U.S. patent application number 15/682380 was filed with the patent office on 2018-07-19 for rapid clearance of antigen complexes using novel antibodies.
The applicant listed for this patent is Xencor, Inc.. Invention is credited to Matthew Bernett, John Desjarlais, Gregory L. Moore.
Application Number | 20180201686 15/682380 |
Document ID | / |
Family ID | 50030555 |
Filed Date | 2018-07-19 |
United States Patent
Application |
20180201686 |
Kind Code |
A1 |
Moore; Gregory L. ; et
al. |
July 19, 2018 |
RAPID CLEARANCE OF ANTIGEN COMPLEXES USING NOVEL ANTIBODIES
Abstract
The present invention relates to rapid clearance molecules that
bind target antigens and Fc.gamma.RIIb with increased affinity as
compared to parent molecules, said compositions being capable of
causing accelerated clearance of such antigens. Such compositions
are useful for treating a variety of disorders, including allergic
diseases, atherosclerosis, and a variety of other conditions.
Inventors: |
Moore; Gregory L.; (Azusa,
CA) ; Desjarlais; John; (Pasadena, CA) ;
Bernett; Matthew; (Monrovia, CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Xencor, Inc. |
Monrovia |
CA |
US |
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|
Family ID: |
50030555 |
Appl. No.: |
15/682380 |
Filed: |
August 21, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14156432 |
Jan 15, 2014 |
9738722 |
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15682380 |
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61752955 |
Jan 15, 2013 |
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61794164 |
Mar 15, 2013 |
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61794386 |
Mar 15, 2013 |
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61833696 |
Jun 11, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 14/435 20130101; C07K 2317/30 20130101; C07K 2317/76 20130101;
C07K 2319/32 20130101; A61K 38/1709 20130101; C07K 2319/30
20130101; C07K 16/46 20130101; A61K 38/1774 20130101; C07K 16/18
20130101; A61K 39/3955 20130101; C07K 16/2878 20130101; A61K 38/177
20130101; C07K 16/2803 20130101; C07K 2317/77 20130101; C07K
2317/92 20130101; A61K 2039/54 20130101; C07K 2317/52 20130101;
C07K 2317/72 20130101; A61K 39/395 20130101; C07K 16/1027 20130101;
C07K 16/4291 20130101; C07K 2317/732 20130101; A61K 38/17 20130101;
C07K 2317/24 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/42 20060101 C07K016/42 |
Claims
1.-32. (canceled)
33. A rapid clearance molecule comprising: i) a domain that binds
said oxLDL; and ii) a variant Fc domain comprising an amino acid
substitution as compared to a parent Fc domain, wherein said
variant Fc domain binds Fc.gamma.RIIb with increased affinity as
compared to said parent Fc domain; wherein said variant Fc domain
comprises amino acid substitutions selected from the group
consisting of S267E, S267D, L328F, P238D, S267E/L328F, G236N/S267E,
and G236D/S267E, wherein numbering is according to EU index as in
Kabat.
34. A rapid clearance molecule according to claim 29, wherein said
rapid clearance molecule is an anti-oxLDL antibody.
35. A rapid clearance molecule according to claim 29, wherein said
rapid clearance molecule is a LOX-1 Fc fusion protein.
36. A rapid clearance molecule according to claim 29, wherein said
rapid clearance molecule is a CD36 Fc fusion protein.
37. A rapid clearance molecule according to claim 1, wherein said
variant Fc domain is a variant of a parent human IgG1 Fc
domain.
38. A complement receptor 2 (CR2) fusion protein comprising: i) a
CR2 polypeptide comprising one or more domains of CR2; and ii) a
variant Fc domain comprising an amino acid substitution as compared
to a parent Fc domain, wherein said variant Fc domain binds
Fc.gamma.RIIb with increased affinity as compared to said parent Fc
domain; wherein said variant Fc domain comprises amino acid
substitutions selected from the group consisting of S267E, S267D,
L328F, P238D, S267E/L328F, G236N/S267E, and G236D/S267E, wherein
numbering is according to EU index as in Kabat.
39. A complement receptor 2 fusion protein according to claim 38,
wherein said CR2 polypeptide comprises short consensus repeat (SCR)
domains SCR1 and SCR2.
40. A complement receptor 2 fusion protein according to claim 38,
wherein said CR2 polypeptide comprises short consensus repeat (SCR)
domains SCR1-4.
Description
PRIORITY CLAIM
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/156,432, filed Jan. 15, 2014, which claims
priority to U.S. Provisional Application Ser. No. 61/752,955, filed
Jan. 15, 2013; 61/794,164, filed Mar. 15, 2013, 61/794,386, filed
Mar. 15, 2013, and 61/833,696, filed Jun. 11, 2013, each of which
is expressly incorporated by reference in the entirety.
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 21, 2017, is named 067461_5160_US03_ST25.txt and is 231,057
bytes in size.
RELATED APPLICATIONS
[0003] U.S. Ser. Nos. 11/124,620, 13/294,103, 12/341,769 and
12/156,183 are all expressly incorporated by reference in their
entirety, particularly for the recitation of amino acid positions
and substitutions, and all data, figures and legends relating
thereto.
TECHNICAL FIELD
[0004] The present disclosure relates to methods of using
polypeptides with two domains, a first domain that bind a ligand
(such as the variable region of an immunoglobulin or a fusion
partner) and a second domain, an Fc domain, that binds
Fc.gamma.RIIb, particularly human Fc.gamma.RIIb, with high
affinity. These methods resulting in rapid and accelerated
clearance of the polypeptide-ligand complexes, e.g. the
antibody-antigen complexes in the case of antibody polypeptides.
Such methods are useful for treating a variety of conditions.
BACKGROUND OF THE INVENTION
[0005] Antigen recognition by B cells is mediated by the B cell
receptor (BCR), a surface-bound immunoglobulin in complex with
signaling components CD79a (Ig.alpha.) and CD79b (Ig.beta.).
Crosslinking of BCR upon engagement of antigen results in
phosphorylation of immunoreceptor tyrosine-based activation motifs
(ITAMs) within CD79a and CD79b, initiating a cascade of
intracellular signaling events that recruit downstream molecules to
the membrane and stimulate calcium mobilization. This leads to the
induction of diverse B cell responses (e.g., cell survival,
proliferation, antibody production, antigen presentation,
differentiation, etc.) which lead to a humoral immune response
(DeFranco, A. L., 1997, Curr. Opin. Immunol. 9, 296-308; Pierce, S.
K., 2002, Nat. Rev. Immunol. 2, 96-105; Ravetch, J. V. &
Lanier, L. L., 2000, Science 290, 84-89). Other components of the
BCR coreceptor complex enhance (e.g., CD19, CD21, and CD81) or
suppress (e.g., CD22 and CD72) BCR activation signals (Doody, G. M.
et al., 1996, Curr. Opin. Immunol. 8, 378-382; L1, D. H. et al.,
2006, J. Immunol. 176, 5321-5328). In this way, the immune system
maintains multiple BCR regulatory mechanisms to ensure that B cell
responses are tightly controlled.
[0006] When antibodies are produced to an antigen, the circulating
level of immune complexes (e.g., antigen bound to antibody)
increases. These immune complexes downregulate antigen-induced B
cell activation. It is believed that these immune complexes
downregulate antigen-induced B cell activation by coengaging
cognate BCR with the low-affinity inhibitory receptor
Fc.gamma.RIIb, the only IgG receptor on B cells (Heyman, B., 2003,
Immunol. Lett. 88, 157-161). It is also believed that this negative
feedback of antibody production requires interaction of the
antibody Fc domain with Fc.gamma.RIIb since immune complexes
containing F(ab').sub.2 antibody fragments are not inhibitory
(Chan, P. L. & Sinclair, N. R., 1973, Immunology 24, 289-301).
The intracellular immunoreceptor tyrosine-based inhibitory motif
(ITIM) of Fc.gamma.RIIb is necessary to inhibit BCR-induced
intracellular signals (Amigorena, S. et al., 1992, Science 256,
1808-1812; Muta, T., et al., 1994, Nature 368, 70-73). This
inhibitory effect occurs through phosphorylation of the
Fc.gamma.RIIb ITIM, which recruits SH2-containing inositol
polyphosphate 5-phosphatase (SHIP) to neutralize ITAM-induced
intracellular calcium mobilization (Kiener, P. A., et al., 1997, J.
Biol. Chem. 272, 3838-3844; Ono, M., et al., 1996, Nature 383,
263-266; Ravetch, J. V. & Lanier, L. L., 2000, Science 290,
84-89). In addition, Fc.gamma.RIIb-mediated SHIP phosphorylation
inhibits the downstream Ras-MAPK proliferation pathway
(Tridandapani, S. et al., 1998, Immunol. 35, 1135-1146).
[0007] A recently recognized function of Fc.gamma.RIIb is to serve
as a scavenger receptor in the liver, clearing antibody:antigen
immune complexes from circulation. Fc.gamma.RIIb is thus an
important component of the classical reticulo-endothelial system.
For example, Anderson and colleagues (Ganesan et al., J Immunol
2012) published a study demonstrating that three quarters of mouse
Fc.gamma.RIIb is expressed in the liver, with 90% of it being
expressed in Liver Sinusoidal Endothelial Cells (LSEC). Moreover,
the authors demonstrated that clearance of radiolabeled small
immune complexes (SIC) is significantly impaired in an
Fc.gamma.RIIb knockout strain compared to wild-type mice. This is
therefore a natural property of the immune system, which can be
accentuated by Fc engineering for enhanced affinity to
Fc.gamma.RIIb.
[0008] Of relevance in the present invention are allergic diseases.
Allergic diseases and conditions, such as asthma, allergic
rhinitis, atopic dermatitis, and food allergy, have become
increasingly prevalent over the past few decades and now affect
10-40% of the population in industrialized countries. Allergic
diseases profoundly affect the quality of life, and can result in
serious complications, including death, as may occur in serious
cases of asthma and anaphylaxis. Allergies are prevalent, and are
the largest cause of time lost from work and school and their
impact on personal lives as well as their direct and indirect costs
to the medical systems and economy are enormous. For example,
allergic rhinitis (hay fever) affects 22% or more of the population
of the USA, whereas allergic asthma is thought to affect at least
20 million residents of the USA. The economic impact of allergic
diseases in the United States, including health care costs and lost
productivity, has been estimated to amount to $6.4 billion in the
early nineties alone.
[0009] Most allergic diseases are caused by immunoglobulin E
(IgE)-mediated hypersensitivity reactions. IgE is a class of
antibody normally present in the serum at minute concentrations. It
is produced by IgE-secreting plasma cells that express the antibody
on their surface at a certain stage of their maturation. Allergic
patients produce elevated levels of IgE with binding specificity
for ordinarily innocuous antigens to which they are sensitive.
These IgE molecules circulate in the blood and bind to IgE-specific
receptors on the surface of basophils in the circulation and mast
cells along mucosal linings and underneath the skin. Binding of
antigen or allergen to IgE on mast cells, basophils, and other cell
types, crosslink the IgE molecules, and aggregate the underlying
receptors, thus triggering the cells to release vasoactive and
neuronal stimulatory mediators such as histamines, leukotrienes,
prostaglandins, bradykinin, and platelet-activating factor. The
rapid reaction of the immune system to antigen caused by antibody
immune complexes has led to the term immediate or antibody-mediated
hypersensitivity reaction, in contrast to delayed or cell-mediated
hypersensitivity reactions that are mediated by T cells.
IgE-mediated immune reactions are specifically referred to as type
I hypersensitivity reactions.
[0010] The high affinity receptor for IgE (Fc RI) is a key mediator
for immediate allergic manifestations. In addition to mast cells
and basophils, the primary mediators of allergic reactions, Fc RI
is found on a number of other cell types including eosinophils,
platelets and on antigen-presenting cells such as monocytes and
dendritic cells. An additional receptor for IgE is Fc RII, also
known as CD23 or the low-affinity IgE Fc receptor. Fc RII is
expressed broadly on B lymphocytes, macrophages, platelets, and
many other cell types such as airway smooth muscle. Fc RII may play
a role in the feedback regulation of IgE expression and
subsequently Fc RII surface expression.
[0011] Since IgE plays a central role in mediating most allergic
reactions, devising treatments to control IgE levels in the body
and regulating IgE synthesis has been of great interest. Several
strategies have been proposed to treat IgE-mediated allergic
diseases by downregulating IgE levels. One strategy involves
neutralizing the IgE molecules by binding the E-chain of IgE in or
near the Fc-receptor binding site. For example, Omalizumab (Xolair)
is a recombinant humanized monoclonal anti-IgE antibody that binds
to IgE on the same Fc site as Fc RI. Omalizumab causes a reduction
in total serum or circulating IgE in atopic patients, which
attenuates the amount of antigen-specific IgE that can bind to and
sensitize tissue mast cells and basophils. This, in turn, leads to
a decrease in symptoms of allergic diseases. Interestingly, serum
IgE levels increase after start of therapy because of
omalizumab-IgE complex formation and may remain high up to a year
after stopping therapy. Consequently, this issue may lead to
false-negatives on diagnostic tests and therefore IgE levels must
be routinely checked. Accordingly, there exists a need for improved
methods and compositions to reduce IgE-mediated diseases and
disease symptoms.
[0012] Of additional relevance in the present invention is the fact
that antibody/antigen immune complexes are well established
mediators of inflammation in various autoimmune diseases. Moreover,
circulating immune complexes can be deposited in the kidney,
ultimately resulting in nephritis, the leading cause of death in
systemic lupus erythematosus (SLE). Finally, nucleic-acid (RNA or
DNA) containing immune complexes, observed most notably in SLE, can
interact with toll-like receptors (TLRs) on immune cells, inducing
the release of inflammatory cytokines such as interferon alpha,
contributing to disease pathogenesis. The complement system
naturally recognizes these antibody-antigen immune complexes (ICs),
resulting in complement-component C3 `tagging` of the immune
complexes with a variety of fragments of C3 (including C3b, C3b(i),
C3d, and C3g). Under healthy conditions, these tagged immune
complexes are cleared through interaction with a variety of
complement receptors and Fc.gamma.Rs. C3b-C3b-IgG covalent
complexes are immediately formed on interaction of serum C3 with
IgG-IC. These C3b-C3b dimers constitute the core for the assembly
of C3/C5-convertase on the IC, which are subsequently converted
into iC3b-iC3b-IgG by the complement regulators. Further processing
of iC3b can occur through interaction with these regulators, to
produce C3d and C3g. ICs tagged with various forms of C3 have been
detected in a variety of autoimmune disease, and C3d-IC levels in
particular have been shown to correlate directly with disease
activity level in SLE. See Toong C, Adelstein S, Phan T G (2011)
Int J Nephrol Renovasc Dis "Clearing the complexity: immune
complexes and their treatment in lupus nephritis," 4:17-28, which
is hereby incorporated by reference in its entirety and in
particular all figures, legends and disclosure related to models of
DNA-anti-DNA immune complex generation and glomerular damage in
lupus nephritis and potential therapeutic targets. See also Sekita
K, Doi T, Muso E, Yoshida H, Kanatsu K, Hamashima Y (1984) Clin Exp
Immunol "Correlation of C3d fixing circulating immune complexes
with disease activity and clinical parameters in patients with
systemic lupus erythematosus," 55(3):487-494, which is hereby
incorporated by reference in its entirety and in particular all
figures, legends and disclosure related to CIC levels and anti-C3d
assays from patients with various diseases.
[0013] The natural receptor for C3d is the complement receptor 2
(CR2), also known as CD21, expressed on the surface of B cells. CR2
serves as a link to from the innate to the adaptive immune system,
and in healthy conditions, the interaction of C3d-tagged immune
complexes leads to an amplified B cell/antibody response to the
offending antigen. Unfortunately, in autoimmune diseases this
amplification can lead to continuation of an auto-antibody response
to autoantigen, further exacerbating the disease.
[0014] Soluble CRs and CR-Fc fusions have been described for
therapeutic purposes. These include CR1, CR2-Fc (U.S. Pat. No.
6,458,360), CR2-fH (CR2-factor H), and others. However, while these
approaches generally block interaction of C3-tagged ICs with their
associated receptors, they do not necessarily remove the immune
complexes from circulation. Most of the complement receptors and
regulatory proteins are composed of one or more so-called short
complement repeat (SCR) domains, also called complement control
protein (CCP) modules or Sushi domains. Typically, only a subset of
the domains is involved in direct recognition of the associated
complement fragment ligand. For example, it has been demonstrated
that only the first two SCRs of CR2 are essential for C3d binding.
The SCR domains are stable and well-behaved, making them suitable
for use in the development of therapeutic proteins.
[0015] Of further relevance to the present invention relates to the
mechanisms of hemophilia. One issue with hemophiliacs is the effect
that Factor VIII (FVI II (not to be confused with "Fv")) inhibitors
play in disease. Currently, these FVIII inhibitors (generally FVIII
antibodies, as shown in FIG. 28) are a huge problem for
hemophiliacs.
SUMMARY OF THE INVENTION
[0016] Accordingly, in one aspect the present invention provides
compositions and methods for rapidly lowering the serum
concentration of an antigen in a patient comprising administering
an antibody comprising a variable region that binds the antigen and
a variant Fc domain comprising an amino acid substitution as
compared to a parent Fc domain wherein said variant Fc domain binds
human Fc.gamma.RIIb with increased affinity as compared to said
parent Fc domain. These antibodies bind to said antigen to form an
antibody-antigen complex and said complex is cleared at least two
fold faster than the antigen alone.
[0017] In a further aspect, the present invention provides
compositions and methods for lowering the free antigen in a patient
comprising administering an antibody comprising a variable region
that binds the antigen and a variant Fc domain comprising an amino
acid substitution as compared to a parent Fc domain wherein said
variant Fc domain binds Fc.gamma.RIIb with increased affinity as
compared to said parent Fc domain. The administration results in
the concentration of said free antigen decreasing at least 50% more
rapidly than the decrease in concentration seen with an antibody
comprising the parent Fc domain.
[0018] In a further aspect, the present invention provides
compositions and methods for differentially clearing an
antibody-antigen complex in a patient compared to antibody alone,
comprising administering an antibody comprising a variable region
that binds the antigen and a variant Fc domain comprising an amino
acid substitution as compared to a parent Fc domain wherein said
variant Fc domain binds Fc.gamma.RIIb with increased affinity as
compared to said parent Fc domain. These antibodies bind to the
antigen to form an antibody-antigen complex and the complex is
cleared at least two fold faster than the antigen alone.
[0019] In one embodiment and in accordance with any of the above,
the invention provides methods wherein the variant Fc domain
comprises amino acid substitutions selected from the group
consisting of those of FIG. 30, FIG. 47, and FIG. 48.
[0020] In a further embodiment and in accordance with any of the
above, the present invention provides compositions and methods
wherein the variant Fc domain further comprises amino acid
substitutions selected from the group consisting of 434S, 434A,
428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,
436V/428L and 259I/308F/428L.
[0021] In a yet further embodiment and in accordance with any of
the above, according to any previous claim, the present invention
provides compositions and methods wherein the increased affinity
seen with the variant Fc domain is at least a 5-fold or a 10-fold
increase as compared to the parent Fc domain as measured by a
Biacore assay.
[0022] In a still further embodiment and in accordance with any of
the above, the present invention provides compositions and methods
that lower serum concentration of antigen, where the antigen is
selected from the group consisting of IgE, oxoLDL, and FVIII
inhibitor.
[0023] In a still further embodiment and in accordance with any of
the above, the present invention provides compositions and methods
in which the antibody includes a variable region VH domain that
comprises a CDR1 of SEQ ID NO:2, a CDR2 of SEQ ID NO:3 and a CDR3
of SEQ ID NO:4 and a variable region VL domain that comprises a
CDR1 of SEQ ID NO:6, a CDR2 of SEQ ID NO:7 and a CDR3 of SEQ ID
NO:8.
[0024] In a still further embodiment and in accordance with any of
the above, the present invention provides compositions and methods
in which the antibody includes a variable region VH domain that
comprises a CDR1 of SEQ ID NO:18, a CDR2 of SEQ ID NO:19 and a CDR3
of SEQ ID NO:20 and a variable region VL domain that comprises a
CDR1 of SEQ ID NO:22, a CDR2 of SEQ ID NO:23 and a CDR3 of SEQ ID
NO:24.
[0025] In a still further embodiment and in accordance with any of
the above, the present invention provides compositions and methods
in which the variant Fc domain comprises amino acid substitutions
selected from the group consisting of S267E, S267D, L328F, P238D,
S267E/L328F, G236N/S267E, G236D/S267E.
[0026] In a further aspect, the present invention provides a method
of rapidly lowering the serum concentration of an antigen in a
patient, where the method includes the step of: administering an Fc
fusion protein comprising: (i) a binding moiety that binds the
antigen; and (ii) a variant Fc domain comprising an amino acid
substitution as compared to a parent Fc domain, wherein the variant
Fc domain binds Fc.gamma.RIIb with increased affinity as compared
to the parent Fc domain and the Fc fusion protein binds to the
antigen to form a protein-antigen complex that is cleared at least
two fold faster than the antigen alone.
[0027] In a further aspect, the present invention provides
compositions and methods for lowering the free antigen in a patient
comprising administering an Fc fusion protein comprising a binding
moiety that binds the antigen and a variant Fc domain comprising an
amino acid substitution as compared to a parent Fc domain wherein
said variant Fc domain binds Fc.gamma.RIIb with increased affinity
as compared to said parent Fc domain. The administration results in
the concentration of said free antigen decreasing at least 50% more
rapidly than the decrease in concentration seen with an antibody
comprising the parent Fc domain.
[0028] In a further aspect, the present invention provides
compositions and methods for clearing an antibody-antigen complex
in a patient compared to antibody alone, by administering an Fc
fusion protein comprising: (i) a binding moiety that binds to the
antigen; and (ii) a variant Fc domain comprising an amino acid
substitution as compared to a parent Fc domain, wherein the variant
Fc domain binds Fc.gamma.RIIb with increased affinity as compared
to said parent Fc domain; and wherein the Fc fusion protein binds
to said antigen to form a protein-antigen complex and said complex
is cleared at least two fold faster than the protein alone.
[0029] In a further embodiment in accordance with any of the above,
the methods and compositions of the invention include the use of a
fusion protein containing a binding moiety that has a sequence
selected from FIG. 33A or 33B.
[0030] In a further embodiment in accordance with any of the above,
the methods and compositions of the invention include the use of an
Fc fusion protein that has a first monomer and a second monomer,
and the first monomer comprises the sequence shown in FIG. 33C and
the second monomer has the sequence shown in FIG. 33D.
[0031] In a further embodiment in accordance with any of the above,
the methods and compositions of the invention include the use of an
Fc fusion protein that has a first monomer and a second monomer,
and the first monomer comprises the sequence shown in FIG. 33E and
the second monomer has the sequence shown in FIG. 33D.
[0032] In a yet further embodiment in accordance with any of the
above, the methods and compositions of the invention include the
use of an Fc fusion protein that has a first domain comprising a
CR2 sequence and a second domain comprising an engineered Fc
domain. In still further embodiments, the fusion protein sequence
is selected from the sequences depicted in FIG. 40.
[0033] In a further aspect, the present invention provides methods
and compositions for treating an IgE-mediated disease in a patient
by rapidly lowering serum concentration of IgE in said patient by
administering an antibody that has (i) a variable region that binds
IgE; and (ii) a variant Fc domain comprising an amino acid
substitution as compared to a parent Fc domain, where variant Fc
domain binds Fc.gamma.RIIb with increased affinity as compared to
the parent Fc domain, and where the antibody binds to the IgE to
form an antibody-IgE complex and the complex is cleared at least
two fold faster than IgE alone. In certain embodiments, the variant
Fc domain comprises amino acid substitutions selected from the
group consisting of S267E, S267D, L238F, P238D, S267E/L328F,
G236N/S267E, G236D/S267E. In further embodiments, the IgE mediated
disease is selected from the group consisting of: asthma, allergic
rhinitis, atopic dermatitis, and food allergy.
[0034] In a further aspect, the present invention provides methods
and compositions for treating an autoimmune disorder in a patient
by rapidly lowering serum concentration of C3d in the patient by
administering a rapid clearance molecule comprising: (i) a variable
region that binds C3d; and (ii) a variant Fc domain comprising an
amino acid substitution as compared to a parent Fc domain, where
the variant Fc domain binds Fc.gamma.RIIb with increased affinity
as compared to said parent Fc domain, and where the rapid clearance
molecule binds to the C3d to form a molecule-C3d complex and the
complex is cleared at least two fold faster than C3d alone. In
certain embodiments, the variant Fc domain comprises amino acid
substitutions selected from the group consisting of S267E, S267D,
L238F, P238D, S267E/L328F, G236N/S267E, G236D/S267E. In further
embodiments, the autoimmune disorder is selected from the group
consisting of: systemic lupus erythematosus and rheumatoid
arthritis. In yet further embodiments, the rapid clearance molecule
is an antibody or an Fc fusion protein.
[0035] In a further aspect, the present invention provides methods
and compositions for treating atherosclerosis in a patient by
rapidly lowering serum concentration of oxLDL in the patient by
administering a rapid clearance molecule that has: (i) a variable
region that binds oxLDL; and (ii) a variant Fc domain comprising an
amino acid substitution as compared to a parent Fc domain, where
the variant Fc domain binds Fc.gamma.RIIb with increased affinity
as compared to the parent Fc domain; and where the rapid clearance
molecule binds to the oxLDL to form a molecule-oxLDL complex that
is cleared at least two fold faster than oxLDL alone. In certain
embodiments, the variant Fc domain comprises amino acid
substitutions selected from the group consisting of S267E, S267D,
L238F, P238D, S267E/L328F, G236N/S267E, G236D/S267E. In yet further
embodiments, the rapid clearance molecule is an antibody or an Fc
fusion protein.
[0036] In a further aspect, the present invention provides methods
and compositions for treating hemophilia in a patient by rapidly
lowering serum concentration of FVIII inhibitor in said patient by
administering a rapid clearance molecule comprising (i) a variable
region that binds said FVIII inhibitor; and (ii) a variant Fc
domain comprising an amino acid substitution as compared to a
parent Fc domain, wherein the variant Fc domain binds Fc.gamma.RIIb
with increased affinity as compared to the parent Fc domain and
wherein the rapid clearance molecule binds to the FVIII inhibitor
to form a molecule-inhibitor complex and the complex is cleared at
least two fold faster than FVIII inhibitor alone. In certain
embodiments, the variant Fc domain comprises amino acid
substitutions selected from the group consisting of S267E, S267D,
L238F, P238D, S267E/L328F, G236N/S267E, G236D/S267E. In yet further
embodiments, the rapid clearance molecule is an antibody or an Fc
fusion protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIGS. 1A and B. FIG. 1A Illustrates the novel mechanistic
approach for inhibiting IgE+Fc.gamma.RIIb+ B cells. Under
appropriate stimuli, naive B cells can differentiate into IgE+ B
cells. Engagement of antigen with the IgE B cell receptor activates
these cells, which can then differentiate into plasma cells that
release circulating IgE. Binding of circulating IgE binds to Fc
R's, for example on mast cells, basophils, and eosinophils,
activates these cells. Release of histamine, prostaglandins, and
other chemical mediators ultimately results in the clinical
symptoms of allergy and asthma. Omalizumab, having a native IgG1 Fc
region, is capable of blocking binding of IgE to Fc R. Anti-IgE
antibodies with high affinity for Fc.gamma.RIIb, referred to as
Anti-IgE-IIbE in the figure, are capable of not only blocking
binding of IgE to Fc R, but also of inhibiting activation of IgE+ B
cells by mIgE Fc.gamma.RIIb coengagement. FIG. 1B shows the rapid
clearance mechanism, outlining the possible mechanisms of action
(MOA): the first is to sequester the free antigen (in the figure
this is IgE), secondly the production of the antigen is suppressed,
in the case of IgE, and finally the complex of the antigen-antibody
is cleared rapidly.
[0038] FIG. 2. Biacore surface plasmon resonance sensorgrams
showing binding of Fc variant anti-CD19 antibodies to human
Fc.gamma.RIIb.
[0039] FIG. 3. Affinities of Fc variant antibodies for human
Fc.gamma.Rs as determined by Biacore. The graph shows the
log(K.sub.A) for binding of variant and WT IgG1 antibodies to human
Fc.gamma.RI (I), H131 Fc.gamma.RIIa (H IIa), Fc.gamma.RIIb (IIb),
and V158 Fc.gamma.RIIIa (V IIIa). Binding of G236D/S267E and
S267E/L328F to V158 Fc.gamma.RIIIa was not detectable. Binding of
G236R/L328R (Fc-KO) to all receptors tested was not detectable.
[0040] FIG. 4. Affinities of Fc variant antibodies for human
Fc.gamma.Rs as determined by Biacore surface plasmon resonance. The
table provides equilibrium K.sub.D'S for binding of variant and WT
IgG1 antibodies to human Fc.gamma.RI, H131 Fc.gamma.RIIa
Fc.gamma.RIIb, and V158 Fc.gamma.RIIIa, and the fold binding for
each relative to native (WT) IgG1. n.d.=not detectable.
[0041] FIGS. 5A-C. Amino acid sequences of the heavy (VH) and light
(VL) chain variable regions and CDRs of anti-IgE antibodies. CDR
boundaries were defined as described previously based on a
structural alignment of antibody variable regions (Lazar et al.,
2007, Mol Immunol 44:1986-1998).
[0042] FIG. 6. Amino acid sequences of the heavy and light chain WT
and variant constant regions.
[0043] FIG. 7. Amino acid sequences of anti-IgE full length
antibodies that may be used to target IgE+ B cells.
[0044] FIG. 8. Table of affinity data for binding of WT and variant
anti-IgE antibodies to the IgE Fc region and Fc.gamma.RIIb.
[0045] FIG. 9. Plot of affinity data for binding of WT and variant
anti-IgE antibodies to the IgE Fc region and Fc.gamma.RIIb.
[0046] FIG. 10. IgE ELISA using commercial (MabTech) and in-house
(Omalizumab and MaE11) anti-IgE antibodies as capture reagents.
[0047] FIG. 11. The variable region of the anti-IgE antibody
omalizumab does not compete with MabTech capture antibody for IgE
detection in the ELISA protocol.
[0048] FIG. 12. Inhibition of class-switched IgE+ B cells with
variant anti-IgE antibodies enhanced for Fc.gamma.RIIb affinity,
but not antibodies lacking Fc.gamma.R binding (Fc variant
G236R/L328R) or lacking binding to IgE (motavizumab). The plot
shows the concentration of IgE released from PBMCs after 12 days
incubation with IL-4, anti-CD40 (.alpha.-CD40) agonist antibody,
and varying concentrations of the antibodies shown.
[0049] FIG. 13. Variant anti-IgE antibodies do not inhibit
class-switched IgG2+ B cells. The plot shows the concentration of
IgG2 released from PBMCs after 12 days incubation with IL-4,
.alpha.-CD40, and varying concentrations of the antibodies
shown.
[0050] FIG. 14. Inhibition of class-switched IgE+ B cells with
variant anti-IgE antibodies enhanced for Fc.gamma.RIIb affinity.
The plot shows the concentration of IgE released from PBMCs after
14 days incubation with IL-4, anti-CD40 (.alpha.-CD40) agonist
antibody, and varying concentrations of the antibodies shown. Data
were normalized to the lowest concentration of antibody.
[0051] FIG. 15. Inhibition of class-switched IgE+ B cells with
variant anti-IgE antibodies enhanced for Fc.gamma.RIIb affinity.
The plot shows the concentration of IgE released from PBMCs after
14 days incubation with IL-4, anti-CD40 (.alpha.-CD40) agonist
antibody, anti-CD79b BCR cross-linking antibody, and varying
concentrations of the antibodies shown. Data were normalized to the
lowest concentration of antibody.
[0052] FIG. 16. Inhibition of class-switched IgE+ B cells with
variant anti-IgE antibodies enhanced for Fc.gamma.RIIb affinity.
The plot shows the concentration of IgE released from PBMCs after
14 days incubation with IL-4, anti-CD40 (.alpha.-CD40) agonist
antibody, anti-mu BCR cross-linking antibody, and varying
concentrations of the antibodies shown. Data were normalized to the
lowest concentration of antibody.
[0053] FIG. 17. Inhibition of class-switched IgE+ B cells with
variant anti-IgE antibodies enhanced for Fc.gamma.RIIb affinity.
The plot shows the concentration of IgE released from PBMCs after
14 days incubation with IL-4, anti-CD40 (.alpha.-CD40) agonist
antibody, anti-CD79b BCR cross-linking antibody, and varying
concentrations of the antibodies shown.
[0054] FIG. 18. Inhibition of class-switched IgE+ B cells with
variant anti-IgE antibodies enhanced for Fc.gamma.RIIb affinity.
The plot shows the concentration of IgE released from PBMCs after
14 days incubation with IL-4, anti-CD40 (.alpha.-CD40) agonist
antibody, anti-mu BCR cross-linking antibody, and varying
concentrations of the antibodies shown.
[0055] FIG. 19. Protocol for huPBL-SCID in vivo study to test
activity of anti-IgE antibodies. The indicated days reflect the
number of days after engraftment of PBMCs from a donor testing
positive for IgE antibodies specific for Der p 1. Derp1 vacc.
indicates vaccination with Der p 1 antigen.
[0056] FIG. 20. Total serum IgG levels from the huPBL-SCID in vivo
model for each treatment group. The indicated days (7, 23, and 37)
reflect the blood draws outlined in the protocol in FIG. 19. PBS
indicates the untreated vehicle group, Omalizumab indicates the
group treated with Omalizumab_IgG1, and the 3 H1 L1 MaE11 groups
indicate groups treated with humanized MaE11 comprising either a WT
IgG1 (IgG1), S267E/L328F variant (IIbE), or G236R/L328R (Fc-KO) Fc
region.
[0057] FIG. 21. Total serum IgE levels from the huPBL-SCID in vivo
model for each treatment group. The indicated days (7, 23, and 37)
reflect the blood draws outlined in the protocol in FIG. 19. PBS
indicates the untreated vehicle group, Omalizumab indicates the
group treated with Omalizumab_IgG1, and the 3 H1 L1 MaE11 groups
indicate groups treated with humanized MaE11 comprising either a WT
IgG1 (IgG1), S267E/L328F variant (IIbE), or G236R/L328R (Fc-KO) Fc
region. The limit of quantitation for the ELISA method was 31.6
ng/mL; samples that were below this limit were reported as 31.6
ng/mL in the plot.
[0058] FIG. 22 A-C. Data from a chimp study of XmAb7195, described
herein, that shows a rapid and unprecedented reduction in total
IgE. The dosage was a single 5 mg/kg dose, mean baseline IgE level
is .about.3 ug/ml. LLOQ is the lower limit of quantification. This
contrasts with a known Xolair side effect that the concentration of
total IgE is increased upon administration.
[0059] FIG. 23. A scatter plot of calculated half-lives for
individual mice treated with variant anti-IgE antibodies
(IIbE=S267E/L328F).
[0060] FIG. 24. Serum total IgE concentration as a function of time
in human Fc.gamma.RIIb transgenic mice treated with anti-mouse IgE
antibodies. The lower limit of quantification of the IgE assay was
13 ng/ml.
[0061] FIG. 25. Plot of test article half-life in human
Fc.gamma.RIIb transgenic mice versus Fc.gamma.RIIb affinity. A
direct relationship is observed.
[0062] FIG. 26. Plot in vitro internalization of antibody:IgE
complexes into LSEC isolated from Fc.gamma.RIIb transgenic
mice.
[0063] FIG. 27. Liver and heart distribution of .sup.89Zr-IgE upon
co-administration of saline, XmAb7195 (S267E/L328F), or XENP6782
(IgG1).
[0064] FIG. 28. A Factor VIII fusion embodiment to "scrub" FVIII
inhibitor antibodies prior to FVIIIa replacement dosing.
[0065] FIG. 29. An illustration of primary structure and domain
organization of FVI II.
[0066] FIG. 30A-B. List of suitable Fc domain Fc.gamma.RIIb amino
acid substitutions for increased Fc.gamma.RIIb binding.
[0067] FIG. 31. The structure of B-domain deleted human Factor
VIII. Domains A1, A2, A3, C1, and C2 are indicated.
[0068] FIG. 32. Diagram showing Factor VIII inhibitor scrubber
constructs consisting of FVIII domains A2 and C2 fused to a rapid
clearance IIb Fc.
[0069] FIG. 33A-E. Sequences of Factor VIII inhibitor
constructs.
[0070] FIG. 34. Reducing and non-reducing SDS-PAGE of Factor VIII
inhibitor scrubber constructs FVIII_A2_C220S/S267E/L328F and
FVIII_C2_C220S/S267E/L328F.
[0071] FIG. 35. Size-exclusion chromatography of Factor VIII
inhibitor scrubber constructs FVIII_A2_C220S/S267E/L328F and
FVIII_C2_C220S/S267E/L328F.
[0072] FIG. 36A-D. Affinities of Fc variant antibodies for human
Fc.gamma.Rs as determined by Biacore surface plasmon resonance.
FIG. 36A is a table listing the dissociation constant (Kd) for
binding anti-CD19 variant antibodies to human Fc.gamma.RI,
Fc.gamma.RIIa (131R), Fc.gamma.RIIa (131H), Fc.gamma.RIIb,
Fc.gamma.RIIa (158V), and Fc.gamma.RIIIa (158F). FIG. 36B is a
continuation of the list in FIG. 36A. FIG. 36C is a continuation of
the list in FIG. 36A and FIG. 36B. FIG. 36D is a continuation of
the list in FIG. 36A, FIG. 36B, and FIG. 36C. Multiple observations
have been averaged. n.d.=no detectable binding.
[0073] FIG. 37A-D. Fold affinities of Fc variant antibodies for
human Fc.gamma.Rs as determined by Biacore surface plasmon
resonance. FIG. 37A is a table listing the fold improvement or
reduction in affinity relative to WT IgG1 for binding of anti-CD19
variant antibodies to human Fc.gamma.RI, Fc.gamma.RIIa (131R),
Fc.gamma.RIIa (131H), Fc.gamma.RIIb, Fc.gamma.RIIIa (158V), and
Fc.gamma.RIIa (158F). FIG. 37B is a continuation of the list in
FIG. 37A. FIG. 37C is a continuation of the list in FIG. 37A and
FIG. 37B. FIG. 37D is a continuation of the list in FIG. 37A, FIG.
37B, and FIG. 37C. Fold=KD(Native IgG1)/KD(variant). n.d.=no
detectable binding.
[0074] FIG. 38. General overview of the CR2-IIbE embodiment, the
"immune complex scrubber" embodiment. As shown, the "rapid
clearance" mechanism, utilizing a CR2-Fc fusion, wherein the Fc
component of the fusion protein has increased Fc.gamma.RIIb binding
as compared to a wild-type Fc domain (particularly an Fc region
from a human IgG1, IgG2, IgG3 or IgG4) and the CR component is as
described herein.
[0075] FIGS. 39A-C. Binding data of CR2-Fc constructs.
[0076] FIGS. 40A-F. Sequences for the CR embodiments of the
invention.
[0077] FIG. 41. Schematic describing the generation of
atherosclerosis via macrophage uptake of oxLDL and its prevention
by Fc-containing oxLDL-binding proteins with enhanced Fc.gamma.RIIb
affinity.
[0078] FIG. 42. Amino acid sequences for oxLDL-binding
proteins.
[0079] FIG. 43A-B. Amino acid sequences for Fc-containing
oxLDL-binding proteins.
[0080] FIG. 44. Size-exclusion chromatograms for expressed and
purified Fc-containing oxLDL-binding proteins.
[0081] FIG. 45. Amino acid sequences for Fc-containing
oxLDL-binding proteins with enhanced Fc.gamma.RIIb affinity.
[0082] FIG. 46. Amino acid sequences for humanized variable regions
derived from the EO6 parental antibody.
[0083] FIG. 47A-D. List of a variety of suitable Fc domain
Fc.gamma.RIIb amino acid substitutions for increased Fc.gamma.RIIb
binding.
[0084] FIG. 48A-B. Matrix of possible combinations of Fc.gamma.RIIb
variants, FcRn variants, Scaffolds, Fvs and combinations, with each
variant being independently and optionally combined from the
appropriate source Legend: Legend A are suitable FcRn variants:
434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I
or V/434S, 436V/428L, 252Y, 252Y/254T/256E, 259I/308F/428L. Legend
B are suitable scaffolds and include IgG1, IgG2, IgG3, IgG4, and
IgG1/2. Sequences for such scaffolds can be found for example in US
Patent Publication No. 2012/0128663, published on May 24, 2012,
which is hereby incorporated by reference in its entirety for all
purposes and in particular for all teachings, figures and legends
related to scaffolds and their sequences. Legend C are suitable
exemplary target antigens: IgE, IL-4, IL-6, IL-13, TNF.alpha.,
MCP-1, RANTES, TARC, MDC, VEGF, HGF, and NGF, immune complexes,
FVIII inhibitors, LDL, oxidized LDL (OxLDL), Lp(a), SOST, and DKK1.
Legend D reflects the following possible combinations, again, with
each variant being independently and optionally combined from the
appropriate source Legend: 1) Fc.gamma.RIIb variants plus FcRn
variants; 2) Fc.gamma.RIIb variants plus FcRn variants plus
Scaffold; 3) Fc.gamma.RIIb variants plus FcRn variants plus
Scaffold plus Fv; 4) Fc.gamma.RIIb variants plus Scaffold 5)
Fc.gamma.RIIb variants plus Fv; 6) FcRn variants plus Scaffold; 7)
FcRn variants plus Fv; 8) Scaffold plus Fv; 9) Fc.gamma.RIIb
variants plus Scaffold plus Fv; and 10) Fc.gamma.RIIb variants plus
FcRn variants plus Fv.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
Overview of Invention
[0085] The in vivo pharmacokinetic properties of therapeutic
antibodies can be altered through modification of their Fc domain.
Such modifications may include amino acid substitutions, deletions,
or additions as well as other modifications such as chemical
modifications. In the present invention, modifications that
increase affinity of molecules such as antibodies for the
inhibitory Fc receptor Fc.gamma.RIIb (CD32b) are utilized to
facilitate rapid in vivo clearance of complexes comprising the
antigen and the molecule of the invention. Incorporation of the
IIb-enhancing affinity modifications (also referred to herein as
"Fc.gamma.RIIb variants" or "Fc.gamma.RIIb variations" or
grammatical equivalents thereof) into various antibodies leads to a
novel phenomenon whereby the antibody-target complex is cleared
extremely rapidly while the antibody alone retains a reasonably
long half-life. Although much of the discussion herein is directed
to antibodies for the sake of clarity, it will be appreciated that
discussion of the Fc.gamma.RIIb variants described herein are
applicable to any of the rapid clearance molecules described
herein, including polypeptides, antibodies, and Fc fusion
proteins.
