies can bind. Different
antibodies binding in different ways and/or on different sites of
the Properdin protein, will inhibit either amplification loop of
the classical pathway or alternative pathway. Properdin is known to
be part of the amplification loop of the classical pathway. Thus,
classical pathway activation can be dampened by the use of specific
anti-properdin antibodies that inhibit the amplification loop (U.S.
Pat. No. 6,333,034). Some antibodies can inhibit the classical
pathway activation where interactions of Properdin to C3b, within
the classical pathway, become important for classical pathway
amplification. (U.S. Pat. No. 6,333,034)
[0202] Properdin binds to itself and generates aggregates.
Depending upon the configuration of the aggregate, antibodies
binding Properdin can bind mono, di-, tri- and tetramer, with each
generating different responses. Thus antibody-to-properdin ratio
can be 1:1, 1:2, 1:3, and 1:4. This means that an antibody can bind
in any configuration. An assay can be used to separate antibodies
in a rank order according to potency, by the ratio at which they
bind Properdin. In other words, antibodies that bind at a 1:1 ratio
can be separated from those that bind at a 1:2 ratio, a 1:3 ratio
and a 1:4 ratio. A binding ratio of 1:1 suggests that the antibody
binding is via one arm and not by two arms. Such antibodies
demonstrate a 1:1 binding ratio regardless of whether or not the
antibody is a Fab (monovalent) or the IgG (divalent).
[0203] Properdin is involved directly in the AP activation but
indirectly in classical pathway activation via the amplification
loop in vivo. Also, Properdin binds both C3b and C5b. An antibody
which disrupts Properdin's interaction with C3b may or may not
interrupt Properdin's interaction with C5b (and vice versa).
Antibodies that prevent one or both may be of distinguishable
clinical significance.
[0204] Thus, some antibodies targeting Properdin a) inhibit both
the classical pathway and alternative pathway, or b) inhibit the
alternative pathway alone. The selected genus of antibodies would
only include those antibodies targeting Properdin which acted on
Properdin in specifically such a way as to only inhibit the AP, and
not the CP.
Anti Human P (Anti-P) Derived from Mouse
[0205] The protein Properdin (P) is a large protein with a
molecular weight of approximately 50,000. A multitude of antibodies
can be produced against various protein motifs of this large
protein. Not all, or even most, of these antibodies will
necessarily have any therapeutic value. Identification and
selection of the appropriate antibody, or antibodies, those with
optimal therapeutic value, is crucial.
[0206] Two mouse anti-human-P antibodies were selected using a
proprietary combination of successive screening methods. The
Screening Method enabled this inventor to identify those antibodies
which 1) bind to human Properdin, 2) selectively inhibit only the
activity of the alternative complement pathway, and 3) interrupt
the alternative pathway in such a way as to not disrupt the
amplification loop of the classical pathway. These antibodies bind
properdin as the target antigen. And they do so in such a way as to
inhibit the formation of the P(C3b)n, PC3bB and PC3bBb, and by
extension, (Bb)n and C3bBb. The inhibition of these specific
complexes is one of the essential and defining common
characteristics of all the antibodies of the selected genus. In
preventing the formation of these complexes, these antibodies all
prevent the alternative pathway's production of C3b, C5a, C5b, and
C5b-9, as well as TNF-.alpha., IL-1.
Anti Human P (Anti-P) Derived from Rabbit
[0207] Three rabbit anti-human-P antibodies were selected using the
Screening Method (the same that was used for selecting the
anti-human-P mouse antibodies). As can be seen in FIG. 2, these
antibodies inhibit the alternative pathway dependent lysis of
rabbit red blood cells (rRBC) in normal human serum (NHS) in buffer
that lacks calcium and therefore there is no contribution from the
classical complement pathway. In doing so, the effect of these
antibodies is targeted, and isolated, to a section of the
alterative pathway which does not overlap with the classical
pathway. These antibodies prevent the formation of C3a, C3b, C5a,
C5b, and C5b-9. The formation of these specific proteins is the
critical step in the alternative pathway wherein a normal immune
system process can become the source of a pathological condition.
It's the overproduction of these proteins from the alternative
pathway that often cause arthritic conditions.
[0208] The selected alternative pathway specific anti-human-P
antibodies generated in rabbits are analogous in effect to those
from the mouse models. They are analogous in effect because both
the mouse and the rabbit derived antibodies were selected using The
Screening Method. They inhibit the formation of C3a, C3b, C5a, C5b,
and C5b-9; thereby inhibiting the activation of monocytes,
neutrophils, platelets, and the formation of TNF-.alpha. (which
also plays a key role in inflammation).
[0209] Sequences of these anti-properdin rabbit antibodies are very
different as shown in the tables noted below. Therefore, looking at
the protein sequences alone would not necessarily yield any
understanding of their effect on Properdin. Unless tested using the
Screening Method, it would be difficult to determine if a given
antibody belongs to the selected genus of antibodies. Accordingly,
the selected genus of antibodies can't be defined by a specific
amino acid sequence. Rather, the genus is defined by the ability of
its member antibodies to 1) selectively inhibit AP activation
without disrupting any function of the CP, and b) act on a specific
part of the AP that is isolated from the CP and which is
responsible for AP production of C3a, C3b, C5a, C5b, and C5b-9.
Ba as Target Protein
[0210] Anti Human Ba (Anti-Ba) Derived from Mouse
[0211] The protein Ba (cleaved from Factor B) is a large protein
with a molecular weight of approximately 33,000. Thus, like
Properdin and C3b, any of a multitude of antibodies can be produced
against various protein motifs of, and locations on, the protein.
With this protein, as with the other proteins of the AP, the
invention is a selected genus of antibodies which bind to the
protein in such a way as to inhibit the formation of C3a, C3b, C5a,
C5b, and C5b-9, which are required for the pathological progression
of the disease.
[0212] As can be seen in FIG. 2, these antibodies inhibit the
alternative pathway dependent lysis of rabbit red blood cells
(rRBC) in normal human serum (NHS) in a buffer that lacks calcium.
The classical pathway can't function in a buffer which lacks
calcium. Thus, in these conditions, there is no contribution from
the classical complement pathway. Such conditions enable one to
observe the effect these antibodies have on the alternative
complement pathway in the complete absence of the classical
pathway. Observing the antibodies under these conditions is one
step of the Screening Method by which the antibodies of the
invention are identified.
[0213] The sequencing of these anti-alternative pathway antibodies
are very different. Thus, here again we observe that the selected
genus of antibodies can't be defined by a specific amino acid
sequence. Rather, the genus is defined by those which are selected
by The Screening Method.
Bb as Target Protein
[0214] Anti Human Bb (Anti-Bb) Derived from Mouse
[0215] The protein Bb (cleaved product of Factor B) is a large
protein with a molecular weight of approximately 64,000. Thus,
where again we find that several antibodies can be produced against
various protein motifs of this protein. Again we apply the
Screening Method in order to produce only those antibodies which
have the desired effects.
[0216] Mouse anti-human-Bb antibodies were raised against factor Bb
and therefore would not bind the Ba fragment of the antibody. These
monoclonal antibodies were also selected using the Screening
Method. They bind Bb and factor B, but not Ba as the target
antigen. The selected anti-human-Bb antibodies share the features
characteristic of the selected anti-C3b, anti-P, anti-Ba
antibodies. Like all of the antibodies from the selected genus of
antibodies, these anti-human-Bb antibodies prevent the formation of
C3a, C3b, C5a, C5b, and C5b-9 by the alternative pathway. In so
doing, these antibodies also prevent the formation of well known
markers of inflammation such as TNF-.alpha., IL-1.
Anti Human Bb (Anti-Bb) Derived from Rabbit
[0217] Three rabbit anti-Human Bb antibodies were selected using
the Screening Method. Members of the selected genus of antibodies
which bind Bb do not also bind Ba. Factor B is an integral
component of the alternative complement pathway but not the
classical complement pathway. Antibodies binding human Bb, which
survive The Screening Method, prevent the formation of complexes
critical for the propagation of the alternative pathway; C3bB,
PC3bB, C3bBb, PC3bBb, P(C3b)n(Bb)n. Like all antibodies of the
invention, they prevent the AP induced formation of C3b, C3a, C5b,
C5a, and C5b-9, and inhibit the AP at a juncture not shared with
the classical pathway. Inhibition of formation of each of these
molecules has physiologic consequences. Inhibition of C3b (aC3b)
will impact extravascular hemolysis. Inhibition of C3a and C5a will
impact cellular activation and subsequent release of inflammatory
mediators. Inflammatory mediators, when over-produced, can cause
any number of disease pathologies in humans.
[0218] As with other antibodies of the selected genus, sequences of
these rabbit anti-Bb antibodies are very different. Therefore,
looking at the protein sequences alone would not enable one to
predict whether such antibodies could have the desired effect.
[0219] Table 1 and Table 2 list the amino acid sequences of the
heavy and light chains of anti-C3b, anti-P, ant-Ba, and anti-Bb
antibodies that were selected using the Screening Method described
herein. The Tables identify the heavy chain and light chain CDR1s,
CDR2s and CDR3s of the antibodies as well as in the respective
frameworks. Accordingly, aspects of the application described
herein, relate to an isolated monoclonal antibody, or antigen
binding portion thereof comprising: (a) a heavy chain variable
region comprising CDR1, CDR2, and CDR3, of the respective
antibodies; and (b) a light chain variable region comprising CDR1,
CDR2, and CDR3 of the respective antibodies. Other embodiments
described herein relate to antibodies that bind to the same epitope
on as the VH and VL sequences described in Tables 1 and 2.
