U.S. patent number RE49,099 [Application Number 16/419,319] was granted by the patent office on 2022-06-07 for humanized monoclonal antibodies against activated protein c and uses thereof.
This patent grant is currently assigned to Bayer Healthcare LLC. The grantee listed for this patent is BAYER HEALTHCARE LLC. Invention is credited to Ji-Yun Kim, Jan Tebbe, Zhuozhi Wang, Xiao-Yan Zhao, Ying Zhu.
United States Patent |
RE49,099 |
Zhao , et al. |
June 7, 2022 |
Humanized monoclonal antibodies against activated protein c and
uses thereof
Abstract
Provided are humanized antibodies that selectively bind to and
inhibit activated protein C without binding to or inhibiting
unactivated protein C. Methods of treatment employing these
antibodies are described herein.
Inventors: |
Zhao; Xiao-Yan (Union City,
CA), Wang; Zhuozhi (Millbrae, CA), Kim; Ji-Yun
(Berkeley, CA), Zhu; Ying (Alamo, CA), Tebbe; Jan
(Cologne, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
BAYER HEALTHCARE LLC |
Whippany |
NJ |
US |
|
|
Assignee: |
Bayer Healthcare LLC (Whippany,
NJ)
|
Family
ID: |
1000006128005 |
Appl.
No.: |
16/419,319 |
Filed: |
May 22, 2019 |
PCT
Filed: |
November 27, 2013 |
PCT No.: |
PCT/US2013/072137 |
371(c)(1),(2),(4) Date: |
May 19, 2015 |
PCT
Pub. No.: |
WO2014/085527 |
PCT
Pub. Date: |
June 05, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
61731368 |
Nov 29, 2012 |
|
|
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Reissue of: |
14443696 |
Nov 27, 2013 |
9657111 |
May 23, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
16/40 (20130101); G01N 33/573 (20130101); C07K
2317/565 (20130101); C07K 2317/24 (20130101); C07K
2317/51 (20130101); C07K 2317/622 (20130101); C07K
2317/515 (20130101); C07K 2317/567 (20130101); G01N
2333/96461 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); C07K 16/40 (20060101); G01N
33/573 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1544214 |
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Jun 2005 |
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EP |
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1544214 |
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Jun 2005 |
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EP |
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2006-197930 |
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Aug 2006 |
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JP |
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WO 1993/000102 |
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Jan 1993 |
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WO |
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WO 1996/005303 |
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Feb 1996 |
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WO |
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WO 2002/029015 |
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Apr 2002 |
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WO |
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WO 2003/091415 |
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Nov 2003 |
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WO |
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WO 2004/073656 |
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Sep 2004 |
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WO |
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WO 2006/052591 |
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May 2006 |
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WO |
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WO 2007/070750 |
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Dec 2006 |
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WO |
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WO 2007/076524 |
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Jul 2007 |
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WO |
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WO 2008/021156 |
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Feb 2008 |
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WO |
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2009/055669 |
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Apr 2009 |
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WO |
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WO 2009/055669 |
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Apr 2009 |
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WO |
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WO 2012/007516 |
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Jan 2012 |
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WO |
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Other References
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|
Primary Examiner: Turner; Sharon
Attorney, Agent or Firm: ParkerHighlander PLLC
Parent Case Text
PRIORITY CLAIM
This application is a national phase application under 35 U.S.C.
.sctn.371 of International Application No. PCT/US2013/072137, filed
Nov. 27, 2013, which claims benefit of priority to U.S. Provisional
Application Ser. No. 61/731,368, filed Nov. 29, 2012. The entire
contents of the above-referenced disclosures are specifically
incorporated herein by reference.
Claims
The invention claimed is:
1. A humanized antibody binding to activated protein C comprising:
(a) a .[.heavy.]. .Iadd.light .Iaddend.chain comprising .[.heavy.].