[0086] The present invention provides methods of rapidly lowering
the serum concentration of an antigen in a subject by administering
an antibody that has both a variable region that binds the antigen
and a variant Fc domain that binds the Fc.gamma.RIIb receptor with
increased affinity as compared to an un-modified Fc domain. Without
being bound by theory, it appears that an antibody of the invention
binds to the antigen to form an antibody-antigen complex that is
cleared more rapidly than the unbound antigen. Thus, the free
antigen concentration in the patient, e.g. the serum concentration
of free antigen in the patient, is rapidly decreased. In other
words, the antigen-antibody complex is differentially cleared (e.g.
clearance of complex/clearance of antigen ratio is greater than 1).
In some cases, the methods and compositions of the present
invention clear an antibody-antigen complex at least 2, 5, 10, 15,
20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300-fold faster
than clearance of the antigen alone. In certain cases the methods
and compositions of the present invention clear an antibody-antigen
complex 5-500, 50-450, 100-400, 200-350, 100-200-fold faster than
clearance of the antigen alone. In other cases, clearance rates of
25.times. faster than antigen alone, 50.times., 75.times. and
100.times. or more are provided by methods and compositions of the
present invention. In some cases, the methods and compositions of
the present invention decrease the clearance rate of an
antibody-antigen complex by at least 30%, 40%, 50%, 60%, 70%, 80%
90%, 95%, or 99% as compared to the clearance rate of the antigen
alone. In some cases, the methods and compositions of the present
invention decrease the clearance rate of an antibody-antigen
complex by at least 30%, 40%, 50%, 60%, 70%, 80% 90%, 95%, or 99%
as compared to the clearance rate mediated by an antibody
comprising a parent (un-modified) Fc domain.
[0087] Thus, compositions of the present invention include such
"rapid clearance" molecules (also referred to as "scrubbers") of
the present invention that lead to clearance of the
antibody-antigen complex more rapidly than the unbound antigen or
antibody alone. Such rapid clearance compositions are generally
polypeptides that comprise two domains: an antigen or ligand
binding portion and an Fc domain that exhibits increased
Fc.gamma.RIIb binding as compared to a non-engineered Fc region. In
some embodiments, as is further described herein, the rapid
clearance molecules are antibodies, comprising a standard antigen
binding Fv region, and a variant Fc.gamma.RIIb binding region, e.g.
an engineered Fc region. In alternative embodiments, the rapid
clearance molecule is an Fc fusion protein, with a binding ligand
or receptor as one domain (e.g. a CR domain) and an Fc region with
increased Fc.gamma.RIIb binding.
[0088] In addition to the faster clearance rates described above as
compared to clearance of antigen alone, rapid clearance molecules
of the invention further clear the antigen-containing complex more
rapidly than IgG antibodies with the same selectivity would
mediate.
[0089] Application of different Fc.gamma.RIIb-enhancing Fc amino
acid substitutions with varying affinities to the Fc.gamma.RIIb
receptor (e.g. S267E, S267D, L328F, P238D, S267E/L328F,
G236N/S267E, and G236D/S267E, as further described herein) can
allow some "tuning" of how fast the complex antigen is cleared
while maintaining significant half life of the rapid clearance
composition of the invention (including antibodies). That is,
different amino acid substitutions that alter Fc.gamma.RIIb binding
affinity may lead to different balances between the complex
clearance rate and the antibody clearance rate, allowing for
tailoring toward optimal therapeutic profile and dosing. This
tuning may be accomplished by using amino acid substitutions in the
Fc domain that increase binding to Fc.gamma.RIIb as compared to the
parent Fc domain. This increase in binding may be tuned by using Fc
variants with 1-100, 5-90, 10-80, 15-70, 20-60, 30-50, 10-20 fold
greater affinity as compared to the parent Fc domain. This increase
in binding may also be tuned by using Fc variants with 50-200,
60-190, 70-180, 80-170, 90-160, 100-150, 110-140, 120-130, 50-100
greater affinity as compared to the parent Fc domain. In some
cases, affinity is measured by Biacore as described in Example
2.
[0090] In certain cases, molecules of the invention incorporate
Fc.gamma.RIIb receptor variants that can range from very tight
differential binding to Fc.gamma.RIIb to variants that display
increased (as compared to wild type Fc domains) binding affinity
but at a lower level. For example, very tight (or heavy) binding to
Fc.gamma.RIIb receptor may include Fc.gamma.RIIb variants that show
at least 50, 75, 100, 125, 150, 175, 200, 225, 250-fold greater
affinity to Fc.gamma.RIIb receptor as compared to the parent Fc
domain. In contrast, a lower level (or light, also referred to
herein as "lite") increase in binding may include Fc.gamma.RIIb
variants that show no more than 50, 40, 30, 20, 10, 5-fold greater
affinity to Fc.gamma.RIIb receptor as compared to the parent Fc
domain.
[0091] The effects of molecules of the invention may be further
tuned by combining amino acid substitutions that alter
Fc.gamma.RIIb binding affinity with amino acid substitutions that
affect binding to FcRn. Proteins with amino acid substitutions that
affect binding to FcRn (also referred to herein as "FcRn variants")
may in certain situations also increase serum half-life in vivo as
compared to the parent protein. As will be appreciated, any
combination of Fc and FcRn variants may be used to tune clearance
of the antigen-antibody complex. Suitable FcRn variants that may be
combined with any of the Fc variants described herein include
without limitation 434A, 434S, 428L, 308F, 259I, 428L/434S,
259I/308F, 436I/428L, 436I/434S, 436V/434S, 436V/428L, 252Y,
252Y/254T/256E, and 259I/308F/428L.
[0092] Without being bound by theory, it appears that the
accelerated clearance of antibodies containing amino acid
substitutions that confer high affinity (as compared to the parent
Fc domain) to the inhibitory receptor Fc.gamma.RIIb is likely
mediated by interaction with Fc.gamma.RIIb-expressing cells,
possibly liver sinusoidal endothelial cells. In addition, it
appears that the accelerated clearance is due to the clearance of
the antigen-antibody complex via interactions with the
Fc.gamma.RIIb receptor.
[0093] In addition, unexpectedly, administration of antibodies
including the Fc.gamma.RIIb binding affinity variants described
herein leads to near instantaneous drops in total antigen levels,
whereas administration of other antibodies to the antigen that lack
modifications that lead to increased Fc.gamma.RIIb binding affinity
often lead to increases in total antigen levels. Furthermore, the
greater reduction in total antigen levels seen with antibodies with
increased Fc.gamma.RIIb binding affinity leads to superior
reduction of free antigen relative to levels seen with antibodies
that lack the Fc.gamma.RIIb variants.
[0094] In addition, in some cases, compositions and methods of the
invention provide sufficient increased affinity to the
Fc.gamma.RIIb receptor to allow for rapid clearance of the
antibody-antigen complex while allowing appropriate serum half
lives of the unbound antibodies.
[0095] The invention is exemplified in the case of XmAb7195.
XmAb7195 is an anti-IgE antibody that sequesters IgE and prevents
its interaction with FceR1 on mast cells and basophils. The
variable region is similar to the variable region of omalizumab
(Xolair, an anti-IgE antibody). The XmAb7195 Fc domain was
engineered with the S267E/L328F substitutions (Kabat numbering) to
confer high affinity to human Fc.gamma.RIIb. A single 5 mg/kg dose
of XmAb7195 was administered via intravenous injection to
chimpanzees (n=3) and its effects on free and total IgE were
compared to that of omalizumab (anti-IgE with native IgG1 Fc
domain). As shown in FIG. 22, the XmAb7195 antibody had a
relatively short half-life of approximately 2 days, and the
omalizumab exhibited a longer half-life of approximately 11
days.
[0096] Unexpectedly, the XmAb7195 antibody also led to a
near-instantaneous drop of total IgE levels, whereas the omalizumab
treatment led to an increase in total IgE (as observed in humans
treated with omalizumab). The XmAb7195-IgE complexes exhibited
greatly accelerated clearance presumably via their interaction with
FcRIIb. Furthermore, the greater reduction of total IgE using
XmAb7195 led to superior reductions of free IgE relative to
omalizumab-treated animals. PK/PD simulations suggest that the
half-life of the XmAb7195/IgE complexes are on the order of 1
hour--versus an 8 day half-life reported for omalizumab/IgE
complexes. Furthermore, the present invention also suggests that
rapid recovery of the antigen can occur after cessation of antibody
administration.
[0097] This surprising and unexpected result, e.g. that adding
Fc.gamma.RIIb variants to existing antibodies can rapidly clear
antigen in patients leads to a number of useful applications. Any
therapeutic target antigen system in which rapid clearance of the
antigen is desired can be subjected to the present invention. For
example, disease systems in which the antigen load is high find
particular use in the present invention. Similarly, disease systems
where rapid recovery of the antigen is desired after antibody
administration can be treated with the antibodies of the invention.
For example, during the use of TNF antibody inhibitors, patients
frequently get infections. Withdrawing the antibody treatment will
allow rapid recovery of the TNF to fight the infection. Similarly,
these antibodies can be used to treat pathogen infection when rapid
pathogen clearance is desired (for example, when a patient
scheduled for surgery gets an infection, the present invention can
be used to clear the infection rapidly, the therapeutic antibody
rapidly clears as well and surgery can progress). In addition,
these antibodies may be particularly useful in situations where
existing antibodies do not neutralize the antigen, or where
pathogens evolve to evade neutralization.
[0098] In addition, the invention finds use in the treatment of
hemophiliacs. One issue with hemophiliacs is the effect that Factor
VIII (FVIII (not to be confused with "Fv")) inhibitors play in
disease. Currently, these FVIII inhibitors (generally FVIII
antibodies, as shown in FIG. 28) are a huge problem for
hemophiliacs. The present invention works as generally outlined in
FIGS. 28 and 29. Fc fusion proteins, comprising an Fc domain with
Fc.gamma.RIIb amino acid variants, fused to FVIIIa components as
outlined herein, will sequester the inhibitor antibodies, rapidly
clear the inhibitor antibodies, and will inhibit FVII-reactive B
cells (to prohibit the further production of the inhibitors).
[0099] The rapid clearance mechanisms of the present invention are
also used to remove oxidized low-density lipoprotein (oxidized LDL
or oxLDL) from the blood. OxLDL is a key facilitator of
atherosclerosis via macrophage uptake and foam cell formation (see
FIG. 42). In this embodiment, increased affinity for the inhibitory
Fc receptor Fc.gamma.RIIb (CD32b) is utilized to facilitate rapid
in vivo clearance of oxLDL via their interaction with Fc-containing
oxLDL-binding proteins. Incorporation of the IIb-enhancing affinity
substitutions into various Fc-containing oxLDL-binding proteins
leads to a novel phenomenon whereby the complex is cleared
extremely rapidly. Application of different IIb-enhancing
substitutions (including without limitation S267E, S267D, L328F,
P238D, S267E/L328F, G236N/S267E, and G236D/S267E, which are useful
for all the rapid clearance molecules herein) may lead to different
balances between the complex clearance rate and the Fc-containing
oxLDL-binding protein clearance rate, allowing for tailoring toward
optimal therapeutic profile and dosing.
[0100] In addition, the invention finds use in a variety of
diseases or situations where plasmapherisis is typically applied to
clear the body of pathogens, autoantibodies, or other pathogenic
factors. Such diseases include, but are not limited to, the
following: Guillain-Barre syndrome; Chronic inflammatory
demyelinating polyneuropathy; Goodpasture's syndrome;
Hyperviscosity syndromes: Cryoglobulinemia; Paraproteinemia;
Waldenstrom macroglobulinemia; Myasthenia gravis; Thrombotic
thrombocytopenic purpura (TTP)/hemolytic uremic syndrome; Wegener's
granulomatosis; Lambert-Eaton Syndrome; Antiphospholipid Antibody
Syndrome (APS or APLS); Microscopic polyangiitis; Recurrent focal
and segmental glomerulosclerosis in the transplanted kidney; HELLP
syndrome; PANDAS syndrome; Refsum disease; Behcet syndrome;
HIV-related neuropathy; Graves' disease in infants and neonates;
Pemphigus vulgaris; Multiple sclerosis; Rhabdomyolysis; Toxic
Epidermal Necrolysis (TEN). That is, by using Fc.gamma.RIIb
antibodies with variable regions specific to these antigens,
clearance of the antigens in a rapid manner can occur.
Definitions
[0101] Described herein are several definitions. Such definitions
are meant to encompass grammatical equivalents.
[0102] By "ablation" herein is meant a decrease or removal of
activity. Thus for example, "ablating Fc.gamma.R binding" means the
Fc region amino acid variant has less than 50% starting binding as
compared to an Fc region not containing the specific variant, with
less than 70-80-90-95-98% loss of activity being preferred, and in
general, with the activity being below the level of detectable
binding in a Biacore assay. Of particular use in the ablation of
Fc.gamma.R binding is the double variant 236R/328R, and 236R and
328R separately as well.
[0103] By "ADCC" or "antibody dependent cell-mediated cytotoxicity"
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. The role of high affinity binding to Fc.gamma.RIIIa to
ADCC activity is well established. In some cases, as described
herein, amino acid substitutions in the Fc domain can be used to
increase or decrease binding to one or more of the Fc.gamma.R
receptors, as is generally outlined in US Publication 2006/0024298,
hereby incorporated by reference in its entirety and in particular
for the amino acid substitutions disclosed therein, FIG. 41 as well
as the other figures and their accompanying legends in particular.
In addition, for some embodiments outlined herein, it may be
desirable to ablate binding to one or more of the Fc.gamma.R
receptors. For example, the L328F variant ablates Fc.gamma.RIIIa
binding, such that ADCC mechanisms are not triggered. In addition,
significant ablatement of Fc.gamma.R binding to all receptors can
be accomplished using 236R/328R variants.
[0104] By "ADCP" or antibody dependent cell-mediated phagocytosis
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause phagocytosis
of the target cell.
[0105] By "amino acid modification" herein is meant an amino acid
substitution, insertion, and/or deletion in a polypeptide sequence.
By "amino acid substitution" or "substitution" herein is meant the
replacement of an amino acid at a particular position in a parent
polypeptide sequence with a different amino acid. In particular, in
some embodiments, the substitution is to an amino acid that is not
naturally occurring at the particular position, either not
naturally occurring within the organism or in any organism. For
example, the substitution E272Y refers to a variant polypeptide, in
this case an Fc variant, in which the glutamic acid at position 272
is replaced with tyrosine. For clarity, a protein which has been
engineered to change the nucleic acid coding sequence but not
change the starting amino acid (for example exchanging CGG
(encoding arginine) to CGA (still encoding arginine) to increase
host organism expression levels) is not an "amino acid
substitution"; that is, despite the recombinant creation of a new
gene encoding the same protein, if the protein has the same amino
acid at the particular position that it started with, it is not an
amino acid substitution. By "amino acid insertion" or "insertion"
as used herein is meant the addition of an amino acid at a
particular position in a parent polypeptide sequence. By "amino
acid deletion" or "deletion" as used herein is meant the removal of
an amino acid at a particular position in a parent polypeptide
sequence.
[0106] By "antibody" herein is meant a protein consisting of one or
more polypeptides substantially encoded by all or part of the
recognized immunoglobulin genes. The recognized immunoglobulin
genes, for example in humans, include the kappa (.kappa.), lambda
(.lamda.), and heavy chain genetic loci, which together comprise
the myriad variable region genes, and the constant region genes mu
(.nu.), delta (.delta.), gamma (.gamma.), sigma (.sigma.), and
alpha (.alpha.) which encode the IgM, IgD, IgG (IgG1, IgG2, IgG3,
and IgG4), IgE, and IgA (IgA1 and IgA2) isotypes respectively.
Antibody herein is meant to include full length antibodies and
antibody fragments, and may refer to a natural antibody from any
organism, an engineered antibody, or an antibody generated
recombinantly for experimental, therapeutic, or other purposes.
[0107] By "amino acid" and "amino acid identity" as used herein is
meant one of the 20 naturally occurring amino acids or any
non-natural analogues that may be present at a specific, defined
position.
[0108] By "CD32b+ cell" or "Fc.gamma.RIIb+ cell" as used herein is
meant any cell or cell type that expresses CD32b (Fc.gamma.RIIb).
CD32b+ cells include but are not limited to B cells, plasma cells,
dendritic cells, macrophages, neutrophils, mast cells, basophils,
or eosinophils.
[0109] By "IgE+ cell" as used herein is meant any cell or cell type
that expresses IgE. In preferred embodiments of the invention, IgE+
cells express membrane-anchored IgE (mIgE). IgE+ cells include but
are not limited to B cells and plasma cells.
[0110] By "CDC" or "complement dependent cytotoxicity" as used
herein is meant the reaction wherein one or more complement protein
components recognize bound antibody on a target cell and
subsequently cause lysis of the target cell.
[0111] By"molecule" or grammatical equivalents is meant a
bifunctional molecule capable of binding both antigen and
Fc.gamma.RIIb wherein the Kd for binding of the molecule to
Fc.gamma.RIIb is less than about 100 nM on a cell surface resulting
in simultaneous binding of both the antigen to which the antibody
is directed and Fc.gamma.RIIb.
[0112] By "constant region" of an antibody as defined herein is
meant the region of the antibody that is encoded by one of the
light or heavy chain immunoglobulin constant region genes. By
"constant light chain" or "light chain constant region" as used
herein is meant the region of an antibody encoded by the kappa
(C.kappa.) or lambda (C.lamda.) light chains. The constant light
chain typically comprises a single domain, and as defined herein
refers to positions 108-214 of C.kappa. or C.lamda., wherein
numbering is according to the EU index. By "constant heavy chain"
or "heavy chain constant region" as used herein is meant the region
of an antibody encoded by the mu, delta, gamma, alpha, or epsilon
genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or
IgE, respectively. For full length IgG antibodies, the constant
heavy chain, as defined herein, refers to the N-terminus of the CH1
domain to the C-terminus of the CH3 domain, thus comprising
positions 118-447, wherein numbering is according to the EU
index.
[0113] By "effector function" as used herein is meant a biochemical
event that results from the interaction of an antibody Fc region
with an Fc receptor or ligand. Effector functions include
Fc.gamma.R-mediated effector functions such as ADCC and ADCP, and
complement-mediated effector functions such as CDC.
[0114] By "effector cell" as used herein is meant a cell of the
immune system that expresses one or more Fc and/or complement
receptors and mediates one or more effector functions. Effector
cells include but are not limited to monocytes, macrophages,
neutrophils, dendritic cells, eosinophils, mast cells, platelets, B
cells, large granular lymphocytes, Langerhans' cells, natural
killer (NK) cells, and .gamma..delta. T cells, and may be from any
organism including but not limited to humans, mice, rats, rabbits,
and monkeys.
[0115] By "Fab" or "Fab region" as used herein is meant the
polypeptides that comprise the VH, CH1, VH, and CL immunoglobulin
domains. Fab may refer to this region in isolation, or this region
in the context of a full length antibody or antibody fragment.
[0116] By "Fc" or "Fc region", as used herein is meant the
polypeptide comprising the constant region of an antibody excluding
the first constant region immunoglobulin domain and in some cases,
part of the hinge. Thus Fc refers to the last two constant region
immunoglobulin domains of IgA, IgD, and IgG, and the last three
constant region immunoglobulin domains of IgE and IgM, and the
flexible hinge N-terminal to these domains. For IgA and IgM, Fc may
include the J chain. For IgG, Fc comprises immunoglobulin domains
Cgamma2 and Cgamma3 (C.gamma.2 and C.gamma.3) and the hinge between
Cgamma1 (C.gamma.1) and Cgamma2 (C.gamma.2). Although the
boundaries of the Fc region may vary, the human IgG heavy chain Fc
region is usually defined to comprise residues C226 or P230 to its
carboxyl-terminus, wherein the numbering is according to the EU
index as in Kabat. Fc may refer to this region in isolation, or
this region in the context of an Fc polypeptide, as described
below.
[0117] By "Fc polypeptide" as used herein is meant a polypeptide
that comprises all or part of an Fc region. Fc polypeptides include
antibodies, Fc fusions, isolated Fcs, and Fc fragments.
Immunoglobulins may be Fc polypeptides.
[0118] By "Fc fusion" as used herein is meant a protein wherein one
or more polypeptides is operably linked to Fc. Fc fusion is herein
meant to be synonymous with the terms "immunoadhesin", "Ig fusion",
"Ig chimera", and "receptor globulin" (sometimes with dashes) as
used in the prior art (Chamow et al., 1996, Trends Biotechnol
14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200, both
hereby entirely incorporated by reference). An Fc fusion combines
the Fc region of an immunoglobulin with a fusion partner, which in
general may be any protein, polypeptide or small molecule. The role
of the non-Fc part of an Fc fusion, i.e., the fusion partner, is to
mediate target binding, and thus it is functionally analogous to
the variable regions of an antibody. Virtually any protein or small
molecule may be linked to Fc to generate an Fc fusion. Protein
fusion partners may include, but are not limited to, the
target-binding region of a receptor, an adhesion molecule, a
ligand, an enzyme, a cytokine, a chemokine, or some other protein
or protein domain. Small molecule fusion partners may include any
therapeutic agent that directs the Fc fusion to a therapeutic
target. Such targets may be any molecule, e.g., an extracellular
receptor that is implicated in disease.
[0119] By "Fc gamma receptor" or "Fc.gamma.R" as used herein is
meant any member of the family of proteins that bind the IgG
antibody Fc region and are substantially encoded by the Fc.gamma.R
genes. In humans this family includes but is not limited to
Fc.gamma.RI (CD64), including isoforms Fc.gamma.RIa, Fc.gamma.RIb,
and Fc.gamma.RIc; Fc.gamma.RII (CD32), including isoforms
Fc.gamma.RIIa (including allotypes H131 and R131), Fc.gamma.RIIb
(including Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2), and Fc.gamma.RIIc;
and Fc.gamma.RIII (CD16), including isoforms Fc.gamma.RIIIa
(including allotypes V158 and F158) and Fc.gamma.RIIIb (including
allotypes Fc.gamma.RIIIb-NA1 and Fc.gamma.RIIIb-NA2) (Jefferis et
al., 2002, Immunol Lett 82:57-65, incorporated entirely by
reference), as well as any undiscovered human Fc.gamma.Rs or
Fc.gamma.R isoforms or allotypes. An Fc.gamma.R may be from any
organism, including but not limited to humans, mice, rats, rabbits,
and monkeys. Mouse Fc.gamma.Rs include but are not limited to
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and
Fc.gamma.RIII-2 (CD16-2), as well as any undiscovered mouse
Fc.gamma.Rs or Fc.gamma.R isoforms or allotypes.
[0120] By "Fc ligand" or "Fc receptor" as used herein is meant a
molecule, e.g., a polypeptide, from any organism that binds to the
Fc region of an antibody to form an Fc-ligand complex. Fc ligands
include but are not limited to Fc.gamma.Rs, Fc.gamma.Rs,
Fc.gamma.Rs, FcRn, C1q, C3, mannan binding lectin, mannose
receptor, staphylococcal protein A, streptococcal protein G, and
viral Fc.gamma.R. Fc ligands also include Fc receptor homologs
(FcRH), which are a family of Fc receptors that are homologous to
the Fc.gamma.Rs (Davis et al., 2002, Immunological Reviews
190:123-136). Fc ligands may include undiscovered molecules that
bind Fc.
[0121] By "full length antibody" as used herein is meant the
structure that constitutes the natural biological form of an
antibody, including variable and constant regions. For example, in
most mammals, including humans and mice, the full length antibody
of the IgG isotype is a tetramer and consists of two identical
pairs of two immunoglobulin chains, each pair having one light and
one heavy chain, each light chain comprising immunoglobulin domains
VL and CL, and each heavy chain comprising immunoglobulin domains
VH, C.gamma.1, C.gamma.2, and C.gamma.3. In some mammals, for
example in camels and llamas, IgG antibodies may consist of only
two heavy chains, each heavy chain comprising a variable domain
attached to the Fc region.
[0122] By "immunoglobulin" herein is meant a protein comprising one
or more polypeptides substantially encoded by immunoglobulin genes.
Immunoglobulins include but are not limited to antibodies
(including bispecific antibodies) and Fc fusions. Immunoglobulins
may have a number of structural forms, including but not limited to
full length antibodies, antibody fragments, and individual
immunoglobulin domains.
[0123] By "immunoglobulin (Ig) domain" as used herein is meant a
region of an immunoglobulin that exists as a distinct structural
entity as ascertained by one skilled in the art of protein
structure. Ig domains typically have a characteristic
.beta.-sandwich folding topology. The known Ig domains in the IgG
isotype of antibodies are VH C.gamma.1, C.gamma.2, C.gamma.3, VL,
and CL.
[0124] By "IgG" or "IgG immunoglobulin" or "immunoglobulin G" as
used herein is meant a polypeptide belonging to the class of
antibodies that are substantially encoded by a recognized
immunoglobulin gamma gene. In humans this class comprises the
subclasses or isotypes IgG1, IgG2, IgG3, and IgG4.
[0125] By "IgE" or "IgE immunoglobulin" or "immunoglobulin E" as
used herein is meant a polypeptide belonging to the class of
antibodies that are substantially encoded by a recognized
immunoglobulin epsilon gene. IgE may be membrane-anchored (mIgE),
or non-membrane-anchored, also referred to herein as circulating
IgE.
[0126] By "inhibition" of cells or grammatical equivalents is meant
preventing or reducing the activation, proliferation, maturation or
differentiation of targeted cells.
[0127] By "isotype" as used herein is meant any of the subclasses
of immunoglobulins defined by the chemical and antigenic
characteristics of their constant regions. The known human
immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2,
IgM, IgD, and IgE.
[0128] By "modification" herein is meant an alteration in the
physical, chemical, or sequence properties of a protein,
polypeptide, antibody, or immunoglobulin.
[0129] Modifications described herein include amino acid
modifications and glycoform modifications.
[0130] By "glycoform modification" or "modified glycoform" or
"engineered glycoform" as used herein is meant a carbohydrate
composition that is covalently attached to a protein, for example
an antibody, wherein said carbohydrate composition differs
chemically from that of a parent protein. Modified glycoform
typically refers to the different carbohydrate or oligosaccharide;
thus for example an Fc variant may comprise a modified glycoform.
Alternatively, modified glycoform may refer to the Fc variant that
comprises the different carbohydrate or oligosaccharide.
[0131] By "parent polypeptide", "parent protein", "parent
immunoglobulin", "parent Fc domain", "precursor polypeptide",
"precursor protein", or "precursor immunoglobulin" as used herein
is meant an unmodified polypeptide, protein, Fc domain, or
immunoglobulin that is subsequently modified to generate a variant,
e.g., any polypeptide, protein or immunoglobulin which serves as a
template and/or basis for at least one amino acid modification
described herein. The parent polypeptide may be a naturally
occurring polypeptide, or a variant or engineered version of a
naturally occurring polypeptide. Parent polypeptide may refer to
the polypeptide itself, compositions that comprise the parent
polypeptide, or the amino acid sequence that encodes it.
Accordingly, by "parent Fc polypeptide" as used herein is meant an
Fc polypeptide that is modified to generate a variant Fc
polypeptide, and by "parent antibody" as used herein is meant an
antibody that is modified to generate a variant antibody (e.g., a
parent antibody may include, but is not limited to, a protein
comprising the constant region of a naturally occurring Ig).
[0132] By "position" as used herein is meant a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example the EU index as
described in Kabat. For example, position 297 is a position in the
human antibody IgG1.
[0133] By "polypeptide" or "protein" as used herein is meant at
least two covalently attached amino acids, which includes proteins,
polypeptides, oligopeptides and peptides.
[0134] By "residue" as used herein is meant a position in a protein
and its associated amino acid identity. For example, Asparagine 297
(also referred to as Asn297, also referred to as N297) is a residue
in the human antibody IgG1.
[0135] By "rapid clearance" or grammatical equivalents herein is
meant that the antigen-antibody complex composition is cleared from
the blood more quickly than either the antigen alone or the
antibody, or a complex between the antigen and a parent analog of
the antibody. As is understood in the art, antibodies with
different Fvs may have different half lives in serum, so the
comparison is to the starting antibody (e.g. an anti-IgE antibody
without the IIb variants outlined herein) to the IIb engineered
antibody. In general, "rapid clearance" are clearance rates of
25.times. faster than parent antibody, 50.times., 75.times. and
100.times. or more. For example, as outlined herein, the anti-IgE
IIb antibody of the examples shows a one hour clearance rate in
chimps as compared to 2 days of either the parent Xolair antibody
or an Fc IIb polypeptide that does not contain a binding moiety for
IgE. Similarly, "clearance" can be measured as a reduction in free
target antigen of 10%, 25%, 50% with 90 to 99% percentage of
starting serum antigen concentration being a preferred
clearance.
[0136] By "target antigen" as used herein is meant the molecule
that is bound by the variable region of a given antibody, or the
fusion partner of an Fc fusion. A target antigen may be a protein,
carbohydrate, lipid, or other chemical compound. An antibody or Fc
fusion is said to be "specific" for a given target antigen based on
having affinity for the target antigen. A variety of target
antigens are listed below.
[0137] Virtually any antigen may be targeted by the polypeptides of
the invention, including but not limited to proteins, subunits,
domains, motifs, and/or epitopes belonging to the following list of
target antigens, which includes both soluble factors such as
cytokines and membrane-bound factors. Proteins that may be target
antigens of the invention include without limitation: IgE (soluble
and/or membrane-bound), cytokines, e.g., IL-4, IL-6, IL-13, and
TNF.alpha.; chemokines, e.g., MCP-1, RANTES, TARC, and MDC; growth
factors, e.g., VEGF, HGF, and NGF; also, immune complexes, blood
factor inhibitors, e.g. FVIII inhibitors, LDL, oxidized LDL, SOST,
and DKK1. Target antigens may also include without limitation:
17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1
Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin
AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin
RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12,
ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5,
Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin,
alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL,
AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrial natriuretic
factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte
Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK,
Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,
BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4
BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2),
BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II
(BRK-3), BMPs, b-NGF, BOK, Bombesin, Bone-derived neurotrophic
factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5,
C5a, C10, CA125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen
(CEA), carcinoma-associated antigen, Cathepsin A, Cathepsin B,
Cathepsin C/DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin
L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL,
CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16,
CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24,
CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8,
CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6,
CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,
CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19,
CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32,
CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46,
CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80
(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147,
CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium
botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV,
CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4,
CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6,
CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,
CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,
cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN,
Decay accelerating factor, des(1-3)-IGF-1 (brain IGF-1), Dhh,
digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1,
EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin
receptor, Enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin
B2/EphB4, EPO, ERCC, E-selectin, ET-1, Factor 10a, Factor VII,
Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas,
FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR,
FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle stimulating
hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,
FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,
GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2),
GDF-7 (BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1),
GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR,
Glucagon, Glut 4, glycoprotein 10b/Illa (GP 10b/IIa), GM-CSF,
gp130, gp72, GRO, Growth hormone releasing factor, Hapten (NP-cap
or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV) gH
envelope glycoprotein, HCMV UL, Hemopoietic growth factor (HGF),
Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3),
Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD
glycoprotein, HGFA, High molecular weight melanoma-associated
antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3 loop, HLA, HLA-DR,
HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human
cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,
IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE,
IGF, IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1,
IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon
(INF)-alpha, INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain,
Insulin B-chain, Insulin-like growth factor 1, integrin alpha2,
integrin alpha3, integrin alpha4, integrin alpha4/beta1, integrin
alpha4/beta7, integrin alpha5 (alphaV), integrin alpha5/beta1,
integrin alpha5/beta3, integrin alpha6, integrin beta1, integrin
beta2, interferon gamma, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein
5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14,
Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3,
Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin
5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bpi, LBP,
LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen,
LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn,
L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lung surfactant, Luteinizing
hormone, Lymphotoxin Beta Receptor, Mac-1, MAdCAM, MAG, MAP2, MARC,
MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF
receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK,
MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15,
MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP,
mucin (Mud), MUC18, Muellerian-inhibitin substance, Mug, MuSK,
NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,
Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),
NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG,
OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone,
PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1,
PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental
alkaline phosphatase (PLAP), P1GF, PLP, PP14, Proinsulin,
Prorelaxin, Protein C, PS, PSA, PSCA, prostate specific membrane
antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES,
RANTES, Relaxin A-chain, Relaxin B-chain, renin, respiratory
syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors, RLIP76,
RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh,
SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,
STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated
glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell
receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT,
testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha,
TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII,
TGF-beta RIIb, TGF-beta RIII, TGF-beta1, TGF-beta2, TGF-beta3,
TGF-beta4, TGF-beta5, Thrombin, Thymus Ck-1, Thyroid stimulating
hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF,
TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII,
TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER,
TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D
(TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),
TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B
(TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R,
TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR
AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF
RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26
(TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35,
TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95),
TNFRSF6B (DcR3M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9
(4-1 BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2),
TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3,
TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11
(TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand,
DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,
THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15
(TL1A/VEGI), TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a
Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb
TNFC, p33), TNFSF4 (OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand
CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand,
APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand
CD153), TNFSF9 (4-1BB Ligand CD137 Ligand), TP-1, t-PA, Tpo, TRAIL,
TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferring receptor, TRF,
Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125,
tumor-associated antigen expressing Lewis Y related carbohydrate,
TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1, VECAD,
VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3
(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR
integrin, von Willebrands factor, WI F-1, WNT1, WNT2, WNT2B/13,
WNT3, WNT3A, WNT4, WNTSA, WNTSB, WNT6, WNT7A, WNT7B, WNT8A, WNT8B,
WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2,
XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and growth
factors.
[0138] As discussed below, target antigens that find use in the
particular use of rapid clearance of antibody-antigen complexes are
listed below, and include cancer antigens, autoantigens, pathogen
antigens, allergy antigens, etc.
[0139] By "target cell" as used herein is meant a cell that
expresses a target antigen.
[0140] By "variable region" as used herein is meant the region of
an immunoglobulin that comprises one or more Ig domains
substantially encoded by any of the V.kappa., V.lamda., and/or VH
genes that make up the kappa, lambda, and heavy chain
immunoglobulin genetic loci respectively.
[0141] By "variant polypeptide", "polypeptide variant", or
"variant" as used herein is meant a polypeptide sequence that
differs from that of a parent polypeptide sequence by virtue of at
least one amino acid modification. The parent polypeptide may be a
naturally occurring or wild-type (WT) polypeptide, or may be a
modified version of a WT polypeptide. Variant polypeptide may refer
to the polypeptide itself, a composition comprising the
polypeptide, or the amino sequence that encodes it. In some
embodiments, variant polypeptides disclosed herein (e.g., variant
immunoglobulins) may have at least one amino acid modification
compared to the parent polypeptide, e.g. from about one to about
ten amino acid modifications, from about one to about five amino
acid modifications, etc. compared to the parent. The variant
polypeptide sequence herein may possess at least about 80% homology
with a parent polypeptide sequence, e.g., at least about 90%
homology, 95% homology, etc. Accordingly, by "Fc variant" or
"variant Fc" as used herein is meant an Fc sequence that differs
from that of a parent Fc sequence by virtue of at least one amino
acid modification. An Fc variant may only encompass an Fc region,
or may exist in the context of an antibody, Fc fusion, isolated Fc,
Fc fragment, or other polypeptide that is substantially encoded by
Fc. Fc variant may refer to the Fc polypeptide itself, compositions
comprising the Fc variant polypeptide, or the amino acid sequence
that encodes it. By "Fc polypeptide variant" or "variant Fc
polypeptide" as used herein is meant an Fc polypeptide that differs
from a parent Fc polypeptide by virtue of at least one amino acid
modification. By "protein variant" or "variant protein" as used
herein is meant a protein that differs from a parent protein by
virtue of at least one amino acid modification. By "antibody
variant" or "variant antibody" as used herein is meant an antibody
that differs from a parent antibody by virtue of at least one amino
acid modification. By "IgG variant" or "variant IgG" as used herein
is meant an antibody that differs from a parent IgG by virtue of at
least one amino acid modification. By "immunoglobulin variant" or
"variant immunoglobulin" as used herein is meant an immunoglobulin
sequence that differs from that of a parent immunoglobulin sequence
by virtue of at least one amino acid modification.
[0142] By "wild type" or "WT" herein is meant an amino acid
sequence or a nucleotide sequence that is found in nature,
including allelic variations. A WT protein, polypeptide, antibody,
immunoglobulin, IgG, etc. has an amino acid sequence or a
nucleotide sequence that has not been intentionally modified.
Rapid Clearance Molecules
[0143] The present invention is directed to the use of rapid
clearance molecules (also referred to herein as "IIb variants")
with high affinity to the Fc.gamma.RIIb receptor that result in the
rapid clearance from serum of the antibody-antigen complex, while
retaining significant if not all the serum half-life of the unbound
antigen or unbound rapid clearance molecules. In certain
embodiments, the rapid clearance molecules of the invention include
antibodies or Fc fusion proteins. Although much of the discussion
herein is in terms of antibodies for ease of discussion, it will be
appreciated that this discussion applies equally to any of the
rapid clearance molecules described herein.
[0144] In general, rapid clearance molecules of the invention
generally comprise a variable region that binds to an antigen and a
variant Fc domain comprising one or more amino acid substitutions
as compared to a parent Fc domain such that the variant Fc domain
binds Fc.gamma.RIIb with increased affinity as compared to the
parent Fc domain.
[0145] In certain aspects, the rapid clearance ("RC") antibodies
incorporate Fc.gamma.RIIb receptor variants that can range from
very tight differential binding to Fc.gamma.RIIb to variants that
display increased (as compared to wild type Fc domains) binding
affinity but at a lower level. For example, very tight (or heavy)
binding to Fc.gamma.RIIb receptor may include Fc.gamma.RIIb
variants that show at least 50, 75, 100, 125, 150, 175, 200, 225,
250-fold greater affinity to Fc.gamma.RIIb receptor as compared to
the parent Fc domain. In contrast, a lower level (or light, also
referred to herein as "lite") increase in binding may include
Fc.gamma.RIIb variants that show no more than 50, 40, 30, 20, 10,
5-fold greater affinity to Fc.gamma.RIIb receptor as compared to
the parent Fc domain. In further embodiments, tighter/heavier
binding Fc.gamma.RIIb variants show 50-300, 60-275, 70-250, 80-225,
90-200, 100-175, 110-150-fold greater affinity to Fc.gamma.RIIb
receptor as compared to the parent Fc domain, whereas lower/lighter
binding Fc.gamma.RIIb variants show 2-40, 4-35, 6-30, 8-25, 9-20,
10-15-fold greater affinity to Fc.gamma.RIIb receptor as compared
to the parent Fc domain. In certain embodiments, affinity is
measured using Biacore, for example as described in Example 2. As
discussed herein, the functional properties of rapid clearance
molecules, including rapid clearance antibodies, can be tuned using
modifications (such as amino acid substitutions, deletions or
additions) that increase binding to Fc.gamma.RIIb receptor by a
tighter/heavier or a lower/lighter degree.
[0146] As shown herein, the binding affinity of the IIb variants
can be manipulated to result in different clearance/half life
ratios. That is, the IIb variant S267E/L328F shows very high
affinity binding to Fc.gamma.RIIb of the variants discussed herein,
and also has a faster clearance rate for antigen-antibody complexes
among the variants discussed herein. The binding affinity, antibody
half lives and clearance rates can be adjusted using high affinity
binding variants or lower affinity binding variants, e.g. S267E,
G236N/S267E, etc.). For example, the binding affinity of S267E is
about 10.times. lower than the S267E/L328F variant, with a
corresponding increase in half-life of roughly 2.times.-4.times.
higher. In certain aspects, higher/heavier binding results in
decreases in half-life. Thus, clearance rate and half-life can be
adjusted by utilizing Fc.gamma.RIIb-enhancing Fc amino acid
substitutions that possess intermediate or lower increases in
binding affinity (e.g., these substitutions still result in
variants with higher affinity for Fc.gamma.RIIb receptor than the
parent molecules, but the affinities for these variants is not as
increased, i.e., is lower/lighter than the heavy binding variants).
Correlations between half-life and binding affinity can be measured
as known in the art and discussed herein--see for example FIG. 25
and Example 6.
[0147] Application of different Fc.gamma.RIIb-enhancing Fc amino
acid substitutions with varying affinities to the Fc.gamma.RIIb
receptor (e.g. S267E/L328F, G236D/S267E, G236N/S267E, and S267E
alone, as further described herein) can allow some "tuning" of how
fast the complex antigen is cleared while maintaining significant
half life of the rapid clearance composition of the invention
(including antibodies). That is, different amino acid substitutions
that alter Fc.gamma.RIIb binding affinity may lead to different
balances between the complex clearance rate and the antibody
clearance rate, allowing for tailoring toward optimal therapeutic
profile and dosing. This tuning may be accomplished by using amino
acid substitutions in the Fc domain that increase binding to
Fc.gamma.RIIb as compared to the parent Fc domain. This increase in
binding may be tuned by using Fc variants with 1-100, 5-90, 10-80,
15-70, 20-60, 30-50, 10-20, 5-15, fold greater affinity as compared
to the parent Fc domain. This increase in binding may also be tuned
by using Fc variants with 20-500, 30-400, 40-300, 50-200, 60-190,
70-180, 80-170, 90-160, 100-150, 110-140, 120-130, 50-100, 25-75
fold greater affinity to Fc.gamma.RIIb receptor as compared to the
parent Fc domain.
[0148] The effects of molecules of the invention may be further
tuned by combining amino acid substitutions that alter
Fc.gamma.RIIb binding affinity with amino acid substitutions that
affect binding to FcRn. Proteins with amino acid substitutions that
affect binding to FcRn (also referred to herein as "FcRn variants")
may in certain situations also increase serum half-life in vivo as
compared to the parent protein. As will be appreciated, any
combination of Fc and FcRn variants may be used to tune clearance
of the antigen-antibody complex. Suitable FcRn variants that may be
combined with any of the Fc variants described herein that alter
binding to Fc.gamma.RIIb include without limitation 434A, 434S,
428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I/434S,
436V/434S, 436V/428L, 252Y, 252Y/254T/256E, and 259I/308F/428L.