TABLE-US-00001 TABLE 1 HEAVY CHAIN TARGET SPECIES CDR1 CDR2 CDR3
Properdin Mouse GYIFTNYPIH (SEQ ID FIDPGGGYDEPDERFRD RGGGYYLDY (SEQ
NO: 1) (SEQ ID NO: 2) ID NO: 3) Properdin Mouse GFSLSTSGMGVG
HIWWDDVKSYNPALKS IGDGYYSFDY (SEQ (SEQ ID NO: 4) (SEQ ID NO: 5) ID
NO: 6) Properdin Mouse GYIFTTYPIH (SEQ ID FIDPGGGYDEPDDKFRD
RGDGYYFDY (SEQ NO: 7) (SEQ ID NO: 8) ID NO: 9) Properdin AMGEN
GDSISSGGHYWS (SEQ YIYYSGSSYYNPSLKS TGDYFDY (SEQ ID ID NO: 10) (SEQ
ID NO: 11) NO: 12) Properdin AMGEN GFTFSNYGIH (SEQ ID
VIWYDGNNKYYADSV GGYYDSRGYYTPYY NO: 13) KG (SEQ ID NO: 14) YYGMDV
(SEQ ID NO: 15) Properdin AMGEN GFTFSCYGMH (SEQ VIWYDGSNKYYADSVK
AGGATAMDV (SEQ ID NO: 16) G (SEQ ID NO: 17) ID NO: 18) Properdin
AMGEN GYTLTELSMH (SEQ GFDPEDGETIYAQMFQ GTYYDILTGPSYYY ID NO: 19) G
(SEQ ID NO: 20) YGLGV (SEQ ID NO: 21) Properdin AMGEN GGSISIYYWS
(SEQ ID YIYYSGSTNYNPSLKS WNYGDAFDI (SEQ NO: 22) (SEQ ID NO: 23) ID
NO: 24) Properdin Rabbit GFSFSSGYWIF (SEQ GIYSGSSGTTYYANWA
SVDGIDSYDAAFNL ID NO: 25) KG (SEQ ID NO: 26) (SEQ ID NO: 27) Factor
Bb Mouse GYTFTNYWIH (SEQ YINPNTGYNDYNQKFK GGQLGLRRAMDY ID NO: 28) D
(SEQ ID NO: 29) (SEQ ID NO: 30) Factor Bb Rabbit GFDLSTYAMS (SEQ
AVSATTGNTYYATWA YASSGVGTYFDL ID NO: 31) KG (SEQ ID NO: 32) (SEQ ID
NO: 33) Factor Bb Rabbit GFSLSNYHLG (SEQ ID VITYGGSTYYASWVKG
RDSGGYHLDL (SEQ NO: 34) (SEQ ID NO: 35) ID NO: 36) Factor Bb Rabbit
GFSLSSNAIN (SEQ ID TIHTNTKTYYATWARG ADL (SEQ ID NO: 39) NO: 37)
(SEQ ID NO: 38) Factor Mouse GYTFTSYWIN (SEQ ID DIYPVRGITNYSEKFKN
GNFGNFDAMDY C3b NO: 40) (SEQ ID NO: 41) (SEQ ID NO: 42)
TABLE-US-00002 TABLE 2 Light Chain TARGET SPECIES CDR1 CDR2 CDR3
Properdin Mouse RASQDISFFLN (SEQ YTSRYHS(SEQ ID NO: QHGNTLPWT (SEQ
ID NO: 43) 44) ID NO: 45) Properdin Mouse KASQDVSDAVA(SEQ SPSYRYT
(SEQ ID NO: QQHYSTPWT (SEQ ID NO: 46) 47) ID NO: 48) Properdin
Mouse RSSQSLVHSNGNTYL RSSQSLVHSNGNTYL SQNTHVPRT (SEQ H (SEQ ID NO:
49) H (SEQ ID NO: 50) ID NO: 51) Properdin AMGEN RASQDISNYLA (SEQ
AASTLQS (SEQ ID NO: QKYNSAPWT (SEQ ID NO: 52) 53) ID NO: 54)
Properdin AMGEN RASQGISNYLA (SEQ AASTLQS (SEQ ID NO: QKYDSAPWT (SEQ
ID NO: 55) 56) ID NO: 57) Properdin Properdin Properdin Properdin
Rabbit QASDNIYSLLA (SEQ RASTLAS (SEQ ID NO: QQHYDYNYLDVA ID NO: 58)
59) (SEQ ID NO: 60) Factor Bb Mouse RASKSISKYLA(SEQ SGSTLQS (SEQ ID
NO: QQHDEYPWT (SEQ ID NO: 61) 62) ID NO: 63) Factor Bb Rabbit
QASENIYSRLA (SEQ YASDLAS (SEQ ID HSYYWNSAYSDNT ID NO: 64) NO: 65)
(SEQ ID NO: 66) Factor Bb Rabbit QASENIYSYLA (SEQ KASYLAS (SEQ ID
LSTIASASNFDA ID NO: 67) NO: 68) (SEQ ID NO: 69) Factor Bb Rabbit
QSSQSVYRSNNVA EASSLAS (SEQ ID NO: AGGYSSSVDFFFA (SEQ ID NO: 70) 71)
(SEQ ID NO: 72) Factor Mouse SATSSITYIH (SEQ ID DTSRLAS (SEQ ID NO:
QQWSSNPPT (SEQ C3b NO: 73) 74) ID NO: 75)
[0220] In other embodiments, an antibody described herein can
comprises heavy and light chain variable regions comprising amino
acid sequences that are homologous to the amino acid sequences of
the preferred antibodies described herein, and wherein the
antibodies retain the desired functional properties. For example,
the invention provides an isolated monoclonal antibody, or antigen
binding portion thereof, comprising a heavy chain variable region
and a light chain variable region, wherein: (a) the heavy chain
variable region comprises an amino acid sequence that is at least
80% homologous to the amino acid sequence of a heavy chain variable
region listed in Table 1 for a respective antibody; (b) the light
chain variable region comprises an amino acid sequence that is at
least 80% homologous to the amino acid sequence of a light chain
variable region listed in Table 2 for the respective antibody; and
(c) the antibody specifically binds to respective protein, C3b, P,
Ba, or Bb.
[0221] In various aspects, the antibody can be, for example, a
human antibody, a humanized antibody or a chimeric antibody. In
other aspects, the V.sub.H and/or V.sub.L amino acid sequences may
be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences
set forth above. An antibody having V.sub.H and V.sub.L regions
having high (i.e., 80% or greater) homology to the V.sub.H and
V.sub.L regions of the sequences set forth above, can be obtained
by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of
nucleic acid molecules encoding the amino acide sequences, followed
by testing of the encoded altered antibody for retained function
using the functional assays described herein.
[0222] As used herein, the percent homology between two amino acid
sequences is equivalent to the percent identity between the two
sequences. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % homology=# of identical positions/total # of
positions.times.100), taking into account the number of gaps, and
the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm, as described in the
non-limiting examples below.
[0223] In certain aspects, an antibody of the invention can include
a heavy chain variable region comprising CDR1, CDR2 and CDR3
sequences and a light chain variable region comprising CDR1, CDR2
and CDR3 sequences, wherein one or more of these CDR sequences
comprise specified amino acid sequences based on the preferred
antibodies described herein, or conservative modifications thereof,
and wherein the antibodies retain the desired functional
properties. Accordingly, the invention provides an isolated
monoclonal antibody, or antigen binding portion thereof, comprising
a heavy chain variable region comprising CDR1, CDR2, and CDR3
sequences and a light chain variable region comprising CDR1, CDR2,
and CDR3 sequences.
[0224] As used herein, the term "conservative sequence
modifications" is intended to refer to amino acid modifications
that do not significantly affect or alter the binding
characteristics of the antibody containing the amino acid sequence.
Such conservative modifications include amino acid substitutions,
additions and deletions. Modifications can be introduced into an
antibody of the invention by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions are ones in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within the CDR regions of an antibody
of the invention can be replaced with other amino acid residues
from the same side chain family and the altered antibody can be
tested for retained function (i.e., the functions set forth in (c)
through (j) above) using the functional assays described
herein.
[0225] An antibody of the invention further can be prepared using
an antibody having one or more of the V.sub.H and/or V.sub.L
sequences disclosed herein as starting material to engineer a
modified antibody, which modified antibody may have altered
properties from the starting antibody. An antibody can be
engineered by modifying one or more residues within one or both
variable regions (i.e., V.sub.H and/or V.sub.L), for example within
one or more CDR regions and/or within one or more framework
regions. Additionally or alternatively, an antibody can be
engineered by modifying residues within the constant region(s), for
example to alter the effector function(s) of the antibody.
[0226] One type of variable region engineering that can be
performed is CDR grafting. Antibodies interact with target antigens
predominantly through amino acid residues that are located in the
six heavy and light chain complementarity determining regions
(CDRs). For this reason, the amino acid sequences within CDRs are
more diverse between individual antibodies than sequences outside
of CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties. Thus, such antibodies contain the
V.sub.H and V.sub.L CDR sequences described in the Tables yet may
contain different framework sequences from these antibodies.
[0227] Another type of variable region modification is to mutate
amino acid residues within the V.sub.H and/or V.sub.K CDR1, CDR2
and/or CDR3 regions to thereby improve one or more binding
properties (e.g., affinity) of the antibody of interest.
Site-directed mutagenesis or PCR-mediated mutagenesis can be
performed to introduce the mutation(s) and the effect on antibody
binding, or other functional property of interest, can be evaluated
in in vitro or in vivo assays as described herein and provided in
the Examples. Conservative modifications (as discussed above) are
introduced. The mutations may be amino acid substitutions,
additions or deletions, but are preferably substitutions. Moreover,
typically no more than one, two, three, four or five residues
within a CDR region are altered.
[0228] In general, therapeutic antibodies, once selected, can be
manipulated, altered and engineered in a variety of ways for
various different reasons. For example, the inactive (non-binding)
parts of an selected antibody may be changed and manipulated in
countless ways which do not at all change the defining functions of
the antibody. In fact, the functional (protein binging part) of the
antibody might be entirely severed from the rest of the antibody.
These alterations may have utility for making the antibody easier
or less costly to produce. Or, such alterations may make the
antibody more chemically stable in human subjects. These
manipulations and derivations of the selected antibodies are not
new or separate inventions. Accordingly, any such manipulations,
alternations and derivations of the selected genus of antibodies
which utilize the same defining characteristics of the genus itself
are within the scope of the invention.