[0149] In further embodiments, combinations of variants that alter
binding to the Fc.gamma.RIIb are combined with a variety of
scaffolds, target antigens and/or FcRn variants to further tune
clearance properties or other functional properties (such as
binding to Fc.gamma.RIIa) of the antibodies. Exemplary
(non-limiting) combinations are provided in FIG. 48, which provides
a matrix of possible combinations, with each variants being
independently and optionally combined from the appropriate source
Legend: Legend A are suitable FcRn variants: 434A, 434S, 428L,
308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,
436V/428L, 252Y, 252Y/254T/256E, 259I/308F/428L. Legend B are
suitable scaffolds and include IgG1, IgG2, IgG3, IgG4, and IgG1/2.
Legend C are suitable exemplary target antigens: IgE (soluble or
membrane-bound), IL-4, IL-6, IL-13, TNF.alpha., MCP-1, RANTES,
TARC, MDC, VEGF, HGF, and NGF, immune complexes, FVIII inhibitors,
LDL, oxidized LDL (OxLDL), Lp(a), SOST, and DKK1. Legend D reflects
the following possible combinations, again, with each variant being
independently and optionally combined from the appropriate source
Legend: 1) Fc.gamma.RIIb variants plus FcRn variants; 2)
Fc.gamma.RIIb variants plus FcRn variants plus Scaffold; 3)
Fc.gamma.RIIb variants plus FcRn variants plus Scaffold plus Fv; 4)
Fc.gamma.RIIb variants plus Scaffold 5) Fc.gamma.RIIb variants plus
Fv; 6) FcRn variants plus Scaffold; 7) FcRn variants plus Fv; 8)
Scaffold plus Fv; 9) Fc.gamma.RIIb variants plus Scaffold plus Fv;
and 10) Fc.gamma.RIIb variants plus FcRn variants plus Fv. Note
that any of these combinations may also include any of the
Fc.gamma.RIIb variants described herein, including those listed in
FIGS. 30, 36, 48 as well as those listed in the first column of
FIG. 48. Any of these combinations may also include any Fc variants
known in the art, including for example variants described in WO
2012/115241; WO2013/125667; U.S. Pat. No. 6,737,056; U.S. Pat. No.
8,435,517; and Mimoto et al., Protein Engineering Design and
Selection, vol. 26, No. 10, pp. 589-598 (2013), each of which is
hereby incorporated by reference in its entirety for all purposes,
and in particular any figures, legends, or discussion related to
variants that affect binding to Fc.gamma. receptors, including the
Fc.gamma.RIIb receptor. The combinations described in FIG. 48 may
further include selections from additional target antigens known in
the art or described herein.
[0150] In still further embodiments and in accordance with any of
the above, the rapid clearance molecules of the invention reduce
the total concentration of free antigen in a patient as compared to
the concentration prior to treatment with the rapid clearance
molecule. In exemplary embodiments, methods and compositions of the
invention reduce the total concentration of antigen by at least 2,
5, 10, 20, 30, 40, 50, 60, 70, 80, 90-fold as compared to the
concentration prior to treatment with the rapid clearance
molecule.
[0151] In yet further embodiments and in accordance with any of the
above, rapid clearance molecules include amino acid substitutions
(including those described herein) that lead to Fc variants with
increased Fc.gamma.RIIb as compared to the parent Fc domain and
further also alates binding to Fc.gamma.RIIa. Such Fc variants may
include the Fc variants described herein as well as Fc variants
described herein combined with further substitutions that ablate
binding to other Fc.gamma.R, including without limitation
Fc.gamma.RIIa. As discussed above, "ablation" herein is meant a
decrease or removal of activity. Thus for example, "ablating
Fc.gamma.R binding" means the Fc region amino acid variant has less
than 50% starting binding as compared to an Fc region not
containing the specific variant, with less than 70-80-90-95-98%
loss of activity being preferred, and in general, with the activity
being below the level of detectable binding in a Biacore assay.
[0152] In certain aspects and in accordance with any of the above,
accelerated clearance of the antigen containing complexes seen with
rapid clearance molecules containing amino acid substitutions that
confer high affinity (as compared to the parent Fc domain) to the
inhibitory receptor Fc.gamma.RIIb is likely mediated by interaction
with Fc.gamma.RIIb-expressing cells, possibly liver sinusoidal
endothelial cells. In certain embodiments, the accelerated
clearance of the antigen containing molecules is not mediated by
changes in pH or ionic conditions, such as those encountered within
lysosomes.
[0153] In general, antibodies of the invention that have engineered
Fc domains that result in higher affinity than wild-type antibodies
to the Fc.gamma.RIIb receptor can be directed to a variety of
antigens as discussed herein, including cancer antigens, pathogen
antigens, allergy antigens, etc.
[0154] In further embodiments, rapid clearance antibodies of the
invention show functional properties as described in U.S.
Provisional Application Ser. No. 61/752,955, filed Jan. 15, 2013;
61/794,164, filed Mar. 15, 2013, 61/794,386, filed Mar. 15, 2013,
and 61/833,696, filed Jun. 11, 2013, each of which is expressly
incorporated by reference in the entirety and in particular the
figures describing such variants, their functional properties, or
models of their functional properties (as shown for example in
FIGS. 23-26 of U.S. Ser. No. 61/752,955).
Antibodies
[0155] The present invention relates to the generation of
heterodimeric antibodies, generally therapeutic antibodies, through
the use of "heterodimerization amino acid variants". As is
discussed below, the term "antibody" is used generally. Antibodies
that find use in the present invention can take on a number of
formats as described herein, including traditional antibodies as
well as antibody derivatives, fragments and mimetics, described
below. In general, the term "antibody" includes any polypeptide
that includes at least one constant domain, including, but not
limited to, CH1, CH2, CH3 and CL.
[0156] Traditional antibody structural units typically comprise a
tetramer. Each tetramer is typically composed of two identical
pairs of polypeptide chains, each pair having one "light"
(typically having a molecular weight of about 25 kDa) and one
"heavy" chain (typically having a molecular weight of about 50-70
kDa). Human light chains are classified as kappa and lambda light
chains. The present invention is directed to the IgG class, which
has several subclasses, including, but not limited to IgG1, IgG2,
IgG3, and IgG4. Thus, "isotype" as used herein is meant any of the
subclasses of immunoglobulins defined by the chemical and antigenic
characteristics of their constant regions. It should be understood
that therapeutic antibodies can also comprise hybrids of isotypes
and/or subclasses. For example, as shown herein, the present
invention covers heterodimers that can contain one or both chains
that are IgG1/G2 hybrids (see SEQ ID NO:6, for example).
[0157] The amino-terminal portion of each chain includes a variable
region of about 100 to 110 or more amino acids primarily
responsible for antigen recognition, generally referred to in the
art and herein as the "Fv domain" or "Fv region". In the variable
region, three loops are gathered for each of the V domains of the
heavy chain and light chain to form an antigen-binding site. Each
of the loops is referred to as a complementarity-determining region
(hereinafter referred to as a "CDR"), in which the variation in the
amino acid sequence is most significant. "Variable" refers to the
fact that certain segments of the variable region differ
extensively in sequence among antibodies. Variability within the
variable region is not evenly distributed. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-15 amino
acids long or longer.
[0158] Each VH and VL is composed of three hypervariable regions
("complementary determining regions," "CDRs") and four FRs,
arranged from amino-terminus to carboxy-terminus in the following
order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
[0159] The hypervariable region generally encompasses amino acid
residues from about amino acid residues 24-34 (LCDR1; "L" denotes
light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain
variable region and around about 31-35B (HCDR1; "H" denotes heavy
chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain
variable region; Kabat et al., SEQUENCES OF PROTEINS OF
IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991) and/or those residues
forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52
(LCDR2) and 91-96 (LCDR3) in the light chain variable region and
26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain
variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.
Specific CDRs of the invention are described below.
[0160] Throughout the present specification, the Kabat numbering
system is generally used when referring to a residue in the
variable domain (approximately, residues 1-107 of the light chain
variable region and residues 1-113 of the heavy chain variable
region) (e.g, Kabat et al., supra (1991)).
[0161] The CDRs contribute to the formation of the antigen-binding,
or more specifically, epitope binding site of antibodies. "Epitope"
refers to a determinant that interacts with a specific antigen
binding site in the variable region of an antibody molecule known
as a paratope. Epitopes are groupings of molecules such as amino
acids or sugar side chains and usually have specific structural
characteristics, as well as specific charge characteristics. A
single antigen may have more than one epitope.
[0162] The epitope may comprise amino acid residues directly
involved in the binding (also called immunodominant component of
the epitope) and other amino acid residues, which are not directly
involved in the binding, such as amino acid residues which are
effectively blocked by the specifically antigen binding peptide; in
other words, the amino acid residue is within the footprint of the
specifically antigen binding peptide.
[0163] Epitopes may be either conformational or linear. A
conformational epitope is produced by spatially juxtaposed amino
acids from different segments of the linear polypeptide chain. A
linear epitope is one produced by adjacent amino acid residues in a
polypeptide chain. Conformational and nonconformational epitopes
may be distinguished in that the binding to the former but not the
latter is lost in the presence of denaturing solvents.
[0164] An epitope typically includes at least 3, and more usually,
at least 5 or 8-10 amino acids in a unique spatial conformation.
Antibodies that recognize the same epitope can be verified in a
simple immunoassay showing the ability of one antibody to block the
binding of another antibody to a target antigen, for example
"binning."
[0165] In some embodiments, the antibodies are full length. By
"full length antibody" herein is meant the structure that
constitutes the natural biological form of an antibody, including
variable and constant regions, including one or more modifications
as outlined herein.
[0166] Alternatively, the antibodies can be a variety of
structures, including, but not limited to, antibody fragments,
monoclonal antibodies, bispecific antibodies, minibodies, domain
antibodies, synthetic antibodies (sometimes referred to herein as
"antibody mimetics"), chimeric antibodies, humanized antibodies,
antibody fusions (sometimes referred to as "antibody conjugates"),
and fragments of each, respectively.
Antibody Fragments
[0167] In one embodiment, the antibody is an antibody fragment. Of
particular interest are antibodies that comprise Fc regions, Fc
fusions, and the constant region of the heavy chain
(CH1-hinge-CH2-CH3), again also including constant heavy region
fusions.
[0168] Specific antibody fragments include, but are not limited to,
(i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii)
the Fd fragment consisting of the VH and CH1 domains, (iii) the Fv
fragment consisting of the VL and VH domains of a single antibody;
(iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546,
entirely incorporated by reference) which consists of a single
variable, (v) isolated CDR regions, (vi) F(ab')2 fragments, a
bivalent fragment comprising two linked Fab fragments (vii) single
chain Fv molecules (scFv), wherein a VH domain and a VL domain are
linked by a peptide linker which allows the two domains to
associate to form an antigen binding site (Bird et al., 1988,
Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci.
U.S.A. 85:5879-5883, entirely incorporated by reference), (viii)
bispecific single chain Fv (WO 03/11161, hereby incorporated by
reference) and (ix) "diabodies" or "triabodies", multivalent or
multispecific fragments constructed by gene fusion (Tomlinson et.
al., 2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et
al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, all entirely
incorporated by reference). The antibody fragments may be modified.
For example, the molecules may be stabilized by the incorporation
of disulphide bridges linking the VH and VL domains (Reiter et al.,
1996, Nature Biotech. 14:1239-1245, entirely incorporated by
reference).
Chimeric and Humanized Antibodies
[0169] In some embodiments, the scaffold components can be a
mixture from different species. As such, if the protein is an
antibody, such antibody may be a chimeric antibody and/or a
humanized antibody. In general, both "chimeric antibodies" and
"humanized antibodies" refer to antibodies that combine regions
from more than one species. For example, "chimeric antibodies"
traditionally comprise variable region(s) from a mouse (or rat, in
some cases) and the constant region(s) from a human. "Humanized
antibodies" generally refer to non-human antibodies that have had
the variable-domain framework regions swapped for sequences found
in human antibodies. Generally, in a humanized antibody, the entire
antibody, except the CDRs, is encoded by a polynucleotide of human
origin or is identical to such an antibody except within its CDRs.
The CDRs, some or all of which are encoded by nucleic acids
originating in a non-human organism, are grafted into the
beta-sheet framework of a human antibody variable region to create
an antibody, the specificity of which is determined by the
engrafted CDRs. The creation of such antibodies is described in,
e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et
al., 1988, Science 239:1534-1536, all entirely incorporated by
reference. "Backmutation" of selected acceptor framework residues
to the corresponding donor residues is often required to regain
affinity that is lost in the initial grafted construct (U.S. Pat.
No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761;
U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No.
5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297; U.S.
Pat. No. 6,407,213, all entirely incorporated by reference). The
humanized antibody optimally also will comprise at least a portion
of an immunoglobulin constant region, typically that of a human
immunoglobulin, and thus will typically comprise a human Fc region.
Humanized antibodies can also be generated using mice with a
genetically engineered immune system. Roque et al., 2004,
Biotechnol. Prog. 20:639-654, entirely incorporated by reference. A
variety of techniques and methods for humanizing and reshaping
non-human antibodies are well known in the art (See Tsurushita
& Vasquez, 2004, Humanization of Monoclonal Antibodies,
Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and
references cited therein, all entirely incorporated by reference).
Humanization methods include but are not limited to methods
described in Jones et al., 1986, Nature 321:522-525; Riechmann et
al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science,
239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA
86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et
al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997,
Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad.
Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8,
all entirely incorporated by reference. Humanization or other
methods of reducing the immunogenicity of nonhuman antibody
variable regions may include resurfacing methods, as described for
example in Roguska et al., 1994, Proc. Natl. Acad. Sci.
[0170] USA 91:969-973, entirely incorporated by reference. In one
embodiment, the parent antibody has been affinity matured, as is
known in the art. Structure-based methods may be employed for
humanization and affinity maturation, for example as described in
U.S. Ser. No. 11/004,590. Selection based methods may be employed
to humanize and/or affinity mature antibody variable regions,
including but not limited to methods described in Wu et al., 1999,
J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem.
272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37):
22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95:
8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759,
all entirely incorporated by reference. Other humanization methods
may involve the grafting of only parts of the CDRs, including but
not limited to methods described in U.S. Ser. No. 09/810,510; Tan
et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002,
J. Immunol. 169:3076-3084, all entirely incorporated by
reference.
[0171] In one embodiment, the antibody is a minibody. Minibodies
are minimized antibody-like proteins comprising a scFv joined to a
CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061, entirely
incorporated by reference. In some cases, the scFv can be joined to
the Fc region, and may include some or the entire hinge region.
Fc Fusion Proteins
[0172] In addition to antibody constructs discussed herein, the
invention further provides Fc fusion proteins where the Fc region
has IIb variants. That is, rather than have the Fc domain of an
antibody joined to an antibody variable region, the Fc domain can
be joined to other moieties, particularly binding moieties such as
ligands. By "Fc fusion" as used herein is meant a protein wherein
one or more polypeptides is operably linked to an Fc region. Fc
fusion is herein meant to be synonymous with the terms
"immunoadhesin", "Ig fusion", "Ig chimera", and "receptor globulin"
(sometimes with dashes) as used in the prior art (Chamow et al.,
1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin
Immunol 9:195-200, both entirely incorporated by reference). An Fc
fusion combines the Fc region of an immunoglobulin with a fusion
partner, which in general can be any protein or small molecule.
Virtually any protein or small molecule may be linked to Fc to
generate an Fc fusion. Protein fusion partners may include, but are
not limited to, the variable region of any antibody, the
target-binding region of a receptor, an adhesion molecule, a
ligand, an enzyme, a cytokine, a chemokine, or some other protein
or protein domain. Small molecule fusion partners may include any
therapeutic agent that directs the Fc fusion to a therapeutic
target. Such targets may be any molecule, preferably an
extracellular receptor, which is implicated in disease. Thus, the
IgG variants can be linked to one or more fusion partners.
Fc.gamma.RIIb Variants
[0173] The compositions and methods of the invention rely on
Fc.gamma.RIIb variants that increase binding to the Fc.gamma.RIIb
receptor. Related applications discuss the Fc.gamma.RIIb variants
in detail. See for example U.S. Ser. Nos. 11/124,620 and
13/294,103, both of which are incorporated by reference in their
entirety, and in particular for the amino acid variant positions,
accompanying specification description, figures and accompanying
legends, and data relating to the variants. Fc variants that find
particular use herein include, but are not limited to, those listed
in FIG. 30.
[0174] Fc.gamma.RIIb receptor variants that are considered "tight"
binding and display the fastest rapid clearance times, include
S267E/L328F.
[0175] Fc.gamma.RIIb receptor variants increased binding as
compared to wild type Fc domains, e.g. IgG1 domains, but are
considered lower affinity and thus can result in longer half lives
include, but are not limited to, S267E and G236N/267E and
G236D/267E (sometimes referred to as "IIb-lite" variants).
Additional Fc.gamma.R Variants
[0176] In addition to Fc.gamma.RIIb receptor variants, there are a
number of useful Fc substitutions that can be made to alter binding
to one or more of the Fc.gamma.R receptors. Substitutions that
result in increased binding as well as decreased binding can be
useful. For example, it is known that increased binding to
Fc.gamma.RIIIa generally results in increased ADCC (antibody
dependent cell-mediated cytotoxicity; the cell-mediated reaction
wherein nonspecific cytotoxic cells that express Fc.gamma.Rs
recognize bound antibody on a target cell and subsequently cause
lysis of the target cell. Amino acid substitutions that find use in
the present invention include those listed in U.S. Ser. No.
11/124,620 (particularly FIG. 41), Ser. Nos. 11/174,287,
11/396,495, 11/538,406, all of which are expressly incorporated
herein by reference in their entirety and specifically for the
variants disclosed therein. Particular variants that find use
include, but are not limited to, 236A, 239D, 239E, 332E, 332D,
239D/332E, 267D, S267E, L328F, S267E/L328F, 236A/332E,
239D/332E/330Y, 239D, 332E/330L, 243L, 298A and 299T. In some cases
ablation variants such as 236R, 328R, and 236R/328R can be made,
although this is not preferred in some embodiments. Additional
suitable Fc variants are found in FIG. 41 of US 2006/0024298, the
figure and legend of which are hereby incorporated by reference in
their entirety.
[0177] In addition, there are additional Fc substitutions that find
use in increased binding to the FcRn receptor and optionally
increased serum half life, as specifically disclosed in U.S. Ser.
No. 12/341,769, hereby incorporated by reference in its entirety,
including, but not limited to, 434S, 434A, 428L, 308F, 259I,
428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L and
259I/308F/428L.
IgE-Fc.gamma.RIIb Molecules
[0178] One example of rapid clearance applications are antibodies
that contain an antigen binding site to IgE and Fc.gamma.RIIb
variants, resulting in "co-engagement". For example, described
herein are variant anti-IgE antibodies engineered such that the Fc
domain binds to Fc.gamma.RIIb with up to .about.430-fold greater
affinity relative to native IgG1. These Fc.gamma.RIIb
binding-enhanced (IIbE) variants strongly inhibit BCR-induced
calcium mobilization and viability in primary human IgE+ B cells.
The use of a single molecule, such as an antibody to suppress B
cell functions of cognate IgE BCR and Fc.gamma.RIIb represents a
novel approach in the treatment of IgE-mediated diseases.
Non-limiting examples of IgE-mediated diseases include allergic
responses and asthma and are described in U.S. Ser. No. 12/156,183,
hereby incorporated by reference in its entirety, and particularly
for the discussion associated with coengagement, and described
below.
Factor VIII-Fc.gamma.RIIb Molecules
[0179] In one embodiment, the rapid clearance mechanisms of the
present invention are used to treat hemophilia. One issue with
hemophiliacs is the effect that Factor VIII (FVIII (not to be
confused with "Fv")) inhibitors play in disease. The most
significant complication of treatment of hemophilia A is the
development of alloantibodies to the therapeutic Factor VIII, that
then inhibit the activity of the Factor VIII. Approximately 30% of
patients with severe hemophilia A develop these alloantibodies,
recognizing the exogenous correct FVIII as "foreign", generally
resulting in bleeding episodes that are more difficult to
manage.
[0180] FIG. 28 shows the structure of the FVIIIa protein,
consisting of heterodimeric protein comprising a heavy chain
(A1-A2-B, not to be confused with the heavy chain of an antibody)
and a light chain (A3-C1-C2) that are associated through a
noncovalent divalent metal ion linkage between the A1 and A3
domains. The alloantibodies generally develop to the A2 and C2
domains, which are the dominant epitopes for the alloantibodies,
generally accounting for roughly 68% of the alloantibody
antigens.
[0181] Due to the rapid clearance of antibodies and Fc fusions
engineered to bind with high affinity to Fc.gamma.RIIb, the present
invention provides "scrubber" or "drug" Fc fusions, comprising some
or all of the domains of FVIIIa, that bind to the FVIIIa inhibitors
thereby clearing out the alloantibodies. That is, the Fc.gamma.RIIb
fusions, directed against these inhibitor antibodies will sequester
the inhibitor antibodies, rapidly clear the inhibitor antibodies,
and will also inhibit FVIII-reactive B cells (to prohibit the
further production of the inhibitors).
CR-Fc Fusion Molecules
[0182] In one embodiment, the rapid clearance mechanisms of the
present invention are used with complement receptor 2 (CR2)-Fc
fusions for immune system modulation and accelerated clearance of
C3d-tagged immune complexes. These fusion proteins in this context
are sometimes referred to herein as "CR2-IIbE". In this embodiment,
increased affinity for the inhibitory Fc receptor FcRIIb (CD32b) is
utilized to facilitate rapid in vivo clearance of C3-tagged immune
complexes via their interaction with CR-IIbE fusions. Incorporation
of the IIb-enhancing affinity substitutions into various fusions
leads to a novel phenomenon whereby the fusion-target complex is
cleared extremely rapidly while the CR-IIbE alone retains a
reasonably long half-life. Application of different IIb-enhancing
substitutions (e.g. S267E/L328F, G236D/S267E, 236N/267E, or S267E
alone, as are useful for all the rapid clearance molecules herein)
may lead to different balances between the complex clearance rate
and the fusion protein clearance rate, allowing for tailoring
toward optimal therapeutic profile and dosing.
[0183] In another aspect of the invention, rapid
clearance-mediating IIb technology can additionally be applied to
other complement system receptors or inhibitors, including but not
limited to, CR1, Factor H (fH), CR3, and CRIg. Typically, only the
SCR domains required for recognition of the appropriate complement
factor will be required, although additional repeats may be
included for stability.
[0184] In yet another aspect of the invention, rapid
clearance-mediating IIb technology can be applied to antibodies
that recognize C3 fragments, thereby mimicking the above-described
CR-Fc fusions, similar to the IgE and Factor VIII antibody
examples. Examples include, but are not limited to, anti-C3d
antibodies with engineered Fc regions. See also U.S. Ser. No.
61/752,955, filed Jan. 15, 2013, which is hereby incorporated by
reference in its entirety for all purposes and in particular for
all teachings, figures and legends related to schemes of complement
and antibodies directed to same.
Anti-IgE Antibodies
[0185] In some embodiments, the immunoglobulins described herein
bind IgE. The anti-IgE antibodies of the invention may comprise any
variable region, known or not yet known, that has specificity for
IgE. Known anti-IgE antibodies include but are not limited to
murine antibodies MaE11, MaE13, and MaE15, humanized and/or
engineered versions of these antibodies including E25, E26, and
E27, particularly E25, also known as rhuMab-E25, also known as
Omalizumab, such as those described in U.S. Pat. No. 6,761,889,
U.S. Pat. No. 6,329,509, US20080003218A1, and Presta, L G et al.,
1993, J Immunol 151:2623-2632, all herein expressly incorporated by
reference. A preferred engineered version of MaE11 is H1 L1 MaE11,
described in the Examples herein. Other anti-IgE that may be useful
for the invention include murine antibody TES-C21, chimeric
TES-C21, also known as CGP51901 (Corne, J et al., 1997, J Clin
Invest 99:879-887; Racine-Poon, A et al., 1997, Clin Pharmcol Ther
62:675-690), and humanized and/or engineered versions of this
antibody including but not limited to CGP56901, also known as
TNX-901, such as those antibodies described in Kolbinger, F et al.,
1993, Protein Eng 6:971-980. Other anti-IgE antibodies that may
find use for the invention are described in U.S. Pat. No.
6,066,718, U.S. Pat. No. 6,072,035, PCT/US04/02894, U.S. Pat. No.
5,342,924, U.S. Pat. No. 5,091,313, U.S. Pat. No. 5,449,760, U.S.
Pat. No. 5,543,144, U.S. Pat. No. 5,342,924, and U.S. Pat. No.
5,614,611, all of which are incorporated herein by reference. Other
useful anti-IgE antibodies include the murine antibody BSW17. Amino
acid sequences of the variable region VH and VL domains and CDRs of
some of these antibodies are provided in FIG. 5.
Fc Variants and Fc Receptor Binding Properties
[0186] Immunoglobulins disclosed herein may comprise an Fc variant.
An Fc variant comprises one or more amino acid modifications
relative to a parent Fc polypeptide, wherein the amino acid
modification(s) provide one or more optimized properties. An Fc
variant disclosed herein differs in amino acid sequence from its
parent by virtue of at least one amino acid modification. Thus Fc
variants disclosed herein have at least one amino acid modification
compared to the parent. Alternatively, the Fc variants disclosed
herein may have more than one amino acid modification as compared
to the parent, for example from about one to fifty amino acid
modifications, e.g., from about one to ten amino acid
modifications, from about one to about five amino acid
modifications, etc. compared to the parent. Thus the sequences of
the Fc variants and those of the parent Fc polypeptide are
substantially homologous. For example, the variant Fc variant
sequences herein will possess about 80% homology with the parent Fc
variant sequence, e.g., at least about 90% homology, at least about
95% homology, at least about 98% homology, at least about 99%
homology, etc. Modifications disclosed herein include amino acid
modifications, including insertions, deletions, and substitutions.
Modifications disclosed herein also include glycoform
modifications. Modifications may be made genetically using
molecular biology, or may be made enzymatically or chemically.
[0187] Fc variants disclosed herein are defined according to the
amino acid modifications that compose them. Thus, for example,
S267E is an Fc variant with the substitution S267E relative to the
parent Fc polypeptide. Likewise, S267E/L328F defines an Fc variant
with the substitutions S267E and L328F relative to the parent Fc
polypeptide. The identity of the WT amino acid may be unspecified,
in which case the aforementioned variant is referred to as
S267E/L328F. It is noted that the order in which substitutions are
provided is arbitrary, that is to say that, for example,
S267E/L328F is the same Fc variant as L328F/267E, and so on. Unless
otherwise noted, positions discussed herein are numbered according
to the EU index or EU numbering scheme (Kabat et al., 1991,
Sequences of Proteins of Immunological Interest, 5th Ed., United
States Public Health Service, National Institutes of Health,
Bethesda, hereby entirely incorporated by reference). The EU index
or EU index as in Kabat or EU numbering scheme refers to the
numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad
Sci USA 63:78-85, hereby entirely incorporated by reference).
[0188] In certain embodiments, the Fc variants disclosed herein are
based on human IgG sequences, and thus human IgG sequences are used
as the "base" sequences against which other sequences are compared,
including but not limited to sequences from other organisms, for
example rodent and primate sequences. Immunoglobulins may also
comprise sequences from other immunoglobulin classes such as IgA,
IgE, IgGD, IgGM, and the like. It is contemplated that, although
the Fc variants disclosed herein are engineered in the context of
one parent IgG, the variants may be engineered in or "transferred"
to the context of another, second parent IgG. This is done by
determining the "equivalent" or "corresponding" residues and
substitutions between the first and second IgG, typically based on
sequence or structural homology between the sequences of the first
and second IgGs. In order to establish homology, the amino acid
sequence of a first IgG outlined herein is directly compared to the
sequence of a second IgG. After aligning the sequences, using one
or more of the homology alignment programs known in the art (for
example using conserved residues as between species), allowing for
necessary insertions and deletions in order to maintain alignment
(i.e., avoiding the elimination of conserved residues through
arbitrary deletion and insertion), the residues equivalent to
particular amino acids in the primary sequence of the first
immunoglobulin are defined. Alignment of conserved residues may
conserve 100% of such residues. However, alignment of greater than
75% or as little as 50% of conserved residues is also adequate to
define equivalent residues. Equivalent residues may also be defined
by determining structural homology between a first and second IgG
that is at the level of tertiary structure for IgGs whose
structures have been determined. In this case, equivalent residues
are defined as those for which the atomic coordinates of two or
more of the main chain atoms of a particular amino acid residue of
the parent or precursor (N on N, CA on CA, C on C and O on O) are
within about 0.13 nm, after alignment. In another embodiment,
equivalent residues are within about 0.1 nm after alignment.
Alignment is achieved after the best model has been oriented and
positioned to give the maximum overlap of atomic coordinates of
non-hydrogen protein atoms of the proteins. Regardless of how
equivalent or corresponding residues are determined, and regardless
of the identity of the parent IgG in which the IgGs are made, what
is meant to be conveyed is that the Fc variants discovered as
disclosed herein may be engineered into any second parent IgG that
has significant sequence or structural homology with the Fc
variant. Thus for example, if a variant antibody is generated
wherein the parent antibody is human IgG1, by using the methods
described above or other methods for determining equivalent
residues, the variant antibody may be engineered in another IgG1
parent antibody that binds a different antigen, a human IgG2 parent
antibody, a human IgA parent antibody, a mouse IgG2a or IgG2b
parent antibody, and the like. Again, as described above, the
context of the parent Fc variant does not affect the ability to
transfer the Fc variants disclosed herein to other parent IgGs.
[0189] The Fc variants disclosed herein may be optimized for a
variety of Fc receptor binding properties. An Fc variant that is
engineered or predicted to display one or more optimized properties
is herein referred to as an "optimized Fc variant".
[0190] Properties that may be optimized include but are not limited
to enhanced or reduced affinity for an Fc.gamma.R. In one
embodiment, the Fc variants disclosed herein are optimized to
possess enhanced affinity for an inhibitory receptor Fc.gamma.RIIb.
In other embodiments, immunoglobulins disclosed herein provide
enhanced affinity for Fc.gamma.RIIb, yet reduced affinity for one
or more activating Fc.gamma.Rs, including for example Fc.gamma.RI,
Fc.gamma.RIIa, Fc.gamma.RIIIa, and/or Fc.gamma.RIIIb. The
Fc.gamma.R receptors may be expressed on cells from any organism,
including but not limited to human, cynomolgus monkeys, and mice.
The Fc variants disclosed herein may be optimized to possess
enhanced affinity for human Fc.gamma.RIIb.
[0191] By "greater affinity" or "improved affinity" or "enhanced
affinity" or "better affinity" than a parent Fc polypeptide, as
used herein is meant that an Fc variant binds to an Fc receptor
with a significantly higher equilibrium constant of association (KA
or Ka) or lower equilibrium constant of dissociation (KD or Kd)
than the parent Fc polypeptide when the amounts of variant and
parent polypeptide in the binding assay are essentially the same.
For example, the Fc variant with improved Fc receptor binding
affinity may display from about 5 fold to about 1000 fold, e.g.
from about 10 fold to about 500 fold improvement in Fc receptor
binding affinity compared to the parent Fc polypeptide, where Fc
receptor binding affinity is determined, for example, by the
binding methods disclosed herein, including but not limited to
Biacore, by one skilled in the art. Accordingly, by "reduced
affinity" as compared to a parent Fc polypeptide as used herein is
meant that an Fc variant binds an Fc receptor with significantly
lower KA or higher KD than the parent Fc polypeptide. Greater or
reduced affinity can also be defined relative to an absolute level
of affinity. For example, according to the data herein, WT (native)
IgG1 binds Fc.gamma.RIIb with an affinity of about 2 .mu.M, or
about 2000 nM. Furthermore, some Fc variants described herein bind
Fc.gamma.RIIb with an affinity about 10-fold greater to WT IgG1. As
disclosed herein, greater or enhanced affinity means having a KD
lower than about 100 nM, for example between about 10 nM-about 100
nM, between about 1-about 100 nM, or less than about 1 nM.
[0192] Anti-IgE antibodies of the invention preferably have high
affinity for Fc.gamma.RIIb. By high affinity herein is meant that
the affinity of the interaction between the antibody and
Fc.gamma.RIIb is stronger than 100 nM. That is to say that the
equilibrium dissociation constant Kd for binding of the antibody to
Fc.gamma.RIIb is lower than 100 nM.
[0193] In one embodiment, the Fc variants provide selectively
enhanced affinity to Fc.gamma.RIIb relative to one or more
activating receptors. Selectively enhanced affinity means either
that the Fc variant has improved affinity for Fc.gamma.RIIb
relative to the activating receptor(s) as compared to the parent Fc
polypeptide but has reduced affinity for the activating receptor(s)
as compared to the parent Fc polypeptide, or it means that the Fc
variant has improved affinity for both Fc.gamma.RIIb and activating
receptor(s) as compared to the parent Fc polypeptide, however the
improvement in affinity is greater for Fc.gamma.RIIb than it is for
the activating receptor(s). In alternate embodiments, the Fc
variants reduce or ablate binding to one or more activating
Fc.gamma.Rs, reduce or ablate binding to one or more complement
proteins, reduce or ablate one or more Fc.gamma.R-mediated effector
functions, and/or reduce or ablate one or more complement-mediated
effector functions.
[0194] The presence of different polymorphic forms of Fc.gamma.Rs
provides yet another parameter that impacts the therapeutic utility
of the Fc variants disclosed herein. Whereas the specificity and
selectivity of a given Fc variant for the different classes of
Fc.gamma.Rs significantly affects the capacity of an Fc variant to
target a given antigen for treatment of a given disease, the
specificity or selectivity of an Fc variant for different
polymorphic forms of these receptors may in part determine which
research or pre-clinical experiments may be appropriate for
testing, and ultimately which patient populations may or may not
respond to treatment. Thus the specificity or selectivity of Fc
variants disclosed herein to Fc receptor polymorphisms, including
but not limited to Fc.gamma.RIIa, Fc.gamma.RIIIa, and the like, may
be used to guide the selection of valid research and pre-clinical
experiments, clinical trial design, patient selection, dosing
dependence, and/or other aspects concerning clinical trials.
[0195] Fc variants disclosed herein may comprise modifications that
modulate interaction with Fc receptors other than Fc.gamma.Rs,
including but not limited to complement proteins, FcRn, and Fc
receptor homologs (FcRHs). FcRHs include but are not limited to
FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002,
Immunol. Reviews 190:123-136).
[0196] An important parameter that determines the most beneficial
selectivity of a given Fc variant to treat a given disease is the
context of the Fc variant. Thus the Fc receptor selectivity or
specificity of a given Fc variant will provide different properties
depending on whether it composes an antibody, Fc fusion, or Fc
variants with a coupled fusion partner. In one embodiment, an Fc
receptor specificity of the Fc variant disclosed herein will
determine its therapeutic utility. The utility of a given Fc
variant for therapeutic purposes will depend on the epitope or form
of the target antigen and the disease or indication being treated.
For some targets and indications, greater Fc.gamma.RIIb affinity
and reduced activating Fc.gamma.R-mediated effector functions may
be beneficial. For other target antigens and therapeutic
applications, it may be beneficial to increase affinity for
Fc.gamma.RIIb, or increase affinity for both Fc.gamma.RIIb and
activating receptors.
Glycoform Modifications
[0197] Many polypeptides, including antibodies, are subjected to a
variety of post-translational modifications involving carbohydrate
moieties, such as glycosylation with oligosaccharides. There are
several factors that can influence glycosylation. The species,
tissue and cell type have all been shown to be important in the way
that glycosylation occurs. In addition, the extracellular
environment, through altered culture conditions such as serum
concentration, may have a direct effect on glycosylation (Lifely et
al., 1995, Glycobiology 5(8): 813-822).
[0198] All antibodies contain carbohydrate at conserved positions
in the constant regions of the heavy chain. Each antibody isotype
has a distinct variety of N-linked carbohydrate structures. Aside
from the carbohydrate attached to the heavy chain, up to 30% of
human IgGs have a glycosylated Fab region. IgG has a single
N-linked biantennary carbohydrate at Asn297 of the CH2 domain. For
IgG from either serum or produced ex vivo in hybridomas or
engineered cells, the IgG are heterogeneous with respect to the
Asn297 linked carbohydrate (Jefferis et al., 1998, Immunol. Rev.
163:59-76; Wright et al., 1997, Trends Biotech 15:26-32). For human
IgG, the core oligosaccharide normally consists of
GlcNAc2Man3GlcNAc, with differing numbers of outer residues.
[0199] The carbohydrate moieties of immunoglobulins disclosed
herein will be described with reference to commonly used
nomenclature for the description of oligosaccharides. A review of
carbohydrate chemistry which uses this nomenclature is found in
Hubbard et al. 1981, Ann. Rev. Biochem. 50:555-583. This
nomenclature includes, for instance, Man, which represents mannose;
GlcNAc, which represents 2-N-acetylglucosamine; Gal which
represents galactose; Fuc for fucose; and Glc, which represents
glucose. Sialic acids are described by the shorthand notation
NeuNAc, for 5-N-acetylneuraminic acid, and NeuNGc for
5-glycolylneuraminic.
[0200] The term "glycosylation" means the attachment of
oligosaccharides (carbohydrates containing two or more simple
sugars linked together e.g. from two to about twelve simple sugars
linked together) to a glycoprotein. The oligosaccharide side chains
are typically linked to the backbone of the glycoprotein through
either N- or O-linkages. The oligosaccharides of immunoglobulins
disclosed herein occur generally are attached to a CH2 domain of an
Fc region as N-linked oligosaccharides. "N-linked glycosylation"
refers to the attachment of the carbohydrate moiety to an
asparagine residue in a glycoprotein chain. The skilled artisan
will recognize that, for example, each of murine IgG1, IgG2a, IgG2b
and IgG3 as well as human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2
domains have a single site for N-linked glycosylation at amino acid
residue 297 (Kabat et al. Sequences of Proteins of Immunological
Interest, 1991).
[0201] For the purposes herein, a "mature core carbohydrate
structure" refers to a processed core carbohydrate structure
attached to an Fc region which generally consists of the following
carbohydrate structure GlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc)2
typical of biantennary oligosaccharides. The mature core
carbohydrate structure is attached to the Fc region of the
glycoprotein, generally via N-linkage to Asn297 of a CH2 domain of
the Fc region. A "bisecting GlcNAc" is a GlcNAc residue attached to
the .beta.1,4 mannose of the mature core carbohydrate structure.
The bisecting GlcNAc can be enzymatically attached to the mature
core carbohydrate structure by a
.beta.(1,4)-N-acetylglucosaminyltransferase III enzyme (GnTIII).
CHO cells do not normally express GnTIII (Stanley et al., 1984, J.
Biol. Chem. 261:13370-13378), but may be engineered to do so (Umana
et al., 1999, Nature Biotech. 17:176-180).
[0202] Described herein are Fc variants that comprise modified
glycoforms or engineered glycoforms. By "modified glycoform" or
"engineered glycoform" as used herein is meant a carbohydrate
composition that is covalently attached to a protein, for example
an antibody, wherein said carbohydrate composition differs
chemically from that of a parent protein. Engineered glycoforms may
be useful for a variety of purposes, including but not limited to
enhancing or reducing Fc.gamma.R-mediated effector function. In one
embodiment, the immunoglobulins disclosed herein are modified to
control the level of fucosylated and/or bisecting oligosaccharides
that are covalently attached to the Fc region.
[0203] A variety of methods are well known in the art for
generating modified glycoforms (Umana et al., 1999, Nat Biotechnol
17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294;
Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al.,
2003, J Biol Chem 278:3466-3473); (U.S. Pat. No. 6,602,684; U.S.
Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1;
PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1);
(Potelligent.TM. technology [Biowa, Inc., Princeton, N.J.];
GlycoMAb.TM. glycosylation engineering technology [GLYCART
biotechnology AG, Zirich, Switzerland]; all of which are expressly
incorporated by reference). These techniques control the level of
fucosylated and/or bisecting oligosaccharides that are covalently
attached to the Fc region, for example by expressing an IgG in
various organisms or cell lines, engineered or otherwise (for
example Lec-13 CHO cells or rat hybridoma YB2/0 cells), by
regulating enzymes involved in the glycosylation pathway (for
example FUT8 [.alpha.1,6-fucosyltranserase] and/or
.beta.1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by
modifying carbohydrate(s) after the IgG has been expressed. Other
methods for modifying glycoforms of the immunoglobulins disclosed
herein include using glycoengineered strains of yeast (Li et al.,
2006, Nature Biotechnology 24(2):210-215), moss (Nechansky et al.,
2007, Mol Immunjol 44(7):1826-8), and plants (Cox et al., 2006, Nat
Biotechnol 24(12):1591-7). The use of a particular method to
generate a modified glycoform is not meant to constrain embodiments
to that method. Rather, embodiments disclosed herein encompass Fc
variants with modified glycoforms irrespective of how they are
produced.
[0204] In one embodiment, immunoglobulins disclosed herein are
glycoengineered to alter the level of sialylation. Higher levels of
sialylated Fc glycans in immunoglobulin G molecules can adversely
impact functionality (Scallon et al., 2007, Mol Immunol.
44(7):1524-34), and differences in levels of Fc sialylation can
result in modified anti-inflammatory activity (Kaneko et al., 2006,
Science 313:670-673). Because antibodies may acquire
anti-inflammatory properties upon sialylation of Fc core
polysaccharide, it may be advantageous to glycoengineer the
immunoglobulins disclosed herein for greater or reduced Fc sialic
acid content.
[0205] Engineered glycoform typically refers to the different
carbohydrate or oligosaccharide; thus for example an immunoglobulin
may comprise an engineered glycoform. Alternatively, engineered
glycoform may refer to the immunoglobulin that comprises the
different carbohydrate or oligosaccharide. In one embodiment, a
composition disclosed herein comprises a glycosylated Fc variant
having an Fc region, wherein about 51-100% of the glycosylated
antibody, e.g., 80-100%, 90-100%, 95-100%, etc. of the antibody in
the composition comprises a mature core carbohydrate structure
which lacks fucose. In another embodiment, the antibody in the
composition both comprises a mature core carbohydrate structure
that lacks fucose and additionally comprises at least one amino
acid modification in the Fc region. In an alternative embodiment, a
composition comprises a glycosylated Fc variant having an Fc
region, wherein about 51-100% of the glycosylated antibody,
80-100%, or 90-100%, of the antibody in the composition comprises a
mature core carbohydrate structure which lacks sialic acid. In
another embodiment, the antibody in the composition both comprises
a mature core carbohydrate structure that lacks sialic acid and
additionally comprises at least one amino acid modification in the
Fc region. In yet another embodiment, a composition comprises a
glycosylated Fc variant having an Fc region, wherein about 51-100%
of the glycosylated antibody, 80-100%, or 90-100%, of the antibody
in the composition comprises a mature core carbohydrate structure
which contains sialic acid. In another embodiment, the antibody in
the composition both comprises a mature core carbohydrate structure
that contains sialic acid and additionally comprises at least one
amino acid modification in the Fc region. In another embodiment,
the combination of engineered glycoform and amino acid modification
provides optimal Fc receptor binding properties to the
antibody.