[0229] The invention includes compounds which constitute the
functional (target protein binding) components of any one or
several of the selected genus of antibodies, as well as the
therapeutic use such compounds. These compounds include, but are
not limited to, whole antibodies of the selected genus,
antigen-binding fragments of antibodies of the selected genus, and
chimeric or humanized manifestations of any antibody or antibody
fragment derived from the selected genus of antibodies. Such
derivations of the inventions may include, but are not limited to,
truncated, linear, single-chained, an IgG fragment, a F(ab)
fragment, a F(ab') fragment, a F(ab)2 fragment, a F(ab')2 fragment,
an Fv fragment or an scFv fragment which may be manifested from any
antibody of the selected genus.
[0230] The invention includes the result of any member of the
antibody genus having its Fc region mutated at the 297 position to
generate an aglycosylated antibody. The invention includes the
results of any antibody of the selected genus being engineered to
elicit reduced Fc-mediated effector functions. Methods of
engineering may include, without limitation, amino acid mutations,
amino acid additions or deletions, glycan modification or removal,
pegylation, and/or truncation.
Methods of Administration
[0231] The invention provides methods of treatment comprising
administering to a subject an effective amount of an embodiment of
the invented genus of antibodies. The subject may be an animal (a
mammal such as a cow, pig, rat, or monkey) but is preferably a
human. Various delivery systems are known and can be used to
administer an embodiment of the invention, (e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis,
construction of a nucleic acid as part of a retroviral or other
vector, etc.). Methods of introduction can be enteral or parenteral
and may include, but are not limited to, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, and oral routes. The compounds may be administered by
any convenient route, for example by infusion or bolus injection,
by absorption through epithelial or mucocutaneous linings (e.g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be
administered together with other biologically active agents.
Administration can be systemic or local. Administration can be
acute or chronic (e.g., daily, weekly, monthly, etc.) or in
combination with other agents.
Dosage
[0232] Administration of the invented genus of antibodies, and/or
any functional derivations thereof, may be by any method known in
the art. Such administration may be subcutaneous, intraarticular,
intramuscular, intradermal, intraperitoneal, intravenous,
intranasal, or via oral routes of administration. In one preferred
embodiment, the antibody is administered by subcutaneous injection
or intravenous injection. In a specific embodiment, the antibody is
administered by subcutaneous injection.
[0233] In one embodiment, the amount of AP antibody administered is
in a dosage range between 0.3 mg/kg to 30 mg/kg. In a more specific
embodiment, the AP antibody is administered once a day in a range
between 0.5 mg/kg to 10 mg/kg. In another embodiment, AP antibody
is administered in a dosage range between 0.3 mg/kg to 30 mg/kg at
least once a week. In yet another embodiment, AP antibody is
administered in a dosage range between 0.3 mg/kg to 30 mg/kg at
least once a month. Thus, depending upon the AP inhibition profile,
administration regimen can be chosen.
Formulation
[0234] The compound can be administered to an individual in a
formulation with a pharmaceutically acceptable excipient(s). A wide
variety of pharmaceutically acceptable excipients are known in the
art and need not be discussed in detail herein.
[0235] The compound can be incorporated into a variety of
formulations for therapeutic administration. In one example, a
subject compound can be formulated into pharmaceutical compositions
by combination with appropriate, pharmaceutically acceptable
carriers or diluents, and can be formulated into preparations in
solid, semi-solid, liquid or gaseous forms, such as tablets,
capsules, powders, granules, ointments, solutions, suppositories,
injections, inhalants and aerosols.
[0236] Formulations can also be developed for subcutaneous,
intraperitoneal, intravenous, and intraarticular
administration.
Dosing Schedule
[0237] A compound of the present invention can be administered to
an individual with a certain frequency and for a period of time so
as to achieve the desired therapeutic effect. For example, an
antibody of the present invention can be administered, for example,
once per month, twice per month, three times per month, every other
week (qow), once per week (qw), twice per week (biw), three times
per week (tiw), four times per week, five times per week, six times
per week, every other day (qod), daily (qd), twice a day (qid), or
three times a day (tid), or substantially continuously, or
continuously, over a period of time ranging from about one day to
about one week, from about two weeks to about four weeks, from
about one month to about two months, from about two months to about
four months, from about four months to about six months, or
longer.
AP Specific Antibodies that Inhibit Alternative Pathway
(AP)-Dependdent Inflammation in Hemolytic Diseases
Alternative Pathway in Whole Blood--Inflammation Model
[0238] There is direct link between C3a/C5a production and
activation of neutrophils, monocytes, and platelets and release of
a battery of inflammatory cytokines, proteases, and peroxides. In
this model, whole blood from a healthy donor is subjected to AP
activation via contact as a stimulus. Anaphylatoxin production,
cellular activation and measurement of inflammatory cytokines were
determined in the presence and absence of antibodies of the current
invention. Activation of cells is related to aggregateformation and
finally removel from circulation causing cytopenia. AP antibodies
of this invention are demonstrated to have a regulatory effect on
prevention of activation and cytopenia.
[0239] In order to demonstrate the effect of the activation of the
alternative pathway in vivo, an ex vivo whole blood inflammation
model was used. This model produces effects similar to those
exhibited by the cells involved in initiating and perpetuating the
inflammatory response. The whole blood system contains the full
array of complement proteins and cells responsible for carrying out
the ultimate inflammatory response which is the end result of
alternative pathway activation. The alternative pathway is
triggered in whole blood by contact of the plasma with the
artificial surfaces of the polypropylene tubing. Even a simple
exposure of the plasma to air can trigger AP activation, and the
resultant cellular activation and release of inflammatory
mediators. In this model, blood circulation in an artificial system
generates complement anaphylatoxins, activated cells, and
inflammatory mediators such as TNF-.alpha. and IL-1. Multiplex
analysis further indicated the production of cytokines such as
VEGF, IL-1, IL-17, and several macrophage derived cytokines. These
effects in vitro can predict the disease outcome if elevated levels
of such components are found in blood or local tissue levels.
[0240] Anti-C3b, Anti-Ba, Anti-Bb, and Anti-P antibodies have the
potential to down regulate the formation of TNF-.alpha. and
therefore prevent the onset and progression of the arthritic
condition. The therapeutic value of a reduction of TNF-.alpha. is a
known phenomenon to those of ordinary skill in the art. However, as
discussed previously, one skilled in the art cannot predict the
outcome of using any given antibody against a given protein in the
alternative pathway. Not all Anti-C3b antibodies will have a
therapeutic effect. The same is true for Anti-Ba, Anti-Bb and
Anti-P antibodies unless selected using the two step process.
AP Specific Antibodies and Cellular Lysis/Inflammation in
Humans
[0241] In addition to the process of developing the invented genus
of antibodies, as well as the resultant genus of antibodies, the
invention additionally consists of a method of treating cellular
lysis, cellular damage, and inflammation in hemolytic disorders.
The method comprises of administering to the afflicted subject a
therapeutically effective amount of a compound which is either a
member of the invented genus of antibodies, and/or has been derived
from such an antibody and utilizes the same AP inhibiting
properties as any antibody from the invented genus of antibodies.
Such a compound, or compounds, would inhibit the AP processes which
lead to the complement activated intravascular and extravascular
hemolysis. The antibodies of the claimed invention do not inhibit
CP amplification loop and therefore only inhibit AP activation
regardless of the target against which they have been made. These
antibodies are claimed to inhibit only alternative pathway derived
C3b but not classical pathway derived C3b.
[0242] In other inventions, the classical pathway can use the
amplification of the alternative pathway amplication loop and
prevent C3b produced via the classical pathway.
[0243] Intravascular Hemolysis--is caused by the deposition of
C5b-9 on cell surfaces of erythrocytes. The MAC causes cell lysis.
Such a lysis is present on cells that are deficient in CD59.
Antibodies of the claimed invention inhibit only AP derived C5b-9
formation and not the classical pathway derived C3b.
[0244] Extravascular Hemolysis--is caused by the deposition of C3b
on the cell surface. The C3b is responsible for effective removal
of cells via extravascular route. The antibodies of the claimed
invention inhibit C3b formation and therefore inhibition of
alternative pathway mediated removal of cells.
[0245] Paroxysmal nocturnal hemoglobinuria ("PNH") and other
hemolytic diseases are treated using a antibody of the claimed
invention which binds to or otherwise blocks the generation and/or
activity of one or more complement components of the alternative
pathway and not the classical pathway. Such compounds include, for
example, antibodies and fragments of the antibodies which bind to
or otherwise block the generation and/or activity of one or more
complement components of the alternative pathway but not classical
pathway, such as, for example, an antibody specific to complement
component C3b, Properdin, Ba, and Bb. The compound is an anti-C3b
antibody, an anti-properdin antibody, an anti Ba antibody, and an
anti-Bb antibody. Such antibodies are further selected from the
group consisting of Anti-C3b (murine, chimeric and humanized), an
anti-P antibody (murine, chimeric and humanized), and anti-Ba
(murine, chimeric and humanized), and anti-Bb (murine, chimeric,
humanized) and other functional fragments of such antibodies. These
antibodies are required to have two major functions; a) ability to
inhibit C3b and C5b-9 formation.
[0246] It was surprising to find that a group of selected
antibodies do not inhibit the amplification loop of the classical
pathway and therefore do not inhibit the classical pathway in 10%
NHS. These antibodies were selected from a set of assays that
specifically isolates antibodies that are specific to the
alternative pathway. These compounds inhibit the pathway upstream
and therefore are a potent inhibitors of C3a, C3b, C5a, C5b, and
C5b-9 formation in vitro and in ex vivo in human blood and
plasma/serum. Inhibition of C3b formation by such antibodies is
important to prevent extravascular hemolysis. Antibodies of this
invention prevent C3b formation produced only via the AP but not CP
in a dose dependent fashion in human serum and whole bood.
Antibodies of this invention also inhibit AP derived C5b-9 and
sC5b-9 formation in whole blood and/or serum. Therefore it is
surprising that these antibodies do not inhibit any amplification
of the classical pathway.
[0247] The AP-inhibiting antibodies can be administered
prophylactically in individuals known to have a hemolytic disease
to prevent, or help prevent the onset of symptoms. Alternatively,
the AP-inhibiting antibodies can be administered as a therapeutic
regimen to an individual experiencing symptoms of a hemolytic
disease.
[0248] In another aspect, a method of increasing the proportion of
damage sensitive type III red blood cells and therefore the total
red blood cell count in a patient afflicted with a hemolytic
disease will increase. The method comprises administering a
compound which binds to a specific AP protein and blocks the AP but
not the CP. By increasing total number of erythrocytes, symptoms
such as fatigue and anemia are alleviated in a patient afflicted
with a hemolytic disease.
[0249] In another aspect, the present invention provides a method
of rendering a subject afflicted with a hemolytic disease,
transfusion-independent by administering a compound to the subject.
The, compound being selected from the group consisting of anti-C3b,
anti-P, anti-Ba, and anti-Bb antibodies and their functionally
active antigen binding fragments which bind the AP specific
protein, compounds which block the formation of C3b and C5b-9 that
block the activity of one or more AP specific proteins.
[0250] It is surprising that AP specific antibodies can reduce the
lysis of erythrocytes and patients are rendered
transfusion-independent in accordance with the present methods.
Less C5b-9 formation is directly related to less cellular damage
means more cells and patients can become transfusion independent
and may not require transfusion.
[0251] In another aspect, the present invention contemplates a
method of reducing the lysis of red blood cells, the present
methods reduce the amount of free hemoglobin in the blood, thereby
increasing nitric oxide (NO) and prevention of kidney damage.
[0252] In another aspect, the present invention contemplates a
method of treating/preventing thrombosis in a subject by
administering the antibodies of the claimed invention to prevent
platelet activation, platelet lysis, removal of platelets, and
formation of platelet aggregates.
[0253] In another aspect, the present invention contemplates a
method of treating pharmacological effects of preventing cell
damage, wherein the cells are selected from the group comprising
neutrophils, monocytes, platelets, and T-lymphocytes.
[0254] In yet another aspect, the present invention contemplates a
method of treating a subject afflicted with a hemolytic disease by
administering: 1) one or more compounds known to increase
hematopoiesis in combination with 2) a compound selected from the
group consisting of compounds that inhibit AP activation by
inhibiting C3b formation and C5b-9 formation in a subject. Suitable
compounds known to increase hematopoiesis include, for example,
steroids, immunosuppressants (such as, cyclosporin),
anti-coagulants (such as, warfarin), folic acid, iron and the like,
erythropoietin (EPO) and antithymocyte globulin (ATG) and
antilymphocyte globulin (ALG). In particularly useful embodiments,
erythropoietin (EPO) is administered in combination with an
antibody selected from the group consisting of anti-C3b, anti-P,
anti-Ba, and anti-Bb antibodies.
[0255] In another aspect, the present disclosure provides a method
of treating one or more symptoms of hemolytic diseases in a subject
where the red cells are subject to complement attack, by
administering a compound selected from the group consisting of
compounds which bind to AP specific complement components,
compounds which block the formation of C3a, C5a, C5b-9 and
compounds which block the activity of one or more complement
components such as P, Ba, Bb, C3a, C5a, C5b, C6, C7, C8, and C9,
said compound being administered alone or in combination with one
or more compounds known to increase hematopoiesis.
[0256] In another aspect, the methods of the present invention can
selectively inhibit the activation of the alternative pathway in a
human. The Type-AP antibody can inhibit activation of the
alternative pathway without affecting activation of the classical
pathway or the amplification loop of the CP.
[0257] In another aspect, the alternative pathway specific antibody
can be selected from the group comprising, but is not limited to,
an anti-C3b antibody, and anti-Factor Ba antibody, an anti-Factor
Bb antibody, anti-factor B antibody, an anti-Factor D antibody, or
an anti-Properdin antibody.
[0258] In a further aspect, the alternative pathway protein that
the alternative specific antibody of the present invention can bind
to can be selected from the group comprising, but is not limited
to, C3b, Factor B, Factor Ba, Factor Bb, Factor D, or
Properdin.
[0259] In yet another aspect, the methods of the present invention
can be used to prevent the formation of byproducts that can form as
a result of activation of the alternative pathway in a human. In
one example, the methods of the present invention can prevent the
formation of anaphylatoxins. Anaphylatoxins include, C3a and C5a.
In another example, the methods of the present invention can
prevent the formation of C5b-9 or sC5b-9 (otherwise known as MAC).
In a further example, the methods of the present invention can
prevent the activation of neutrophils, macrophages, and platelets
in a subject. In yet another example, the methods of the present
invention can prevent the formation of cytokines. Cytokines can
include, but are not limited to, IL-1, TNF-.alpha., VEGF,
GM-CSF.
[0260] In one aspect, the alternative pathway specific antibody of
the present invention can be a monoclonal antibody, a polyclonal
antibody, an aglycosylated antibody, or an antibody that has one or
more mutations.
[0261] In another aspect, the alternative pathway specific antibody
of the present invention can be selected from the group including,
but not limited to, human, humanized, recombinant, chimeric,
de-immunized, truncated, aglycosylated, linear, single-chained, an
IgG fragment, a F(ab) fragment, a F(ab') fragment, a F(ab)2
fragment, a F(ab')2 fragment, an Fv fragment or an scFv
fragment.
[0262] In another aspect, the methods of the present invention can
include an alternative pathway specific antibody that can have a
reduced effector function. Reduced effector functions can include,
but are not limited to, reduced Fc binding, lack of Fc activation,
an Fc region that contains mutations that prevent the Fc effector
functions, or the prevention of activation of platelets and cells
that bear Fc receptors.
[0263] In another aspect, an effective amount of the alternative
pathway specific antibody can be administered to the subject. In
one example, the alternative pathway specific antibody or antigen
binding fragment thereof can be administered to the subject in a
therapeutically effective amount. In another example, the
alternative pathway specific antibody or antigen binding fragment
thereof can be administered to the subject in a prophylactically
effective amount. In a further example the alternative pathway
specific antibody can be effective in a therapeutic setting in vivo
or ex vivo. In yet another example, the alternative pathway
specific antibody can be effective in a prophylactic setting in
vivo or ex vivo.
[0264] In yet another aspect, the alternative pathway specific
antibody of the present invention can contain antigen binding
regions termed as complementarity determining regions, or CDRs. In
one example, the CDRs of the alternative pathway specific antibody
can be present in a fusion protein. In another example, the CDRs of
the alternative pathway specific antibody can be derived from a
rabbit alternative pathway specific monoclonal antibody or a mouse
alternative pathway specific monoclonal antibody. In a further
example, the CDRs of the alternative pathway specific antibody can
have greater than 50% homology to the native CDRs of the
alternative pathway specific antibody.
AP Specific Antibodies and Other Diseases
[0265] The invention genus of antibodies may be used to treat any
disease, or disease condition, associated with inappropriate
activation, or over activation, of the alternative pathway.
Examples of alternative complement pathway associated disorders are
numerous. The following is a list of some, but not all, of the
diseases, and/or disease symptoms and conditions, which may be
ameliorated through administration of the invention genus of
antibodies.
[0266] Pathologies of the Auditory System--Meniere's disease, in
which complement factors H and B are over-expressed
[0267] Pathologies of the Cardiovascular System--Kawasaki's disease
(arteritis) Cardiac surgery complications Henoch -Schonlein purpura
nephritis, wherein studies suggest that generation of MAC may be
involved in the pathogenesis of vascular injury in a significantly
large number of skin lesions and of HSP nephritis, Vascular leakage
syndrome (associated with elevated c3a), Percutaneous coronary
intervention (PCI)/coronary angioplasty, Ischemia-reperfusion
following acute myocardial infarction, Myocardial infarction, which
elevates C3 and C4, Atherosclerosis, where C5a is present in
atherosclerotic plaques, Immune complex vasculitis, in which MAC
alters the membrane integrity of endothelial cells, Arteritis,
which contain C3 and C4 deposits, Aneurysm, where it has been shown
that C5 inhibition attenuates injury in abdominal aortic aneurysm
model, Cardiomyopathy, where c5b-9 activates TNF-.alpha.,
vasculitis, where it has been shown that C5-/- mice and factor B-/-
mice do not develop disease, Takayasu's arteritis, Dilated
cardiomyopathy, where c5b-9 activates TNF-.alpha., Venous gas
embolus (VGE), Wegener's granulomatosis, Behcet's syndrome,
Autoimmune cardiomyopathy, Balloon angioplasty, in which high
levels of C5a are associated with restenosis, Myocarditis, where
C3a and TNF-.alpha. are present, Percutaneous transluminal coronary
angioplasty (PTCA), IL-2 induced vascular leakage syndrome,
Coronary artery disease (CAD), where there are high C3 levels,
Dressler's syndrome (postmyocardial infarction syndrome), in which
C3d levels are elevated, Giant cell arteritis (temporal or cranial
arteritis), Ischemic heart disease, Ischemia-reperfusion injury,
which generates C3a and c5a, Leukocytoclastic vasculitis, in which
c3d,g and Terminal complement complexes are present, Mesenteric
artery reperfusion, where it has been shown binding C3b attenuates
injury, Microscopic polyangiitis, Pauci-immune vasculitis,
associated with MAC, c3d, factor P, and factor B, Pulmonary
vasculitis, Raynaud phenomenon, Post-ischemic reperfusion
conditions, Pulmonary embolisms and infarcts, Restenosis following
stent placement, Subacute bacterial endocarditis, where C3d is
present Vasculitis associated with rheumatoid arthritis and C3
deposits.
[0268] Pathologies of Connective Tissue--Mixed connective tissue
disease and Polymyalgia rheumatica, which C3 and C4 are
deposited.
[0269] Pathologies of the Skin--Pemphigoid, Epidermolysis bullosa
acquisita, in which Factor B deficient mice display delayed and
less severe blistering in a disease model, Autoimmune bullous
dermatoses, Bullous pemphigoid, which is associated with C3 and C5,
scleroderma, where C5b-9 and C5a receptors are activated,
Angioedema, Hereditary angioneurotic edema (HAE), Erythema
multiforme, Herpes gestationis, Sjogren's syndrome, with activated
c5b-9,Psoriasis, Alopecia areata, Atopic dermatitis (eczema), where
levels of C3 and C4 are increased, Cicatricial pemphigoid,
Dermatitis herpetiformis, Diffuse systemic sclerosis, Discoid lupus
erythematosus, Eosinophilic spongiosis, Erythema nodosum, Lichen
planus, Linear iga disease, Localized systemic sclerosis(morphea),
Mucha-Habermann disease, Occular cicatricial pemphigoid, Pemphigus,
Pemphigus vulgaris, Pyoderma gangrenosum,VitiligoUrticaria.