Other Modifications
[0206] Immunoglobulins disclosed herein may comprise one or more
modifications that provide optimized properties that are not
specifically related to Fc.gamma.R- or complement-mediated effector
functions per se. Said modifications may be amino acid
modifications, or may be modifications that are made enzymatically
or chemically. Such modification(s) likely provide some improvement
in the immunoglobulin, for example an enhancement in its stability,
solubility, function, or clinical use. Disclosed herein are a
variety of improvements that may be made by coupling the
immunoglobulins disclosed herein with additional modifications.
[0207] In one embodiment, the variable region of an antibody
disclosed herein may be affinity matured, that is to say that amino
acid modifications have been made in the VH and/or VL domains of
the antibody to enhance binding of the antibody to its target
antigen. Such types of modifications may improve the association
and/or the dissociation kinetics for binding to the target antigen.
Other modifications include those that improve selectivity for
target antigen vs. alternative targets. These include modifications
that improve selectivity for antigen expressed on target vs.
non-target cells. Other improvements to the target recognition
properties may be provided by additional modifications. Such
properties may include, but are not limited to, specific kinetic
properties (i.e. association and dissociation kinetics),
selectivity for the particular target versus alternative targets,
and selectivity for a specific form of target versus alternative
forms. Examples include full-length versus splice variants,
cell-surface vs. soluble forms, selectivity for various polymorphic
variants, or selectivity for specific conformational forms of the
target antigen. Immunoglobulins disclosed herein may comprise one
or more modifications that provide reduced or enhanced
internalization of an immunoglobulin.
[0208] In one embodiment, modifications are made to improve
biophysical properties of the immunoglobulins disclosed herein,
including but not limited to stability, solubility, and oligomeric
state. Modifications can include, for example, substitutions that
provide more favorable intramolecular interactions in the
immunoglobulin such as to provide greater stability, or
substitution of exposed nonpolar amino acids with polar amino acids
for higher solubility. Other modifications to the immunoglobulins
disclosed herein include those that enable the specific formation
or homodimeric or homomultimeric molecules. Such modifications
include but are not limited to engineered disulfides, as well as
chemical modifications or aggregation methods which may provide a
mechanism for generating covalent homodimeric or homomultimers.
Additional modifications to the variants disclosed herein include
those that enable the specific formation or heterodimeric,
heteromultimeric, bifunctional, and/or multifunctional molecules.
Such modifications include, but are not limited to, one or more
amino acid substitutions in the CH3 domain, in which the
substitutions reduce homodimer formation and increase heterodimer
formation. Additional modifications include modifications in the
hinge and CH3 domains, in which the modifications reduce the
propensity to form dimers.
[0209] In further embodiments, the immunoglobulins disclosed herein
comprise modifications that remove proteolytic degradation sites.
These may include, for example, protease sites that reduce
production yields, as well as protease sites that degrade the
administered protein in vivo. In one embodiment, additional
modifications are made to remove covalent degradation sites such as
deamidation (i.e. deamidation of glutaminyl and asparaginyl
residues to the corresponding glutamyl and aspartyl residues),
oxidation, and proteolytic degradation sites. Deamidation sites
that are particular useful to remove are those that have enhance
propensity for deamidation, including, but not limited to
asparaginyl and glutamyl residues followed by glycines (NG and QG
motifs, respectively). In such cases, substitution of either
residue can significantly reduce the tendency for deamidation.
Common oxidation sites include methionine and cysteine residues.
Other covalent modifications, that can either be introduced or
removed, include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the "-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983), incorporated entirely by reference), acetylation of
the N-terminal amine, and amidation of any C-terminal carboxyl
group. Additional modifications also may include but are not
limited to posttranslational modifications such as N-linked or
O-linked glycosylation and phosphorylation.
[0210] Modifications may include those that improve expression
and/or purification yields from hosts or host cells commonly used
for production of biologics. These include, but are not limited to
various mammalian cell lines (e.g. CHO), yeast cell lines,
bacterial cell lines, and plants. Additional modifications include
modifications that remove or reduce the ability of heavy chains to
form inter-chain disulfide linkages. Additional modifications
include modifications that remove or reduce the ability of heavy
chains to form intra-chain disulfide linkages.
[0211] The immunoglobulins disclosed herein may comprise
modifications that include the use of unnatural amino acids
incorporated using, for example, the technologies developed by
Schultz and colleagues, including but not limited to methods
described by Cropp & Shultz, 2004, Trends Genet. 20(12):625-30,
Anderson et al., 2004, Proc. Natl. Acad. Sci. U.S.A.
101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et
al., 2003, Science 301(5635):964-7, all incorporated entirely by
reference. In some embodiments, these modifications enable
manipulation of various functional, biophysical, immunological, or
manufacturing properties discussed above. In additional
embodiments, these modifications enable additional chemical
modification for other purposes. Other modifications are
contemplated herein. For example, the immunoglobulin may be linked
to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes,
or copolymers of polyethylene glycol and polypropylene glycol.
Additional amino acid modifications may be made to enable specific
or non-specific chemical or posttranslational modification of the
immunoglobulins. Such modifications, include, but are not limited
to PEGylation and glycosylation. Specific substitutions that can be
utilized to enable PEGylation include, but are not limited to,
introduction of novel cysteine residues or unnatural amino acids
such that efficient and specific coupling chemistries can be used
to attach a PEG or otherwise polymeric moiety. Introduction of
specific glycosylation sites can be achieved by introducing novel
N-X-T/S sequences into the immunoglobulins disclosed herein.
[0212] Modifications to reduce immunogenicity may include
modifications that reduce binding of processed peptides derived
from the parent sequence to MHC proteins. For example, amino acid
modifications would be engineered such that there are no or a
minimal number of immune epitopes that are predicted to bind, with
high affinity, to any prevalent MHC alleles. Several methods of
identifying MHC-binding epitopes in protein sequences are known in
the art and may be used to score epitopes in an antibody disclosed
herein. See for example U.S. Ser. No. 09/903,378, U.S. Ser. No.
10/754,296, U.S. Ser. No. 11/249,692, and references cited therein,
all expressly incorporated by reference.
[0213] In some embodiments, immunoglobulins disclosed herein may be
combined with immunoglobulins that alter FcRn binding. Such
variants may provide improved pharmacokinetic properties to the
immunoglobulins. Preferred variants that increase binding to FcRn
and/or improve pharmacokinetic properties include but are not
limited to substitutions at positions 259, 308, 428, and 434,
including but not limited to for example 259I, 308F, 428L, 428M,
434S, 434H, 434F, 434Y, and 434M (PCT/US2008/088053, filed Dec. 22,
2008, entitled "Fc Variants with Altered Binding to FcRn", entirely
incorporated by reference). Other variants that increase Fc binding
to FcRn include but are not limited to: 250E, 250Q, 428L, 428F,
250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216,
Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A,
286A, 305A, 307A, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields
et al, Journal of Biological Chemistry, 2001, 276(9):6591-6604,
entirely incorporated by reference), 252F, 252T, 252Y, 252W, 254T,
256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 4331,
433P, 433Q, 434H, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H,
308T/309P/311 S (Dall Acqua et al. Journal of Immunology, 2002,
169:5171-5180, Dall'Acqua et al., 2006, The Journal of biological
chemistry 281:23514-23524, entirely incorporated by reference).
[0214] Covalent modifications of antibodies are included within the
scope of immunoglobulins disclosed herein, and are generally, but
not always, done post-translationally. For example, several types
of covalent modifications of the antibody are introduced into the
molecule by reacting specific amino acid residues of the antibody
with an organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues.
[0215] In some embodiments, the covalent modification of the
antibodies disclosed herein comprises the addition of one or more
labels. The term "labeling group" means any detectable label. In
some embodiments, the labeling group is coupled to the antibody via
spacer arms of various lengths to reduce potential steric
hindrance. Various methods for labeling proteins are known in the
art and may be used in generating immunoglobulins disclosed
herein.
Antibody-Drug Conjugates
[0216] In some embodiments, the multispecific antibodies of the
invention are conjugated with drugs to form antibody-drug
conjugates (ADCs). In general, ADCs are used in oncology
applications, where the use of antibody-drug conjugates for the
local delivery of cytotoxic or cytostatic agents allows for the
targeted delivery of the drug moiety to tumors, which can allow
higher efficacy, lower toxicity, etc. An overview of this
technology is provided in Ducry et al., Bioconjugate Chem., 21:5-13
(2010), Carter et al., Cancer J. 14(3):154 (2008) and Senter,
Current Opin. Chem. Biol. 13:235-244 (2009), all of which are
hereby incorporated by reference in their entirety.
[0217] Thus the invention provides multispecific antibodies
conjugated to drugs. Generally, conjugation is done by covalent
attachment to the antibody, as further described below, and
generally relies on a linker, often a peptide linkage (which, as
described below, may be designed to be sensitive to cleavage by
proteases at the target site or not). In addition, as described
above, linkage of the linker-drug unit (LU-D) can be done by
attachment to cysteines within the antibody. As will be appreciated
by those in the art, the number of drug moieties per antibody can
change, depending on the conditions of the reaction, and can vary
from 1:1 to 10:1 drug:antibody. As will be appreciated by those in
the art, the actual number is an average.
[0218] Thus the invention provides multispecific antibodies
conjugated to drugs. As described below, the drug of the ADC can be
any number of agents, including but not limited to cytotoxic agents
such as chemotherapeutic agents, growth inhibitory agents, toxins
(for example, an enzymatically active toxin of bacterial, fungal,
plant, or animal origin, or fragments thereof), or a radioactive
isotope (that is, a radioconjugate) are provided. In other
embodiments, the invention further provides methods of using the
ADCs.
[0219] Drugs for use in the present invention include cytotoxic
drugs, particularly those which are used for cancer therapy. Such
drugs include, in general, DNA damaging agents, anti-metabolites,
natural products and their analogs. Exemplary classes of cytotoxic
agents include the enzyme inhibitors such as dihydrofolate
reductase inhibitors, and thymidylate synthase inhibitors, DNA
intercalators, DNA cleavers, topoisomerase inhibitors, the
anthracycline family of drugs, the vinca drugs, the mitomycins, the
bleomycins, the cytotoxic nucleosides, the pteridine family of
drugs, diynenes, the podophyllotoxins, dolastatins, maytansinoids,
differentiation inducers, and taxols.
[0220] Members of these classes include, for example, methotrexate,
methopterin, dichloromethotrexate, 5-fluorouracil,
6-mercaptopurine, cytosine arabinoside, melphalan, leurosine,
leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C,
mitomycin A, caminomycin, aminopterin, tallysomycin,
podophyllotoxin and podophyllotoxin derivatives such as etoposide
or etoposide phosphate, vinblastine, vincristine, vindesine,
taxanes including taxol, taxotere retinoic acid, butyric acid,
N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin,
ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,
camptothecin, maytansinoids (including DM1), monomethylauristatin E
(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and
their analogues.
[0221] Toxins may be used as antibody-toxin conjugates and include
bacterial toxins such as diphtheria toxin, plant toxins such as
ricin, small molecule toxins such as geldanamycin (Mandler et al
(2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000)
Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al
(2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213;
Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and
calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al
(1993) Cancer Res. 53:3336-3342). Toxins may exert their cytotoxic
and cytostatic effects by mechanisms including tubulin binding, DNA
binding, or topoisomerase inhibition.
[0222] Conjugates of a multispecific antibody and one or more small
molecule toxins, such as a maytansinoids, dolastatins, auristatins,
a trichothecene, calicheamicin, and CC1065, and the derivatives of
these toxins that have toxin activity, are contemplated.
Maytansinoids
[0223] Maytansine compounds suitable for use as maytansinoid drug
moieties are well known in the art, and can be isolated from
natural sources according to known methods, produced using genetic
engineering techniques (see Yu et al (2002) PNAS 99:7968-7973), or
maytansinol and maytansinol analogues prepared synthetically
according to known methods. As described below, drugs may be
modified by the incorporation of a functionally active group such
as a thiol or amine group for conjugation to the antibody.
[0224] Exemplary maytansinoid drug moieties include those having a
modified aromatic ring, such as: C-19-dechloro (U.S. Pat. No.
4,256,746) (prepared by lithium aluminum hydride reduction of
ansamytocin P2); C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro
(U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation
using Streptomyces or Actinomyces or dechlorination using LAH); and
C-20-demethoxy, C-20-acyloxy (--OCOR), +/-dechloro (U.S. Pat. No.
4,294,757) (prepared by acylation using acyl chlorides) and those
having modifications at other positions
[0225] Exemplary maytansinoid drug moieties also include those
having modifications such as: C-9-SH (U.S. Pat. No. 4,424,219)
(prepared by the reaction of maytansinol with H2S or P2S5);
C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);
C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat.
No. 4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S.
Pat. No. 4,364,866) (prepared by the conversion of maytansinol by
Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and
4,315,929) (isolated from Trewia nudlflora); C-18-N-demethyl (U.S.
Pat. Nos. 4,362,663 and 4,322,348) (prepared by the demethylation
of maytansinol by Streptomyces); and 4,5-deoxy (U.S. Pat. No.
4,371,533) (prepared by the titanium trichloride/LAH reduction of
maytansinol).
[0226] Of particular use are DM1 (disclosed in U.S. Pat. No.
5,208,020, incorporated by reference) and DM4 (disclosed in U.S.
Pat. No. 7,276,497, incorporated by reference). See also a number
of additional maytansinoid derivatives and methods in U.S. Pat. No.
5,416,064, WO/01/24763, 7,303,749, 7,601,354, U.S. Ser. No.
12/631,508, WO02/098883, 6,441,163, 7,368,565, WO02/16368 and
WO04/1033272, all of which are expressly incorporated by reference
in their entirety.
[0227] ADCs containing maytansinoids, methods of making same, and
their therapeutic use are disclosed, for example, in U.S. Pat. Nos.
5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235
B1, the disclosures of which are hereby expressly incorporated by
reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described ADCs comprising a maytansinoid designated DM1
linked to the monoclonal antibody C242 directed against human
colorectal cancer. The conjugate was found to be highly cytotoxic
towards cultured colon cancer cells, and showed antitumor activity
in an in vivo tumor growth assay.
[0228] Chari et al., Cancer Research 52:127-131 (1992) describe
ADCs in which a maytansinoid was conjugated via a disulfide linker
to the murine antibody A7 binding to an antigen on human colon
cancer cell lines, or to another murine monoclonal antibody TA.1
that binds the HER-2/neu oncogene. The cytotoxicity of the
TA.1-maytansonoid conjugate was tested in vitro on the human breast
cancer cell line SK-BR-3, which expresses 3.times.105 HER-2 surface
antigens per cell. The drug conjugate achieved a degree of
cytotoxicity similar to the free maytansinoid drug, which could be
increased by increasing the number of maytansinoid molecules per
antibody molecule. The A7-maytansinoid conjugate showed low
systemic cytotoxicity in mice.
Auristatins and Dolastatins
[0229] In some embodiments, the ADC comprises a multispecific
antibody conjugated to dolastatins or dolostatin peptidic analogs
and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483;
5,780,588). Dolastatins and auristatins have been shown to
interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al (2001) Antimicrob. Agents and
Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.
5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob.
Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug
moiety may be attached to the antibody through the N (amino)
terminus or the C (carboxyl) terminus of the peptidic drug moiety
(WO 02/088172).
[0230] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in
"Senter et al, Proceedings of the American Association for Cancer
Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004
and described in United States Patent Publication No. 2005/0238648,
the disclosure of which is expressly incorporated by reference in
its entirety.
[0231] An exemplary auristatin embodiment is MMAE (see U.S. Pat.
No. 6,884,869 expressly incorporated by reference in its
entirety).
[0232] Another exemplary auristatin embodiment is MMAF (see US
2005/0238649, U.S. Pat. Nos. 5,767,237 and 6,124,431, expressly
incorporated by reference in their entirety).
[0233] Additional exemplary embodiments comprising MMAE or MMAF and
various linker components (described further herein) have the
following structures and abbreviations (wherein Ab means antibody
and p is 1 to about 8):
[0234] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;
Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al
(1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.
Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin
Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol
21(7):778-784.
Calicheamicin
[0235] In other embodiments, the ADC comprises an antibody of the
invention conjugated to one or more calicheamicin molecules. For
example, Mylotarg is the first commercial ADC drug and utilizes
calicheamicin .gamma.1 as the payload (see U.S. Pat. No. 4,970,198,
incorporated by reference in its entirety). Additional
calicheamicin derivatives are described in U.S. Pat. Nos.
5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001, 5,767,285
and 5,877,296, all expressly incorporated by reference. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma.1I, .alpha.2I, .alpha.2I,
N-acetyl-.gamma.1I, PSAG and .theta.I1 (Hinman et al., Cancer
Research 53:3336-3342 (1993), Lode et al., Cancer Research
58:2925-2928 (1998) and the aforementioned U.S. patents to American
Cyanamid). Another anti-tumor drug that the antibody can be
conjugated is QFA which is an antifolate. Both calicheamicin and
QFA have intracellular sites of action and do not readily cross the
plasma membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
Duocarmycins
[0236] CC-1065 (see U.S. Pat. No. 4,169,888, incorporated by
reference) and duocarmycins are members of a family of antitumor
antibiotics utilized in ADCs. These antibiotics appear to work
through sequence-selectively alkylating DNA at the N3 of adenine in
the minor groove, which initiates a cascade of events that result
in apoptosis.
[0237] Important members of the duocarmycins include duocarmycin A
(U.S. Pat. No. 4,923,990, incorporated by reference) and
duocarmycin SA (U.S. Pat. No. 5,101,038, incorporated by
reference), and a large number of analogues as described in U.S.
Pat. Nos. 7,517,903, 7,691,962, 5,101,038; 5,641,780; 5,187,186;
5,070,092; 5,070,092; 5,641,780; 5,101,038; 5,084,468, 5,475,092,
5,585,499, 5,846,545, WO2007/089149, WO2009/017394A1, 5,703,080,
6,989,452, 7,087,600, 7,129,261, 7,498,302, and U.S. Pat. No.
7,507,420, all of which are expressly incorporated by
reference.
Other Cytotoxic Agents
[0238] Other antitumor agents that can be conjugated to the
antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0239] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0240] The present invention further contemplates an ADC formed
between an antibody and a compound with nucleolytic activity (e.g.,
a ribonuclease or a DNA endonuclease such as a deoxyribonuclease;
DNase).
[0241] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At211, I131, I125, Y90, Re186, Re188,
Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu.
[0242] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as Tc99m or
I123, Re186, Re188 and In111 can be attached via a cysteine residue
in the peptide. Yttrium-90 can be attached via a lysine residue.
The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res.
Commun. 80: 49-57 can be used to incorporate Iodine-123.
"Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press
1989) describes other methods in detail.
[0243] For compositions comprising a plurality of antibodies, the
drug loading is represented by p, the average number of drug
molecules per Antibody. Drug loading may range from 1 to 20 drugs
(D) per Antibody. The average number of drugs per antibody in
preparation of conjugation reactions may be characterized by
conventional means such as mass spectroscopy, ELISA assay, and
HPLC. The quantitative distribution of Antibody-Drug-Conjugates in
terms of p may also be determined.
[0244] In some instances, separation, purification, and
characterization of homogeneous Antibody-Drug-conjugates where p is
a certain value from Antibody-Drug-Conjugates with other drug
loadings may be achieved by means such as reverse phase HPLC or
electrophoresis. In exemplary embodiments, p is 2, 3, 4, 5, 6, 7,
or 8 or a fraction thereof.
[0245] The generation of Antibody-drug conjugate compounds can be
accomplished by any technique known to the skilled artisan.
Briefly, the Antibody-drug conjugate compounds can include a
multispecific antibody as the Antibody unit, a drug, and optionally
a linker that joins the drug and the binding agent.
[0246] A number of different reactions are available for covalent
attachment of drugs and/or linkers to binding agents. This is can
be accomplished by reaction of the amino acid residues of the
binding agent, for example, antibody molecule, including the amine
groups of lysine, the free carboxylic acid groups of glutamic and
aspartic acid, the sulfhydryl groups of cysteine and the various
moieties of the aromatic amino acids. A commonly used non-specific
methods of covalent attachment is the carbodiimide reaction to link
a carboxy (or amino) group of a compound to amino (or carboxy)
groups of the antibody. Additionally, bifunctional agents such as
dialdehydes or imidoesters have been used to link the amino group
of a compound to amino groups of an antibody molecule.
[0247] Also available for attachment of drugs to binding agents is
the Schiff base reaction. This method involves the periodate
oxidation of a drug that contains glycol or hydroxy groups, thus
forming an aldehyde which is then reacted with the binding agent.
Attachment occurs via formation of a Schiff base with amino groups
of the binding agent. Isothiocyanates can also be used as coupling
agents for covalently attaching drugs to binding agents. Other
techniques are known to the skilled artisan and within the scope of
the present invention.
[0248] In some embodiments, an intermediate, which is the precursor
of the linker, is reacted with the drug under appropriate
conditions. In other embodiments, reactive groups are used on the
drug and/or the intermediate. The product of the reaction between
the drug and the intermediate, or the derivatized drug, is
subsequently reacted with a multispecific antibody of the invention
under appropriate conditions.
[0249] It will be understood that chemical modifications may also
be made to the desired compound in order to make reactions of that
compound more convenient for purposes of preparing conjugates of
the invention. For example a functional group e.g. amine, hydroxyl,
or sulfhydryl, may be appended to the drug at a position which has
minimal or an acceptable effect on the activity or other properties
of the drug
Linker Units
[0250] Typically, the antibody-drug conjugate compounds comprise a
Linker unit between the drug unit and the antibody unit. In some
embodiments, the linker is cleavable under intracellular or
extracellular conditions, such that cleavage of the linker releases
the drug unit from the antibody in the appropriate environment. For
example, solid tumors that secrete certain proteases may serve as
the target of the cleavable linker; in other embodiments, it is the
intracellular proteases that are utilized. In yet other
embodiments, the linker unit is not cleavable and the drug is
released, for example, by antibody degradation in lysosomes.
[0251] In some embodiments, the linker is cleavable by a cleaving
agent that is present in the intracellular environment (for
example, within a lysosome or endosome or caveolea). The linker can
be, for example, a peptidyl linker that is cleaved by an
intracellular peptidase or protease enzyme, including, but not
limited to, a lysosomal or endosomal protease. In some embodiments,
the peptidyl linker is at least two amino acids long or at least
three amino acids long or more.
[0252] Cleaving agents can include, without limitation, cathepsins
B and D and plasmin, all of which are known to hydrolyze dipeptide
drug derivatives resulting in the release of active drug inside
target cells (see, e.g., Dubowchik and Walker, 1999, Pharm.
Therapeutics 83:67-123). Peptidyl linkers that are cleavable by
enzymes that are present in CD38-expressing cells. For example, a
peptidyl linker that is cleavable by the thiol-dependent protease
cathepsin-B, which is highly expressed in cancerous tissue, can be
used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker (SEQ ID NO: X)).
Other examples of such linkers are described, e.g., in U.S. Pat.
No. 6,214,345, incorporated herein by reference in its entirety and
for all purposes.
[0253] In some embodiments, the peptidyl linker cleavable by an
intracellular protease is a Val-Cit linker or a Phe-Lys linker
(see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis
of doxorubicin with the val-cit linker).
[0254] In other embodiments, the cleavable linker is pH-sensitive,
that is, sensitive to hydrolysis at certain pH values. Typically,
the pH-sensitive linker hydrolyzable under acidic conditions. For
example, an acid-labile linker that is hydrolyzable in the lysosome
(for example, a hydrazone, semicarbazone, thiosemicarbazone,
cis-aconitic amide, orthoester, acetal, ketal, or the like) may be
used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929;
Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville
et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers are
relatively stable under neutral pH conditions, such as those in the
blood, but are unstable at below pH 5.5 or 5.0, the approximate pH
of the lysosome. In certain embodiments, the hydrolyzable linker is
a thioether linker (such as, e.g., a thioether attached to the
therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat.
No. 5,622,929).
[0255] In yet other embodiments, the linker is cleavable under
reducing conditions (for example, a disulfide linker). A variety of
disulfide linkers are known in the art, including, for example,
those that can be formed using SATA
(N-succinimidyl-5-acetylthioacetate), SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB
(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT
(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-
-, SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.
47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody
Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,
Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)
[0256] In other embodiments, the linker is a malonate linker
(Johnson et al., 1995, Anticancer Res. 15:1387-93), a
maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem.
3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995,
Bioorg-Med-Chem. 3(10):1305-12).
[0257] In yet other embodiments, the linker unit is not cleavable
and the drug is released by antibody degradation. (See U.S.
Publication No. 2005/0238649 incorporated by reference herein in
its entirety and for all purposes).
[0258] In many embodiments, the linker is self-immolative. As used
herein, the term "self-immolative Spacer" refers to a bifunctional
chemical moiety that is capable of covalently linking together two
spaced chemical moieties into a stable tripartite molecule. It will
spontaneously separate from the second chemical moiety if its bond
to the first moiety is cleaved. See for example, WO 2007059404A2,
WO06110476A2, WO005112919A2, WO2010/062171, WO09/017394,
WO07/089149, WO 07/018431, WO04/043493 and WO02/083180, which are
directed to drug-cleavable substrate conjugates where the drug and
cleavable substrate are optionally linked through a self-immolative
linker and which are all expressly incorporated by reference.
[0259] Often the linker is not substantially sensitive to the
extracellular environment. As used herein, "not substantially
sensitive to the extracellular environment," in the context of a
linker, means that no more than about 20%, 15%, 10%, 5%, 3%, or no
more than about 1% of the linkers, in a sample of antibody-drug
conjugate compound, are cleaved when the antibody-drug conjugate
compound presents in an extracellular environment (for example, in
plasma).
[0260] Whether a linker is not substantially sensitive to the
extracellular environment can be determined, for example, by
incubating with plasma the antibody-drug conjugate compound for a
predetermined time period (for example, 2, 4, 8, 16, or 24 hours)
and then quantitating the amount of free drug present in the
plasma.
[0261] In other, non-mutually exclusive embodiments, the linker
promotes cellular internalization. In certain embodiments, the
linker promotes cellular internalization when conjugated to the
therapeutic agent (that is, in the milieu of the linker-therapeutic
agent moiety of the antibody-drug conjugate compound as described
herein). In yet other embodiments, the linker promotes cellular
internalization when conjugated to both the auristatin compound and
the multispecific antibodies of the invention.
[0262] A variety of exemplary linkers that can be used with the
present compositions and methods are described in WO 2004-010957,
U.S. Publication No. 2006/0074008, U.S. Publication No.
20050238649, and U.S. Publication No. 2006/0024317 (each of which
is incorporated by reference herein in its entirety and for all
purposes).
Drug Loading
[0263] Drug loading is represented by p and is the average number
of Drug moieties per antibody in a molecule. Drug loading ("p") may
be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20 or more moieties (D) per antibody, although frequently the
average number is a fraction or a decimal. Generally, drug loading
of from 1 to 4 is frequently useful, and from 1 to 2 is also
useful. ADCs of the invention include collections of antibodies
conjugated with a range of drug moieties, from 1 to 20. The average
number of drug moieties per antibody in preparations of ADC from
conjugation reactions may be characterized by conventional means
such as mass spectroscopy and, ELISA assay.
[0264] The quantitative distribution of ADC in terms of p may also
be determined. In some instances, separation, purification, and
characterization of homogeneous ADC where p is a certain value from
ADC with other drug loadings may be achieved by means such as
electrophoresis.
[0265] For some antibody-drug conjugates, p may be limited by the
number of attachment sites on the antibody. For example, where the
attachment is a cysteine thiol, as in the exemplary embodiments
above, an antibody may have only one or several cysteine thiol
groups, or may have only one or several sufficiently reactive thiol
groups through which a linker may be attached. In certain
embodiments, higher drug loading, e.g. p>5, may cause
aggregation, insolubility, toxicity, or loss of cellular
permeability of certain antibody-drug conjugates. In certain
embodiments, the drug loading for an ADC of the invention ranges
from 1 to about 8; from about 2 to about 6; from about 3 to about
5; from about 3 to about 4; from about 3.1 to about 3.9; from about
3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to
about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about
3.7. Indeed, it has been shown that for certain ADCs, the optimal
ratio of drug moieties per antibody may be less than 8, and may be
about 2 to about 5. See US 2005-0238649 A1 (herein incorporated by
reference in its entirety).
[0266] In certain embodiments, fewer than the theoretical maximum
of drug moieties are conjugated to an antibody during a conjugation
reaction. An antibody may contain, for example, lysine residues
that do not react with the drug-linker intermediate or linker
reagent, as discussed below. Generally, antibodies do not contain
many free and reactive cysteine thiol groups which may be linked to
a drug moiety; indeed most cysteine thiol residues in antibodies
exist as disulfide bridges. In certain embodiments, an antibody may
be reduced with a reducing agent such as dithiothreitol (DTT) or
tricarbonylethylphosphine (TCEP), under partial or total reducing
conditions, to generate reactive cysteine thiol groups. In certain
embodiments, an antibody is subjected to denaturing conditions to
reveal reactive nucleophilic groups such as lysine or cysteine.
[0267] The loading (drug/antibody ratio) of an ADC may be
controlled in different ways, e.g., by: (i) limiting the molar
excess of drug-linker intermediate or linker reagent relative to
antibody, (ii) limiting the conjugation reaction time or
temperature, (iii) partial or limiting reductive conditions for
cysteine thiol modification, (iv) engineering by recombinant
techniques the amino acid sequence of the antibody such that the
number and position of cysteine residues is modified for control of
the number and/or position of linker-drug attachments (such as
thioMab or thioFab prepared as disclosed herein and in
WO2006/034488 (herein incorporated by reference in its
entirety)).
[0268] It is to be understood that where more than one nucleophilic
group reacts with a drug-linker intermediate or linker reagent
followed by drug moiety reagent, then the resulting product is a
mixture of ADC compounds with a distribution of one or more drug
moieties attached to an antibody. The average number of drugs per
antibody may be calculated from the mixture by a dual ELISA
antibody assay, which is specific for antibody and specific for the
drug. Individual ADC molecules may be identified in the mixture by
mass spectroscopy and separated by HPLC, e.g. hydrophobic
interaction chromatography.
[0269] In some embodiments, a homogeneous ADC with a single loading
value may be isolated from the conjugation mixture by
electrophoresis or chromatography.
Methods of Determining Cytotoxic Effect of ADCs
[0270] Methods of determining whether a Drug or Antibody-Drug
conjugate exerts a cytostatic and/or cytotoxic effect on a cell are
known. Generally, the cytotoxic or cytostatic activity of an
Antibody Drug conjugate can be measured by: exposing mammalian
cells expressing a target protein of the Antibody Drug conjugate in
a cell culture medium; culturing the cells for a period from about
6 hours to about 5 days; and measuring cell viability. Cell-based
in vitro assays can be used to measure viability (proliferation),
cytotoxicity, and induction of apoptosis (caspase activation) of
the Antibody Drug conjugate.
[0271] For determining whether an Antibody Drug conjugate exerts a
cytostatic effect, a thymidine incorporation assay may be used. For
example, cancer cells expressing a target antigen at a density of
5,000 cells/well of a 96-well plated can be cultured for a 72-hour
period and exposed to 0.5 .mu.Ci of 3H-thymidine during the final 8
hours of the 72-hour period. The incorporation of 3H-thymidine into
cells of the culture is measured in the presence and absence of the
Antibody Drug conjugate.
[0272] For determining cytotoxicity, necrosis or apoptosis
(programmed cell death) can be measured. Necrosis is typically
accompanied by increased permeability of the plasma membrane;
swelling of the cell, and rupture of the plasma membrane. Apoptosis
is typically characterized by membrane blebbing, condensation of
cytoplasm, and the activation of endogenous endonucleases.
Determination of any of these effects on cancer cells indicates
that an Antibody Drug conjugate is useful in the treatment of
cancers.
[0273] Cell viability can be measured by determining in a cell the
uptake of a dye such as neutral red, trypan blue, or ALAMAR.TM.
blue (see, e.g., Page et al., 1993, Intl. J. Oncology 3:473-476).
In such an assay, the cells are incubated in media containing the
dye, the cells are washed, and the remaining dye, reflecting
cellular uptake of the dye, is measured spectrophotometrically. The
protein-binding dye sulforhodamine B (SRB) can also be used to
measure cytoxicity (Skehan et al., 1990, J. Natl. Cancer Inst.
82:1107-12).
[0274] Alternatively, a tetrazolium salt, such as MTT, is used in a
quantitative colorimetric assay for mammalian cell survival and
proliferation by detecting living, but not dead, cells (see, e.g.,
Mosmann, 1983, J. Immunol. Methods 65:55-63).
[0275] Apoptosis can be quantitated by measuring, for example, DNA
fragmentation. Commercial photometric methods for the quantitative
in vitro determination of DNA fragmentation are available. Examples
of such assays, including TUNEL (which detects incorporation of
labeled nucleotides in fragmented DNA) and ELISA-based assays, are
described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular
Biochemicals).
[0276] Apoptosis can also be determined by measuring morphological
changes in a cell. For example, as with necrosis, loss of plasma
membrane integrity can be determined by measuring uptake of certain
dyes (e.g., a fluorescent dye such as, for example, acridine orange
or ethidium bromide). A method for measuring apoptotic cell number
has been described by Duke and Cohen, Current Protocols in
Immunology (Coligan et al. eds., 1992, pp. 3.17.1-3.17.16). Cells
also can be labeled with a DNA dye (e.g., acridine orange, ethidium
bromide, or propidium iodide) and the cells observed for chromatin
condensation and margination along the inner nuclear membrane.
Other morphological changes that can be measured to determine
apoptosis include, e.g., cytoplasmic condensation, increased
membrane blebbing, and cellular shrinkage.
[0277] The presence of apoptotic cells can be measured in both the
attached and "floating" compartments of the cultures. For example,
both compartments can be collected by removing the supernatant,
trypsinizing the attached cells, combining the preparations
following a centrifugation wash step (e.g., 10 minutes at 2000
rpm), and detecting apoptosis (e.g., by measuring DNA
fragmentation). (See, e.g., Piazza et al., 1995, Cancer Research
55:3110-16).
[0278] In vivo, the effect of a therapeutic composition of the
multispecific antibody of the invention can be evaluated in a
suitable animal model. For example, xenogenic cancer models can be
used, wherein cancer explants or passaged xenograft tissues are
introduced into immune compromised animals, such as nude or SCID
mice (Klein et al., 1997, Nature Medicine 3: 402-408). Efficacy can
be measured using assays that measure inhibition of tumor
formation, tumor regression or metastasis, and the like.
[0279] The therapeutic compositions used in the practice of the
foregoing methods can be formulated into pharmaceutical
compositions comprising a carrier suitable for the desired delivery
method. Suitable carriers include any material that when combined
with the therapeutic composition retains the anti-tumor function of
the therapeutic composition and is generally non-reactive with the
patient's immune system. Examples include, but are not limited to,
any of a number of standard pharmaceutical carriers such as sterile
phosphate buffered saline solutions, bacteriostatic water, and the
like (see, generally, Remington's Pharmaceutical Sciences 16th
Edition, A. Osal., Ed., 1980).
Conjugates
[0280] In one embodiment, the molecules disclosed herein are
antibody "fusion proteins", sometimes referred to herein as
"antibody conjugates". The fusion partner or conjugate partner can
be proteinaceous or non-proteinaceous; the latter generally being
generated using functional groups on the antibody and on the
conjugate partner. Conjugate and fusion partners may be any
molecule, including small molecule chemical compounds and
polypeptides. For example, a variety of antibody conjugates and
methods are described in Trail et al., 1999, Curr. Opin. Immunol.
11:584-588, incorporated entirely by reference. Possible conjugate
partners include but are not limited to cytokines, cytotoxic
agents, toxins, radioisotopes, chemotherapeutic agent,
anti-angiogenic agents, a tyrosine kinase inhibitors, and other
therapeutically active agents. In some embodiments, conjugate
partners may be thought of more as payloads, that is to say that
the goal of a conjugate is targeted delivery of the conjugate
partner to a targeted cell, for example a cancer cell or immune
cell, by the immunoglobulin. Thus, for example, the conjugation of
a toxin to an immunoglobulin targets the delivery of said toxin to
cells expressing the target antigen. As will be appreciated by one
skilled in the art, in reality the concepts and definitions of
fusion and conjugate are overlapping. The designation of a fusion
or conjugate is not meant to constrain it to any particular
embodiment disclosed herein. Rather, these terms are used loosely
to convey the broad concept that any immunoglobulin disclosed
herein may be linked genetically, chemically, or otherwise, to one
or more polypeptides or molecules to provide some desirable
property.
[0281] Suitable conjugates include, but are not limited to, labels
as described below, drugs and cytotoxic agents including, but not
limited to, cytotoxic drugs (e.g., chemotherapeutic agents) or
toxins or active fragments of such toxins. Suitable toxins and
their corresponding fragments include diptheria A chain, exotoxin A
chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin,
enomycin and the like. Cytotoxic agents also include radiochemicals
made by conjugating radioisotopes to antibodies, or binding of a
radionuclide to a chelating agent that has been covalently attached
to the antibody. Additional embodiments utilize calicheamicin,
auristatins, geldanamycin, maytansine, and duocarmycins and
analogs.
[0282] In one embodiment, the molecules disclosed herein are fused
or conjugated to a cytokine. By "cytokine" as used herein is meant
a generic term for proteins released by one cell population that
act on another cell as intercellular mediators. For example, as
described in Penichet et al., 2001, J. Immunol. Methods 248:91-101,
incorporated entirely by reference, cytokines may be fused to
antibody to provide an array of desirable properties. Examples of
such cytokines are lymphokines, monokines, and traditional
polypeptide hormones. Included among the cytokines are growth
hormone such as human growth hormone, N-methionyl human growth
hormone, and bovine growth hormone; parathyroid hormone; thyroxine;
insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones
such as follicle stimulating hormone (FSH), thyroid stimulating
hormone (TSH), and luteinizing hormone (LH); hepatic growth factor;
fibroblast growth factor; prolactin; placental lactogen; tumor
necrosis factor-alpha and -beta; mullerian-inhibiting substance;
mouse gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-beta; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;
insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-alpha, beta,
and -gamma; colony stimulating factors (CSFs) such as
macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and
granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1,
IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or
TNF-beta; C5a; and other polypeptide factors including LIF and kit
ligand (KL). As used herein, the term cytokine includes proteins
from natural sources or from recombinant cell culture, and
biologically active equivalents of the native sequence
cytokines.
[0283] In yet another embodiment, an molecules disclosed herein may
be conjugated to a "receptor" (such streptavidin) for utilization
in tumor pretargeting wherein the immunoglobulin-receptor conjugate
is administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g. a radionucleotide). In an alternate
embodiment, the immunoglobulin is conjugated or operably linked to
an enzyme in order to employ Antibody Dependent Enzyme Mediated
Prodrug Therapy (ADEPT). ADEPT may be used by conjugating or
operably linking the immunoglobulin to a prodrug-activating enzyme
that converts a prodrug (e.g. a peptidyl chemotherapeutic
agent.
[0284] When immunoglobulin partners are used as conjugates,
conjugate partners may be linked to any region of an immunoglobulin
disclosed herein, including at the N- or C-termini, or at some
residue in-between the termini. A variety of linkers may find use
in immunoglobulins disclosed herein to covalently link conjugate
partners to an immunoglobulin. By "linker", "linker sequence",
"spacer", "tethering sequence" or grammatical equivalents thereof,
herein is meant a molecule or group of molecules (such as a monomer
or polymer) that connects two molecules and often serves to place
the two molecules in one configuration. Linkers are known in the
art; for example, homo- or hetero-bifunctional linkers as are well
known (see, 1994 Pierce Chemical Company catalog, technical section
on cross-linkers, pages 155-200, incorporated entirely by
reference). A number of strategies may be used to covalently link
molecules together. These include, but are not limited to
polypeptide linkages between N- and C-termini of proteins or
protein domains, linkage via disulfide bonds, and linkage via
chemical cross-linking reagents. In one aspect of this embodiment,
the linker is a peptide bond, generated by recombinant techniques
or peptide synthesis. The linker peptide may predominantly include
the following amino acid residues: Gly, Ser, Ala, or Thr. The
linker peptide should have a length that is adequate to link two
molecules in such a way that they assume the correct conformation
relative to one another so that they retain the desired activity.
Suitable lengths for this purpose include at least one and not more
than 50 amino acid residues. In one embodiment, the linker is from
about 1 to 30 amino acids in length, e.g., a linker may be 1 to 20
amino acids in length. Useful linkers include glycine-serine
polymers (including, for example, (GS)n, (GSGGS)n (Set forth as SEQ
ID NO:1), (GGGGS)n (Set forth as SEQ ID NO:2) and (GGGS)n (Set
forth as SEQ ID NO:3), where n is an integer of at least one),
glycine-alanine polymers, alanine-serine polymers, and other
flexible linkers, as will be appreciated by those in the art.
Alternatively, a variety of nonproteinaceous polymers, including
but not limited to polyethylene glycol (PEG), polypropylene glycol,
polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol, may find use as linkers.
Production
[0285] Also disclosed herein are methods for producing and
experimentally testing the antibodies used in the methods described
herein. The disclosed methods are not meant to constrain
embodiments to any particular application or theory of operation.
Rather, the provided methods are meant to illustrate generally that
one or more immunoglobulins may be produced and experimentally
tested to obtain immunoglobulins. General methods for antibody
molecular biology, expression, purification, and screening are
described in Antibody Engineering, edited by Duebel &
Kontermann, Springer-Verlag, Heidelberg, 2001; and Hayhurst &
Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard &
Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; Antibodies: A
Laboratory Manual by Harlow & Lane, New York: Cold Spring
Harbor Laboratory Press, 1988, all incorporated entirely by
reference.
[0286] In one embodiment disclosed herein, nucleic acids are
created that encode the molecules, and that may then be cloned into
host cells, expressed and assayed, if desired. Thus, nucleic acids,
and particularly DNA, may be made that encode each protein
sequence. These practices are carried out using well-known
procedures. For example, a variety of methods that may find use in
generating immunoglobulins disclosed herein are described in
Molecular Cloning--A Laboratory Manual, 3rd Ed. (Maniatis, Cold
Spring Harbor Laboratory Press, New York, 2001), and Current
Protocols in Molecular Biology (John Wiley & Sons), both
incorporated entirely by reference. As will be appreciated by those
skilled in the art, the generation of exact sequences for a library
comprising a large number of sequences is potentially expensive and
time consuming. By "library" herein is meant a set of variants in
any form, including but not limited to a list of nucleic acid or
amino acid sequences, a list of nucleic acid or amino acid
substitutions at variable positions, a physical library comprising
nucleic acids that encode the library sequences, or a physical
library comprising the variant proteins, either in purified or
unpurified form. Accordingly, there are a variety of techniques
that may be used to efficiently generate libraries disclosed
herein. Such methods include but are not limited to gene assembly
methods, PCR-based method and methods which use variations of PCR,
ligase chain reaction-based methods, pooled oligo methods such as
those used in synthetic shuffling, error-prone amplification
methods and methods which use oligos with random mutations,
classical site-directed mutagenesis methods, cassette mutagenesis,
and other amplification and gene synthesis methods. As is known in
the art, there are a variety of commercially available kits and
methods for gene assembly, mutagenesis, vector subcloning, and the
like, and such commercial products find use in for generating
nucleic acids that encode immunoglobulins.
[0287] The molecules disclosed herein may be produced by culturing
a host cell transformed with nucleic acid, e.g., an expression
vector, containing nucleic acid encoding the molecules, under the
appropriate conditions to induce or cause expression of the
protein. The conditions appropriate for expression will vary with
the choice of the expression vector and the host cell, and will be
easily ascertained by one skilled in the art through routine
experimentation. A wide variety of appropriate host cells may be
used, including but not limited to mammalian cells, bacteria,
insect cells, and yeast. For example, a variety of cell lines that
may find use in generating immunoglobulins disclosed herein are
described in the ATCC.RTM. cell line catalog, available from the
American Type Culture Collection.
[0288] In one embodiment, the molecules are expressed in mammalian
expression systems, including systems in which the expression
constructs are introduced into the mammalian cells using virus such
as retrovirus or adenovirus. Any mammalian cells may be used, e.g.,
human, mouse, rat, hamster, and primate cells. Suitable cells also
include known research cells, including but not limited to Jurkat T
cells, NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2/0, NSO
cells and variants thereof. In an alternate embodiment, library
proteins are expressed in bacterial cells. Bacterial expression
systems are well known in the art, and include Escherichia coli (E.
coli), Bacillus subtilis, Streptococcus cremoris, and Streptococcus
lividans. In alternate embodiments, immunoglobulins are produced in
insect cells (e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast
cells (e.g. S. cerevisiae, Pichia, etc). In an alternate
embodiment, molecules are expressed in vitro using cell free
translation systems. In vitro translation systems derived from both
prokaryotic (e.g. E. coli) and eukaryotic (e.g. wheat germ, rabbit
reticulocytes) cells are available and may be chosen based on the
expression levels and functional properties of the protein of
interest. For example, as appreciated by those skilled in the art,
in vitro translation is required for some display technologies, for
example ribosome display. In addition, the immunoglobulins may be
produced by chemical synthesis methods. Also transgenic expression
systems both animal (e.g. cow, sheep or goat milk, embryonated
hen's eggs, whole insect larvae, etc.) and plant (e.g. corn,
tobacco, duckweed, etc.)
[0289] The nucleic acids that encode the molecules disclosed herein
may be incorporated into an expression vector in order to express
the protein. A variety of expression vectors may be utilized for
protein expression. Expression vectors may comprise
self-replicating extra-chromosomal vectors or vectors which
integrate into a host genome. Expression vectors are constructed to
be compatible with the host cell type. Thus expression vectors
which find use in generating immunoglobulins disclosed herein
include but are not limited to those which enable protein
expression in mammalian cells, bacteria, insect cells, yeast, and
in in vitro systems. As is known in the art, a variety of
expression vectors are available, commercially or otherwise, that
may find use for expressing molecules disclosed herein.
[0290] Expression vectors typically comprise a protein operably
linked with control or regulatory sequences, selectable markers,
any fusion partners, and/or additional elements. By "operably
linked" herein is meant that the nucleic acid is placed into a
functional relationship with another nucleic acid sequence.
Generally, these expression vectors include transcriptional and
translational regulatory nucleic acid operably linked to the
nucleic acid encoding the molecule, and are typically appropriate
to the host cell used to express the protein. In general, the
transcriptional and translational regulatory sequences may include
promoter sequences, ribosomal binding sites, transcriptional start
and stop sequences, translational start and stop sequences, and
enhancer or activator sequences. As is also known in the art,
expression vectors typically contain a selection gene or marker to
allow the selection of transformed host cells containing the
expression vector. Selection genes are well known in the art and
will vary with the host cell used.
[0291] molecules may be operably linked to a fusion partner to
enable targeting of the expressed protein, purification, screening,
display, and the like. Fusion partners may be linked to the
immunoglobulin sequence via a linker sequences. The linker sequence
will generally comprise a small number of amino acids, typically
less than ten, although longer linkers may also be used. Typically,
linker sequences are selected to be flexible and resistant to
degradation. As will be appreciated by those skilled in the art,
any of a wide variety of sequences may be used as linkers. For
example, a common linker sequence comprises the amino acid sequence
GGGGS. A fusion partner may be a targeting or signal sequence that
directs immunoglobulin and any associated fusion partners to a
desired cellular location or to the extracellular media. As is
known in the art, certain signaling sequences may target a protein
to be either secreted into the growth media, or into the
periplasmic space, located between the inner and outer membrane of
the cell. A fusion partner may also be a sequence that encodes a
peptide or protein that enables purification and/or screening. Such
fusion partners include but are not limited to polyhistidine tags
(His-tags) (for example H6 and H10 or other tags for use with
Immobilized Metal Affinity Chromatography (IMAC) systems (e.g. Ni+2
affinity columns)), GST fusions, MBP fusions, Strep-tag, the BSP
biotinylation target sequence of the bacterial enzyme BirA, and
epitope tags which are targeted by antibodies (for example c-myc
tags, flag-tags, and the like). As will be appreciated by those
skilled in the art, such tags may be useful for purification, for
screening, or both. For example, an immunoglobulin may be purified
using a His-tag by immobilizing it to a Ni+2 affinity column, and
then after purification the same His-tag may be used to immobilize
the antibody to a Ni+2 coated plate to perform an ELISA or other
binding assay (as described below). A fusion partner may enable the
use of a selection method to screen immunoglobulins (see below).
Fusion partners that enable a variety of selection methods are
well-known in the art. For example, by fusing the members of an
immunoglobulin library to the gene III protein, phage display can
be employed (Kay et al., Phage display of peptides and proteins: a
laboratory manual, Academic Press, San Diego, Calif., 1996; Lowman
et al., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science
228:1315-1317, incorporated entirely by reference). Fusion partners
may enable immunoglobulins to be labeled. Alternatively, a fusion
partner may bind to a specific sequence on the expression vector,
enabling the fusion partner and associated immunoglobulin to be
linked covalently or noncovalently with the nucleic acid that
encodes them. The methods of introducing exogenous nucleic acid
into host cells are well known in the art, and will vary with the
host cell used. Techniques include but are not limited to
dextran-mediated transfection, calcium phosphate precipitation,
calcium chloride treatment, polybrene mediated transfection,
protoplast fusion, electroporation, viral or phage infection,
encapsulation of the polynucleotide(s) in liposomes, and direct
microinjection of the DNA into nuclei. In the case of mammalian
cells, transfection may be either transient or stable.
[0292] In one embodiment, molecules are purified or isolated after
expression. Proteins may be isolated or purified in a variety of
ways known to those skilled in the art. Standard purification
methods include chromatographic techniques, including ion exchange,
hydrophobic interaction, affinity, sizing or gel filtration, and
reversed-phase, carried out at atmospheric pressure or at high
pressure using systems such as FPLC and HPLC. Purification methods
also include electrophoretic, immunological, precipitation,
dialysis, and chromatofocusing techniques. Ultrafiltration and
diafiltration techniques, in conjunction with protein
concentration, are also useful. As is well known in the art, a
variety of natural proteins bind Fc and antibodies, and these
proteins can find use for purification of immunoglobulins disclosed
herein. For example, the bacterial proteins A and G bind to the Fc
region. Likewise, the bacterial protein L binds to the Fab region
of some antibodies, as of course does the antibody's target
antigen. Purification can often be enabled by a particular fusion
partner. For example, immunoglobulins may be purified using
glutathione resin if a GST fusion is employed, Ni+2 affinity
chromatography if a His-tag is employed, or immobilized anti-flag
antibody if a flag-tag is used. For general guidance in suitable
purification techniques, see, e.g. incorporated entirely by
reference Protein Purification: Principles and Practice, 3rd Ed.,
Scopes, Springer-Verlag, N Y, 1994, incorporated entirely by
reference. The degree of purification necessary will vary depending
on the screen or use of the immunoglobulins. In some instances no
purification is necessary. For example in one embodiment, if the
immunoglobulins are secreted, screening may take place directly
from the media. As is well known in the art, some methods of
selection do not involve purification of proteins. Thus, for
example, if a library of immunoglobulins is made into a phage
display library, protein purification may not be performed.
In Vitro Experimentation
[0293] molecules may be screened using a variety of methods,
including but not limited to those that use in vitro assays, in
vivo and cell-based assays, and selection technologies. Automation
and high-throughput screening technologies may be utilized in the
screening procedures. Screening may employ the use of a fusion
partner or label. The use of fusion partners has been discussed
above. By "labeled" herein is meant that the immunoglobulins
disclosed herein have one or more elements, isotopes, or chemical
compounds attached to enable the detection in a screen. In general,
labels fall into three classes: a) immune labels, which may be an
epitope incorporated as a fusion partner that is recognized by an
antibody, b) isotopic labels, which may be radioactive or heavy
isotopes, and c) small molecule labels, which may include
fluorescent and colorimetric dyes, or molecules such as biotin that
enable other labeling methods. Labels may be incorporated into the
compound at any position and may be incorporated in vitro or in
vivo during protein expression.
[0294] In one embodiment, the functional and/or biophysical
properties of molecules are screened in an in vitro assay. In vitro
assays may allow a broad dynamic range for screening properties of
interest. Properties that may be screened include but are not
limited to stability, solubility, and affinity for Fc ligands, for
example Fc.gamma.Rs. Multiple properties may be screened
simultaneously or individually. Proteins may be purified or
unpurified, depending on the requirements of the assay. In one
embodiment, the screen is a qualitative or quantitative binding
assay for binding of molecules to a protein or nonprotein molecule
that is known or thought to bind the molecule. In one embodiment,
the screen is a binding assay for measuring binding to the target
antigen. In an alternate embodiment, the screen is an assay for
binding of molecules to an Fc ligand, including but are not limited
to the family of Fc.gamma.Rs, the neonatal receptor FcRn, the
complement protein C1q, and the bacterial proteins A and G. Said Fc
ligands may be from any organism. In one embodiment, Fc ligands are
from humans, mice, rats, rabbits, and/or monkeys. Binding assays
can be carried out using a variety of methods known in the art,
including but not limited to FRET (Fluorescence Resonance Energy
Transfer) and BRET (Bioluminescence Resonance Energy
Transfer)-based assays, AlphaScreen.TM. (Amplified Luminescent
Proximity Homogeneous Assay), Scintillation Proximity Assay, ELISA
(Enzyme-Linked Immunosorbent Assay), SPR (Surface Plasmon
Resonance, also known as BIACORE.RTM.), isothermal titration
calorimetry, differential scanning calorimetry, gel
electrophoresis, and chromatography including gel filtration. These
and other methods may take advantage of some fusion partner or
label of the immunoglobulin. Assays may employ a variety of
detection methods including but not limited to chromogenic,
fluorescent, luminescent, or isotopic labels.
[0295] The biophysical properties of molecules, for example
stability and solubility, may be tested using a variety of methods
known in the art. Protein stability may be determined by measuring
the thermodynamic equilibrium between folded and unfolded states.
For example, molecules disclosed herein may be unfolded using
chemical denaturant, heat, or pH, and this transition may be
monitored using methods including but not limited to circular
dichroism spectroscopy, fluorescence spectroscopy, absorbance
spectroscopy, NMR spectroscopy, calorimetry, and proteolysis. As
will be appreciated by those skilled in the art, the kinetic
parameters of the folding and unfolding transitions may also be
monitored using these and other techniques. The solubility and
overall structural integrity of an molecule may be quantitatively
or qualitatively determined using a wide range of methods that are
known in the art. Methods which may find use for characterizing the
biophysical properties of molecules disclosed herein include gel
electrophoresis, isoelectric focusing, capillary electrophoresis,
chromatography such as size exclusion chromatography, ion-exchange
chromatography, and reversed-phase high performance liquid
chromatography, peptide mapping, oligosaccharide mapping, mass
spectrometry, ultraviolet absorbance spectroscopy, fluorescence
spectroscopy, circular dichroism spectroscopy, isothermal titration
calorimetry, differential scanning calorimetry, analytical
ultra-centrifugation, dynamic light scattering, proteolysis, and
cross-linking, turbidity measurement, filter retardation assays,
immunological assays, fluorescent dye binding assays,
protein-staining assays, microscopy, and detection of aggregates
via ELISA or other binding assay. Structural analysis employing
X-ray crystallographic techniques and NMR spectroscopy may also
find use. In one embodiment, stability and/or solubility may be
measured by determining the amount of protein solution after some
defined period of time. In this assay, the protein may or may not
be exposed to some extreme condition, for example elevated
temperature, low pH, or the presence of denaturant. Because
function typically requires a stable, soluble, and/or
well-folded/structured protein, the aforementioned functional and
binding assays also provide ways to perform such a measurement. For
example, a solution comprising an immunoglobulin could be assayed
for its ability to bind target antigen, then exposed to elevated
temperature for one or more defined periods of time, then assayed
for antigen binding again. Because unfolded and aggregated protein
is not expected to be capable of binding antigen, the amount of
activity remaining provides a measure of the molecule's stability
and solubility.
[0296] In one embodiment, molecules may be tested using one or more
cell-based or in vitro assays. For such assays, immunoglobulins,
purified or unpurified, are typically added exogenously such that
cells are exposed to individual variants or groups of variants
belonging to a library. These assays are typically, but not always,
based on the biology of the ability of the immunoglobulin to bind
to the target antigen and mediate some biochemical event, for
example effector functions like cellular lysis, phagocytosis,
ligand/receptor binding inhibition, inhibition of growth and/or
proliferation, apoptosisand the like. Such assays often involve
monitoring the response of cells to immunoglobulin, for example
cell survival, cell death, cellular phagocytosis, cell lysis,
change in cellular morphology, or transcriptional activation such
as cellular expression of a natural gene or reporter gene. For
example, such assays may measure the ability of molecules to elicit
ADCC, ADCP, or CDC. For some assays additional cells or components,
that is in addition to the target cells, may need to be added, for
example serum complement, or effector cells such as peripheral
blood monocytes (PBMCs), NK cells, macrophages, and the like. Such
additional cells may be from any organism, e.g., humans, mice, rat,
rabbit, and monkey. Crosslinked or monomeric antibodies may cause
apoptosis of certain cell lines expressing the antibody's target
antigen, or they may mediate attack on target cells by immune cells
which have been added to the assay. Methods for monitoring cell
death or viability are known in the art, and include the use of
dyes, fluorophores, immunochemical, cytochemical, and radioactive
reagents. For example, caspase assays or annexin-flourconjugates
may enable apoptosis to be measured, and uptake or release of
radioactive substrates (e.g. Chromium-51 release assays) or the
metabolic reduction of fluorescent dyes such as alamar blue may
enable cell growth, proliferation or activation to be monitored. In
one embodiment, the DELFIA.RTM. EuTDA-based cytotoxicity assay
(Perkin Elmer, MA) is used. Alternatively, dead or damaged target
cells may be monitored by measuring the release of one or more
natural intracellular proteins, for example lactate dehydrogenase.
Transcriptional activation may also serve as a method for assaying
function in cell-based assays. In this case, response may be
monitored by assaying for natural genes or proteins which may be
upregulated or down-regulated, for example the release of certain
interleukins may be measured, or alternatively readout may be via a
luciferase or GFP-reporter construct. Cell-based assays may also
involve the measure of morphological changes of cells as a response
to the presence of an immunoglobulin. Cell types for such assays
may be prokaryotic or eukaryotic, and a variety of cell lines that
are known in the art may be employed. Alternatively, cell-based
screens are performed using cells that have been transformed or
transfected with nucleic acids encoding the molecules.
[0297] In vitro assays include but are not limited to binding
assays, ADCC, CDC, cytotoxicity, proliferation, peroxide/ozone
release, chemotaxis of effector cells, inhibition of such assays by
reduced effector function antibodies; ranges of activities such as
>100.times. improvement or >100.times. reduction, blends of
receptor activation and the assay outcomes that are expected from
such receptor profiles.
In Vivo Experimentation
[0298] The biological properties of the molecules disclosed herein
may be characterized in cell, tissue, and whole organism
experiments. As is known in the art, drugs are often tested in
animals, including but not limited to mice, rats, rabbits, dogs,
cats, pigs, and monkeys, in order to measure a drug's efficacy for
treatment against a disease or disease model, or to measure a
drug's pharmacokinetics, toxicity, and other properties. Said
animals may be referred to as disease models. With respect to the
molecules disclosed herein, a particular challenge arises when
using animal models to evaluate the potential for in-human efficacy
of candidate polypeptides--this is due, at least in part, to the
fact that molecules that have a specific effect on the affinity for
a human Fc receptor may not have a similar affinity effect with the
orthologous animal receptor. These problems can be further
exacerbated by the inevitable ambiguities associated with correct
assignment of true orthologues (Mechetina et al., Immunogenetics,
2002 54:463-468, incorporated entirely by reference), and the fact
that some orthologues simply do not exist in the animal (e.g.
humans possess an Fc.gamma.RIIa whereas mice do not). Therapeutics
are often tested in mice, including but not limited to mouse
strains NZB, NOD, BXSB, MRL/Ipr, K/BxN and transgenics (including
knockins and knockouts). Such mice can develop various autoimmune
conditions that resemble human organ specific, systemic autoimmune
or inflammatory disease pathologies such as systemic lupus
erythematosus (SLE) and rheumatoid arthritis (RA). For example, an
immunoglobulin disclosed herein intended for autoimmune diseases
may be tested in such mouse models by treating the mice to
determine the ability of the immunoglobulin to reduce or inhibit
the development of the disease pathology. Because of the
incompatibility between the mouse and human Fc.gamma. receptor
system, an alternative approach is to use a murine SCID model in
which immune deficient mice are engrafted with human PBLs or PBMCs
(huPBL-SCID, huPBMC-SCID) providing a semi-functional human immune
system with human effector cells and Fc receptors. In such a model,
an antigen challenge (such as tetanus toxoid) activates the B cells
to differentiate into plasma cells and secrete immunoglobulins,
thus reconstituting antigen specific humoral immunity. Therefore, a
dual targeting immunoglobulin disclosed herein that specifically
binds to IgE and Fc.gamma.RIIb on B cells may be tested to examine
the ability to specifically inhibit B cell differentiation. Such
experimentation may provide meaningful data for determination of
the potential of said immunoglobulin to be used as a therapeutic.
Other organisms, e.g., mammals, may also be used for testing. For
example, because of their genetic similarity to humans, monkeys can
be suitable therapeutic models, and thus may be used to test the
efficacy, toxicity, pharmacokinetics, or other property of the
immunoglobulins disclosed herein. Tests of the immunoglobulins
disclosed herein in humans are ultimately required for approval as
drugs, and thus of course these experiments are contemplated. Thus
the immunoglobulins disclosed herein may be tested in humans to
determine their therapeutic efficacy, toxicity, pharmacokinetics,
and/or other clinical properties.
[0299] The molecules disclosed herein may confer superior
performance on Fc-containing therapeutics in animal models or in
humans. The receptor binding profiles of such immunoglobulins, as
described in this specification, may, for example, be selected to
increase the potency of cytotoxic drugs or to target specific
effector functions or effector cells to improve the selectivity of
the drug's action. Further, receptor binding profiles can be
selected that may reduce some or all effector functions thereby
reducing the side-effects or toxicity of such Fc-containing drug.
For example, an immunoglobulin with reduced binding to
Fc.gamma.RIIIa, Fc.gamma.RI and Fc.gamma.RIIa can be selected to
eliminate most cell-mediated effector function, or an
immunoglobulin with reduced binding to C1q may be selected to limit
complement-mediated effector functions. In some contexts, such
effector functions are known to have potential toxic effects.
Therefore eliminating them may increase the safety of the
Fc-bearing drug and such improved safety may be characterized in
animal models. In some contexts, such effector functions are known
to mediate the desirable therapeutic activity. Therefore enhancing
them may increase the activity or potency of the Fc-bearing drug
and such improved activity or potency may be characterized in
animal models.
[0300] In some embodiments, molecules disclosed herein may be
assessed for efficacy in clinically relevant animal models of
various human diseases. In many cases, relevant models include
various transgenic animals for specific antigens and receptors.
[0301] Relevant transgenic models such as those that express human
Fc receptors (e.g., CD32b) could be used to evaluate and test
immunoglobulins and Fc-fusions in their efficacy. The evaluation of
molecules by the introduction of human genes that directly or
indirectly mediate effector function in mice or other rodents may
enable physiological studies of efficacy in autoimmune disorders
(including SLE, RA and MS). Human Fc receptors such as
Fc.gamma.RIIb may possess polymorphisms such as that in gene
promoter (-343 from G to C) or transmembrane domain of the receptor
187 I or T which would further enable the introduction of specific
and combinations of human polymorphisms into rodents. The various
studies involving polymorphism-specific FcRs is not limited to this
section, however encompasses all discussions and applications of
FcRs in general as specified in throughout this application.
Immunoglobulins disclosed herein may confer superior activity on
Fc-containing drugs in such transgenic models, in particular
variants with binding profiles optimized for human Fc.gamma.RIIb
mediated activity may show superior activity in transgenic CD32b
mice. Similar improvements in efficacy in mice transgenic for the
other human Fc receptors, e.g. Fc.gamma.RIIa, Fc.gamma.RI, etc.,
may be observed for molecules with binding profiles optimized for
the respective receptors. Mice transgenic for multiple human
receptors would show improved activity for immunoglobulins with
binding profiles optimized for the corresponding multiple
receptors.
[0302] Because of the difficulties and ambiguities associated with
using animal models to characterize the potential efficacy of
candidate therapeutic antibodies in a human patient, some variant
polypeptides disclosed herein may find utility as proxies for
assessing potential in-human efficacy. Such proxy molecules may
mimic--in the animal system--the FcR and/or complement biology of a
corresponding candidate human immunoglobulin. This mimicry is most
likely to be manifested by relative association affinities between
specific immunoglobulins and animal vs. human receptors. For
example, if one were using a mouse model to assess the potential
in-human efficacy of an Fc variant that has reduced affinity for
the inhibitory human Fc.gamma.RIIb, an appropriate proxy variant
would have reduced affinity for mouse Fc.gamma.RII. It should also
be noted that the proxy Fc variants could be created in the context
of a human Fc variant, an animal Fc variant, or both.
[0303] In one embodiment, the testing of molecules may include
study of efficacy in primates (e.g. cynomolgus monkey model) to
facilitate the evaluation of depletion of specific target cells
harboring the target antigen. Additional primate models include but
are not limited to use of the rhesus monkey to assess Fc
polypeptides in therapeutic studies of autoimmune, transplantation
and cancer.
[0304] Toxicity studies are performed to determine antibody or
Fc-fusion related-effects that cannot be evaluated in standard
pharmacology profiles, or occur only after repeated administration
of the agent. Most toxicity tests are performed in two species--a
rodent and a non-rodent--to ensure that any unexpected adverse
effects are not overlooked before new therapeutic entities are
introduced into man. In general, these models may measure a variety
of toxicities including genotoxicity, chronic toxicity,
immunogenicity, reproductive/developmental toxicity and
carcinogenicity. Included within the aforementioned parameters are
standard measurement of food consumption, bodyweight, antibody
formation, clinical chemistry, and macro- and microscopic
examination of standard organs/tissues (e.g. cardiotoxicity).
Additional parameters of measurement are injection site trauma and
the measurement of neutralizing antibodies, if any. Traditionally,
monoclonal antibody therapeutics, naked or conjugated, are
evaluated for cross-reactivity with normal tissues,
immunogenicity/antibody production, conjugate or linker toxicity
and "bystander" toxicity of radiolabelled species. Nonetheless,
such studies may have to be individualized to address specific
concerns and following the guidance set by ICH S6 (Safety studies
for biotechnological products, also noted above). As such, the
general principles are that the products are sufficiently well
characterized, impurities/contaminants have been removed, that the
test material is comparable throughout development, that GLP
compliance is maintained.
[0305] The pharmacokinetics (PK) of the molecules disclosed herein
may be studied in a variety of animal systems, with the most
relevant being non-human primates such as the cynomolgus and rhesus
monkeys. Single or repeated i.v./s.c. administrations over a dose
range of 6000-fold (0.05-300 mg/kg) can be evaluated for half-life
(days to weeks) using plasma concentration and clearance. Volume of
distribution at a steady state and level of systemic absorbance can
also be measured. Examples of such parameters of measurement
generally include maximum observed plasma concentration (Cmax), the
time to reach Cmax (Tmax), the area under the plasma
concentration-time curve from time 0 to infinity [AUC(0-inf] and
apparent elimination half-life (T1/2). Additional measured
parameters could include compartmental analysis of
concentration-time data obtained following i.v. administration and
bioavailability.
[0306] The molecules disclosed herein may confer superior
pharmacokinetics on Fc-containing therapeutics in animal systems or
in humans. For example, increased binding to FcRn may increase the
half-life and exposure of the Fc-containing drug. Alternatively,
decreased binding to FcRn may decrease the half-life and exposure
of the Fc-containing drug in cases where reduced exposure is
favorable such as when such drug has side-effects.
[0307] It is known in the art that the array of Fc receptors is
differentially expressed on various immune cell types, as well as
in different tissues. Differential tissue distribution of Fc
receptors may ultimately have an impact on the pharmacodynamic (PD)
and pharmacokinetic (PK) properties of molecules disclosed herein.
Because molecules of the present invention have varying affinities
for the array of Fc receptors, further screening of the
polypeptides for PD and/or PK properties may be extremely useful
for defining the optimal balance of PD, PK, and therapeutic
efficacy conferred by each candidate polypeptide.
[0308] Pharmacodynamic studies may include, but are not limited to,
targeting specific cells or blocking signaling mechanisms,
measuring inhibition of antigen-specific antibodies etc. The
molecules disclosed herein may target particular effector cell
populations and thereby direct Fc-containing drugs to induce
certain activities to improve potency or to increase penetration
into a particularly favorable physiological compartment. For
example, neutrophil activity and localization can be targeted by an
molecule that targets Fc.gamma.RIIIb. Such pharmacodynamic effects
may be demonstrated in animal models or in humans.
Use
[0309] Once made the molecules as described herein find use in a
variety of methods. In a preferred embodiment the method includes
contacting a cell that coexpresses IgE and Fc.gamma.RIIb with a
molecule such that both IgE and Fc.gamma.RIIb are bound by the
molecule and the cell is inhibited. By "inhibited" in this context
is meant that the molecule is preventing or reducing activation
and/or proliferation of IgE+ B cells.
[0310] The molecules disclosed herein may find use in a wide range
of products. In one embodiment a molecule disclosed herein is a
therapeutic, a diagnostic, or a research reagent. The molecules may
find use in a composition that is monoclonal or polyclonal. The
molecules disclosed herein may be used for therapeutic purposes. As
will be appreciated by those in the art, the molecules disclosed
herein may be used for any therapeutic purpose that antibodies, and
the like may be used for. The molecules may be administered to a
patient to treat disorders including but not limited to autoimmune
and inflammatory diseases, infectious diseases, and cancer.
[0311] A "patient" for the purposes disclosed herein includes both
humans and other animals, e.g., other mammals. Thus the molecules
disclosed herein have both human therapy and veterinary
applications. The term "treatment" or "treating" as disclosed
herein is meant to include therapeutic treatment, as well as
prophylactic, or suppressive measures for a disease or disorder.
Thus, for example, successful administration of an molecule prior
to onset of the disease results in treatment of the disease. As
another example, successful administration of an optimized molecule
after clinical manifestation of the disease to combat the symptoms
of the disease comprises treatment of the disease. "Treatment" and
"treating" also encompasses administration of an optimized
immunoglobulin after the appearance of the disease in order to
eradicate the disease. Successful administration of an agent after
onset and after clinical symptoms have developed, with possible
abatement of clinical symptoms and perhaps amelioration of the
disease, comprises treatment of the disease. Those "in need of
treatment" include mammals already having the disease or disorder,
as well as those prone to having the disease or disorder, including
those in which the disease or disorder is to be prevented.
[0312] In one embodiment, a molecule disclosed herein is
administered to a patient having a disease involving inappropriate
expression of a protein or other molecule. Within the scope
disclosed herein this is meant to include diseases and disorders
characterized by aberrant proteins, due for example to alterations
in the amount of a protein present, protein localization,
posttranslational modification, conformational state, the presence
of a mutant or pathogen protein, etc. Similarly, the disease or
disorder may be characterized by alterations molecules including
but not limited to polysaccharides and gangliosides. An
overabundance may be due to any cause, including but not limited to
overexpression at the molecular level, prolonged or accumulated
appearance at the site of action, or increased activity of a
protein relative to normal. Included within this definition are
diseases and disorders characterized by a reduction of a protein.
This reduction may be due to any cause, including but not limited
to reduced expression at the molecular level, shortened or reduced
appearance at the site of action, mutant forms of a protein, or
decreased activity of a protein relative to normal. Such an
overabundance or reduction of a protein can be measured relative to
normal expression, appearance, or activity of a protein, and said
measurement may play an important role in the development and/or
clinical testing of the immunoglobulins disclosed herein.
[0313] Disclosed herein are novel methods of treating IgE-mediated
disorders, e.g., food and environmental allergies and allergic
asthma. In preferred embodiments, allergic diseases that may be
treated by the products and methods of the invention include
allergic and atopic asthma, atopic dermatitis and eczema, allergic
rhinitis, allergic conjunctivitis and rhinoconjunctivitis, allergic
encephalomyelitis, allergic rhinitis, allergic vasculitis, and
anaphylactic shock. Environmental and food allergies that may be
treated include allergies to dustmite, cockroach, cat and other
animals, pollen (including ragweed, Bermuda grass, Russian thistle,
oak, rye, and others), molds and fungi (e.g., Alternaria alternata,
Aspergillus and others), latex, insect stings (bee, wasp, and
others), penicillin and other drugs, strawberries and other fruits
and vegetables, peanuts, soy, and other legumes, walnuts and other
treenuts, shellfish and other seafood, milk and other dairy
products, wheat and other grains, and eggs. Indeed, any food
allergen, aeroallergen, occupational allergen, or other
IgE-mediated environmental allergen may be treated by a therapeutic
amount of the products disclosed in this invention. For examples of
common allergens, see Arbes et al., Prevalences of positive skin
test responses to 10 common allergens in the US population: Results
from the Third National Health and Nutrition Examination Survey,
Clinical Gastroenterology 116(2), 377-383 (2005).
[0314] Also disclosed are diagnostic tests to identify patients who
are likely to show a favorable clinical response to a molecule
disclosed herein, or who are likely to exhibit a significantly
better response when treated with an molecule disclosed herein
versus one or more currently used therapeutics. Any of a number of
methods for determining Fc.gamma.R polymorphisms in humans known in
the art may be used. Furthermore, also disclosed are prognostic
tests performed on clinical samples such as blood and tissue
samples. Such tests may assay for activity, regardless of
mechanism. Such information may be used to identify patients for
inclusion or exclusion in clinical trials, or to inform decisions
regarding appropriate dosages and treatment regemins. Such
information may also be used to select a drug that contains a
particular molecule that shows superior activity in such assay.
Formulation
[0315] Pharmaceutical compositions are contemplated wherein an
molecule disclosed herein and one or more therapeutically active
agents are formulated. Formulations of the molecules disclosed
herein are prepared for storage by mixing said immunoglobulin
having the desired degree of purity with optional pharmaceutically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980,
incorporated entirely by reference), in the form of lyophilized
formulations or aqueous solutions. Acceptable carriers, excipients,
or stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, acetate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl
orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners
and other flavoring agents; fillers such as microcrystalline
cellulose, lactose, corn and other starches; binding agents;
additives; coloring agents; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG). In one embodiment, the pharmaceutical
composition that comprises the immunoglobulin disclosed herein may
be in a water-soluble form, such as being present as
pharmaceutically acceptable salts, which is meant to include both
acid and base addition salts. "Pharmaceutically acceptable acid
addition salt" refers to those salts that retain the biological
effectiveness of the free bases and that are not biologically or
otherwise undesirable, formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid and the like, and organic acids such as acetic
acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like. "Pharmaceutically acceptable base
addition salts" include those derived from inorganic bases such as
sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc, copper, manganese, aluminum salts and the like. Some
embodiments include at least one of the ammonium, potassium,
sodium, calcium, and magnesium salts. Salts derived from
pharmaceutically acceptable organic non-toxic bases include salts
of primary, secondary, and tertiary amines, substituted amines
including naturally occurring substituted amines, cyclic amines and
basic ion exchange resins, such as isopropylamine, trimethylamine,
diethylamine, triethylamine, tripropylamine, and ethanolamine. The
formulations to be used for in vivo administration may be sterile.
This is readily accomplished by filtration through sterile
filtration membranes or other methods.
[0316] The molecules disclosed herein may also be formulated as
immunoliposomes. A liposome is a small vesicle comprising various
types of lipids, phospholipids and/or surfactant that is useful for
delivery of a therapeutic agent to a mammal. Liposomes containing
the immunoglobulin are prepared by methods known in the art. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes. Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0317] The molecule and other therapeutically active agents may
also be entrapped in microcapsules prepared by methods including
but not limited to coacervation techniques, interfacial
polymerization (for example using hydroxymethylcellulose or
gelatin-microcapsules, or poly-(methylmethacylate) microcapsules),
colloidal drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules), and
macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980,
incorporated entirely by reference. Sustained-release preparations
may be prepared. Suitable examples of sustained-release
preparations include semipermeable matrices of solid hydrophobic
polymer, which matrices are in the form of shaped articles, e.g.
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (for example
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides, copolymers of L-glutamic acid and gamma
ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the Lupron
Depot.RTM. (which are injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate),
poly-D-(-)-3-hydroxybutyric acid, and ProLease.RTM. (commercially
available from Alkermes), which is a microsphere-based delivery
system composed of the desired bioactive molecule incorporated into
a matrix of poly-DL-lactide-co-glycolide (PLG).
Administration
[0318] Administration of the pharmaceutical composition comprising
an molecule disclosed herein, e.g., in the form of a sterile
aqueous solution, may be done in a variety of ways, including, but
not limited to orally, subcutaneously, intravenously, intranasally,
intraotically, transdermally, topically (e.g., gels, salves,
lotions, creams, etc.), intraperitoneally, intramuscularly,
intrapulmonary, vaginally, parenterally, rectally, or
intraocularly. In some instances, for example for the treatment of
wounds, inflammation, etc., the immunoglobulin may be directly
applied as a solution or spray. As is known in the art, the
pharmaceutical composition may be formulated accordingly depending
upon the manner of introduction.
[0319] Subcutaneous administration may be used in circumstances
where the patient may self-administer the pharmaceutical
composition. Many protein therapeutics are not sufficiently potent
to allow for formulation of a therapeutically effective dose in the
maximum acceptable volume for subcutaneous administration.
[0320] This problem may be addressed in part by the use of protein
formulations comprising arginine-HCl, histidine, and polysorbate.
Antibodies disclosed herein may be more amenable to subcutaneous
administration due to, for example, increased potency, improved
serum half-life, or enhanced solubility.
[0321] As is known in the art, protein therapeutics are often
delivered by IV infusion or bolus. The antibodies disclosed herein
may also be delivered using such methods. For example,
administration may be by intravenous infusion with 0.9% sodium
chloride as an infusion vehicle.
[0322] Pulmonary delivery may be accomplished using an inhaler or
nebulizer and a formulation comprising an aerosolizing agent. For
example, AERx.RTM. inhalable technology commercially available from
Aradigm, or Inhance.TM. pulmonary delivery system commercially
available from Nektar Therapeutics may be used. Antibodies
disclosed herein may be more amenable to intrapulmonary delivery.
FcRn is present in the lung, and may promote transport from the
lung to the bloodstream (e.g. Syntonix WO 04004798, Bitonti et al.
(2004) Proc. Nat. Acad. Sci. 101:9763-8, both incorporated entirely
by reference). Accordingly, antibodies that bind FcRn more
effectively in the lung or that are released more efficiently in
the bloodstream may have improved bioavailability following
intrapulmonary administration. Antibodies disclosed herein may also
be more amenable to intrapulmonary administration due to, for
example, improved solubility or altered isoelectric point.
[0323] Furthermore, molecules disclosed herein may be more amenable
to oral delivery due to, for example, improved stability at gastric
pH and increased resistance to proteolysis. Furthermore, FcRn
appears to be expressed in the intestinal epithelia of adults, so
antibodies disclosed herein with improved FcRn interaction profiles
may show enhanced bioavailability following oral administration.
FcRn mediated transport of antibodies may also occur at other mucus
membranes such as those in the gastrointestinal, respiratory, and
genital tracts.
[0324] In addition, any of a number of delivery systems are known
in the art and may be used to administer the antibodies disclosed
herein. Examples include, but are not limited to, encapsulation in
liposomes, microparticles, microspheres (e.g., PLA/PGA
microspheres), and the like. Alternatively, an implant of a porous,
non-porous, or gelatinous material, including membranes or fibers,
may be used. Sustained release systems may comprise a polymeric
material or matrix such as polyesters, hydrogels,
poly(vinylalcohol), polylactides, copolymers of L-glutamic acid and
ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolic acid
copolymers such as the Lupron Depot.RTM., and
poly-D-(-)-3-hydroxyburyric acid. It is also possible to administer
a nucleic acid encoding an immunoglobulin disclosed herein, for
example by retroviral infection, direct injection, or coating with
lipids, cell surface receptors, or other transfection agents. In
all cases, controlled release systems may be used to release the
immunoglobulin at or close to the desired location of action.
Dosing
[0325] The dosing amounts and frequencies of administration are, in
one embodiment, selected to be therapeutically or prophylactically
effective. As is known in the art, adjustments for protein
degradation, systemic versus localized delivery, and rate of new
protease synthesis, as well as the age, body weight, general
health, sex, diet, time of administration, drug interaction and the
severity of the condition may be necessary, and will be
ascertainable with routine experimentation by those skilled in the
art.
[0326] The concentration of the therapeutically active molecule in
the formulation may vary from about 0.1 to 100 weight %. In one
embodiment, the concentration of the molecule is in the range of
0.003 to 1.0 molar. In order to treat a patient, a therapeutically
effective dose of the immunoglobulin disclosed herein may be
administered. By "therapeutically effective dose" herein is meant a
dose that produces the effects for which it is administered. The
exact dose will depend on the purpose of the treatment, and will be
ascertainable by one skilled in the art using known techniques.
Dosages may range from 0.0001 to 100 mg/kg of body weight or
greater, for example 0.1, 1, 10, or 50 mg/kg of body weight. In one
embodiment, dosages range from 1 to 10 mg/kg.
[0327] In some embodiments, only a single dose of the molecule is
used. In other embodiments, multiple doses of the molecule are
administered. The elapsed time between administrations may be less
than 1 hour, about 1 hour, about 1-2 hours, about 2-3 hours, about
3-4 hours, about 6 hours, about 12 hours, about 24 hours, about 48
hours, about 2-4 days, about 4-6 days, about 1 week, about 2 weeks,
or more than 2 weeks.
[0328] In other embodiments the molecules disclosed herein are
administered in metronomic dosing regimes, either by continuous
infusion or frequent administration without extended rest periods.
Such metronomic administration may involve dosing at constant
intervals without rest periods. Typically such regimens encompass
chronic low-dose or continuous infusion for an extended period of
time, for example 1-2 days, 1-2 weeks, 1-2 months, or up to 6
months or more. The use of lower doses may minimize side effects
and the need for rest periods.
[0329] In certain embodiments the molecules disclosed herein and
one or more other prophylactic or therapeutic agents are cyclically
administered to the patient. Cycling therapy involves
administration of a first agent at one time, a second agent at a
second time, optionally additional agents at additional times,
optionally a rest period, and then repeating this sequence of
administration one or more times. The number of cycles is typically
from 2-10. Cycling therapy may reduce the development of resistance
to one or more agents, may minimize side effects, or may improve
treatment efficacy.
Combination Therapies
[0330] The molecules disclosed herein may be administered
concomitantly with one or more other therapeutic regimens or
agents. Additional therapeutic regimes or agents may be used to
treat the same disease, to treat an accompanying complication, or
may be used to improve the efficacy or safety of the
immunoglobulin
[0331] Particularly preferred co-therapies include those that are
approved or are being clinically evaluated for the treatment of
IgE-mediated disorders such as allergies and asthma. In particular,
the therapeutic compositions of the invention may be used in
combination with anti-inflammatories such as corticosteroids,
and/or brochodilators such as inhaled 32-agonists, the two major
groups of medications. Inhaled corticosteroids include fluticasone,
budesonide, flunisolide, mometasone, triamcinolone, and
beclomethasone, whereas oral corticosteroids include prednisone,
methylprednisolone, and prednisolone. Other steroids include
glucocorticoids, dexamethasone, cortisone, hydroxycortisone,
azulfidineicosanoids such as prostaglandins, thromboxanes, and
leukotrienes, as well as topical steroids such as anthralin,
calcipotriene, clobetasol, and tazarotene. Bronchodilators increase
the diameter of the air passages and ease the flow to and from the
lungs. Brochodilators that may be combined with the therapies of
the invention include short-acting bronchodilators such as
metaproterenol, ephedrine, terbutaline, and albuterol, and
long-acting bronchodilators such as salmeterol, metaproterenol, and
theophylline.