[0270] Pathologies of the Endocrine System--Hashimoto's
thyroiditis, Diabetes mellitus type 1, in which C3, c3d, and C4
levels are increased, Stress anxiety, Pancreatitis, Addison's
disease, Insulin resistance, which increases factor H, Diabetic
angiopathyGraves' disease.
[0271] Conditions Associated with Extracorporeal
Procedures--Post-cardiopulmonary bypass inflammation,
Heparin-induced extracorporeal LDL precipitation (HELP), where C5a
is increased, Postperfusion syndrome, Post-operative pulmonary
dysfunction, Post-pump syndrome in cardiopulmonary bypass or renal
bypass, which increases c5b-9, and complement activation during
cardiopulmonary bypass operations, hemodialysis, cardiopulmonary
bypass, leukopheresis, plasmapheresis, plateletpheresis, and
extracorporeal membrane oxygenation (ECMO), which can activate
SC5b-9 via alternative pathway.
[0272] Pathologies of the Gastrointestinal System--Crohn's disease,
Celiac Disease/gluten-sensitive enteropathy, associated with c3b,
Intestinal ischemia, Inflammatory bowel disease (IBD), associated
with c5a, Ulcerative colitis, where it has been shown a C5a
antibody attenuates damage in colitis model, Eosinophilic
gastroenteritis, Gastritis, where levels of c3b, ic3b, and C3c are
increasedPancreatitis.
[0273] Hematologic Disorders--Catastrophic anti-phospholipid
syndrome (CAPS)[96], Cold Agglutinin Disease (CAD), which increases
c3b, Thrombotic thrombocytopenic purpura (TTP), which increases
CD46, factor H, and factor I, Idiopathic thrombocytopenic purpura,
where C3 and C4 detected are on platelets, Serum sickness, where
abnormal factor H leads to increased glomerular C3 deposition,
Endotoxemia, Sepsis, Atypical hemolytic uremic syndrome (ahus),
where there is enhanced formation of c3bbb convertase and
resistance to complement regulators, Paroxysmal Nocturnal
Hemoglobinuria (PNH), where it has been shown a C5 antibody
treatment reduced thromboembolism risk, Septic shock, sickle cell
anemia, which elevates c3b, Hypereosinophilic syndrome, which
increases c5a, anti-phospholipid, Autoimmune lymphoproliferative
syndrome, Dego's disease, where c5b-9 is activated, Evan's
syndrome, essential mixed cryoglobulinemia, and pure red cell
aplasia.
[0274] Pathologies of the Hepatic System--Autoimmune chronic active
hepatitis, which increase c3d, Infectious hepatitis, Primary
biliary cirrhosis inflammation (PBC), associated with higher clq,
C3, factor B, and properdin levels, Primary sclerosing cholangitis,
where C3 is increased Autoimmune hepatitis.
[0275] Pathologies of Hypersensitivity--Anaphylactic shock, in
which blocking C3a and C5a has shown to be effective therapy,
Anaphylactoid reactions from use of radiographic contrast media,
adverse drug reaction,Allergy.
[0276] Pathologies of the Musculoskeletal System--Osteoarthritis,
Osteoporosis, Acute gouty arthritis, where C6 and MAC are
activated, Spondyloarthropathy, Polymyositis, Dermatomyositis,
which increases C3b and c5b-C9, Ankylosing spondylitis, associated
with increased c3b, Arthritis, where C5a levels rise,
Enthesitis-related arthritis, Eosinophilic fasciitis, Juvenile
rheumatoid (idiopathic) arthritis, with increased clq, C4, and MAC,
Myositis, Psoriatic arthritis, where it has been shown that
anti-05a prevents arthritis, Reiter's syndrome (reactive arthritis)
Relapsing polychondritis.
[0277] Pathologies of the Nervous System--Myasthenia gravis,
Multiple sclerosis (MS), Guillain Barre syndrome, which activates
C3a and c5a, stroke, where C4 and sC3b-5 is elevated, Cranial nerve
damage in meningitis, Variant Creutzfeldt-Jakob disease (vcjd),
Neuropathic pain, Alzheimer's disease (AD), where it has been shown
that treatment with C5a receptor antagonist reduced pathology,
Multifocal motor neuropathy (MMN), Huntington's disease (HD)where
there is deposition of C3 and C9 and upregulation of C5a receptors,
Amyotrophic lateral sclerosis (ALS), which increases C5a and c5a,
Parkinson's disease, degenerative disc disease (DDD), Idiopathic
polyneuropathy, allergic neuritis, where C3 depletion can result in
less injury, Acute disseminated encephalomyelitis, Acute
hemorrhagic leukoencephalitis, Autoimmune peripheral neuropathy,
Chronic inflammatory demyelinating polyneuropathy, demyelination ,
where reduction in C3 and C4 has shown to prevent demyelination,
Idiopathic inflammatory demyelinating diseases, Lambert-Eaton
myasthenic syndrome, Meningitis, in which C5a is correlated with
prognosis and c5ar deficient mice suffered less brain damage,
Miller-Fisher syndrome, Neuromyelitis optica (NMO), Perivenous
encephalomyelitis, where it has been shown C6 deficient mice are
unable to form MAC and exhibit no demyelination, progressive
inflammatory neuropathy, opsoclonus myoclonus syndrome, Rasmussen's
encephalitis, pediatric autoimmune neuropsychiatric disorders
associated with streptococcus, stiff person syndrome, Susac
syndromeanxiety.
[0278] Pathologies of Vision--Endophthalmitis, where there is
higher levels of C3a and C4a in the vitreous, Diabetic retinopathy,
where there are C3d and c5b-9 deposits in choriocapillaris,
Diabetic retinal microangiopathy, with C5b-9 in the retina,
Histoplasmosis of the eye, Purtscher's retinopathy, Age-related
macular degeneration (AMD), Dry Age-Related Macular Degeneration
(AMD), with elevated c3a, choroidal neurovascularization (CNV),
Uveitis, Diabetic macular edema, Pathological myopia, Central
retinal vein occlusion (CRVO), Retinal neovascularization, Retinal
pigment epithelium (RPE), Choroidal neovascularization (CNY),
Dominant drusen, where C3a and C5a promote coronial
neurovasculiaztion, Photoreceptor and/or Retinal Pigmented
Epithelial (RPE) loss, Stargardt's diseaseScleritis.
[0279] Oncological Pathologies--Hemangiomas, Tumor cell
metastasis.
[0280] Pathologies of the Renal System--Glomerulonephritis,
Poststreptococcal glomerulonephritis (PSGN), Goodpasture's disease,
Membranous nephritis, Berger's Disease/iga nephropathy,
Mesangioproliferative glomerulonephritis, where c5b-9 is elevated,
Membranoproliferative glomerulonephritis(Dense Deposit Disease),
Membranous glomerulonephritis, Renal cortical necrosis (RCN), Renal
reperfusion injury, where it has been shown C5 inhibition protects
from renal injury, Cryoglobulinemic glomerulonephritis, ABO
Incompatible Renal Transplant, Atypical hemolytic uremic syndrome
(ahus), Lupus (SLE) nephritis.
[0281] Pathologies of the Respiratory System--Eosinophilic
pneumonia, Hypersensitivity pneumonitis, Bronchiecstasis, Reactive
airway disease syndrome, where it has been shown C5 and c5ar
deficient mice show no airway hyperreactivity, Respiratory
syncytial virus (RSV) infection, Parainfluenza virus infection,
Rhinovirus infection, Adenovirus infection, Allergic
bronchopulmonary aspergillosis (ABPA), Tuberculosis, Parasitic lung
disease, Pollution-induced asthma, in which higher C3c and C4 in
serum has been shown in children living in polluted areas, Airway
hyperresponsiveness (AHR), Adult respiratory distress syndrome,
which elevates C3 and c3a, Exercise-induced asthma, Cough variant
asthma, occupational asthma, Allergic asthma, Pollen-induced
asthma, Severe asthma, Chronic obstructive pulmonary disease
(COPD), Emphysema, bronchitis, Cystic fibrosis, Interstitial lung
disease, Acute respiratory distress syndrome (ARDS),
Transfusion-related acute lung injury (TRALI), Acute lung injury,
Byssinosis, Asbestos-induced inflammation, Bronchoconstriction,
Fibrosing alveolitis (idiopathic pulmonary fibrosis), which
elevates factor Ba, Ischemia/reperfusion acute lung injury, Organic
dust diseases, where C3, c3d, and factor B levels increase,
Pneumonia, Pathologies caused by inert dusts and minerals (e.g.,
silicon, coal dust, beryllium, and asbestos).
[0282] Systemic Disorders--Systemic lupus erythematosis (SLE),
Rheumatoid arthritis, Acquired Immune Deficiency Syndrome (AIDS),
Sarcoid, Systemic inflammatory response syndrome (SIRS), Systemic
juvenile rheumatoid arthritis, which elevates Factor Bb and SC5b-9,
Castleman's disease, Complement component 2 deficiency, Multiple
organ failure, Interleukin-2 induced toxicity during IL-2 therapy,
Barraquer-Simons Syndrome (acquired partial lipodystrophy).
[0283] Complications of Organ and Tissue Transplants--Transplant
rejection, where it has been shown anti-05 antibodies improved
graft survival, Xenograft rejection, Allotransplantation of organs
or grafts, where it has been shown C5 inhibition reduces antibody
mediated rejection, Hyperacute rejection, Graft versus host
disease, Hyperacute allograft rejection, Presensitized Renal
Transplant - Living Donor, Revascularization to transplants and/or
replants.