[0332] The therapies of the invention may be combined with
non-steroidal anti-inflammatory drugs (NSAIDs) such as asprin,
ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen,
indomethacin, ketoralac, oxaprozin, nabumentone, sulindac,
tolmentin, rofecoxib, naproxen, ketoprofen, and nabumetone.
Co-therapies may include antihistamines such as loratadine,
fexofenadine, cetirizine, diphenhydramine, chlorpheniramine
maleate, clemastine, and azelastine. Co-therapy may include
cromoglycate, cromolyn sodium, and nedrocromil, as well as
decongestants, spray or oral, such as oxymetazoline, phenylephrine,
and pseudoephedrine. The therapies of the invention may be combined
with a class of anti-inflammatories called leukotriene-receptor
antagonists such as pranlukast, zafirlukast, and montelukast, and
leukotriene-receptor synthesis-inhibitors such as zileuton.
[0333] The therapies of the invention may be combined with other
immunotherapies, including allergy shots, as well as other
antagonists of IgE or Fc Rs. The therapies of the invention may be
combined with antagonists of chemokines or cytokines, including but
not limited to antibodies and Fc fusions, including but not limited
to inhibitors of chemokines CCR3, CCR4, CCR8, and CRTH2, and CCR5,
and inhibitors of cytokines IL-13, IL-4, IL-5, IL-6, IL-9, IL-10,
IL-12, IL-15, IL-18, IL-19, IL-21, Class II family of cytokine
receptors, IL-22, IL-23, IL-25, IL-27, IL-31, and IL-33. The
therapies of the invention may be combined with modulators of
adhesion, transcription factors, and/or intracellular signaling.
For example, the immunoglobulins of the invention may be combined
with modulators of NF-.kappa.b, AP-1, GATA-3, Stat1, Stat-6, c-maf,
NFATs, suppressors of cytokine signaling (SOCS), peroxisome
proliferator-activated receptors (PPARs), MAP kinase, p38 MAPK,
JNK, and sphingosine I-phosphate receptors. The therapies of the
invention may be administered with suplatast tolilate, inhibitors
of phosphodiesterase 4 (PDE4), calcium channel blockers, and
heparin-like molecules. Possible co-therapies for the invention are
described further in detail in Caramori et al., 2008, Journal of
Occupational Medicine and Toxicology 3-S1-S6.
[0334] The therapies of the invention may also be used in
conjuction with one or more antibiotics, anti-fungal agents, or
antiviral agents. The antibodies disclosed herein may also be
combined with other therapeutic regimens such as surgery.
EXAMPLES
[0335] Examples are provided below are for illustrative purposes
only. These examples are not meant to constrain any embodiment
disclosed herein to any particular application or theory of
operation.
Example 1. Novel Methods for Inhibiting IE+Fc.gamma.RIIb+ Cells
[0336] Immunoglobulin IgE is a central initiator and propagator of
allergic response in affected tissue. IgE binds the high affinity
receptor for IgE (Fc RI), a key receptor involved in immediate
allergic manifestations that is expressed on a variety of effector
cells, including mast cells, basophils, eosinophils, as well as
other cell types. Cross-linking of Fc RI by immune-complexed
IgE-allergen activates these cells, releasing chemical mediators
such as histamine, prostaglandins, and leukotrienes, which may lead
to the development of a type I hypersensitivity reaction. The
approved monoclonal antibody Omalizumab (Xolair) neutralizes IgE by
binding to it and blocking engagement with Fc R's. Omalizumab
reduces bioactive IgE through sequestration, attenuating the amount
of antigen-specific IgE that can bind to and sensitize tissue mast
cells and basophils. This neutralization of free circulating IgE,
in turn, leads to a decrease in symptoms of allergic diseases.
Interestingly, serum IgE levels increase after start of therapy
because of omalizumab-IgE complex formation and may remain high up
to a year after stopping therapy. Consequently, this issue may lead
to false-negatives on diagnostic tests and therefore IgE levels
must be routinely checked.
[0337] A novel approach to targeting the IgE pathway involves not
only blocking free circulating IgE from engaging Fc Rs on effector
cells, but targeting the source of IgE production. IgE is secreted
by IgE-producing plasma cells located in lymph nodes draining the
site of antigen entry or locally at the sites of allergic
reactions. IgE-producing plasma cells are differentiated from IgE+
B cells. Class switching of B cells to IgE production is induced by
two separate signals, both of which can be provided by TH2
cells.
[0338] There are two forms of immunoglobulins: the secreted and the
membrane-anchored form. The membrane-anchored form differs from the
secreted form in that the former has a membrane-anchoring peptide
extending from the C terminus of the heavy-chain. Membrane-anchored
immunoglobulin on B-cells, also referred to as the B cell receptor
(BCR) complex, is critical for B-cell functions. It can transduce
signals for resting B cells to differentiate into activated
lymphoblasts and Ig-secreting plasma cells.
[0339] Differentiated B cells expressing membrane-anchored IgE,
referred to here as mIgE+ B cells, possess a natural negatively
regulating feedback mechanism--the inhibitory Fc receptor
Fc.gamma.RIIb. Fc.gamma.RIIb is expressed on a variety of immune
cells, including B cells, dendritic cells, monocytes, and
macrophages, where it plays a critical role in immune regulation.
In its normal role on B cells, Fc.gamma.RIIb serves as a feedback
mechanism to modulate B cell activation through the B cell receptor
(BCR). Engagement of BCR by immune complexed antigen on mature B
cells activates an intracellular signaling cascade, including
calcium mobilization, which leads to cell proliferation and
differentiation. However, as IgG antibodies with specificity to the
antigen are produced, the associated immune complexes (ICs) can
crosslink the BCR with Fc.gamma.RIIb, whereupon the activation of
BCR is inhibited by engagement of Fc.gamma.RIIb and associated
intracellular signaling pathways that interfere with the downstream
pathways of BCR activation. The expression of Fc.gamma.RIIb on the
surface of mIgE+ B cells, which use mIgE as their BCR, serves as a
negative regulator of these cell types.
[0340] A novel strategy for inhibiting IgE-mediated disease,
illustrated in FIG. 1, is to inhibit IgE+ B cells (i.e. B cells
expressing membrane anchored IgE) by coengaging membrane anchored
IgE and the inhibitory receptor Fc.gamma.RIIb. In B cells that have
class-switched to express IgE, mIgE serves as the BCR (referred to
herein as mIgE BCR). This approach would potentially mimic the
natural biological mechanism of immune complex-mediated suppression
of B cell activation, thereby preventing differentiation into
IgE-producing plasma cells. IgE-producing plasma cells reside in
the bone marrow and probably have a life span of several weeks to
several months. Since new IgE-secreting plasma cells go through
mIgE-expressing B- cell stages during differentiation, if their
generation is abrogated by inhibiting their mIgE+ B cell precursors
with this anti-IgE treatment, the existing plasma cells will die
off within weeks to months, and thus the production of IgE will
also gradually abate. Importantly, inhibition of IgE+ memory B
cells, which bear mIgE, would also be inhibited by anti-IgE
immunoglobulins that coengage Fc.gamma.RIIb with high affinity. If
this occurs, therapy may have long-term impact on the fundamental
disease.
Example 2. Anti-IgE Antibodies with High Affinity for
Fc.gamma.RIIb
[0341] Under physiological conditions, bridging of the BCR with
Fc.gamma.RIIb and subsequent B cell suppression occurs via immune
complexes of IgGs and cognate antigen. The design strategy was to
reproduce this effect using a single crosslinking antibody. Human
IgG binds human Fc.gamma.RIIb with weak affinity (greater than 100
nM for IgG1), and Fc.gamma.RIIb-mediated inhibition occurs in
response to immune-complexed but not monomeric IgG. It was reasoned
that high affinity to this receptor (less than 100 nM) would be
required for maximal inhibition of B cell activation. In order to
enhance the inhibitory activity of the anti-IgE antibodies of the
invention, the Fc region was engineered with variants that improve
binding to Fc.gamma.RIIb. Engineered Fc variants have been
described that bind to Fc.gamma.RIIb with improved affinity
relative to native IgG1 (U.S. Ser. No. 12/156,183, filed May 30,
2008, entitled "Methods and Compositions for Inhibiting CD32b
Expressing cells", herein incorporated expressly by reference).
[0342] Variants were originally generated in the context of an
antibody targeting the antigen CD19, a regulatory component of the
BCR coreceptor complex. The Fv region of this antibody is a
humanized and affinity matured version of antibody 4G7, and is
referred to herein as HuAM4G7. The Fv genes for this antibody were
subcloned into the mammalian expression vector pTT5 (National
Research Council Canada). Mutations in the Fc domain were
introduced using site-directed mutagenesis (QuikChange, Stratagene,
Cedar Creek, Tex.). In addition, control knock out variants with
ablated affinity for Fc receptors were generated that comprise the
substitutions G236R and L328R (G236R/L328R). This variant is
referred to as Fc-KO or Fc knockout. Heavy and light chain
constructs were cotransfected into HEK293E cells for expression,
and antibodies were purified using protein A affinity
chromatography (Pierce Biotechnology, Rockford, Ill.).
[0343] Recombinant human Fc.gamma.RIIb protein for binding studies
was obtained from R&D Systems (Minneapolis, Minn.). Genes
encoding Fc.gamma.RIIa and Fc.gamma.RIIIa receptor proteins were
obtained from the Mammalian Gene Collection (ATCC), and subcloned
into pTT5 vector (National Research Council Canada) containing
6.times. His tags. Allelic forms of the receptors (H131 and R131
for Fc.gamma.RIIa and V158 and F158 for Fc.gamma.RIIIa) were
generated using QuikChange mutagenesis. Vectors encoding the
receptors were transfected into HEK293T cells, and proteins were
purified using nickel affinity chromatography.
[0344] Variants were tested for receptor affinity using Biacore
technology, also referred to as Biacore herein, a surface plasmon
resonance (SPR) based technology for studying biomolecular
interactions in real time. SPR measurements were performed using a
Biacore 3000 instrument (Biacore, Piscataway, N.J.). A protein A/G
(Pierce Biotechnology) CM5 biosensor chip (Biacore) was generated
using a standard primary amine coupling protocol. All measurements
were performed using HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M
NaCl, 3 mM EDTA, 0.005% vol/vol surfactant P20, Biacore).
Antibodies at 20 nM or 50 nM in HBS-EP buffer were immobilized on
the protein A/G surface and Fc.gamma.Rs were injected. After each
cycle, the surface was regenerated by injecting glycine buffer (10
mM, pH 1.5). Data were processed by zeroing time and response
before the injection of Fc.gamma.R and by subtracting appropriate
nonspecific signals (response of reference channel and injection of
running buffer). Kinetic analyses were performed by global fitting
of binding data with a 1:1 Langmuir binding model using
BIAevaluation software (Biacore).
[0345] A representative set of sensorgrams for binding of select
variant anti-CD19 antibodies to Fc.gamma.RIIb is shown in FIG. 2.
The affinities of all variants and WT (native) IgG1 to all of the
Fc.gamma.Rs, obtained from fits of the Biacore binding data, are
plotted in FIG. 3 and provided numerically in FIG. 4. Whereas WT
IgG1 Fc binds with Fc.gamma.RIIb with .mu.M affinity (KD=1.8 .mu.M
in FIG. 4), a number of variants, for example G236D/S267E,
S239D/S267E, and S267E/L328F, have been engineered that bind the
inhibitory receptor more tightly. The S239D/1332E variant, as
described in U.S. Ser. No. 11/124,620, also has improved affinity
for the activating receptors Fc.gamma.RIIa and Fc.gamma.RIIIa, and
therefore is capable of mediated enhanced antibody-dependent
cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP). The
G236R/L328R variant, also referred to Fc-knockout or Fc-KO, lacks
binding to the Fc receptors, and is used as a control in the
experiments described herein.
[0346] Select variants were constructed in antibodies that target
IgE. The heavy and light chain variable regions (VH and VL) of
anti-IgE antibodies are provided in FIG. 5. Omalizumab is a
humanized antibody that is currently approved for the treatment of
allergic asthma, and is marketed under the name Xolair. MaE11 is
the murine precursor of Omalizumab. H1L1_MaE11 is a novel humanized
version of MaE11. Genes encoding the heavy and light VH and VL
domains of these anti-IgE antibodies were synthesized commercially
(Blue Heron Biotechnologies). Also synthesized were the variable
region VH and VL genes of the anti-respiratory syncytial virus
(RSV) antibody motavizumab, used in the experiments described
herein as a negative control. VL genes were subcloned into the
mammalian expression vector pTT5 (NRC-BRI, Canada) encoding the
Ckappa constant chain. VH genes were subcloned into the pTT5 vector
encoding native IgG1 and variant constant chains. Amino acid
sequences of select constant chains are provided in FIG. 6. All DNA
was sequenced to confirm the fidelity of the sequences. The amino
acid sequences of the full length heavy and light chains of select
antibodies are provided in FIG. 7. As shown in FIG. 9 and discussed
in further detail herein, H1L1_MaE11 shows higher affinity to IgE
than Omalizumab.
[0347] Plasmids containing heavy and light chain genes were
co-transfected into HEK293E cells using lipofectamine (Invitrogen)
and grown in FreeStyle 293 media (Invitrogen). After 5 days of
growth, the antibodies were purified from the culture supernatant
by protein A affinity using MabSelect resin (GE Healthcare).
[0348] Variant and native IgG1 anti-IgE antibodies were tested for
binding to IgE and to Fc.gamma.RIIb using Biacore. DNA encoding the
Fc region of IgE, which contains the binding site for the anti-IgE
antibodies used, was sythesized (Blue Heron Biotechnologies) and
subcloned into the pTT5 vector. IgE Fc was expressed in 293E cells
and purified using protein A as described above. SPR measurements
were performed using the protein A/antibody capture method
described above, except that analyte was either Fc.gamma.RIIb or
the Fc region of IgE. Data acquisition and fitting are as described
above. FIG. 8 provides the resulting equilibrium binding constants
(KDs) obtained from these binding experiments, as well as the fold
affinity relative to native IgG1 for binding to Fc.gamma.RIIb. FIG.
9 shows plots of these data. The results confirm the high of
affinity of the antibodies for IgE, and that the S267E/L328F
variant improves binding to Fc.gamma.RIIb over two orders of
magnitude, consistent with previous results.
[0349] The use of particular variants, for example S267E/L328F and
S239D/1332E, are meant here as proof of concept for the mechanism
as described herein, and are not meant to constrain the invention
to their particular use. The data provided in U.S. Ser. No.
12/156,183 and U.S. Ser. No. 11/124,620 indicate that a number of
engineered variants, at specific Fc positions, provide the targeted
properties. Substitutions to enhance Fc.gamma.R affinity, in
particular to Fc.gamma.RIIb, include: 234, 235, 236, 237, 239, 266,
267, 268, 325, 326, 327, 328, and 332. In some embodiments,
substitutions are made to at least one or more of the nonlimiting
following positions to enhance affinity to Fc.gamma.RIIb: 235, 236,
239, 266, 267, 268, and 328.
[0350] Nonlimiting combinations of positions for making
substitutions to enhance affinity to Fc.gamma.RIIb include:
234/239, 234/267, 234/328, 235/236, 235/239, 235/267, 235/268,
235/328, 236/239, 236/267, 236/268, 236/328, 237/267, 239/267,
239/268, 239/327, 239/328, 239/332, 266/267, 267/268, 267/325,
267/327, 267/328, 267/332, 268/327, 268/328, 268/332, 326/328,
327/328, and 328/332. In some embodiments, combinations of
positions for making substitutions to enhance affinity to
Fc.gamma.RIIb include, but are not limited to: 235/267, 236/267,
239/268, 239/267, 267/268, and 267/328.
[0351] Substitutions for enhancing affinity to Fc.gamma.RIIb
include: 234D, 234E, 234W, 235D, 235F, 235R, 235Y, 236D, 236N,
237D, 237N, 239D, 239E, 266M, 267D, S267E, 268D, 268E, 327D, 327E,
L328F, 328W, 328Y, and 332E. In some embodiments, combination of
positions for making substitutions for enhancing affinity to
Fc.gamma.RIIb include, but are not limited to: 235Y, 236D, 239D,
266M, S267E, 268D, 268E, L328F, 328W, and 328Y.
[0352] Combinations of substitutions for enhancing affinity to
Fc.gamma.RIIb include: L234D/S267E, L234E/S267E, L234F/S267E,
L234E/L328F, L234W/S239D, L234W/S239E, L234W/S267E, L234W/L328Y,
L235D/S267E, L235D/L328F, L235F/S239D, L235F/S267E, L235F/L328Y,
L235Y/G236D, L235Y/S239D, L235Y/S267D, L235Y/S267E, L235Y/H268E,
L235Y/L328F, G236D/S239D, G236D/S267E, G236D/H268E, G236D/L328F,
G236N/S267E, G237D/S267E, G237N/S267E, S239D/S267D, S239D/S267E,
S239D/H268D, S239D/H268E, S239D/A327D, S239D/L328F, S239D/L328W,
S239D/L328Y, S239D/1332E, S239E/S267E, V266M/S267E, S267D/H268E,
S267E/H268D, S267E/H268E, S267E/N325L, S267E/A327D, S267E/A327E,
S267E/L328F, S267E/L3281, S267E/L328Y, S267E/1332E, H268D/A327D,
H268D/L328F, H268D/L328W, H268D/L328Y, H268D/1332E, H268E/L328F,
H268E/L328Y, A327D/L328Y, L328F/1332E, L328W/1332E, and
L328Y/1332E. In some embodiments, combinations of substitutions for
enhancing affinity to Fc.gamma.RIIb include, but are not limited
to: L235Y/S267E, G236D/S267E, S239D/H268D, S239D/S267E,
S267E/H268D, S267E/H268E, and S267E/L328F.
Example 3. In Vitro Inhibition of IE+ B Cells by Anti-IgE
Antibodies with High Affinity to Fc.gamma.RIIb
[0353] An enzyme-linked immunosorbent assay (ELISA) was established
to detect IgE. Flat bottom plates were prepared by coating with pH
9.4 Na Bicarbonate buffer, followed by adherence with anti-IgE
capture antibodies at 10 ug/ml overnight in pH 9.4 (0.1 M
NaBicarbonate buffer). After overnight, the plate was blocked with
3% BSA/PBS, and serial dilutions of IgE (from a human IgE ELISA
kit, Bethyl Laboratories) was added 3.times. to 1 ug/ml. After 3
hours, plates were washed 3.times.(200 .mu.l) with TTBS, and bound
IgE was measured. HRP-conjugated goat polyclonal anti-human IgE
antibody (Bethyl Laboratories) was added at (1:5000) for 1 hour in
1% BSA/PBS. Samples were washed 3.times. and IgE was detected with
TMB peroxidase substrate (KPL, Inc 50-76-00). Reactions were
stopped with 50 .mu.l 2N H2SO.sub.4 and read at 450 nm.
[0354] FIG. 10 shows capture of IgE with various anti-human IgE
antibodies, including a pool of three monoclonal anti-IgE
antibodies (MabTech; 107/182/101), MaE11_IgG1_G236R/L328R, and
Omalizumab_IgG1_G236R/L328R. The data show that the commercial
anti-IgE antibody reagent (MabTech), Omalizumab, and its parent
chimeric antibody MaE11 are able to capture IgE. In order to use
this assay to detect IgE, it was necessary to determine whether
MaE11 and omalizumab antibodies would interfere with IgE capture by
the MabTech anti-IgE reagent. The assay was repeated as described
above, and concentration of IgE from absorbance was calculated
using a standard curve. FIG. 11 shows that anti-IgE antibody
omalizumab_G236R/L328R does not compete with the MabTech anti-IgE
antibody in the current ELISA protocol.
[0355] Fc variant anti-IgE antibodies were tested for their
capacity to inhibit IgE+ B cells. Human PBMCs were induced to class
switch to IgE producing B cells by adding 5 ng/ml interleukin-4
(IL-4) and 100 ng/ml anti-CD40 antibody (clone G28.5 IgG1). The
anti-CD40 antibody is an agonist of CD40, and thus mimics the
activity of the co-activator CD40L. Varying concentration of
anti-IgE antibodies were added, and the samples were incubated for
12 days. ELISA plates were prepared and blocked as described above,
using 5 ug/ml Mabtech anti-IgE as the capture antibody. 100 .mu.l
of the PBMC samples were added and incubated >3 hours, and then
washed with TTBS 3.times.(200 .mu.l). Antibody-HRP conjugated
antibody was added and detected as described above. Absorbance at
450 nm was converted to IgE concentration using a standard curve.
The results are shown in FIG. 12. Antibodies lacking Fc.gamma.R
binding (G236R/L328R variants) or having no specificity for IgE
(Motavizumab anti-RSV antibody) had no effect on IgE production
from differentiated B cells. In contrast, variant antibodies with
greater affinity for Fc.gamma.RIIb inhibited IgE production. These
data suggest that co-engagement of surface IgE and the inhibitory
Fc.gamma.R receptor Fc.gamma.RIIb inhibits class-switched B cells
of that immunoglobulin type. Inhibition of IgE+ B cells reduces the
number of IgE expressing plasma cells, which in turn reduces the
amount of IgE detected. To evaluate the selectivity of this
activity for IgE producing B cells, human IgG2 was measured from
the same samples using an IgG2 ELISA (Bethyl Laboratories). FIG. 13
shows that IgG2 secretion was not inhibited, indicating that the
inhibitory activity of anti-IgE antibodies with high Fc.gamma.RIIb
affinity is selective for IgE+ class-switched cells. Repeat of this
experiment using variant versions of the approved anti-IgE antibody
Omalizumab showed similar inhibitory results by the variant with
high Fc.gamma.RIIb affinity (FIG. 14).
[0356] The capacity of anti-IgE antibodies with high Fc.gamma.RIIb
affinity to inhibit IgE production was evaluated in the presence of
mIgE BCR stimulation. The above assay was repeated, with
class-switching to IgE promoted by IL-4 and .alpha.-CD40 agonist
antibody, and in addition the B cells were activated using either
anti-mu or anti-CD79b antibody. These antibodies cross-link the
BCR, thereby providing a signal similar to immune-complexed
antigen. Anti-mu antibody cross-links membrane-anchored IgM, and
anti-CD79b cross-links CD79b, which is a signaling component of the
BCR complex. PBMCs were incubated for 14 days with IL-4,
.alpha.-CD40, and either anti-CD79b or anti-mu, and IgE was
detected as described above. The results for anti-CD79b (FIG. 15)
and anti-mu (FIG. 16) show that the anti-IgE antibodies with high
affinity for Fc.gamma.RIIb are capable of inhibiting IgE production
when B cells are stimulated via BCR cross-linking.
[0357] An additional strategy for inhibiting IgE+ B cells is to
deplete them. This may be carried out using an anti-IgE antibody
that is enhanced for effector function. The variant S239D/1332E
increases binding to activating receptor Fc.gamma.RIIa and
Fc.gamma.RIIIa (FIG. 3 and FIG. 4), and thus improves ADCC and ADCP
effector functions. The above B cell assay was carried out using a
S239D/1332E variant of the anti-IgE antibody Omalizumab. PBMCs were
incubated for 14 days with IL-4, .alpha.-CD40, and either
anti-CD79b (FIG. 17) or anti-mu (FIG. 18), and IgE was detected as
described above. The results (FIGS. 17 and 18) show that anti-IgE
antibodies with optimized effector function are able to inhibit IgE
production from class-switched IgE+ B cells.
Example 4. In Vivo Inhibition of IgE+ B Cells by Anti-IgE
Antibodies with High Affinity to Fc.gamma.RIIb
[0358] The immunoglobulins disclosed herein were assessed using a
huPBL-SCID mouse model as a proxy for therapeutic activity in
humans. This study examined the capacity of the anti-IgE antibodies
described here to inhibit B cell activity and plasma cell
development in response to a common human allergen--dust mite
protein Der p 1. In this method, human peripheral blood leukocytes
(PBLs) from a blood donor with allergic response to Der p 1 were
engrafted to immune-deficient SCID mice and treated with the native
or variant anti-IgE antibodies. The mice were challenged with an
antigen to stimulate an immune response, and production of
immunoglobulins was measured to examine the course of B cell
development into plasma cells.
[0359] Blood donors were screened for allergy to dust mite antigen
based on the presence of anti-IgE antibodies against Der p 1. A
donor with positive reactivity was leukapheresed to obtained
peripheral blood mononuclear cells (PBMCs). The protocol for the
study is provided in FIG. 20. One day prior to PBMC injection, mice
were given intraperitoneal (i.p.) injections with 100 .mu.l of
anti-asialo GM antibody (Wako, Richmond, Va.) to deplete murine
natural killer (NK) cells. The next day, mice were injected i.p.
with 3.times.10.sup.7 PBLs in a 0.5 ml volume. After PBMC
injection, mice were assigned to 5 different groups of mice with 7
mice in each group. On day 7 post PBMC injection, blood was
collected from all mice via retro-orbital sinus/plexus (OSP)
puncture for determination of human IgG and IgE levels by ELISA
(ZeptoMetrix, Buffalo, N.Y.). Two days later (day 9), mice were
injected i.p. with 10 mg/kg antibody or PBS. On day 11, mice were
injected i.p. with 15 ug dustmite antigen Der p 1 (LoTox Natural
Der p 1, Indoor Biotechnologies, Charlottesville, Va.). On day 23
(12 days post antigen vaccination), blood was collected from all
mice for determination of human IgG and IgE antibodies. On the same
day, mice received a second injection i.p. with 10 mg/kg antibody
or PBS. Two days later (day 25), mice received a boost vaccination
i.p. of 10 ug dustmite antigen Der p 1. On day 37 (12 days post
antigen boost), blood was collected by OSP for human immunoglobulin
determination. Human IgG and IgE concentrations were measured using
ELISA methods similar to those described above.
[0360] The results are shown in FIGS. 20 and 21 for serum IgG and
IgE levels respectively. Before the allergen challenge, the levels
of human IgG and IgE antibodies were low in all the groups. After
Der p 1 immunization, all groups showed high levels of human IgG,
indicating a robust immune response by engrafted human B cells to
either the vaccinated Der p 1 antigen or endogenous mouse antigens.
In contrast to IgG response, the treatment groups differed
significantly in their production of IgE antibodies. Omalizumab and
the IgG1 version of H1 L1 MaE11 were equivalent to vehicle in their
capacity to inhibit production of human IgE. However the
Fc.gamma.RIIb-enhanced (IIbE, S267E/L328F) version of H1L1 MaE11
showed no detectable levels of human IgE. The Fc-KO (variant
G236R/L328R) version of H1 L1 MaE11, which lacks binding to all
Fc.gamma.Rs, showed an enhancement in human IgE production. This is
possibly due to its ability to cross-link human mIgE and thus
activate IgE+ B cells, yet its complete lack of Fc.gamma.RIIb
inhibitory or Fc.gamma.RIIa/IIIa cytotoxic activities such as those
possessed by the IgG1 and IIbE versions of the antibody. These in
vivo data show that anti-IgE antibodies with high affinity for
Fc.gamma.RIIb are capable of inhibiting human IgE+ B cell
activation and immunoglobulin secreting plasma cell
differentiation, and thus support the potential of the
immunoglobulins disclosed herein for treating IgE-mediated
disorders.
Example 5. Comparative PK/PD Model of XmAb7195 vs. Omalizumab
Effect on Free and Total IgE in Chimpanzees
[0361] XmAb7195 (anti-human IgE, S267E/L328F) was evaluated for its
pharmacokinetics and pharmacodynamics (free and total IgE) in
chimpanzees following a single intravenous dose of 5 mg/kg.
Chimpanzees and humans have similar Fc.gamma.RIIb structure at the
critical binding region (Arginine at position 131, or 131-R), in
contrast to macaques which do not have the relevant contact amino
acid. The comparator antibody in this study was commercially
available omalizumab (Xolair.RTM.. Genentech, USA), an anti-human
IgE antibody with a wild type human IgG1 Fc domain.
[0362] The purpose of this study was two-fold. The first objective
was to evaluate the pharmacokinetic behavior of XmAb7195 in
chimpanzees. Sequence differences among primate species lead to
significant differences in receptor affinities for XmAb7195. The
receptor-mediated clearance of XmAb7195 may involve Fc-gamma
receptors type II (a and b). PK experiments in other non-human
primates have been performed, but may not be predictive since
macaques do not have arginine at position 131 of the Fc gamma type
II receptors. PK in chimpanzees, which have the appropriate
genotype, may be more predictive of the PK/PD profile expected in
human clinical studies. The second purpose of the study was to
evaluate the pharmacodynamic effect of a single dose of XmAb7195 on
the sequestration, production, and clearance of IgE. In each of
these objectives, we used omalizumab as a comparator molecule in
order to evaluate the effect that the engineered Fc had on PK/PD
parameters.
[0363] The results of free drug concentrations as a function of
time are presented in FIG. 22A. Notably, XmAb7195 has a shorter
half-life of approximately 2 days compared to the approximately 11
days observed for omalizumab. Analysis of free and total IgE was
undertaken on serum samples at each of the PK time points. Free IgE
levels exhibited a rapid drop immediately after dosing. The nadir
of free IgE concentrations for the omalizumab-treated chimps
averaged approximately 50 ng/ml at one hour post dosing. XmAb7195
caused a more significant reduction of free IgE, reaching levels
below the lower limit of quantitation (LLOQ) of 4 ng/ml almost
immediately after dosing and remaining below the LLOQ up to day 10.
(FIG. 22B) Omalizumab increased total IgE for a period of weeks,
similar to its observed effects in humans. XmAb7195 caused a rapid
disappearance of total IgE--reaching the LLOQ within 1 hour
post-dosing and lasting for 10 days --followed by a gradual return
to baseline levels over a period of weeks. FIG. 22C shows group
mean total IgE levels versus time for chimpanzees treated with
omalizumab or XmAb7195 (anti-IgE, S267E/L328F). The lower limit of
quantification was 0.2 .mu.g/ml.
Example 6. PK/PD of Anti-IgE Antibodies in Human Fc.gamma.RIIb
Transgenic Mice
[0364] Several anti-mouse IgE antibodies with different
Fc.gamma.RIIb-enhancing Fc substitutions were produced for
comparison of their ability to modulate total IgE concentrations in
vivo. The first, XENP8253, comprises the R1 E4 Fv domain
(anti-mouse IgE) and an Fc domain containing the S267E/L328F
substitutions. The second, XENP8252, is a surrogate for omalizumab,
comprising the R1 E4 Fv domain with a native human IgG1 backbone.
Additional Fc variants--S267E, G236D/S267E, and G236N/S267E were
also characterized to examine the relationship between human
Fc.gamma.RIIb affinity and pharmacokinetics and
pharmacodynamics.
[0365] A single 2 mg/kg dose of all anti-IgE antibodies sequestered
serum IgE and reduced free IgE serum levels by several orders of
magnitude within hours of the treatment. Their effect on total IgE,
however, was very different. The omalizumab surrogate (XENP8252),
which contains an unmodified IgG1 Fc domain, had no discernible
effect on total IgE relative to the PBS control. In contrast, the
high Fc.gamma.RIIb affinity variant S267E/L328F reduced total IgE
within hours, and caused sustained reduction of total IgE relative
to both the PBS group and the XENP8252-treated mice. The extremely
rapid onset of the total IgE reduction in this model system
indicates (without being bound by theory) that anti-IgE antibodies
with enhanced affinity for Fc.gamma.RIIb increase the rate of
drug:IgE complex clearance. This hypothesis is consistent with the
observation that the S267E/L328F (IIbE) variant has reduced
half-life (approximately 2.5 days) relative to the IgG1 antibody
(XENP8252) (approximately 11 days). (FIG. 23). FIG. 24 shows serum
total IgE concentration as a function of time in the human
Fc.gamma.RIIb transgenic mice treated with anti-mouse IgE
antibodies. The lower limit of quantification of this IgE assay was
13 ng/ml.
[0366] The additional variant antibodies--S267E, G236D/S267E, and
G236N/S267E, with Fc.gamma.RIIb affinities intermediate between
IgG1 and S267E/L328, have intermediate half-lives and intermediate
effects on total IgE, revealing a direct relationship between
Fc.gamma.RIIb affinity and clearance rates of either antibody alone
or antibody complexed with antigen. (See FIG. 25).
Example 7. Antibody:IgE Complex Internalization by Liver Sinusoidal
Endothelial Cells (LSEC) from Fc.gamma.RIb Transgenic Mice
[0367] A hypothesis tested was that much of the in vivo accelerated
clearance of antibody and antibody:IgE complexes is mediated by
liver sinusoidal endothelial cells (LSEC). Anderson and colleagues
(Ganesan et al., J Immunol 2012) published a study demonstrating
that three quarters of mouse Fc.gamma.RIIb is expressed in the
liver, with 90% of it being expressed in LSEC. Moreover, the
authors demonstrated that clearance of radiolabeled small immune
complexes (SIC) is significantly impaired in an Fc.gamma.RIIb
knockout strain compared to wild-type mice.
[0368] An LSEC enrichment protocol was adopted from Katz et al,
(Katz et al., (2004) J Immunol "Liver sinusoidal endothelial cells
are insufficient to activate T cells," 173(1): 230-235) for
non-parenchymal cell isolation. Briefly, mouse liver was infused
with 1 ml of 1% (w/v) collagenase D in 1.times.HBSS using syringe
and needle. Then, the liver was quickly minced in 20 ml of 1%
collagenase D and incubated at 37.degree. C. for 20 minutes with
stirring to keep the cells in suspension. The cell suspension was
passed through a 100 .mu.m cell strainer filter mesh and spun
3.times. at low speed (30.times.g, 10 minutes) to remove the bulk
of parenchymal hepatocytes. The final enriched non-parenchymal
liver cell pellet (300.times.g, 10 minutes) was washed twice with
PBS (300.times.g, 10 minutes) and used in internalization
assays.
[0369] FITC conjugated anti-IgE antibodies plus human IgE
pre-formed IC were incubated with enriched LSEC's for 60 minutes at
37.degree. C., washed with low pH "Acid" wash buffer (glycine,
NaCl, pH=2.7) and stained with anti-CD146 and anti-CD45 antibodies.
The internalized signal (FITC MFI) was quantified from
CD146+CD45low LSEC's and MESF normalized values are plotted. As
shown in FIG. 26, IgE complexes formed with the anti-IgE antibody
containing the high IIb affinity variant S267E/L328 internalize
into LSEC more substantially than either anti-IgE IgG1 or anti-IgE
with Fc knockout substitutions (G236R/L328R). Variants with
intermediate IIb affinity--S267E, G236D/S267E, and
G236N/S267E--displayed intermediate internalization corresponding
with their relative affinities.
Example 8. Whole Body Imaging Study of IgE Biodistribution
[0370] A study was designed to evaluate the biodistribution and
specific hepatic uptake of XmAb7195 (anti-IgE-S267E/L328F) as
compared to saline controls and control antibody XENP6728
(anti-IgE-IgG1), when administered intravenously together with
.sup.89Zr-IgE to female Fc.gamma.RIIb transgenic mice.
[0371] Treatment began on Day 0. Animals were first injected
intravenously with .sup.89Zr-labeled IgE in the range of 0.10 to
0.13 mCi. This was immediately followed by an intravenous injection
of saline, 10 mg/kg XmAb7195, or 10 mg/kg XENP6728.
[0372] Animals were induced with 3.0% isoflurane in air (2.0 L/min)
and were maintained during imaging procedures at 1.0-2.0%
isoflurane in air (2.0 L/min). Animals were positioned inside the
Siemens Inveon PET ring, and the PET acquisition was initiated and
data was acquired continuously for 2 hours for the first
acquisition.
[0373] Administration of XmAb7195 was associated with significantly
greater, more rapid and more sustained accumulation of
.sup.89Zr-IgE in the liver, compared with saline and compared with
the IgG1 analog XENP6728. Correspondingly, administration of
XmAb7195 was associated with significantly reduced accumulation of
.sup.89Zr-IgE in the heart, which is representative of the amount
of labeled IgE in bulk circulation. (FIG. 27).
Example 9. CR2-Fc Fusions with High Affinity for Fc.gamma.RIIb
[0374] Soluble CRs and CR-Fc fusions have been described for
therapeutic purposes. These include CR1, CR2-Fc (U.S. Pat. No.
6,458,360), CR2-fH (CR2-factor H), and others. However, while these
approaches generally block interaction of C3-tagged ICs with their
associated receptors, they do not necessarily remove the immune
complexes from circulation. Most of the complement receptors and
regulatory proteins are composed of one or more so-called short
complement repeat (SCR) domains, also called complement control
protein (CCP) modules or Sushi domains. Typically, only a subset of
the domains is involved in direct recognition of the associated
complement fragment ligand. For example, it has been demonstrated
that only the first two SCRs of CR2 are essential for C3d binding.
The SCR domains are stable and well-behaved, making them suitable
for use in the development of therapeutic proteins.
[0375] Genes encoding the first two (SCR1-2) or first four (SCR1-4)
SCR domains from human and mouse were synthesized commercially
(Blue Heron Biotechnologies). Genes were subcloned into the
mammalian expression vector pTT5 (NRC-BRI, Canada) encoding the
human IgG1 Fc (hinge-CH2-CH3 domains). The SCR domains were also
subcloned into pTT5 vectors encoding variant IgG1 Fc domains
containing S267E, G236D/L328F, G236N/L328F, S267E/L328F (high
FcRIIb binding), or G236R/L328R (ablated FcR binding or Fc
knockout; also shown as FcKO) substitutions. All DNA was sequenced
to confirm the fidelity of the sequences. Amino acid sequences of
select CR2-Fc variants are provided in FIG. 40. Plasmids containing
heavy and light chain genes were co-transfected into HEK293E cells
using lipofectamine (Invitrogen) and grown in FreeStyle 293 media
(Invitrogen). After 5 days of growth, the antibodies were purified
from the culture supernatant by protein A affinity using MabSelect
resin (GE Healthcare).
[0376] SPR measurements were performed using a Biacore 3000
instrument (Biacore, Piscataway, N.J.). A protein A (Pierce
Biotechnology) CM5 biosensor chip (Biacore) was generated using a
standard primary amine coupling protocol. All measurements were
performed using HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3
mM EDTA, 0.005% vol/vol surfactant P20, Biacore). CR2-Fc variants
at 25 nM in HBS-EP buffer were immobilized on the protein A surface
and then 100 nM purified human C3d (Alpha Diagnostic cat#
C3D18-N-25) was injected. After each cycle, the surface was
regenerated by injecting glycine buffer (10 mM, pH 1.5). Data were
processed by zeroing time and response before the injection of C3d
and by subtracting appropriate nonspecific signals (response of
reference channel and injection of running buffer). Kinetic
analyses were performed by global fitting of binding data with a
1:1 Langmuir binding model using BIAevaluation software (Biacore).
Example binding curves are shown in FIG. 39A.
[0377] Binding of CR2-Fc constructs to recombinant C3d-Fc was
evaluated using ELISA. XENP12561 or XENP12562 (hSCR1-2 or hSCR1-4
Fc fusion) were coated to plates followed by adding varying
concentrations of XENP12704, which is an anti-IgE antibody
containing human C3d fused to the C-terminus of Ckappa. Anti-C1q
antibody and no plate coating were used as controls. Plates were
incubated overnight at 4.degree. C. and an
anti-IgG-F(ab')2-specific-HRP antibody was used for ELISA
detection. Results are shown in FIG. 39B. XENP12561 and XENP12562
showed clear binding to recombinant C3d, with XENP12562 (containing
hSCR1-4 domains) showing slightly stronger binding. A similar ELISA
format was also used to evaluate the binding of CR2-Fc constructs
to C3d-tagged immune complexes (IC) present in normal and
rheumatoid arthritis (RA) patient sera. RA patients have autoimmune
antibodies present and their sera are expected to contain a higher
amount of C3d-tagged IC of these antibodies compared to normal
subjects. XENP12561 or XENP12562 (hSCR1-2 or hSCR1-4 Fc fusion)
were coated to plates followed by adding varying concentrations of
normal or RA patient sera. Plates were incubated overnight at
4.degree. C. and an anti-IgG-F(ab')2-specific-HRP antibody was used
for ELISA detection. Results are shown in FIG. 39C. XENP12561 and
XENP12562 showed strong binding to c3d-tagged IC present in both
normal and RA patient sera. These results show that there is
approximately 100-fold higher amount of IC in RA patient sera
compared to normal sera.
Example 10. Design of Fc-Containing oxLDL-Binding Proteins with
Enhanced Fc.gamma.RIIb Affinity
[0378] OxLDL is bound naturally by scavenger receptors such as
LOX-1 (also known as OLR1) and CD36. Amino acid sequences for human
and mouse versions of these receptors are listed in FIG. 43. LOX-1
and CD36 Fc fusions can be designed (XENP13516, XENP13517,
XENP13518, sequences are listed in FIG. 44A-B). An Fc region is
desirable to increase serum half-life, stability, and expression
yields, while also serving as a scaffold for the inclusion of Fc
variants for enhancing Fc.gamma.RIIb affinity. Also, monoclonal
antibodies that bind oxLDL are known in the art (XENP13514 and
XENP13515, sequences are listed in FIG. 44A-B).
[0379] Plasmids containing appropriate genes were transfected or
co-transfected into HEK293E cells using lipofectamine (Invitrogen)
and grown in FreeStyle 293 media (Invitrogen). After 5 days of
growth, the proteins were purified from the culture supernatant by
protein A affinity using MabSelect resin (GE Healthcare). The
resulting proteins were examined by size-exclusion chromatography
(see FIG. 45).
[0380] Based on these results, amino acid sequences were designed
for Fc-containing oxLDL-binding proteins containing the S267E/L328F
Fc variant that confers enhanced Fc.gamma.RIIb affinity (FIG. 46).
Alternative Fc variants with various levels of Fc.gamma.RIIb
affinity can also be used as mentioned above, e.g., G236N/S236E,
G236D/S267E, and S267E. These variants allow for the rapid
clearance of oxLDL from the blood.
[0381] The EO6 antibody can be humanized to reduce its
immunogenicity as a therapeutic in humans. Sequences of humanized
variable regions derived from the EO6 parental sequence can be
found in FIG. 47.
[0382] All cited references are herein expressly incorporated by
reference in their entirety.