[0284] Associated with Trauma--Hemorrhagic shock, where it has been
shown C5a receptor antagonist attenuates multiple organ injury,
Hypovolemic shock, Spinal cord injury, Cerebral trauma and/or
hemorrhage, Severe burns, where it has been shown C5a blockade
improves burn-induced cardiac dysfunction, Frostbite, Crush injury,
Wound healing,Brain trauma, Cerebral ischemia reperfusion, which
elevates C5 Smoke injury.
[0285] Pathologies of the Urogenital and/or Reproductive
System--Spontaneous abortion, Sensory bladder disease, Interstitial
cystitis(painful bladder syndrome), Fetomaternal tolerance,
Preeclampsia, Sinusitis, Complications of pregnancy, Chronic
abacterial cystitis, Hemolysis, elevated liver enzymes, and low
platelets (HELLP) syndrome, Infertility, Placental dysfunction and
miscarriage and pre-eclampsia, Recurrent fetal loss.
[0286] Other relevant diseases and conditions--Agammaglobulinemia,
antisynthetase syndrome, atopic allergy, autoimmune enteropathy,
autoimmune inner ear disease, autoimmune polyendocrine syndrome
type 1 (Whitaker's syndrome), autoimmune polyendocrine syndrome
type 2 (Schmidt syndrome), autoimmune progesterone dermatitis, Balo
disease/Balo concentric sclerosis, Vitelliform macular dystrophy
(best disease), Bickerstaff's encephalitis, Blau syndrome, Cancer,
chemical injury (due to irritant gasses and chemicals, e.g.,
chlorine, phosgene, sulfur dioxide, hydrogen sulfide, nitrogen
dioxide, ammonia, and hydrochloric acid), chronic recurrent
multifocal osteomyelitis, Churg-Strauss syndrome, Cogan syndrome,
corneal neovascularization, Cushing's syndrome, cutaneous
leukocytoclastic angiitis, Dercum's disease, fibrodysplasia
ossificans progressiva, fibrogenic dust diseases, gastrointestinal
pemphigoid, Hashimoto's encephalitis, hemolytic uremic syndrome
(HUS), hemoptysis, hypogammaglobulinemia, immune complex-associated
inflammation, ischemia-related retinopathies, lichen sclerosus,
lupoid hepatitis, juvenile lymphocytic thyroiditis, Majeed
syndrome, malattia leventinese (radial drusen), neuromyotonia,
North Carolina macular dystrophy, ord's thyroiditis, palindromic
rheumatism, paraneoplastic cerebellar degeneration, parasitic
diseases, Parry Romberg syndrome, pars planitis, Parsonage-Turner
syndrome, pattern dystrophy, pernicious anaemia, POEMS syndrome,
polyarteritis nodosa, proliferative nephritis, restless leg
syndrome, retroperitoneal fibrosis, rheumatic fever, rotational
atherectomy, Schnitzler syndrome, Sorsby's fundus dystrophy,
Still's disease, Surgical trauma, Sydenham chorea, sympathetic
ophthalmia, Tolosa-Hunt syndrome, transverse myelitis,
undifferentiated spondyloarthropathy, vasculitis associated with
systemic lupus erythematosus, vasculitis associated with hepatitis
A, von Hippel-Lindau disease (VHL), Whipple's disease, Autoimmune
Neutropenia, Chemotherapy, Hemodialysis, Human Immunodeficiency
Virus (HIV), Malaria, Epstein Barr Virus, Vitamin Deficiencies,
Hypersplenism, Idiopathic Thrombocytopenic Purpura (ITP),
Disseminated Intravascular Coagulation(DIC), Post-Transfusion
Purpura, Neonatal Allo-Immune Thrombocytopenia, Onyalai, Cyclic
Neutropenia, Snake bites, Administration of Interferon,
Administration of Tumor Necrosis Factor, administration of
Radiotherapy, and application of Corticosteroids.
[0287] ITP--ITP is a relatively common hematological disorder
defined by low platelet count, normal bone marrow and the absence
of other causes of thrombocytopenia. ITP can be diagnosed using
standard clinical laboratory tests are used, including: urinalysis,
CBC with differential, hematology, coagulation, serum chemistry
(includes determining concentration of GM-CSF and soluble GM-CSF),
surfactant D, erythrocyte sedimentation rate, and C-reactive
protein.
[0288] Patients with chronic ITP are identified as at risk for
bleeding if platelet the count is less than 30.times.10.sup.9/1 for
those patients not receiving corticosteroids or less than
50.times.10.sup.9/L for those patients receiving
corticosteroids.
[0289] Antibody mediated rejection in organ transplantation,
antineutrophil cytoplasmic autoantibody (ANCA) vasculitis,
catastrophic antiphospholipid antibody syndrome, dense deposit
disease or C3 nephropathy, hemolytic cold agglutinin disease,
neuromyelitis optica, nonexudative (dry) macular degeneration,
Shiga toxin E. coli-related hemolytic uremic syndrome (STEC-HUS),
systemic lupus erythematosus (SLE), thrombotic thrombocytopenic
purpura (TTP)
EXAMPLE 1
Cellular Assay to Demonstrate Inhibition of AP Activation in a
Subject using Exemplary Compounds of the Invention Genus of
Antibodies
[0290] To assess the ability of the exemplary compounds of the
present invention to inhibit AP activation in a in vivo-like
system, an erythrocyte hemolysis assay was used. Rabbit red blood
cells (rRBCs) were incubated with normal human serum (NHS) in an AP
enabling buffer. The presence of rRBCs ("the foreign body")
preferentially induces activation of the AP, resulting in C5b-9
deposition on the erythrocytes and ultimately causing cell lysis.
The extent of cell lysis is detected based on light scattering at
optical density of 700 nm. Exemplary compounds of the invention
genus of antibodies inhibited hemolysis of rRBCs in a dose
dependent manner, as shown in FIGS. 2 and 3.
[0291] Introducing rabbit Erythrocytes (rRBC) into 10% human serum
(with Mg.sup.2+/EGTA) represent the introduction of a foreign cell
surface which initiates the alternative complement cascade.
Activation of the AP results in the formation of MAC which causes
lysis of the foreign cells (the rRBCs). The selected antibodies of
the present invention prevent lysis of these erythrocytes. This
process was quantified after examining the light scattering caused
by intact red blood cells.
[0292] It is well established that rabbit erythrocytes specifically
activate the AP, with a resulting lysis of the rRBCs by the C5b-9
(MAC) complex. A progressive decrease in light scatter (due to
lysis of intact cells) was measured at 700 nm as a function of time
in a temperature-controlled ELISA plate reader. The data were
recorded and analyzed with a SpectraMax 190 plate reader and
SoftMax Pro software. The results were plotted with MicroCal Origin
Software.
[0293] As shown in FIG. 4, anti-C3b, anti-Ba, anti-Bb, and anti-P
antibodies of the present invention inhibit AP activation and
therefore lysis of rRBC in human serum only under conditions that
promotes alternative pathway dependent lysis.
[0294] Lysis of cells occurs in several diseases including
hemolytic diseases. Inhibition of lysis would provide significant
benefit in disease conditions where cell death occurs as a result
of production of C5b-9 (FIG. 4). Lysis of cells also is indicative
of tissue injury seen in other diseases where role of complement in
tissue injury appears to be well established.
EXAMPLE 2
The Antibody of the Present Invention Does Not Inhibit the
Classical Pathway
[0295] To test the activity of the antibodies for CP inhibition,
antibody-sensitized, sheep erythrocytes (sRBC) were incubated in 1%
normal human serum in CP buffer (Ca.sup.2+/Mg.sup.2+). These sRBCs
activate the CP, which induces lysis of cell membranes. Lysis of
the cell membranes results in a gradual decrease in light scattered
by cells. When an alternative pathway specific antibody of the
present invention was incubated with sRBCs at 37.degree. C. in 1%
NHS with a buffer containing Ca.sup.2+ and Mg.sup.2+ ("the CP
buffer") no effect on hemolysis was observed within the time period
beginning with the start of hemolysis and concluding with maximal
hemolysis. This implies that the alternative pathway specific
antibody of the present invention does not affect CP hemolytic
activity in NHS (FIG. 5) and is not expected to compromise the CP's
expected contribution to host defense against pathogens.
[0296] Monoclonal antibodies of the present invention, irrespective
of the target antigen against which they have been raised, do not
inhibit the classical pathway. In a typical assay, antibody
sensitized sheep erythrocytes are incubated with Normal Human
Serum, with CP buffer containing Ca.sup.++. These conditions allow
for selective activation of the classical pathway. Mechanistically,
the antigen-Antibody complex on the surface of the sheep cells
activates the classical complement pathway which causes erythrocyte
lysis.
[0297] As shown in FIG. 5, the representative antibody of the
present invention that inhibits the AP but not the CP or the
amplification loop of the CP. Development of monoclonal antibodies
of this invention will leave the classical pathway intact for host
defense against infection.
[0298] Lack of inhibition of CP activation by the antibodies of the
current genus suggests that host defense will not be compromised as
classical pathway is required for host defense. Classical pathway,
upon activation, generates C3b which is required for opsonization.
In a disease state during AP activation. Thus C3b mediated
opsonization is not inhibited by the antibody of this
invention.
EXAMPLE 3
The Antibody of the Present Invention Does Not Inhibit the
Amplification Process Required for the Full Potential of the
Classical Pathway
[0299] A specifically designed assay was used in order to test
candidate antibodies for any inhibitory effect on any amplification
process which may affect the full potential of the Classical
Pathway. In this assay, antibody-sensitized sheep erythrocytes
(sRBC) were incubated in 10% normal human serum in CP buffer
(Ca.sup.2+/Mg.sup.2+). These sRBCs activate the CP via an
antibody-antigen bond complex, which induces lysis of cell
membranes. Lysis of cell membranes results in a gradual decrease in
light scattered by intact cells. When the alternative pathway
specific antibody of the present invention was incubated with sRBCs
at 37.degree. C. in Ca.sup.2+ and Mg.sup.2+ containing buffer ("the
CP buffer") in 10% normal human serum (NHS), no effect on hemolysis
was observed (FIG. 2 Panel B) within the time period beginning with
the start of hemolysis and concluding with maximal hemolysis. This
implies that the alternative pathway specific antibody of the
present invention does not affect CP hemolytic activity in NHS and
is not expected to compromise the CP's expected contribution to
host defense against pathogens. It also implies that the
alternative pathway specific antibodies of the present invention do
not affect any amplification process which may be required for the
full potential of the CP. Accordingly, antibodies of the invention
genus are not expected to compromise the CP's full contribution to
normal host defense to pathogens.
[0300] Monoclonal antibodies of the present invention were
evaluated for their effect on the on amplification of the
alternative pathway. This was done using an assay of normal human
serum (10% NHS with AP isolating Mg.sup.2+ only buffer) at 37
degree C. with a fixed number of rabbit erythrocytes (Covance) in a
temperature controlled ELISA plate reader capable of reading at 700
nm. A progressive decrease in light scatter (due to lysis of intact
cells) was measured at 700 nm as a function of time. The data were
recorded and analyzed with a SpectraMax 190 plate reader and
SoftMax Pro software.
[0301] As shown in FIG. 2, panel B, the alternative pathway
specific antibody of the present invention does not inhibit
amplification of the CP which might be initiated by the AP
amplification loop. The antibody of the present invention does not
inhibit any amplification of the CP (or the CP amplification loop,
FIG. 2, panel A) and therefore is a specific inhibitor of the AP.
Host defense will remain intact.
EXAMPLE 4
The Antibody of the Present Invention Inhibits C3b Formation when
AP is Activated
[0302] Alternative pathway activation generates C3b via the
cleaving of C3 by AP C3 convertase. C3 is thereby split into C3b
and C3a. Antibodies were evaluated for inhibition of C3b using LPS
to activate the Alternative Pathway. Microtiter plates were coated
with LPS (Lipopolysaccharide from Salmonella Typhosa) 2 .mu.g/50
.mu.l in PBS overnight. The wells were incubated with 1% BSA in PBS
to block the unoccupied sites on the plate. Following 2 hour
incubation at 37 degree, the plate was rinsed with PBS and
incubated with Normal human serum (10% final concentration in AP
buffer) was mixed with antibodies of the invention and incubated
with LPS coated wells. The plate was again incubated for 2 hours
37.degree. C. to allow C3b formation to occur. The plates were
extensively washed with PBS, and components of the C3 convertase
were detected appropriately with antibodies. We detected C3b with
rabbit anti-human C3c at 1:2000 in blocking solution. Following
incubation, the plates were rinsed with PBS and prepared with
peroxidase labeled goat anti-rabbit at 1:2000 in blocking solution
for C3b detection. All plates were developed with TMB following
extensive washing with PBS. In the presence of an AP specific
antibody of the present invention inhibition of C3b formation was
observed.
[0303] The alternative pathway specific antibodies of the present
invention inhibit formation of C3b produced in excess via the
alternative complement pathway. C3b coated cells are generally
destroyed via what is known as extravascular hemolysis in PNH
disease. Other nucleated cells can be removed as well via the same
mechanism. Thus neutropenia, leokopenia and thrombocytopenia are
some examples where the end result is the reduction in the number
of cells. The genus of antibodies claimed in the current
application is expected to prevent the formation of C3b responsible
for removal of cells via extravascular route. Extravascular lysis
is important in indications such as paroxysmal nocturnal
hemoglobinuria where C3b coated erythrocytes are removed from
circulation via the unwanted extravascular route.
[0304] Shown in FIG. 18 is blood from PNH patient. T lymphocytes
are shown in yellow, monocytes are shown in blue, and neutrophils
are shown in red. These cells atin with FITC labeled CD45 to stain
all leukocytes. Platelets are shown in green. As shown, all cells
carry C3b suggesting that it is CD55 may be partly absent on all
cells to allow C3b deposition. Antibodies of the current invention
would inhibit C3b deposition as shown in FIG. 6.
EXAMPLE 5
The Antibody of the Present Invention Inhibits C5b-9 formation in
AP buffer in 10% NHS
[0305] Alternative pathway activation generates C3b via the
cleaving of C3 by AP C3 convertase. C3 is thereby split into C3b
and C3a. AP C5 convertase cleaves C5 into C5a and C5b. The C5b
molecule inserts itself into the plasma membrane and generates
C5b-9 molecules on the cell surface leading to cellular laysis and
damage of the cell wall. Antibodies were evaluated for inhibition
of C5b-9 using LPS to activate the Alternative Pathway. Microtiter
plates were coated with LPS (Lipopolysaccharide from Salmonella
Typhosa) 2 .mu.g/50 .mu.l in PBS overnight. Following 2 hour
incubation at room temperature, the plate was rinsed with PBS and
incubated with Normal human serum (10% final concentration in AP
buffer) was mixed with antibodies of the invention and incubated
with LPS coated wells. The plate was again incubated for 2 hours
37.degree. C. to allow C5b-9 formation to occur. The plates were
extensively washed with PBS, and components of the C5b-9 were
detected appropriately with neo antibody to C5b-9. We detected
C5b-9 with mouse anti-MAC at 1:2000 in blocking solution. All
plates were developed with TMB following extensive washing with
PBS. In the presence of an AP specific antibody of the present
invention inhibition of C5b-9 formation was observed.
[0306] As shown in FIG. 7, the alternative pathway specific
antibodies of the present invention inhibit formation of C5b-9
produced in excess via the alternative complement pathway. C5b-9
coated cells are destroyed via intraysacular hemolysis in PNH
disease. Other nucleated cells can be removed as well via the same
mechanism. Thus neutropenia, leokopenia and thrombocytopenia are
some examples where the end result is the reduction in the number
of cells. The genus of antibodies claimed in the current
application is expected to prevent the formation of C5b-9
responsible for removal of cells via extravascular route.
Extravascular lysis is important in indications such as paroxysmal
nocturnal hemoglobinuria where C5b-9 coated erythrocytes are
removed from circulation via the intravascular lysis.
[0307] As shown in FIG. 10, all Blood cells were stained with CD59
and C5b-9 antibodies. Cells including platelets, neutrophil,
monocytes and T lymphocytes are attacked by C5b-9. As shown for
each cell type are the plots for CD59 and MAC. In this donor all
cells demonstrated a similar pattern and therefore all or one cell
type is sufficient to demonstrate the value of this assay for PNH
detection and drug monitoring. The ratio of C5b-9 carring cells
versus CD59 deficient cells appear to be similar. These cells will
be attacked and would be dead. The antibody of the current
invention inhibit the formation of C5b-9.
EXAMPLE 6
Inhibition of Formation of Inflammatory Mediators in Whole Blood
Inflammation Model by Compounds (Antibodies of the Current
Genus)
[0308] Alternative pathway activation generates C3b, which is
cleaved from C3 by AP C3 convertase. C3 is cleaved into C3b and
C3a. Inhibition of C3b formation has been addressed in Example 5.
Formation of C3a is measured using an ELISA (Quidel Corp).
Antibodies of the present invention inhibit the formation of C3a.
C3a receptors, which bind C3a, are found on monocytes. C3a is known
to activate monocyte which release TNF-.alpha., a potent
inflammatory cytokine and an inflammatory mediator. TNF-.alpha.
plays a role in the development and progression of arthritis.
Anti-TNF-.alpha. therapies alone have provided significant, though
incomplete, benefits for patients with various arthritic conditions
and diseases, including rheumatoid arthritis and osteoarthritis.
Inhibition of C3a formation is directly linked to the inhibition of
monocyte activation and inhibition of TNF-.alpha. formation and
arthritis inflammation.
[0309] AP activation in whole blood replicates conditions that are
primary to disease induction and progression. Blood inflammationis
linked to AP activation and production of inflammatory cytokines.
When whole human blood is subjected to AP activation via an
artificial trigger, inflammation in whole blood forwards to
completion. This includes the formation of anaphylatoxins (e.g.,
C3a, C5a), the MAC complex (C5b-9/sC5b-9), activation of
pro-inflammatory cells such as neutrophils, monocytes and
platelets, and formation and release of pro-inflammatory cytokines
including TNF-.alpha., IL-1 .beta., IL-6, IL-8, and IL-17.
[0310] In this blood inflammation (BI) model, a 2 mL aliquot of
freshly isolated heparinized human blood was circulated in
polyvinyl chloride tubing at 37.degree. C. for 2 hours. Blood
samples following the tubing loop rotation were evaluated for C3a,
C5a, and C5b-9/sC5b-9 formation. Additionally inflammation markers
such as TNF-.alpha. and neutrophil elastase were also measured.
[0311] The results shown in FIG. 8 demonstrate that antibodies of
the invention inhibit C3a formation. Elevated levels of C3a have
been found in several diseases where significant pathology exists.
Excessive C3a production results in excessive monocyte activation
and a progressively severe pathology. Many diseases where C3a is
found elevated can be treated with the antibodies of this
invention. Inhibition of C3a suggests inhibition of monocytes
activation and inhibition of inflammation in vivo. Thus ex vivo
assays are relective of in vivo inflammation which occurs in other
diseases.
[0312] Results shown in FIG. 9 demonstrate that antibodies of the
invention inhibit C5a formation. C5a activates neutrophils and
monocytes by binding their respective receptors on each of these
cell types. Activated neutrophils express CD1lb and release
elastase and are responsible for edema in several models of
inflammation. As shown in FIG. 11, the alternative pathway specific
antibodies of the present invention inhibit neutrophil activation
and, consequently, neutrophil mediated pathological outcomes in
vivo. As shown in FIG. 19-25 inhibition f AP activation prevents
tissue inflammation, synovitis, bone and cartilage degradation.
[0313] Results from FIGS. 11, 12, and 13 show that the antibody of
the current invention prevent cellular activation. Activation of
all three major cell types is inhibited. Aggregate formation and
thrombosis is also inhibited by these antibodies as shown in FIG.
4.