[0383] Whereas particular embodiments have been described above for
purposes of illustration, it will be appreciated by those skilled
in the art that numerous variations of the details may be made
without departing from the invention as described in the appended
claims.
Sequence CWU 1
1
961121PRTArtificial SequenceOmalizumab variable heavy chain
sequence 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser
Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Ser Ile Thr Tyr Asp Gly
Ser Thr Asn Tyr Asn Pro Ser Val 50 55 60 Lys Gly Arg Ile Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 29PRTArtificial
SequenceOmalizumab variable heavy chain sequence CDR1 2Tyr Ser Ile
Thr Ser Gly Tyr Ser Trp 1 5 35PRTArtificial SequenceOmalizumab
variable heavy chain sequence CDR2 3Thr Tyr Asp Gly Ser 1 5
412PRTArtificial SequenceOmalizumab variable heavy chain sequence
CDR3 4Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val 1 5 10
5111PRTArtificial SequenceOmalizumab variable light chain sequence
5Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr
Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu
Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110
610PRTArtificial SequenceOmalizumab variable light chain sequence
CDR1 6Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr 1 5 10
77PRTArtificial SequenceOmalizumab variable light chain sequence
CDR2 7Ala Ala Ser Tyr Leu Glu Ser 1 5 87PRTArtificial
SequenceOmalizumab variable light chain sequence CDR3 8Ser His Glu
Asp Pro Tyr Thr 1 5 9121PRTArtificial SequenceMaE11 variable heavy
chain sequence 9Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Ala Cys Ser Val Thr Gly Tyr
Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Ile Arg Gln Phe
Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Ser Ile Thr Tyr Asp
Gly Ser Ser Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser
Val Thr Arg Asp Thr Ser Gln Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu
Asn Ser Ala Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala
Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105
110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 109PRTArtificial
SequenceMaE11 variable heavy chain sequence CDR1 10Tyr Ser Ile Thr
Ser Gly Tyr Ser Trp 1 5 115PRTArtificial SequenceMaE11 variable
heavy chain sequence CDR2 11Thr Tyr Asp Gly Ser 1 5
1212PRTArtificial SequenceMaE11 variable heavy chain sequence CDR3
12Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val 1 5 10
13111PRTArtificial SequenceMaE11 variable light chain sequence
13Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr
Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly
Gln Pro Pro 35 40 45 Ile Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gly
Ser Glu Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala
Ala Thr Phe Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr
Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110
1410PRTArtificial SequenceMaE11 variable light chain sequence CDR1
14Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr 1 5 10 157PRTArtificial
SequenceMaE11 variable light chain sequence CDR2 15Ala Ala Ser Tyr
Leu Gly Ser 1 5 167PRTArtificial SequenceMaE11 variable light chain
sequence CDR3 16Ser His Glu Asp Pro Tyr Thr 1 5 17121PRTArtificial
SequenceH1L1MaE11 variable heavy chain sequence 17Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30
Tyr Ser Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp 35
40 45 Ile Gly Ser Ile Thr Tyr Asp Gly Ser Ser Asn Tyr Asn Pro Ser
Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn
Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Tyr Phe Gly His
Trp His Phe Ala Val Trp Gly 100 105 110 Ala Gly Thr Leu Val Thr Val
Ser Ser 115 120 189PRTArtificial SequenceH1L1MaE11 variable heavy
chain sequence CDR1 18Tyr Ser Ile Thr Ser Gly Tyr Ser Trp 1 5
195PRTArtificial SequenceH1L1MaE11 variable heavy chain sequence
CDR2 19Thr Tyr Asp Gly Ser 1 5 2012PRTArtificial SequenceH1L1MaE11
variable heavy chain sequence CDR3 20Gly Ser His Tyr Phe Gly His
Trp His Phe Ala Val 1 5 10 21111PRTArtificial SequenceH1L1MaE11
variable light chain sequence 21Asp Ile Gln Leu Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Met
Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu
Ile Tyr Ala Ala Ser Tyr Leu Gly Ser Glu Ile Pro Ala 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70
75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu
Ile Lys 100 105 110 2210PRTArtificial SequenceH1L1MaE11 variable
light chain sequence CDR1 22Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr
1 5 10 237PRTArtificial SequenceH1L1MaE11 variable light chain
sequence CDR2 23Ala Ala Ser Tyr Leu Gly Ser 1 5 247PRTArtificial
SequenceH1L1MaE11 variable light chain sequence CDR3 24Ser His Glu
Asp Pro Tyr Thr 1 5 25123PRTArtificial SequenceTES-C21 variable
heavy chain sequence 25Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Thr
Gly Tyr Thr Phe Ser Met Tyr 20 25 30 Trp Leu Glu Trp Val Lys Gln
Arg Pro Gly His Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Ser Pro
Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys
Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90
95 Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr
100 105 110 Trp Gly Gln Gly Thr Ser Leu Thr Val Ser Ser 115 120
267PRTArtificial SequenceTES-C21 variable heavy chain sequence CDR1
26Tyr Thr Phe Ser Met Tyr Trp 1 5 276PRTArtificial SequenceTES-C21
variable heavy chain sequence CDR2 27Ser Pro Gly Thr Phe Thr 1 5
2814PRTArtificial SequenceTES-C21 variable heavy chain sequence
CDR3 28Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 1 5
10 29107PRTArtificial SequenceTES-C21 variable light chain sequence
29Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1
5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr
Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asp Gly Ser Pro Arg
Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu
Asn Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr
Cys Gln Gln Ser Asp Ser Trp Pro Thr 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105 306PRTArtificial SequenceTES-C21
variable light chain sequence CDR1 30Gln Ser Ile Gly Thr Asn 1 5
317PRTArtificial SequenceTES-C21 variable light chain sequence CDR2
31Tyr Ala Ser Glu Ser Ile Ser 1 5 327PRTArtificial SequenceTES-C21
variable light chain sequence CDR3 32Ser Asp Ser Trp Pro Thr Thr 1
5 33107PRTArtificial SequenceCkappa light chain 33Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35
40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 105 34330PRTArtificial SequenceNative IgG1 constant
chain 34Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245
250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 325 330 35330PRTArtificial SequenceS267E/L328F IgG1
constant chain 35Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100
105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys 130 135 140 Val Val Val Asp Val Glu His Glu Asp Pro Glu
Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala
Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225
230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 325 330 36330PRTArtificial
SequenceG236D/S267E IgG1 constant chain 36Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70
75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Asp Gly Pro Ser Val
Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Glu His
Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195
200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315
320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
37218PRTArtificial SequenceOmalizumab light chain (VH-C ) 37Asp Ile
Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp 20
25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly
Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150
155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215 38451PRTArtificial SequenceOmalizumab IgG1 heavy chain
38Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser
Gly 20 25 30 Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp 35 40 45 Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn
Tyr Asn Pro Ser Val 50 55 60 Lys Gly Arg Ile Thr Ile Ser Arg Asp
Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His
Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260
265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385
390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450
39451PRTArtificial SequenceOmalizumab S267E/L328F heavy chain 39Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly
20 25 30 Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp 35 40 45 Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr
Asn Pro Ser Val 50 55 60 Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Tyr
Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145
150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu His 260 265
270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Phe Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390
395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 40218PRTArtificial
SequenceH1L1 MaE11 light chain (VH-C ) 40Asp Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp
Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gly Ser Glu Ile Pro Ala 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180
185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
41451PRTArtificial SequenceH1L1 MaE11 IgG1 heavy chain 41Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20
25 30 Tyr Ser Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu
Trp 35 40 45 Ile Gly Ser Ile Thr Tyr Asp Gly Ser Ser Asn Tyr Asn
Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser
Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Tyr Phe
Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Ala Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150
155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275
280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395
400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 42451PRTArtificial
SequenceH1L1 MaE11 S267E/L328F heavy chain 42Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr
Ser Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp 35 40
45 Ile Gly Ser Ile Thr Tyr Asp Gly Ser Ser Asn Tyr Asn Pro Ser Leu
50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Tyr Phe Gly His Trp
His Phe Ala Val Trp Gly 100 105 110 Ala Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro
Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Glu His 260 265 270 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305
310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala
Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425
430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445 Pro Gly Lys 450 43573PRTArtificial
SequenceFVIII_A2_IgG1_C220S/S267E/L328F 43Ser Val Ala Lys Lys His
Pro Lys Thr Trp Val His Tyr Ile Ala Ala 1 5 10 15 Glu Glu Glu Asp
Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp 20 25 30 Arg Ser
Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly 35 40 45
Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe 50
55 60 Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro
Leu 65 70 75 80 Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe
Lys Asn Gln 85 90 95 Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly
Ile Thr Asp Val Arg 100 105 110 Pro Leu Tyr Ser Arg Arg Leu Pro Lys
Gly Val Lys His Leu Lys Asp 115 120 125 Phe Pro Ile Leu Pro Gly Glu
Ile Phe Lys Tyr Lys Trp Thr Val Thr 130 135 140 Val Glu Asp Gly Pro
Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr 145 150 155 160 Tyr Ser
Ser Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile 165 170 175
Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn 180
185 190 Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe
Asp 195 200 205 Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg
Phe Leu Pro 210 215 220 Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu
Phe Gln Ala Ser Asn 225 230 235 240 Ile Met His Ser Ile Asn Gly Tyr
Val Phe Asp Ser Leu Gln Leu Ser 245 250 255 Val Cys Leu His Glu Val
Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala 260 265 270 Gln Thr Asp Phe
Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His 275 280 285 Lys Met
Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu 290 295 300
Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys 305
310 315 320 His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu
Lys Val 325 330 335 Ser Ser Cys Asp Lys Glu Pro Lys Ser Ser Asp Lys
Thr His Thr Cys 340 345 350 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu 355 360 365 Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu 370 375 380 Val Thr Cys Val Val Val
Asp Val Glu His Glu Asp Pro Glu Val Lys 385 390 395 400 Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 405 410 415 Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 420 425
430 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
435 440 445 Val Ser Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 450 455 460 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 465 470 475 480 Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys 485 490 495 Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln 500 505 510 Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 515 520 525 Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 530 535 540 Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 545 550
555 560 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 565 570
44395PRTArtificial SequenceFVIII_C2_IgG1_C220S/S267E/L328F 44Asp
Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile 1 5 10
15 Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala
20 25 30 Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg
Ser Asn 35 40 45 Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp
Leu Gln Val Asp 50 55 60 Phe Gln Lys Thr Met Lys Val Thr Gly Val
Thr Thr Gln Gly Val Lys 65 70 75 80 Ser Leu Leu Thr Ser Met Tyr Val
Lys Glu Phe Leu Ile Ser Ser Ser 85 90 95 Gln Asp Gly His Gln Trp
Thr Leu Phe Phe Gln Asn Gly Lys Val Lys 100 105 110 Val Phe Gln Gly
Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu 115 120 125 Asp Pro
Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 130 135 140
Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln 145
150 155 160 Asp Leu Tyr Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
Pro Pro 165 170 175 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro 180 185 190 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr 195 200 205 Cys Val Val Val Asp Val Glu His
Glu Asp Pro Glu Val Lys Phe Asn 210 215 220 Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg 225 230 235 240 Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 245 250 255 Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 260 265
270 Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
275 280 285 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu 290 295 300 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe 305 310 315 320 Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu 325 330 335 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 340 345 350 Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 355 360 365 Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 370 375 380 Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395
45572PRTArtificial Sequenceheavy chain sequence 45Ser Val Ala Lys
Lys His Pro Lys Thr Trp Val His Tyr Ile Ala Ala 1 5 10 15 Glu Glu
Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp 20 25 30
Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly 35
40 45 Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr
Phe 50 55 60 Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile Leu
Gly Pro Leu 65 70 75 80 Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile
Ile Phe Lys Asn Gln 85 90 95 Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
His Gly Ile Thr Asp Val Arg 100 105 110 Pro Leu Tyr Ser Arg Arg Leu
Pro Lys Gly Val Lys His Leu Lys Asp 115 120 125 Phe Pro Ile Leu Pro
Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr 130 135 140 Val Glu Asp
Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr 145 150 155 160
Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile 165
170 175 Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly
Asn 180 185 190 Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe Ser
Val Phe Asp 195 200 205 Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile
Gln Arg Phe Leu Pro 210 215 220 Asn Pro Ala Gly Val Gln Leu Glu Asp
Pro Glu Phe Gln Ala Ser Asn 225 230 235 240 Ile Met His Ser Ile Asn
Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser 245 250 255 Val Cys Leu His
Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala 260 265 270 Gln Thr
Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His 275 280 285
Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu 290
295 300 Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly
Cys 305 310 315 320 His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala
Leu Leu Lys Val 325 330 335 Ser Ser Cys Asp Lys Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Cys 340 345 350 Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu 355 360 365 Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 370 375 380 Val Thr Cys Val
Val Val Asp Val Glu His Glu Asp Pro Glu Val Gln 385 390 395 400 Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 405 410
415 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
420 425 430 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys 435 440 445 Val Ser Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys 450 455 460 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser 465 470 475 480 Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 485 490 495 Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln 500 505 510 Pro Glu Asn
Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 515 520 525 Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 530 535
540 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
545 550 555 560 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565
570 46395PRTArtificial Sequenceheavy chain sequence 46Asp Leu Asn
Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile 1 5 10 15 Ser
Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala 20 25
30 Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn
35 40 45 Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln
Val Asp 50 55 60 Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr
Gln Gly Val Lys 65 70 75 80 Ser Leu Leu Thr Ser Met Tyr Val Lys Glu
Phe Leu Ile Ser Ser Ser 85 90 95 Gln Asp Gly His Gln Trp Thr Leu
Phe Phe Gln Asn Gly Lys Val Lys 100 105 110 Val Phe Gln Gly Asn Gln
Asp Ser Phe Thr Pro Val Val Asn Ser Leu 115 120 125 Asp Pro Pro Leu
Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 130 135 140 Val His
Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln 145 150 155
160 Asp Leu Tyr Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
165 170 175 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro 180 185 190 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr 195 200 205 Cys Val Val Val Asp Val Glu His Glu Asp
Pro Glu Val Lys Phe Lys 210 215 220 Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg 225 230 235 240 Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 245 250 255 Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 260 265 270 Asn
Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 275 280
285 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
290 295 300 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe 305 310 315 320 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu 325 330 335 Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe 340 345 350 Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly 355 360 365 Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr 370 375 380 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 47747PRTArtificial
SequenceFVIII_A2-C2_IgG1_C220S/S267E/L328F 47Ser Val Ala Lys Lys
His Pro Lys Thr Trp Val His Tyr Ile Ala Ala 1 5 10 15 Glu Glu Glu
Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp 20 25 30 Arg
Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly 35 40
45 Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe
50 55 60 Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly
Pro Leu 65 70 75 80 Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile
Phe Lys Asn Gln 85 90
95 Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg
100 105 110 Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu
Lys Asp 115 120 125 Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys
Trp Thr Val Thr 130 135 140 Val Glu Asp Gly Pro Thr Lys Ser Asp Pro
Arg Cys Leu Thr Arg Tyr 145 150 155 160 Tyr Ser Ser Phe Val Asn Met
Glu Arg Asp Leu Ala Ser Gly Leu Ile 165 170 175 Gly Pro Leu Leu Ile
Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn 180 185 190 Gln Ile Met
Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp 195 200 205 Glu
Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro 210 215
220 Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn
225 230 235 240 Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu
Gln Leu Ser 245 250 255 Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile
Leu Ser Ile Gly Ala 260 265 270 Gln Thr Asp Phe Leu Ser Val Phe Phe
Ser Gly Tyr Thr Phe Lys His 275 280 285 Lys Met Val Tyr Glu Asp Thr
Leu Thr Leu Phe Pro Phe Ser Gly Glu 290 295 300 Thr Val Phe Met Ser
Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys 305 310 315 320 His Asn
Ser Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val 325 330 335
Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu 340
345 350 Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala
Ile 355 360 365 Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn
Met Phe Ala 370 375 380 Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu
Gln Gly Arg Ser Asn 385 390 395 400 Ala Trp Arg Pro Gln Val Asn Asn
Pro Lys Glu Trp Leu Gln Val Asp 405 410 415 Phe Gln Lys Thr Met Lys
Val Thr Gly Val Thr Thr Gln Gly Val Lys 420 425 430 Ser Leu Leu Thr
Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser 435 440 445 Gln Asp
Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys 450 455 460
Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu 465
470 475 480 Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln
Ser Trp 485 490 495 Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly
Cys Glu Ala Gln 500 505 510 Asp Leu Tyr Glu Pro Lys Ser Ser Asp Lys
Thr His Thr Cys Pro Pro 515 520 525 Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro 530 535 540 Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr 545 550 555 560 Cys Val Val
Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe Asn 565 570 575 Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 580 585
590 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
595 600 605 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser 610 615 620 Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys 625 630 635 640 Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu 645 650 655 Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe 660 665 670 Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 675 680 685 Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 690 695 700 Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 705 710
715 720 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr 725 730 735 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 740 745
48148PRTArtificial SequenceHuman CR2 SCR1-2 domains 48Met Gly Ala
Ala Gly Leu Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1 5 10 15 Gly
Val Leu Gly Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly 20 25
30 Arg Ile Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg
35 40 45 Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser
Leu Leu 50 55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly Thr Trp Asp
Lys Pro Ala Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser
Cys Pro Glu Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys Ile Arg Gly
Ser Thr Pro Tyr Arg His Gly Asp 100 105 110 Ser Val Thr Phe Ala Cys
Lys Thr Asn Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser Val Trp Cys
Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140 Thr Cys
Val Ser 145 49273PRTArtificial SequenceHuman CR2 SCR1-4 domains
49Met Gly Ala Ala Gly Leu Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1
5 10 15 Gly Val Leu Gly Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn
Gly 20 25 30 Arg Ile Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr
Val Ile Arg 35 40 45 Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly
Glu Lys Ser Leu Leu 50 55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly
Thr Trp Asp Lys Pro Ala Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys
Tyr Ser Ser Cys Pro Glu Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys
Ile Arg Gly Ser Thr Pro Tyr Arg His Gly Asp 100 105 110 Ser Val Thr
Phe Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser
Val Trp Cys Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135
140 Thr Cys Val Ser Val Phe Pro Leu Glu Cys Pro Ala Leu Pro Met Ile
145 150 155 160 His Asn Gly His His Thr Ser Glu Asn Val Gly Ser Ile
Ala Pro Gly 165 170 175 Leu Ser Val Thr Tyr Ser Cys Glu Ser Gly Tyr
Leu Leu Val Gly Glu 180 185 190 Lys Ile Ile Asn Cys Leu Ser Ser Gly
Lys Trp Ser Ala Val Pro Pro 195 200 205 Thr Cys Glu Glu Ala Arg Cys
Lys Ser Leu Gly Arg Phe Pro Asn Gly 210 215 220 Lys Val Lys Glu Pro
Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe 225 230 235 240 Phe Cys
Asp Glu Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys 245 250 255
Val Ile Ala Gly Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys Glu 260
265 270 Glu 50140PRTArtificial SequenceMouse CR2 SCR1-2 domains
50Met Leu Thr Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1
5 10 15 Pro Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro
Ile 20 25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser
Tyr Arg Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu
Asn Gln Val His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys
Glu Ser Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val
Pro Gly Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg
His Gly Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr
Met Lys Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met
Trp Gly Pro Thr Ala Leu Pro Val Cys Glu Ser 130 135 140
51265PRTArtificial SequenceMouse CR2 SCR1-4 domains 51Met Leu Thr
Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10 15 Pro
Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile 20 25
30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser Tyr Arg Leu
35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu Asn Gln Val
His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys Glu Ser Val
Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val Pro Gly Gly
Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg His Gly Asp
Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr Met Lys Gly
Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met Trp Gly Pro
Thr Ala Leu Pro Val Cys Glu Ser Asp Phe Pro Leu 130 135 140 Glu Cys
Pro Ser Leu Pro Thr Ile His Asn Gly His His Thr Gly Gln 145 150 155
160 His Val Asp Gln Phe Val Ala Gly Leu Ser Val Thr Tyr Ser Cys Glu
165 170 175 Pro Gly Tyr Leu Leu Thr Gly Lys Lys Thr Ile Lys Cys Leu
Ser Ser 180 185 190 Gly Asp Trp Asp Gly Val Ile Pro Thr Cys Lys Glu
Ala Gln Cys Glu 195 200 205 His Pro Gly Lys Phe Pro Asn Gly Gln Val
Lys Glu Pro Leu Ser Leu 210 215 220 Gln Val Gly Thr Thr Val Tyr Phe
Ser Cys Asn Glu Gly Tyr Gln Leu 225 230 235 240 Gln Gly Gln Pro Ser
Ser Gln Cys Val Ile Val Glu Gln Lys Ala Ile 245 250 255 Trp Thr Lys
Lys Pro Val Cys Lys Glu 260 265 52380PRTArtificial SequenceHuman
CR2 SCR1-2 IgG1 52Met Gly Ala Ala Gly Leu Leu Gly Val Phe Leu Ala
Leu Val Ala Pro 1 5 10 15 Gly Val Leu Gly Ile Ser Cys Gly Ser Pro
Pro Pro Ile Leu Asn Gly 20 25 30 Arg Ile Ser Tyr Tyr Ser Thr Pro
Ile Ala Val Gly Thr Val Ile Arg 35 40 45 Tyr Ser Cys Ser Gly Thr
Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu 50 55 60 Cys Ile Thr Lys
Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro 65 70 75 80 Lys Cys
Glu Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val 85 90 95
Pro Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly Asp 100
105 110 Ser Val Thr Phe Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn
Lys 115 120 125 Ser Val Trp Cys Gln Ala Asn Asn Met Trp Gly Pro Thr
Arg Leu Pro 130 135 140 Thr Cys Val Ser Glu Pro Lys Ser Ser Asp Lys
Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225
230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345
350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
380 53380PRTArtificial SequenceHuman CR2 SCR1-2 IgG1 S267E 53Met
Gly Ala Ala Gly Leu Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1 5 10
15 Gly Val Leu Gly Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly
20 25 30 Arg Ile Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val
Ile Arg 35 40 45 Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu
Lys Ser Leu Leu 50 55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly Thr
Trp Asp Lys Pro Ala Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys Tyr
Ser Ser Cys Pro Glu Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys Ile
Arg Gly Ser Thr Pro Tyr Arg His Gly Asp 100 105 110 Ser Val Thr Phe
Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser Val
Trp Cys Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140
Thr Cys Val Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145
150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Glu His Glu
Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265
270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 54380PRTArtificial
SequenceHuman CR2 SCR1-2 IgG1 S267E/L328F 54Met Gly Ala Ala Gly Leu
Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1 5 10 15 Gly
Val Leu Gly Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly 20 25
30 Arg Ile Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg
35 40 45 Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser
Leu Leu 50 55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly Thr Trp Asp
Lys Pro Ala Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser
Cys Pro Glu Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys Ile Arg Gly
Ser Thr Pro Tyr Arg His Gly Asp 100 105 110 Ser Val Thr Phe Ala Cys
Lys Thr Asn Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser Val Trp Cys
Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140 Thr Cys
Val Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155
160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Glu His Glu Asp Pro
Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys
Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280
285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 370 375 380 55380PRTArtificial
SequenceHuman CR2 SCR1-2 IgG1 G236N/S267E 55Met Gly Ala Ala Gly Leu
Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1 5 10 15 Gly Val Leu Gly
Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly 20 25 30 Arg Ile
Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg 35 40 45
Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu 50
55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala
Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu
Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro
Tyr Arg His Gly Asp 100 105 110 Ser Val Thr Phe Ala Cys Lys Thr Asn
Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser Val Trp Cys Gln Ala Asn
Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140 Thr Cys Val Ser Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys
Pro Ala Pro Glu Leu Leu Asn Gly Pro Ser Val Phe Leu Phe 165 170 175
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180
185 190 Thr Cys Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys
Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305
310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 370 375 380 56380PRTArtificial SequenceHuman CR2
SCR1-2 IgG1 G236D/S267E 56Met Gly Ala Ala Gly Leu Leu Gly Val Phe
Leu Ala Leu Val Ala Pro 1 5 10 15 Gly Val Leu Gly Ile Ser Cys Gly
Ser Pro Pro Pro Ile Leu Asn Gly 20 25 30 Arg Ile Ser Tyr Tyr Ser
Thr Pro Ile Ala Val Gly Thr Val Ile Arg 35 40 45 Tyr Ser Cys Ser
Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu 50 55 60 Cys Ile
Thr Lys Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro 65 70 75 80
Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val 85
90 95 Pro Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly
Asp 100 105 110 Ser Val Thr Phe Ala Cys Lys Thr Asn Phe Ser Met Asn
Gly Asn Lys 115 120 125 Ser Val Trp Cys Gln Ala Asn Asn Met Trp Gly
Pro Thr Arg Leu Pro 130 135 140 Thr Cys Val Ser Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu
Leu Leu Asp Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys
Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe 195 200 205
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210
215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330
335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380 57380PRTArtificial SequenceHuman CR2 SCR1-2 IgG1
G236R/L328R 57Met Gly Ala Ala Gly Leu Leu Gly Val Phe Leu Ala Leu
Val Ala Pro 1 5 10 15 Gly Val Leu Gly Ile Ser Cys Gly Ser Pro Pro
Pro Ile Leu Asn Gly 20 25 30 Arg Ile Ser Tyr Tyr Ser Thr Pro Ile
Ala Val Gly Thr Val Ile Arg 35 40 45 Tyr Ser Cys Ser Gly Thr Phe
Arg Leu Ile Gly Glu Lys Ser Leu Leu 50 55 60 Cys Ile Thr Lys Asp
Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro 65 70 75 80 Lys Cys Glu
Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val 85 90 95 Pro
Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly Asp 100 105
110 Ser Val Thr Phe Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys
115 120 125 Ser Val Trp Cys Gln Ala Asn Asn Met Trp Gly Pro Thr Arg
Leu Pro 130 135 140 Thr Cys Val Ser Glu Pro Lys Ser Ser Asp Lys Thr
His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Arg
Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230
235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val 245 250 255 Ser Asn Lys Ala Arg Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355
360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380
58505PRTArtificial SequenceHuman CR2 SCR1-4 IgG1 58Met Gly Ala Ala
Gly Leu Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1 5 10 15 Gly Val
Leu Gly Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly 20 25 30
Arg Ile Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg 35
40 45 Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu
Leu 50 55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly Thr Trp Asp Lys
Pro Ala Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser Cys
Pro Glu Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys Ile Arg Gly Ser
Thr Pro Tyr Arg His Gly Asp 100 105 110 Ser Val Thr Phe Ala Cys Lys
Thr Asn Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser Val Trp Cys Gln
Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140 Thr Cys Val
Ser Val Phe Pro Leu Glu Cys Pro Ala Leu Pro Met Ile 145 150 155 160
His Asn Gly His His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly 165
170 175 Leu Ser Val Thr Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly
Glu 180 185 190 Lys Ile Ile Asn Cys Leu Ser Ser Gly Lys Trp Ser Ala
Val Pro Pro 195 200 205 Thr Cys Glu Glu Ala Arg Cys Lys Ser Leu Gly
Arg Phe Pro Asn Gly 210 215 220 Lys Val Lys Glu Pro Pro Ile Leu Arg
Val Gly Val Thr Ala Asn Phe 225 230 235 240 Phe Cys Asp Glu Gly Tyr
Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys 245 250 255 Val Ile Ala Gly
Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys Glu 260 265 270 Glu Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 275 280 285
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 290
295 300 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 305 310 315 320 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr 325 330 335 Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu 340 345 350 Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His 355 360 365 Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 370 375 380 Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 385 390 395 400 Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 405 410
415 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
420 425 430 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 435 440 445 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 450 455 460 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 465 470 475 480 Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 485 490 495 Lys Ser Leu Ser Leu
Ser Pro Gly Lys 500 505 59505PRTArtificial SequenceHuman CR2 SCR1-4
IgG1 S267E 59Met Gly Ala Ala Gly Leu Leu Gly Val Phe Leu Ala Leu
Val Ala Pro 1 5 10 15 Gly Val Leu Gly Ile Ser Cys Gly Ser Pro Pro
Pro Ile Leu Asn Gly 20 25 30 Arg Ile Ser Tyr Tyr Ser Thr Pro Ile
Ala Val Gly Thr Val Ile Arg 35 40 45 Tyr Ser Cys Ser Gly Thr Phe
Arg Leu Ile Gly Glu Lys Ser Leu Leu 50 55 60 Cys Ile Thr Lys Asp
Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro 65 70 75 80 Lys Cys Glu
Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val 85 90 95 Pro
Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly Asp 100 105
110 Ser Val Thr Phe Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys
115 120 125 Ser Val Trp Cys Gln Ala Asn Asn Met Trp Gly Pro Thr Arg
Leu Pro 130 135 140 Thr Cys Val Ser Val Phe Pro Leu Glu Cys Pro Ala
Leu Pro Met Ile 145 150 155 160 His Asn Gly His His Thr Ser Glu Asn
Val Gly Ser Ile Ala Pro Gly 165 170 175 Leu Ser Val Thr Tyr Ser Cys
Glu Ser Gly Tyr Leu Leu Val Gly Glu 180 185 190 Lys Ile Ile Asn Cys
Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro 195 200 205 Thr Cys Glu
Glu Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly 210 215 220 Lys
Val Lys Glu Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe 225 230
235 240 Phe Cys Asp Glu Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg
Cys 245 250
255 Val Ile Ala Gly Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys Glu
260 265 270 Glu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
Cys Pro 275 280 285 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 290 295 300 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 305 310 315 320 Val Val Asp Val Glu His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 325 330 335 Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 340 345 350 Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 355 360 365 Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 370 375
380 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
385 390 395 400 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met 405 410 415 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 420 425 430 Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 435 440 445 Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 450 455 460 Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 465 470 475 480 Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 485 490 495
Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 60505PRTArtificial
SequenceHuman CR2 SCR1-4 IgG1 S267E/L328F 60Met Gly Ala Ala Gly Leu
Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1 5 10 15 Gly Val Leu Gly
Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly 20 25 30 Arg Ile
Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg 35 40 45
Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu 50
55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala
Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu
Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro
Tyr Arg His Gly Asp 100 105 110 Ser Val Thr Phe Ala Cys Lys Thr Asn
Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser Val Trp Cys Gln Ala Asn
Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140 Thr Cys Val Ser Val
Phe Pro Leu Glu Cys Pro Ala Leu Pro Met Ile 145 150 155 160 His Asn
Gly His His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly 165 170 175
Leu Ser Val Thr Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu 180
185 190 Lys Ile Ile Asn Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro
Pro 195 200 205 Thr Cys Glu Glu Ala Arg Cys Lys Ser Leu Gly Arg Phe
Pro Asn Gly 210 215 220 Lys Val Lys Glu Pro Pro Ile Leu Arg Val Gly
Val Thr Ala Asn Phe 225 230 235 240 Phe Cys Asp Glu Gly Tyr Arg Leu
Gln Gly Pro Pro Ser Ser Arg Cys 245 250 255 Val Ile Ala Gly Gln Gly
Val Ala Trp Thr Lys Met Pro Val Cys Glu 260 265 270 Glu Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 275 280 285 Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 290 295 300
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 305
310 315 320 Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr 325 330 335 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu 340 345 350 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His 355 360 365 Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 370 375 380 Ala Phe Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 385 390 395 400 Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 405 410 415 Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 420 425
430 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
435 440 445 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 450 455 460 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val 465 470 475 480 Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln 485 490 495 Lys Ser Leu Ser Leu Ser Pro
Gly Lys 500 505 61505PRTArtificial SequenceHuman CR2 SCR1-4 IgG1
G236N/S267E 61Met Gly Ala Ala Gly Leu Leu Gly Val Phe Leu Ala Leu
Val Ala Pro 1 5 10 15 Gly Val Leu Gly Ile Ser Cys Gly Ser Pro Pro
Pro Ile Leu Asn Gly 20 25 30 Arg Ile Ser Tyr Tyr Ser Thr Pro Ile
Ala Val Gly Thr Val Ile Arg 35 40 45 Tyr Ser Cys Ser Gly Thr Phe
Arg Leu Ile Gly Glu Lys Ser Leu Leu 50 55 60 Cys Ile Thr Lys Asp
Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro 65 70 75 80 Lys Cys Glu
Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val 85 90 95 Pro
Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly Asp 100 105
110 Ser Val Thr Phe Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys
115 120 125 Ser Val Trp Cys Gln Ala Asn Asn Met Trp Gly Pro Thr Arg
Leu Pro 130 135 140 Thr Cys Val Ser Val Phe Pro Leu Glu Cys Pro Ala
Leu Pro Met Ile 145 150 155 160 His Asn Gly His His Thr Ser Glu Asn
Val Gly Ser Ile Ala Pro Gly 165 170 175 Leu Ser Val Thr Tyr Ser Cys
Glu Ser Gly Tyr Leu Leu Val Gly Glu 180 185 190 Lys Ile Ile Asn Cys
Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro 195 200 205 Thr Cys Glu
Glu Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly 210 215 220 Lys
Val Lys Glu Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe 225 230
235 240 Phe Cys Asp Glu Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg
Cys 245 250 255 Val Ile Ala Gly Gln Gly Val Ala Trp Thr Lys Met Pro
Val Cys Glu 260 265 270 Glu Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys Pro 275 280 285 Ala Pro Glu Leu Leu Asn Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 290 295 300 Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val 305 310 315 320 Val Val Asp Val
Glu His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 325 330 335 Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 340 345 350
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 355
360 365 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 370 375 380 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln 385 390 395 400 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met 405 410 415 Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro 420 425 430 Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 435 440 445 Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 450 455 460 Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 465 470 475
480 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
485 490 495 Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505
62505PRTArtificial SequenceHuman CR2 SCR1-4 IgG1 G236D/S267E 62Met
Gly Ala Ala Gly Leu Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1 5 10
15 Gly Val Leu Gly Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly
20 25 30 Arg Ile Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val
Ile Arg 35 40 45 Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu
Lys Ser Leu Leu 50 55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly Thr
Trp Asp Lys Pro Ala Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys Tyr
Ser Ser Cys Pro Glu Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys Ile
Arg Gly Ser Thr Pro Tyr Arg His Gly Asp 100 105 110 Ser Val Thr Phe
Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser Val
Trp Cys Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140
Thr Cys Val Ser Val Phe Pro Leu Glu Cys Pro Ala Leu Pro Met Ile 145
150 155 160 His Asn Gly His His Thr Ser Glu Asn Val Gly Ser Ile Ala
Pro Gly 165 170 175 Leu Ser Val Thr Tyr Ser Cys Glu Ser Gly Tyr Leu
Leu Val Gly Glu 180 185 190 Lys Ile Ile Asn Cys Leu Ser Ser Gly Lys
Trp Ser Ala Val Pro Pro 195 200 205 Thr Cys Glu Glu Ala Arg Cys Lys
Ser Leu Gly Arg Phe Pro Asn Gly 210 215 220 Lys Val Lys Glu Pro Pro
Ile Leu Arg Val Gly Val Thr Ala Asn Phe 225 230 235 240 Phe Cys Asp
Glu Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys 245 250 255 Val
Ile Ala Gly Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys Glu 260 265
270 Glu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
275 280 285 Ala Pro Glu Leu Leu Asp Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys 290 295 300 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 305 310 315 320 Val Val Asp Val Glu His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr 325 330 335 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 340 345 350 Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His 355 360 365 Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 370 375 380 Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 385 390
395 400 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 405 410 415 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 420 425 430 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn 435 440 445 Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu 450 455 460 Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 465 470 475 480 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 485 490 495 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 500 505 63505PRTArtificial
SequenceHuman CR2 SCR1-4 IgG1 G236R/L328R 63Met Gly Ala Ala Gly Leu
Leu Gly Val Phe Leu Ala Leu Val Ala Pro 1 5 10 15 Gly Val Leu Gly
Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly 20 25 30 Arg Ile
Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg 35 40 45
Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu 50
55 60 Cys Ile Thr Lys Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala
Pro 65 70 75 80 Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu
Pro Ile Val 85 90 95 Pro Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro
Tyr Arg His Gly Asp 100 105 110 Ser Val Thr Phe Ala Cys Lys Thr Asn
Phe Ser Met Asn Gly Asn Lys 115 120 125 Ser Val Trp Cys Gln Ala Asn
Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140 Thr Cys Val Ser Val
Phe Pro Leu Glu Cys Pro Ala Leu Pro Met Ile 145 150 155 160 His Asn
Gly His His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly 165 170 175
Leu Ser Val Thr Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu 180
185 190 Lys Ile Ile Asn Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro
Pro 195 200 205 Thr Cys Glu Glu Ala Arg Cys Lys Ser Leu Gly Arg Phe
Pro Asn Gly 210 215 220 Lys Val Lys Glu Pro Pro Ile Leu Arg Val Gly
Val Thr Ala Asn Phe 225 230 235 240 Phe Cys Asp Glu Gly Tyr Arg Leu
Gln Gly Pro Pro Ser Ser Arg Cys 245 250 255 Val Ile Ala Gly Gln Gly
Val Ala Trp Thr Lys Met Pro Val Cys Glu 260 265 270 Glu Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 275 280 285 Ala Pro
Glu Leu Leu Arg Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 290 295 300
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 305
310 315 320 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr 325 330 335 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu 340 345 350 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His 355 360 365 Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 370 375 380 Ala Arg Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 385 390 395 400 Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 405 410 415 Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 420 425
430 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
435 440 445 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 450 455 460 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val 465 470 475 480 Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln 485 490 495 Lys Ser Leu Ser Leu Ser Pro
Gly Lys 500 505
64372PRTArtificial SequenceMouse CR2 SCR1-2 IgG1 64Met Leu Thr Trp
Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10 15 Pro Pro
Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile 20 25 30
Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser Tyr Arg Leu 35
40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu Asn Gln Val His
Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys Glu Ser Val Asn
Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val Pro Gly Gly Phe
Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg His Gly Asp Ser
Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr Met Lys Gly Ser
Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met Trp Gly Pro Thr
Ala Leu Pro Val Cys Glu Ser Glu Pro Lys Ser 130 135 140 Ser Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 145 150 155 160
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 165
170 175 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 180 185 190 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu 195 200 205 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr 210 215 220 Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 225 230 235 240 Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 245 250 255 Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 260 265 270 Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 275 280 285
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 290
295 300 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro 305 310 315 320 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 325 330 335 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 340 345 350 Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 355 360 365 Ser Pro Gly Lys 370
65372PRTArtificial SequenceMouse CR2 SCR1-2 IgG1 S267E 65Met Leu
Thr Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10 15
Pro Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile 20
25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser Tyr Arg
Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu Asn Gln
Val His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys Glu Ser
Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val Pro Gly
Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg His Gly
Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr Met Lys
Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met Trp Gly
Pro Thr Ala Leu Pro Val Cys Glu Ser Glu Pro Lys Ser 130 135 140 Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 145 150
155 160 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 165 170 175 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Glu 180 185 190 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu 195 200 205 Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr 210 215 220 Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn 225 230 235 240 Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 245 250 255 Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 260 265 270
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 275
280 285 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val 290 295 300 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro 305 310 315 320 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr 325 330 335 Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val 340 345 350 Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 355 360 365 Ser Pro Gly Lys
370 66372PRTArtificial SequenceMouse CR2 SCR1-2 IgG1 S267E/L328F
66Met Leu Thr Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1
5 10 15 Pro Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro
Ile 20 25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser
Tyr Arg Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu
Asn Gln Val His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys
Glu Ser Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val
Pro Gly Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg
His Gly Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr
Met Lys Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met
Trp Gly Pro Thr Ala Leu Pro Val Cys Glu Ser Glu Pro Lys Ser 130 135
140 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
145 150 155 160 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu 165 170 175 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Glu 180 185 190 His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu 195 200 205 Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr 210 215 220 Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 225 230 235 240 Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro 245 250 255
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 260
265 270 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val 275 280 285 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val 290 295 300 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro 305 310 315 320 Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr 325 330 335 Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val 340 345 350 Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 355 360 365 Ser Pro
Gly Lys 370 67372PRTArtificial SequenceMouse CR2 SCR1-2 IgG1
G236N/S267E 67Met Leu Thr Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser
Cys Asp Pro 1 5 10 15 Pro Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr
Tyr Ser Leu Pro Ile 20 25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr
Cys Ser Pro Ser Tyr Arg Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe
Cys Ile Ser Glu Asn Gln Val His Ala 50 55 60 Thr Trp Asp Lys Ala
Pro Pro Ile Cys Glu Ser Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser
Asp Pro Ile Val Pro Gly Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys
Ala Pro Phe Arg His Gly Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105
110 Asn Phe Thr Met Lys Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu
115 120 125 Met Trp Gly Pro Thr Ala Leu Pro Val Cys Glu Ser Glu Pro
Lys Ser 130 135 140 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu 145 150 155 160 Asn Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 165 170 175 Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Glu 180 185 190 His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 195 200 205 Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 210 215 220 Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 225 230
235 240 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 245 250 255 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 260 265 270 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val 275 280 285 Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 290 295 300 Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 305 310 315 320 Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 325 330 335 Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 340 345 350
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 355
360 365 Ser Pro Gly Lys 370 68372PRTArtificial SequenceMouse CR2
SCR1-2 IgG1 G236D/S267E 68Met Leu Thr Trp Phe Leu Phe Tyr Phe Ser
Glu Ile Ser Cys Asp Pro 1 5 10 15 Pro Pro Glu Val Lys Asn Ala Arg
Lys Pro Tyr Tyr Ser Leu Pro Ile 20 25 30 Val Pro Gly Thr Val Leu
Arg Tyr Thr Cys Ser Pro Ser Tyr Arg Leu 35 40 45 Ile Gly Glu Lys
Ala Ile Phe Cys Ile Ser Glu Asn Gln Val His Ala 50 55 60 Thr Trp
Asp Lys Ala Pro Pro Ile Cys Glu Ser Val Asn Lys Thr Ile 65 70 75 80
Ser Cys Ser Asp Pro Ile Val Pro Gly Gly Phe Met Asn Lys Gly Ser 85
90 95 Lys Ala Pro Phe Arg His Gly Asp Ser Val Thr Phe Thr Cys Lys
Ala 100 105 110 Asn Phe Thr Met Lys Gly Ser Lys Thr Val Trp Cys Gln
Ala Asn Glu 115 120 125 Met Trp Gly Pro Thr Ala Leu Pro Val Cys Glu
Ser Glu Pro Lys Ser 130 135 140 Ser Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu 145 150 155 160 Asp Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 165 170 175 Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu 180 185 190 His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 195 200 205
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 210
215 220 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn 225 230 235 240 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro 245 250 255 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 260 265 270 Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val 275 280 285 Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 290 295 300 Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 305 310 315 320 Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 325 330
335 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
340 345 350 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 355 360 365 Ser Pro Gly Lys 370 69372PRTArtificial
SequenceMouse CR2 SCR1-2 IgG1 G236R/L328R 69Met Leu Thr Trp Phe Leu
Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10 15 Pro Pro Glu Val
Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile 20 25 30 Val Pro
Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser Tyr Arg Leu 35 40 45
Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu Asn Gln Val His Ala 50
55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys Glu Ser Val Asn Lys Thr
Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val Pro Gly Gly Phe Met Asn
Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg His Gly Asp Ser Val Thr
Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr Met Lys Gly Ser Lys Thr
Val Trp Cys Gln Ala Asn Glu 115 120 125 Met Trp Gly Pro Thr Ala Leu
Pro Val Cys Glu Ser Glu Pro Lys Ser 130 135 140 Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 145 150 155 160 Arg Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 165 170 175
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 180
185 190 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu 195 200 205 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr 210 215 220 Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 225 230 235 240 Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Arg Pro Ala Pro 245 250 255 Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 260 265 270 Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 275 280 285 Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 290 295 300
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 305
310 315 320 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr 325 330 335 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val 340 345 350 Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 355 360 365 Ser Pro Gly Lys 370
70497PRTArtificial SequenceMouse CR2 SCR1-4 IgG1 70Met Leu Thr Trp
Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10 15 Pro Pro
Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro
Ile 20 25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser
Tyr Arg Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu
Asn Gln Val His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys
Glu Ser Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val
Pro Gly Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg
His Gly Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr
Met Lys Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met
Trp Gly Pro Thr Ala Leu Pro Val Cys Glu Ser Asp Phe Pro Leu 130 135
140 Glu Cys Pro Ser Leu Pro Thr Ile His Asn Gly His His Thr Gly Gln
145 150 155 160 His Val Asp Gln Phe Val Ala Gly Leu Ser Val Thr Tyr
Ser Cys Glu 165 170 175 Pro Gly Tyr Leu Leu Thr Gly Lys Lys Thr Ile
Lys Cys Leu Ser Ser 180 185 190 Gly Asp Trp Asp Gly Val Ile Pro Thr
Cys Lys Glu Ala Gln Cys Glu 195 200 205 His Pro Gly Lys Phe Pro Asn
Gly Gln Val Lys Glu Pro Leu Ser Leu 210 215 220 Gln Val Gly Thr Thr
Val Tyr Phe Ser Cys Asn Glu Gly Tyr Gln Leu 225 230 235 240 Gln Gly
Gln Pro Ser Ser Gln Cys Val Ile Val Glu Gln Lys Ala Ile 245 250 255
Trp Thr Lys Lys Pro Val Cys Lys Glu Glu Pro Lys Ser Ser Asp Lys 260
265 270 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro 275 280 285 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser 290 295 300 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 305 310 315 320 Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 325 330 335 Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 340 345 350 Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355 360 365 Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 370 375 380
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 385
390 395 400 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr 405 410 415 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 420 425 430 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu 435 440 445 Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys 450 455 460 Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu 465 470 475 480 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485 490 495 Lys
71497PRTArtificial SequenceMouse CR2 SCR1-4 IgG1 S267E 71Met Leu
Thr Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10 15
Pro Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile 20
25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser Tyr Arg
Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu Asn Gln
Val His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys Glu Ser
Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val Pro Gly
Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg His Gly
Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr Met Lys
Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met Trp Gly
Pro Thr Ala Leu Pro Val Cys Glu Ser Asp Phe Pro Leu 130 135 140 Glu
Cys Pro Ser Leu Pro Thr Ile His Asn Gly His His Thr Gly Gln 145 150
155 160 His Val Asp Gln Phe Val Ala Gly Leu Ser Val Thr Tyr Ser Cys
Glu 165 170 175 Pro Gly Tyr Leu Leu Thr Gly Lys Lys Thr Ile Lys Cys
Leu Ser Ser 180 185 190 Gly Asp Trp Asp Gly Val Ile Pro Thr Cys Lys
Glu Ala Gln Cys Glu 195 200 205 His Pro Gly Lys Phe Pro Asn Gly Gln
Val Lys Glu Pro Leu Ser Leu 210 215 220 Gln Val Gly Thr Thr Val Tyr
Phe Ser Cys Asn Glu Gly Tyr Gln Leu 225 230 235 240 Gln Gly Gln Pro
Ser Ser Gln Cys Val Ile Val Glu Gln Lys Ala Ile 245 250 255 Trp Thr
Lys Lys Pro Val Cys Lys Glu Glu Pro Lys Ser Ser Asp Lys 260 265 270
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 275
280 285 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 290 295 300 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu
His Glu Asp 305 310 315 320 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 325 330 335 Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val 340 345 350 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355 360 365 Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 370 375 380 Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 385 390 395
400 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
405 410 415 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 420 425 430 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 435 440 445 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys 450 455 460 Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 465 470 475 480 Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485 490 495 Lys
72497PRTArtificial SequenceMouse CR2 SCR1-4 IgG1 S267E/L328F 72Met
Leu Thr Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10
15 Pro Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile
20 25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser Tyr
Arg Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu Asn
Gln Val His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys Glu
Ser Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val Pro
Gly Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg His
Gly Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr Met
Lys Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met Trp
Gly Pro Thr Ala Leu Pro Val Cys Glu Ser Asp Phe Pro Leu 130 135 140
Glu Cys Pro Ser Leu Pro Thr Ile His Asn Gly His His Thr Gly Gln 145
150 155 160 His Val Asp Gln Phe Val Ala Gly Leu Ser Val Thr Tyr Ser
Cys Glu 165 170 175 Pro Gly Tyr Leu Leu Thr Gly Lys Lys Thr Ile Lys
Cys Leu Ser Ser 180 185 190 Gly Asp Trp Asp Gly Val Ile Pro Thr Cys
Lys Glu Ala Gln Cys Glu 195 200 205 His Pro Gly Lys Phe Pro Asn Gly
Gln Val Lys Glu Pro Leu Ser Leu 210 215 220 Gln Val Gly Thr Thr Val
Tyr Phe Ser Cys Asn Glu Gly Tyr Gln Leu 225 230 235 240 Gln Gly Gln
Pro Ser Ser Gln Cys Val Ile Val Glu Gln Lys Ala Ile 245 250 255 Trp
Thr Lys Lys Pro Val Cys Lys Glu Glu Pro Lys Ser Ser Asp Lys 260 265
270 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
275 280 285 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 290 295 300 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Glu His Glu Asp 305 310 315 320 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 325 330 335 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 340 345 350 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355 360 365 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys 370 375 380 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 385 390
395 400 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr 405 410 415 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 420 425 430 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 435 440 445 Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 450 455 460 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 465 470 475 480 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485 490 495 Lys
73497PRTArtificial SequenceMouse CR2 SCR1-4 IgG1 G236N/S267E 73Met
Leu Thr Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10
15 Pro Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile
20 25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser Tyr
Arg Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu Asn
Gln Val His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys Glu
Ser Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val Pro
Gly Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg His
Gly Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr Met
Lys Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met Trp
Gly Pro Thr Ala Leu Pro Val Cys Glu Ser Asp Phe Pro Leu 130 135 140
Glu Cys Pro Ser Leu Pro Thr Ile His Asn Gly His His Thr Gly Gln 145
150 155 160 His Val Asp Gln Phe Val Ala Gly Leu Ser Val Thr Tyr Ser
Cys Glu 165 170 175 Pro Gly Tyr Leu Leu Thr Gly Lys Lys Thr Ile Lys
Cys Leu Ser Ser 180 185 190 Gly Asp Trp Asp Gly Val Ile Pro Thr Cys
Lys Glu Ala Gln Cys Glu 195 200 205 His Pro Gly Lys Phe Pro Asn Gly
Gln Val Lys Glu Pro Leu Ser Leu 210 215 220 Gln Val Gly Thr Thr Val
Tyr Phe Ser Cys Asn Glu Gly Tyr Gln Leu 225 230 235 240 Gln Gly Gln
Pro Ser Ser Gln Cys Val Ile Val Glu Gln Lys Ala Ile 245 250 255 Trp
Thr Lys Lys Pro Val Cys Lys Glu Glu Pro Lys Ser Ser Asp Lys 260 265
270 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Asn Gly Pro
275 280 285 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 290 295 300 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Glu His Glu Asp 305 310 315 320 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 325 330 335 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 340 345 350 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355 360 365 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 370 375 380 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 385 390
395 400 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr 405 410 415 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 420 425 430 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 435 440 445 Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 450 455 460 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 465 470 475 480 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485 490 495 Lys
74497PRTArtificial SequenceMouse CR2 SCR1-4 IgG1 G236D/S267E 74Met
Leu Thr Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10
15 Pro Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile
20 25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser Tyr
Arg Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu Asn
Gln Val His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys Glu
Ser Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val Pro
Gly Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg His
Gly Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr Met
Lys Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met Trp
Gly Pro Thr Ala Leu Pro Val Cys Glu Ser Asp Phe Pro Leu 130 135 140
Glu Cys Pro Ser Leu Pro Thr Ile His Asn Gly His His Thr Gly Gln 145
150 155 160 His Val Asp Gln Phe Val Ala Gly Leu Ser Val Thr Tyr Ser
Cys Glu 165 170 175 Pro Gly Tyr Leu Leu Thr Gly Lys Lys Thr Ile Lys
Cys Leu Ser Ser 180 185 190 Gly Asp Trp Asp Gly Val Ile Pro Thr Cys
Lys Glu Ala Gln Cys Glu 195 200 205 His Pro Gly Lys Phe Pro Asn Gly
Gln Val Lys Glu Pro Leu Ser Leu 210 215 220 Gln Val Gly Thr Thr Val
Tyr Phe Ser Cys Asn Glu Gly Tyr Gln Leu 225 230 235 240 Gln Gly Gln
Pro Ser Ser Gln Cys Val Ile Val Glu Gln Lys Ala Ile 245 250 255 Trp
Thr Lys Lys Pro Val Cys Lys Glu Glu Pro Lys Ser Ser Asp Lys 260 265
270 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Asp Gly Pro
275 280 285 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 290 295 300 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val
Glu His Glu Asp 305 310 315 320 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 325 330 335 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 340 345 350 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355 360 365 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 370 375 380 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 385 390
395 400 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr 405 410 415 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 420 425 430 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 435 440 445 Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 450 455 460 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 465 470 475 480 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485 490 495 Lys
75497PRTArtificial SequenceMouse CR2 SCR1-4 IgG1 G236R/L328R 75Met
Leu Thr Trp Phe Leu Phe Tyr Phe Ser Glu Ile Ser Cys Asp Pro 1 5 10
15 Pro Pro Glu Val Lys Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile
20 25 30 Val Pro Gly Thr Val Leu Arg Tyr Thr Cys Ser Pro Ser Tyr
Arg Leu 35 40 45 Ile Gly Glu Lys Ala Ile Phe Cys Ile Ser Glu Asn
Gln Val His Ala 50 55 60 Thr Trp Asp Lys Ala Pro Pro Ile Cys Glu
Ser Val Asn Lys Thr Ile 65 70 75 80 Ser Cys Ser Asp Pro Ile Val Pro
Gly Gly Phe Met Asn Lys Gly Ser 85 90 95 Lys Ala Pro Phe Arg His
Gly Asp Ser Val Thr Phe Thr Cys Lys Ala 100 105 110 Asn Phe Thr Met
Lys Gly Ser Lys Thr Val Trp Cys Gln Ala Asn Glu 115 120 125 Met Trp
Gly Pro Thr Ala Leu Pro Val Cys Glu Ser Asp Phe Pro Leu 130 135 140
Glu Cys Pro Ser Leu Pro Thr Ile His Asn Gly His His Thr Gly Gln 145
150 155 160 His Val Asp Gln Phe Val Ala Gly Leu Ser Val Thr Tyr Ser
Cys Glu 165 170 175 Pro Gly Tyr Leu Leu Thr Gly Lys Lys Thr Ile Lys
Cys Leu Ser Ser 180 185 190 Gly Asp Trp Asp Gly Val Ile Pro Thr Cys
Lys Glu Ala Gln Cys Glu 195 200 205 His Pro Gly Lys Phe Pro Asn Gly
Gln Val Lys Glu Pro Leu Ser Leu 210 215 220 Gln Val Gly Thr Thr Val
Tyr Phe Ser Cys Asn Glu Gly Tyr Gln Leu 225 230 235 240 Gln Gly Gln
Pro Ser Ser Gln Cys Val Ile Val Glu Gln Lys Ala Ile 245 250 255 Trp
Thr Lys Lys Pro Val Cys Lys Glu Glu Pro Lys Ser Ser Asp Lys 260 265
270 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Arg Gly Pro
275 280 285 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 290 295 300 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp 305 310 315 320 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 325 330 335 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 340 345 350 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355 360 365 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Arg Pro Ala Pro Ile Glu Lys 370 375 380 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 385 390
395 400 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr 405 410 415 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 420 425 430 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 435 440 445 Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 450 455 460 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 465 470 475 480 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485 490 495 Lys
76273PRTArtificial SequenceHuman LOX-1 76Met Thr Phe Asp Asp Leu
Lys Ile Gln Thr Val Lys Asp Gln Pro Asp 1 5 10 15 Glu Lys Ser Asn
Gly Lys Lys Ala Lys Gly Leu Gln Phe Leu Tyr Ser 20 25 30 Pro Trp
Trp Cys Leu Ala Ala Ala Thr Leu Gly Val Leu Cys Leu Gly 35 40 45
Leu Val Val Thr Ile Met Val Leu Gly Met Gln Leu Ser Gln Val Ser 50
55 60 Asp Leu Leu Thr Gln Glu Gln Ala Asn Leu Thr His Gln Lys Lys
Lys 65 70 75 80 Leu Glu Gly Gln Ile Ser Ala Arg Gln Gln Ala Glu Glu
Ala Ser Gln 85 90 95 Glu Ser Glu Asn Glu Leu Lys Glu Met Ile Glu
Thr Leu Ala Arg Lys 100 105 110 Leu Asn Glu Lys Ser Lys Glu Gln Met
Glu Leu His His Gln Asn Leu 115 120 125 Asn Leu Gln Glu Thr Leu Lys
Arg Val Ala Asn Cys Ser Ala Pro Cys 130 135 140 Pro Gln Asp Trp Ile
Trp His Gly Glu Asn Cys Tyr Leu Phe Ser Ser 145 150 155 160 Gly Ser
Phe Asn Trp Glu Lys Ser Gln Glu Lys Cys Leu Ser Leu Asp 165 170 175
Ala Lys Leu Leu Lys Ile Asn Ser Thr Ala Asp Leu Asp Phe Ile Gln 180
185 190 Gln Ala Ile Ser Tyr Ser Ser Phe Pro Phe Trp Met Gly Leu Ser
Arg 195 200 205 Arg Asn Pro Ser Tyr Pro Trp Leu Trp Glu Asp Gly Ser
Pro Leu Met 210 215 220 Pro His Leu Phe Arg Val Arg Gly Ala Val Ser
Gln Thr Tyr Pro Ser 225 230 235 240 Gly Thr Cys Ala Tyr Ile Gln Arg
Gly Ala Val Tyr Ala Glu Asn Cys 245 250 255 Ile Leu Ala Ala Phe Ser
Ile Cys Gln Lys Lys Ala Asn Leu Arg Ala 260 265 270 Gln
77363PRTArtificial SequenceMouse LOX-1 77Met Thr Phe Asp Asp Lys
Met Lys Pro Ala Asn Asp Glu Pro Asp Gln 1 5 10 15 Lys Ser Cys Gly
Lys Lys Pro Lys Gly Leu His Leu Leu Ser Ser Pro 20 25 30 Trp Trp
Phe Pro Ala Ala Met Thr Leu Val Ile Leu Cys Leu Val Leu 35 40 45
Ser Val Thr Leu Ile Val Gln Trp Thr Gln Leu Arg Gln Val Ser Asp 50
55 60 Leu Leu Lys Gln Tyr Gln Ala Asn Leu Thr Gln Gln Asp Arg Ile
Leu 65 70 75 80 Glu Gly Gln Met Leu Ala Gln Gln Lys Ala Glu Asn Ala
Ser Gln Glu 85 90 95 Ser Lys Lys Glu Leu Lys Gly Lys Ile Asp Thr
Leu Thr Gln Lys Leu 100 105 110 Asn Glu Lys Ser Lys Glu Gln Glu Glu
Leu Leu Gln Lys Asn Gln Asn 115 120 125 Leu Gln Glu Ala Leu Gln Arg
Ala Ala Asn Ser Ser Glu Glu Ser Gln 130 135 140 Arg Glu Leu Lys Gly
Lys Ile Asp Thr Ile Thr Arg Lys Leu Asp Glu 145 150 155 160 Lys Ser
Lys Glu Gln Glu Glu Leu Leu Gln Met Ile Gln Asn Leu Gln 165 170 175
Glu Ala Leu Gln Arg Ala Ala Asn Ser Ser Glu Glu Ser Gln Arg Glu 180
185 190 Leu Lys Gly Lys Ile Asp Thr Leu Thr Leu Lys Leu Asn Glu Lys
Ser 195 200 205 Lys Glu Gln Glu Glu Leu Leu Gln Lys Asn Gln Asn Leu
Gln Glu Ala 210 215 220 Leu Gln Arg Ala Ala Asn Phe Ser Gly Pro Cys
Pro Gln Asp Trp Leu 225 230 235 240 Trp His Lys Glu Asn Cys Tyr Leu
Phe His Gly Pro Phe Ser Trp Glu 245 250 255 Lys Asn Arg Gln Thr Cys
Gln Ser Leu Gly Gly Gln Leu Leu Gln Ile 260 265 270 Asn Gly Ala Asp
Asp Leu Thr Phe Ile Leu Gln Ala Ile Ser His Thr 275 280 285 Thr Ser
Pro Phe Trp Ile Gly Leu His Arg Lys Lys Pro Gly Gln Pro 290 295 300
Trp Leu Trp Glu Asn Gly Thr Pro Leu Asn Phe Gln Phe Phe Lys Thr 305
310 315 320 Arg Gly Val Ser Leu Gln Leu Tyr Ser Ser Gly Asn Cys Ala
Tyr Leu 325 330 335 Gln Asp Gly Ala Val Phe Ala Glu Asn Cys Ile Leu
Ile Ala Phe Ser 340 345 350 Ile Cys Gln Lys Lys Thr Asn His Leu Gln
Ile 355 360 78472PRTArtificial SequenceHuman CD36 78Met Gly Cys Asp
Arg Asn Cys Gly Leu Ile Ala Gly Ala Val Ile Gly 1 5 10 15 Ala Val
Leu Ala Val Phe Gly Gly Ile Leu Met Pro Val Gly Asp Leu 20 25 30
Leu Ile Gln Lys Thr Ile Lys Lys Gln Val Val Leu Glu Glu Gly Thr 35
40 45 Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Glu Val Tyr Arg
Gln 50 55 60 Phe Trp Ile Phe Asp Val Gln Asn Pro Gln Glu Val Met
Met Asn Ser 65 70 75 80 Ser Asn Ile Gln Val Lys Gln Arg Gly Pro Tyr
Thr Tyr Arg Val Arg 85 90 95 Phe Leu Ala Lys Glu Asn Val Thr Gln
Asp Ala Glu Asp Asn Thr Val 100 105 110 Ser Phe Leu Gln Pro Asn Gly
Ala Ile Phe Glu Pro Ser Leu Ser Val 115 120 125 Gly Thr Glu Ala Asp
Asn Phe Thr Val Leu Asn Leu Ala Val Ala Ala 130 135 140 Ala Ser His
Ile Tyr Gln Asn Gln Phe Val Gln Met Ile Leu Asn Ser 145 150 155 160
Leu Ile Asn Lys Ser Lys Ser Ser Met Phe Gln Val Arg Thr Leu Arg 165
170 175 Glu Leu Leu Trp Gly Tyr Arg Asp Pro Phe Leu Ser Leu Val Pro
Tyr 180 185 190 Pro Val Thr Thr Thr Val Gly Leu Phe Tyr Pro Tyr Asn
Asn Thr Ala 195 200 205 Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp
Asn Ile Ser Lys Val 210 215 220 Ala Ile Ile Asp Thr Tyr Lys Gly Lys
Arg Asn Leu Ser Tyr Trp Glu 225 230 235 240 Ser His Cys Asp Met Ile
Asn Gly Thr Asp Ala Ala Ser Phe Pro Pro 245 250 255 Phe Val Glu Lys
Ser Gln Val Leu Gln Phe Phe Ser Ser Asp Ile Cys 260 265 270 Arg Ser
Ile Tyr Ala Val Phe Glu Ser Asp Val Asn Leu Lys Gly Ile 275 280 285
Pro Val Tyr Arg Phe Val Leu Pro Ser Lys Ala Phe Ala Ser Pro Val 290
295 300 Glu Asn Pro Asp Asn Tyr Cys Phe Cys Thr Glu Lys Ile Ile Ser
Lys 305 310 315 320 Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Ser Lys
Cys Lys Glu Gly 325 330 335 Arg Pro Val Tyr Ile Ser Leu Pro His Phe
Leu Tyr Ala Ser Pro Asp 340 345 350 Val Ser Glu Pro Ile Asp Gly Leu
Asn Pro Asn Glu Glu Glu His Arg 355 360 365 Thr Tyr Leu Asp Ile Glu
Pro Ile Thr Gly Phe Thr Leu Gln Phe Ala 370 375 380 Lys Arg Leu Gln
Val Asn Leu Leu Val Lys Pro Ser Glu Lys Ile Gln 385 390 395 400 Val
Leu Lys Asn Leu Lys Arg Asn Tyr Ile Val Pro Ile Leu Trp Leu 405 410
415 Asn Glu Thr Gly Thr Ile Gly Asp Glu Lys Ala Asn Met Phe Arg Ser
420 425 430 Gln Val Thr Gly Lys Ile Asn Leu Leu Gly Leu Ile Glu Met
Ile Leu 435 440 445 Leu Ser Val Gly Val Val Met Phe Val Ala Phe Met
Ile Ser Tyr Cys 450 455 460 Ala Cys Arg Ser Lys Thr Ile Lys 465 470
79472PRTArtificial SequenceMouse CD36 79Met Gly Cys Asp Arg Asn Cys
Gly Leu Ile Ala Gly Ala Val Ile Gly 1 5 10 15 Ala Val Leu Ala Val
Phe Gly Gly Ile Leu Met Pro Val Gly Asp Met 20 25 30 Leu Ile Glu
Lys Thr Ile Lys Arg Glu Val Val Leu Glu Glu Gly Thr 35 40 45 Thr
Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Thr Val Tyr Arg Gln 50 55
60 Phe Trp Ile Phe Asp Val Gln Asn Pro Asp Asp Val Ala Lys Asn Ser
65 70 75 80 Ser Lys Ile Lys Val Lys Gln Arg Gly Pro Tyr Thr Tyr Arg
Val Arg 85 90 95 Tyr Leu Ala Lys Glu Asn Ile Thr Gln Asp Pro Glu
Asp His Thr Val 100 105 110 Ser Phe Val Gln Pro Asn Gly Ala Ile Phe
Glu Pro Ser Leu Ser Val 115 120 125 Gly Thr Glu Asp Asp Asn Phe Thr
Val Leu Asn Leu Ala Val Ala Ala 130 135 140 Ala Pro His Ile Tyr Gln
Asn Ser Phe Val Gln Val Val Leu Asn Ser 145 150 155 160 Leu Ile Lys
Lys Ser Lys Ser Ser Met Phe Gln Thr Arg Ser Leu Lys 165 170 175 Glu
Leu Leu Trp Gly Tyr Lys Asp Pro Phe Leu Ser Leu Val Pro Tyr 180 185
190 Pro Ile Ser Thr Thr Val Gly Val Phe Tyr Pro Tyr Asn Asp Thr Val
195 200 205 Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Asn Ile Ser
Lys Val 210 215 220 Ala Ile Ile Glu Ser Tyr Lys Gly Lys Arg Asn Leu
Ser Tyr Trp Pro 225 230 235 240 Ser Tyr Cys Asp Met Ile Asn Gly Thr
Asp Ala Ala Ser Phe Pro Pro 245 250 255 Phe Val Glu Lys Ser Arg Thr
Leu Arg Phe Phe Ser Ser Asp Ile Cys 260 265 270 Arg Ser Ile Tyr Ala
Val Phe Gly Ser Glu Ile Asp Leu Lys Gly Ile 275 280 285 Pro Val Tyr
Arg Phe Val Leu Pro Ala Asn Ala Phe Ala Ser Pro Leu 290 295 300 Gln
Asn Pro Asp Asn His Cys Phe Cys Thr Glu Lys Val Ile Ser Asn 305 310
315 320 Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Gly Lys Cys Lys Glu
Gly 325 330 335 Lys Pro Val Tyr Ile Ser Leu Pro His Phe Leu His Ala
Ser Pro Asp 340 345 350 Val Ser Glu Pro Ile Glu Gly Leu His Pro Asn
Glu Asp Glu His Arg 355 360 365 Thr Tyr Leu Asp Val Glu Pro Ile Thr
Gly Phe Thr Leu Gln Phe Ala 370 375 380 Lys Arg Leu Gln Val Asn Ile
Leu Val Lys Pro Ala Arg Lys Ile Glu 385 390 395 400 Ala Leu Lys Asn
Leu Lys Arg Pro Tyr Ile Val Pro Ile Leu Trp Leu 405 410 415 Asn Glu
Thr Gly Thr Ile Gly Asp Glu Lys Ala Glu Met Phe Lys Thr 420 425 430
Gln Val Thr Gly Lys Ile Lys Leu Leu Gly Met Val Glu Met Ala Leu 435
440 445 Leu Gly Ile Gly Val Val Met Phe Val Ala Phe Met Ile Ser Tyr
Cys 450 455 460 Ala Cys Lys Ser Lys Asn Gly Lys 465 470
80453PRTArtificial SequenceEO6_H0L0_IgG1_WT heavy chain 80Glu Val
Lys Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30
Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35
40 45 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser
Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser
Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu
Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser
Ser Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165
170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290
295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410
415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 81220PRTArtificial
SequenceEO6_H0L0_IgG1_WT light chain 81Asp Ile Val Met Thr Gln Ser
Pro Thr Phe Leu Ala Val Thr Ala Ser 1 5 10 15 Lys Lys Val Thr Ile
Ser Cys Thr Ala Ser Glu Ser Leu Tyr Ser Ser 20 25 30 Lys His Lys
Val His Tyr Leu Ala Trp Tyr Gln Lys Lys Pro Glu Gln 35 40 45 Ser
Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Ile Gly Val 50 55
60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80 Ile Ser Ser Val Gln Val Glu Asp Leu Thr His Tyr Tyr Cys
Ala Gln 85 90 95 Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr
Lys Leu Glu Leu 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185
190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
220 82451PRTArtificial Sequence2D03_H0L0_IgG1_WT heavy chain 82Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Ser Val Gly Gly His Arg Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Arg Val Gly
Pro Ser Gly Gly Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145
150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265
270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390
395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 83216PRTArtificial
Sequence2D03_H0L0_IgG1_WT light chain 83Gln Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser
Cys Ser Gly Ser Asn Thr Asn Ile Gly Lys Asn 20 25 30 Tyr Val Ser
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Ala Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Ala
Ser Leu 85 90 95 Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185
190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215
84379PRTArtificial
SequencehuLOX1(137-237)-(G4S)2-Fc(216)_IgG1_C220S 84Val Ala Asn Cys
Ser Ala Pro Cys Pro Gln Asp Trp Ile Trp His Gly 1 5 10 15 Glu Asn
Cys Tyr Leu Phe Ser Ser Gly Ser Phe Asn Trp Glu Lys Ser 20 25 30
Gln Glu Lys Cys Leu Ser Leu Asp Ala Lys Leu Leu Lys Ile Asn Ser 35
40 45 Thr Ala Asp Leu Asp Phe Ile Gln Gln Ala Ile Ser Tyr Ser Ser
Phe 50 55 60 Pro Phe Trp Met Gly Leu Ser Arg Arg Asn Pro Ser Tyr
Pro Trp Leu 65 70 75 80 Trp Glu Asp Gly Ser Pro Leu Met Pro His Leu
Phe Arg Val Arg Gly 85 90 95 Ala Val Ser Gln Thr Tyr Pro Ser Gly
Thr Cys Ala Tyr Ile Gln Arg 100 105 110 Gly Ala Val Tyr Ala Glu Asn
Cys Ile Leu Ala Ala Phe Ser Ile Cys 115 120 125 Gln Lys Lys Ala Asn
Leu Arg Ala Gln Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 145 150 155 160
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 165
170 175 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr 180 185 190 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn 195 200 205 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg 210 215 220 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val 225 230 235 240 Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 245 250 255 Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 260 265 270 Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 275 280 285
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 290
295 300 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 305 310 315 320 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe 325 330 335 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly 340 345 350 Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr 355 360 365 Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 370 375 85652PRTArtificial
SequencehuCD36(30-439)-(G4S)2-Fc(216)_IgG1_C220S 85Gly Asp Leu Leu
Ile Gln Lys Thr Ile Lys Lys Gln Val Val Leu Glu 1 5 10 15 Glu Gly
Thr Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Glu Val 20 25 30
Tyr Arg Gln Phe Trp Ile Phe Asp Val Gln Asn Pro Gln Glu Val Met 35
40 45 Met Asn Ser Ser Asn Ile Gln Val Lys Gln Arg Gly Pro Tyr Thr
Tyr 50 55 60 Arg Val Arg Phe Leu Ala Lys Glu Asn Val Thr Gln Asp
Ala Glu Asp 65 70 75 80 Asn Thr Val Ser Phe Leu Gln Pro Asn Gly Ala
Ile Phe Glu Pro Ser 85 90 95 Leu Ser Val Gly Thr Glu Ala Asp Asn
Phe Thr Val Leu Asn Leu Ala 100 105 110 Val Ala Ala Ala Ser His Ile
Tyr Gln Asn Gln Phe Val Gln Met Ile 115 120 125 Leu Asn Ser Leu Ile
Asn Lys Ser Lys Ser Ser Met Phe Gln Val Arg 130 135 140 Thr Leu Arg
Glu Leu Leu Trp Gly Tyr Arg Asp Pro Phe Leu Ser Leu 145 150 155 160
Val Pro Tyr Pro Val Thr Thr Thr Val Gly Leu Phe Tyr Pro Tyr Asn 165
170 175 Asn Thr Ala Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Asn
Ile 180 185 190 Ser Lys Val Ala Ile Ile Asp Thr Tyr Lys Gly Lys Arg
Asn Leu Ser 195 200 205 Tyr Trp Glu Ser His Cys Asp Met Ile Asn Gly
Thr Asp Ala Ala Ser 210 215 220 Phe Pro Pro Phe Val Glu Lys Ser Gln
Val Leu Gln Phe Phe Ser Ser 225 230 235 240 Asp Ile Cys Arg Ser Ile
Tyr Ala Val Phe Glu Ser Asp Val Asn Leu 245 250 255 Lys Gly Ile Pro
Val Tyr Arg Phe Val Leu Pro Ser Lys Ala Phe Ala 260 265 270 Ser Pro
Val Glu Asn Pro Asp Asn Tyr Cys Phe Cys Thr Glu Lys Ile 275 280 285
Ile Ser Lys Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Ser Lys Cys 290
295 300 Lys Glu Gly Arg Pro Val Tyr Ile Ser Leu Pro His Phe Leu Tyr
Ala 305 310 315 320 Ser Pro Asp Val Ser Glu Pro Ile Asp Gly Leu Asn
Pro Asn Glu Glu 325 330 335 Glu His Arg Thr Tyr Leu Asp Ile Glu Pro
Ile Thr Gly Phe Thr Leu 340 345 350 Gln Phe Ala Lys Arg Leu Gln Val
Asn Leu Leu Val Lys Pro Ser Glu 355 360 365 Lys Ile Gln Val Leu Lys
Asn Leu Lys Arg Asn Tyr Ile Val Pro Ile 370 375 380 Leu Trp Leu Asn
Glu Thr Gly Thr Ile Gly Asp Glu Lys Ala Asn Met 385 390 395 400 Phe
Arg Ser Gln Val Thr Gly Lys Ile Asn Gly Gly Gly Gly Ser Gly 405 410
415 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
420 425 430 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe 435 440 445 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val 450 455 460 Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe 465 470 475 480 Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 485 490 495 Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 500 505 510 Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 515 520 525 Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 530 535
540
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 545
550 555 560 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly 565 570 575 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro 580 585 590 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser 595 600 605 Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln 610 615 620 Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His 625 630 635 640 Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 645 650 86379PRTArtificial
Sequenceempty-Fc(216)_IgG1_C220S-(G4S)2-huLOX1(137-237) 86Glu Pro
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20
25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150
155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly
Lys Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Val Ala
Asn Cys Ser Ala Pro Cys Pro Gln Asp Trp Ile Trp 245 250 255 His Gly
Glu Asn Cys Tyr Leu Phe Ser Ser Gly Ser Phe Asn Trp Glu 260 265 270
Lys Ser Gln Glu Lys Cys Leu Ser Leu Asp Ala Lys Leu Leu Lys Ile 275
280 285 Asn Ser Thr Ala Asp Leu Asp Phe Ile Gln Gln Ala Ile Ser Tyr
Ser 290 295 300 Ser Phe Pro Phe Trp Met Gly Leu Ser Arg Arg Asn Pro
Ser Tyr Pro 305 310 315 320 Trp Leu Trp Glu Asp Gly Ser Pro Leu Met
Pro His Leu Phe Arg Val 325 330 335 Arg Gly Ala Val Ser Gln Thr Tyr
Pro Ser Gly Thr Cys Ala Tyr Ile 340 345 350 Gln Arg Gly Ala Val Tyr
Ala Glu Asn Cys Ile Leu Ala Ala Phe Ser 355 360 365 Ile Cys Gln Lys
Lys Ala Asn Leu Arg Ala Gln 370 375 87453PRTArtificial
SequenceEO6_H0L0_IgG1_S267E/L328F heavy chain sequence 87Glu Val
Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20
25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp
Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu
Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp
Thr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg
Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Tyr Tyr
Gly Ser Ser Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150
155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270
Glu His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275
280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Phe Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395
400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450
88451PRTArtificial Sequence2D03_H0L0_IgG1_S267E/L328F heavy chain
sequence 88Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Val Gly Gly His
Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Ser Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Ile Arg Val Gly Pro Ser Gly Gly Ala Phe Asp Tyr Trp Gly 100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115
120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235
240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Glu His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Phe Pro Ala Pro Ile 325 330 335 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360
365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450
89379PRTArtificial
SequencehuLOX1(137-237)-(G4S)2-Fc(216)_IgG1_C220S/ S267E/L328F
89Val Ala Asn Cys Ser Ala Pro Cys Pro Gln Asp Trp Ile Trp His Gly 1
5 10 15 Glu Asn Cys Tyr Leu Phe Ser Ser Gly Ser Phe Asn Trp Glu Lys
Ser 20 25 30 Gln Glu Lys Cys Leu Ser Leu Asp Ala Lys Leu Leu Lys
Ile Asn Ser 35 40 45 Thr Ala Asp Leu Asp Phe Ile Gln Gln Ala Ile
Ser Tyr Ser Ser Phe 50 55 60 Pro Phe Trp Met Gly Leu Ser Arg Arg
Asn Pro Ser Tyr Pro Trp Leu 65 70 75 80 Trp Glu Asp Gly Ser Pro Leu
Met Pro His Leu Phe Arg Val Arg Gly 85 90 95 Ala Val Ser Gln Thr
Tyr Pro Ser Gly Thr Cys Ala Tyr Ile Gln Arg 100 105 110 Gly Ala Val
Tyr Ala Glu Asn Cys Ile Leu Ala Ala Phe Ser Ile Cys 115 120 125 Gln
Lys Lys Ala Asn Leu Arg Ala Gln Gly Gly Gly Gly Ser Gly Gly 130 135
140 Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
145 150 155 160 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro 165 170 175 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr 180 185 190 Cys Val Val Val Asp Val Glu His Glu
Asp Pro Glu Val Lys Phe Asn 195 200 205 Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg 210 215 220 Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 225 230 235 240 Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 245 250 255
Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 260
265 270 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu 275 280 285 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe 290 295 300 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu 305 310 315 320 Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe 325 330 335 Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 340 345 350 Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 355 360 365 Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 90652PRTArtificial
SequencehuCD36(30-439)-(G4S)2-Fc(216)_IgG1_C220S/S267E/ L328F 90Gly
Asp Leu Leu Ile Gln Lys Thr Ile Lys Lys Gln Val Val Leu Glu 1 5 10
15 Glu Gly Thr Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Glu Val
20 25 30 Tyr Arg Gln Phe Trp Ile Phe Asp Val Gln Asn Pro Gln Glu
Val Met 35 40 45 Met Asn Ser Ser Asn Ile Gln Val Lys Gln Arg Gly
Pro Tyr Thr Tyr 50 55 60 Arg Val Arg Phe Leu Ala Lys Glu Asn Val
Thr Gln Asp Ala Glu Asp 65 70 75 80 Asn Thr Val Ser Phe Leu Gln Pro
Asn Gly Ala Ile Phe Glu Pro Ser 85 90 95 Leu Ser Val Gly Thr Glu
Ala Asp Asn Phe Thr Val Leu Asn Leu Ala 100 105 110 Val Ala Ala Ala
Ser His Ile Tyr Gln Asn Gln Phe Val Gln Met Ile 115 120 125 Leu Asn
Ser Leu Ile Asn Lys Ser Lys Ser Ser Met Phe Gln Val Arg 130 135 140
Thr Leu Arg Glu Leu Leu Trp Gly Tyr Arg Asp Pro Phe Leu Ser Leu 145
150 155 160 Val Pro Tyr Pro Val Thr Thr Thr Val Gly Leu Phe Tyr Pro
Tyr Asn 165 170 175 Asn Thr Ala Asp Gly Val Tyr Lys Val Phe Asn Gly
Lys Asp Asn Ile 180 185 190 Ser Lys Val Ala Ile Ile Asp Thr Tyr Lys
Gly Lys Arg Asn Leu Ser 195 200 205 Tyr Trp Glu Ser His Cys Asp Met
Ile Asn Gly Thr Asp Ala Ala Ser 210 215 220 Phe Pro Pro Phe Val Glu
Lys Ser Gln Val Leu Gln Phe Phe Ser Ser 225 230 235 240 Asp Ile Cys
Arg Ser Ile Tyr Ala Val Phe Glu Ser Asp Val Asn Leu 245 250 255 Lys
Gly Ile Pro Val Tyr Arg Phe Val Leu Pro Ser Lys Ala Phe Ala 260 265
270 Ser Pro Val Glu Asn Pro Asp Asn Tyr Cys Phe Cys Thr Glu Lys Ile
275 280 285 Ile Ser Lys Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Ser
Lys Cys 290 295 300 Lys Glu Gly Arg Pro Val Tyr Ile Ser Leu Pro His
Phe Leu Tyr Ala 305 310 315 320 Ser Pro Asp Val Ser Glu Pro Ile Asp
Gly Leu Asn Pro Asn Glu Glu 325 330 335 Glu His Arg Thr Tyr Leu Asp
Ile Glu Pro Ile Thr Gly Phe Thr Leu 340 345 350 Gln Phe Ala Lys Arg
Leu Gln Val Asn Leu Leu Val Lys Pro Ser Glu 355 360 365 Lys Ile Gln
Val Leu Lys Asn Leu Lys Arg Asn Tyr Ile Val Pro Ile 370 375 380 Leu
Trp Leu Asn Glu Thr Gly Thr Ile Gly Asp Glu Lys Ala Asn Met 385 390
395 400 Phe Arg Ser Gln Val Thr Gly Lys Ile Asn Gly Gly Gly Gly Ser
Gly 405 410 415 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro 420 425 430 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe 435 440 445 Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val 450 455 460 Thr Cys Val Val Val Asp Val
Glu His Glu Asp
Pro Glu Val Lys Phe 465 470 475 480 Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro 485 490 495 Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr 500 505 510 Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 515 520 525 Ser Asn
Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 530 535 540
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 545
550 555 560 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly 565 570 575 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro 580 585 590 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser 595 600 605 Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln 610 615 620 Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His 625 630 635 640 Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 645 650 91379PRTArtificial
Sequenceempty-Fc(216)_IgG1_C220S/S267E/L328F-(G4S)2-
huLOX1(137-237) 91Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Glu His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100
105 110 Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly 225
230 235 240 Gly Ser Val Ala Asn Cys Ser Ala Pro Cys Pro Gln Asp Trp
Ile Trp 245 250 255 His Gly Glu Asn Cys Tyr Leu Phe Ser Ser Gly Ser
Phe Asn Trp Glu 260 265 270 Lys Ser Gln Glu Lys Cys Leu Ser Leu Asp
Ala Lys Leu Leu Lys Ile 275 280 285 Asn Ser Thr Ala Asp Leu Asp Phe
Ile Gln Gln Ala Ile Ser Tyr Ser 290 295 300 Ser Phe Pro Phe Trp Met
Gly Leu Ser Arg Arg Asn Pro Ser Tyr Pro 305 310 315 320 Trp Leu Trp
Glu Asp Gly Ser Pro Leu Met Pro His Leu Phe Arg Val 325 330 335 Arg
Gly Ala Val Ser Gln Thr Tyr Pro Ser Gly Thr Cys Ala Tyr Ile 340 345
350 Gln Arg Gly Ala Val Tyr Ala Glu Asn Cys Ile Leu Ala Ala Phe Ser
355 360 365 Ile Cys Gln Lys Lys Ala Asn Leu Arg Ala Gln 370 375
92123PRTArtificial SequenceEO6_H1 variable heavy chain sequence
92Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp
Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Gly Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr
Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Thr
Arg Asp Thr Ser Ala Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser
Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp
Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala
Gly Thr Leu Val Thr Val Ser Ser 115 120 93123PRTArtificial
SequenceEO6_H2 variable heavy chain sequence 93Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr
Met Glu Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Val 35 40
45 Gly Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala
50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys
Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser
Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Leu Val Thr
Val Ser Ser 115 120 94113PRTArtificial SequenceEO6_L1 variable
light chain sequence 94Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ala
Ser Glu Ser Leu Tyr Ser Ser 20 25 30 Lys His Lys Val His Tyr Leu
Ala Trp Tyr Gln Lys Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu
Ile Tyr Gly Ala Ser Asn Arg Tyr Ile Gly Val 50 55 60 Pro Asp Arg
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln 85 90
95 Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110 Lys 95113PRTArtificial SequenceEO6_L2 variable light
chain sequence 95Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Glu Ser Leu Tyr Ser Ser 20 25 30 Lys His Lys Val His Tyr Leu Ala
Trp Tyr Gln Lys Lys Pro Gly Lys 35 40 45 Ser Pro Lys Leu Leu Ile
Tyr Gly Ala Ser Asn Arg Tyr Ile Gly Val 50 55 60 Pro Asp Arg Phe
Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln 85 90 95
Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100
105 110 Lys 964PRTArtificial Sequencesequence linker 96Gly Phe Leu
Gly 1
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