EXAMPLE 7
The Antibody of the Present Invention Inhibit hemolysis & LDH
in vivo
[0314] Rabbits were injected with PNH cells. The cells lysed over
time and released hemoglobin and LDH which was measured by hemoQ
and LDH was measured using a kit that measures Lactate dehrogenase
was evaluated. Cytotox LDH Kit Cat#G1781 and G1782
(Promega,Madison, Wis.) was used for LDH measurement. As shown in
FIGS. 16 and 17, the antibody of the current invention inhibits
hemoglobin release and release of LDH in vivo. The antibodies of
the current invention inhibited wrytheocyte lysis and LDH formation
in vivo in animal.
[0315] It is to be understood that this invention is not limited to
particular embodiments described, as such may, of course, vary. It
is also to be understood that the terminology used herein is for
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims. All patents, patent applications,
publications listed or identified in this disclosure are herein
incorporated by reference in their entirety.
Sequence CWU 1
1
75110PRTMus musculus 1Gly Tyr Ile Phe Thr Asn Tyr Pro Ile His 1 5
10 217PRTMus musculus 2Phe Ile Asp Pro Gly Gly Gly Tyr Asp Glu Pro
Asp Glu Arg Phe Arg 1 5 10 15 Asp 39PRTMus musculus 3Arg Gly Gly
Gly Tyr Tyr Leu Asp Tyr 1 5 412PRTMus musculus 4Gly Phe Ser Leu Ser
Thr Ser Gly Met Gly Val Gly 1 5 10 516PRTMus musculus 5His Ile Trp
Trp Asp Asp Val Lys Ser Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15
610PRTMus musculus 6Ile Gly Asp Gly Tyr Tyr Ser Phe Asp Tyr 1 5 10
710PRTMus musculus 7Gly Tyr Ile Phe Thr Thr Tyr Pro Ile His 1 5 10
817PRTMus musculus 8Phe Ile Asp Pro Gly Gly Gly Tyr Asp Glu Pro Asp
Asp Lys Phe Arg 1 5 10 15 Asp 99PRTMus musculus 9Arg Gly Asp Gly
Tyr Tyr Phe Asp Tyr 1 5 1012PRTHomo sapiens 10Gly Asp Ser Ile Ser
Ser Gly Gly His Tyr Trp Ser 1 5 10 1116PRTHomo sapiens 11Tyr Ile
Tyr Tyr Ser Gly Ser Ser Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15
127PRTHomo sapiens 12Thr Gly Asp Tyr Phe Asp Tyr 1 5 1310PRTHomo
sapiens 13Gly Phe Thr Phe Ser Asn Tyr Gly Ile His 1 5 10
1417PRTHomo sapiens 14Val Ile Trp Tyr Asp Gly Asn Asn Lys Tyr Tyr
Ala Asp Ser Val Lys 1 5 10 15 Gly 1520PRTHomo sapiens 15Gly Gly Tyr
Tyr Asp Ser Arg Gly Tyr Tyr Thr Pro Tyr Tyr Tyr Tyr 1 5 10 15 Gly
Met Asp Val 20 1610PRTHomo sapiens 16Gly Phe Thr Phe Ser Cys Tyr
Gly Met His 1 5 10 1717PRTHomo sapiens 17Val Ile Trp Tyr Asp Gly
Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 189PRTHomo
sapiens 18Ala Gly Gly Ala Thr Ala Met Asp Val 1 5 1910PRTHomo
sapiens 19Gly Tyr Thr Leu Thr Glu Leu Ser Met His 1 5 10
2017PRTHomo sapiens 20Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr
Ala Gln Met Phe Gln 1 5 10 15 Gly 2119PRTHomo sapiens 21Gly Thr Tyr
Tyr Asp Ile Leu Thr Gly Pro Ser Tyr Tyr Tyr Tyr Gly 1 5 10 15 Leu
Gly Val 2210PRTHomo sapiens 22Gly Gly Ser Ile Ser Ile Tyr Tyr Trp
Ser 1 5 10 2316PRTHomo sapiens 23Tyr Ile Tyr Tyr Ser Gly Ser Thr
Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 249PRTHomo sapiens 24Trp
Asn Tyr Gly Asp Ala Phe Asp Ile 1 5 2511PRTOryctolagus cuniculus
25Gly Phe Ser Phe Ser Ser Gly Tyr Trp Ile Phe 1 5 10
2618PRTOryctolagus cuniculus 26Gly Ile Tyr Ser Gly Ser Ser Gly Thr
Thr Tyr Tyr Ala Asn Trp Ala 1 5 10 15 Lys Gly 2714PRTOryctolagus
cuniculus 27Ser Val Asp Gly Ile Asp Ser Tyr Asp Ala Ala Phe Asn Leu
1 5 10 2810PRTMus musculus 28Gly Tyr Thr Phe Thr Asn Tyr Trp Ile
His 1 5 10 2917PRTMus musculus 29Tyr Ile Asn Pro Asn Thr Gly Tyr
Asn Asp Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp 3012PRTMus musculus
30Gly Gly Gln Leu Gly Leu Arg Arg Ala Met Asp Tyr 1 5 10
3110PRTOryctolagus cuniculus 31Gly Phe Asp Leu Ser Thr Tyr Ala Met
Ser 1 5 10 3217PRTOryctolagus cuniculus 32Ala Val Ser Ala Thr Thr
Gly Asn Thr Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15 Gly
3312PRTOryctolagus cuniculus 33Tyr Ala Ser Ser Gly Val Gly Thr Tyr
Phe Asp Leu 1 5 10 3410PRTOryctolagus cuniculus 34Gly Phe Ser Leu
Ser Asn Tyr His Leu Gly 1 5 10 3516PRTOryctolagus cuniculus 35Val
Ile Thr Tyr Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Val Lys Gly 1 5 10
15 3610PRTOryctolagus cuniculus 36Arg Asp Ser Gly Gly Tyr His Leu
Asp Leu 1 5 10 3710PRTOryctolagus cuniculus 37Gly Phe Ser Leu Ser
Ser Asn Ala Ile Asn 1 5 10 3816PRTOryctolagus cuniculus 38Thr Ile
His Thr Asn Thr Lys Thr Tyr Tyr Ala Thr Trp Ala Arg Gly 1 5 10 15
393PRTOryctolagus cuniculus 39Ala Asp Leu 1 4010PRTMus musculus
40Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn 1 5 10 4117PRTMus
musculus 41Asp Ile Tyr Pro Val Arg Gly Ile Thr Asn Tyr Ser Glu Lys
Phe Lys 1 5 10 15 Asn 4211PRTMus musculus 42Gly Asn Phe Gly Asn Phe
Asp Ala Met Asp Tyr 1 5 10 4311PRTMus musculus 43Arg Ala Ser Gln
Asp Ile Ser Phe Phe Leu Asn 1 5 10 447PRTMus musculus 44Tyr Thr Ser
Arg Tyr His Ser 1 5 459PRTMus musculus 45Gln His Gly Asn Thr Leu
Pro Trp Thr 1 5 4611PRTMus musculus 46Lys Ala Ser Gln Asp Val Ser
Asp Ala Val Ala 1 5 10 477PRTMus musculus 47Ser Pro Ser Tyr Arg Tyr
Thr 1 5 489PRTMus musculus 48Gln Gln His Tyr Ser Thr Pro Trp Thr 1
5 4916PRTMus musculus 49Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly
Asn Thr Tyr Leu His 1 5 10 15 5016PRTMus musculus 50Arg Ser Ser Gln
Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 519PRTMus
musculus 51Ser Gln Asn Thr His Val Pro Arg Thr 1 5 5211PRTHomo
sapiens 52Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ala 1 5 10
537PRTHomo sapiens 53Ala Ala Ser Thr Leu Gln Ser 1 5 549PRTHomo
sapiens 54Gln Lys Tyr Asn Ser Ala Pro Trp Thr 1 5 5511PRTHomo
sapiens 55Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala 1 5 10
567PRTHomo sapiens 56Ala Ala Ser Thr Leu Gln Ser 1 5 579PRTHomo
sapiens 57Gln Lys Tyr Asp Ser Ala Pro Trp Thr 1 5
5811PRTOryctolagus cuniculus 58Gln Ala Ser Asp Asn Ile Tyr Ser Leu
Leu Ala 1 5 10 597PRTOryctolagus cuniculus 59Arg Ala Ser Thr Leu
Ala Ser 1 5 6012PRTOryctolagus cuniculus 60Gln Gln His Tyr Asp Tyr
Asn Tyr Leu Asp Val Ala 1 5 10 6111PRTMus musculus 61Arg Ala Ser
Lys Ser Ile Ser Lys Tyr Leu Ala 1 5 10 627PRTMus musculus 62Ser Gly
Ser Thr Leu Gln Ser 1 5 639PRTMus musculus 63Gln Gln His Asp Glu
Tyr Pro Trp Thr 1 5 6411PRTOryctolagus cuniculus 64Gln Ala Ser Glu
Asn Ile Tyr Ser Arg Leu Ala 1 5 10 657PRTOryctolagus cuniculus
65Tyr Ala Ser Asp Leu Ala Ser 1 5 6613PRTOryctolagus cuniculus
66His Ser Tyr Tyr Trp Asn Ser Ala Tyr Ser Asp Asn Thr 1 5 10
6711PRTOryctolagus cuniculus 67Gln Ala Ser Glu Asn Ile Tyr Ser Tyr
Leu Ala 1 5 10 687PRTOryctolagus cuniculus 68Lys Ala Ser Tyr Leu
Ala Ser 1 5 6912PRTOryctolagus cuniculus 69Leu Ser Thr Ile Ala Ser
Ala Ser Asn Phe Asp Ala 1 5 10 7013PRTOryctolagus cuniculus 70Gln
Ser Ser Gln Ser Val Tyr Arg Ser Asn Asn Val Ala 1 5 10
717PRTOryctolagus cuniculus 71Glu Ala Ser Ser Leu Ala Ser 1 5
7213PRTOryctolagus cuniculus 72Ala Gly Gly Tyr Ser Ser Ser Val Asp
Phe Phe Phe Ala 1 5 10 7310PRTMus musculus 73Ser Ala Thr Ser Ser
Ile Thr Tyr Ile His 1 5 10 747PRTMus musculus 74Asp Thr Ser Arg Leu
Ala Ser 1 5 759PRTMus musculus 75Gln Gln Trp Ser Ser Asn Pro Pro
Thr 1 5
* * * * *