.Iadd.light .Iaddend.chain CDRs represented by SEQ ID NOS: 1, 2 and
3; and .Iadd.wherein the light chain framework regions are
represented by amino acid residues 1 to 23, 39 to 53, 61 to 92 and
102 to 112 of SEQ ID NO: 26, or having 5 or fewer conservative
amino acid substitutions to SEQ ID NO: 26 framework regions,
and.Iaddend. (b) a .[.light.]. .Iadd.heavy .Iaddend.chain
comprising .[.light.]. .Iadd.heavy .Iaddend.chain CDRs represented
by SEQ ID NOS: 4, 5 and 6 .Iadd.and wherein the heavy chain
framework regions are represented by amino acid residues 1 to 30,
36 to 49, 69 to 100 and 108 to 118 of SEQ ID NO: 16, or having 5 or
fewer conservative amino acid substitutions to SEQ ID NO: 16
framework regions.Iaddend..
.[.2. The antibody of claim 1, wherein the heavy chain framework
regions are represented by SEQ ID NOS: 7, 8, 9 and 10, or having 5
or fewer conservative amino acid substitutions..].
.[.3. The antibody of claim 1, wherein the light chain framework
regions are represented by SEQ ID NOS: 11, 12, 13 and 14, or having
5 or fewer conservative amino acid substitutions..].
4. The antibody of claim .[.2.]. .Iadd.1.Iaddend., wherein residue
.[.14.]. .Iadd.49 .Iaddend.of SEQ ID NO: .[.8.]. .Iadd.16.Iaddend.
is substituted with Alanine.
5. The antibody of claim .[.2.]. .Iadd.1.Iaddend., wherein residues
.[.11, 13 and 31 of SEQ ID NO: 9.]. .Iadd.79, 81 and 99 of SEQ ID
NO: 16 .Iaddend.are .[.is.]. substituted with one or more of Serine
(residue .[.11.]. .Iadd.79.Iaddend.), Valine (residue .[.13.].
.Iadd.81.Iaddend.) and Isoleucine (residue .[.31.].
.Iadd.99.Iaddend.).
6. The antibody of claim 1, wherein said heavy chain comprises SEQ
ID .[.NOS: 16-24.]. .Iadd.NO: 16, 17, 18, 19, 20, 21 or
22.Iaddend..
7. The antibody of claim .[.3.]. .Iadd.1.Iaddend., wherein residue
4 of SEQ ID NO: .[.11.]. .Iadd.26 .Iaddend.is substituted with
Leucine.
8. The antibody of claim .[.3.]. .Iadd.1.Iaddend., wherein residue
.[.12.]. .Iadd.72 .Iaddend.of SEQ ID NO: .[.13.]. .Iadd.26
.Iaddend.is substituted with Arginine.
9. The antibody of claim 1, wherein said light chain comprises SEQ
ID .[.NOS: 26-30.]. .Iadd.NO: 26, 27, 28 or 29.Iaddend..
10. The antibody of claim 1, wherein the antibody is a single-chain
antibody or antibody fragment binding activated protein C.
11. The antibody of claim 10, wherein the antibody fragment is
further defined as Fab', Fab, F(ab').sub.2, a single domain
antibody, Fv, or scFv.
12. A cell or cell line comprising a nucleic acid encoding a
humanized antibody binding to activated protein C comprising: (a) a
.[.heavy.]. .Iadd.light .Iaddend.chain comprising .[.heavy.].
.Iadd.light .Iaddend.chain CDRs represented by SEQ ID NOS: 1, 2 and
3; and .Iadd.wherein the light chain framework regions are
represented by amino acid residues 1 to 23, 39 to 53, 61 to 92 and
102 to 112 of SEQ ID NO: 26, or having 5 or fewer conservative
amino acid substitutions to SEQ ID NO: 26 framework regions,
and.Iaddend. (b) a .[.light.]. .Iadd.heavy .Iaddend.chain
comprising .[.light.]. .Iadd.heavy .Iaddend.chain CDRs represented
by SEQ ID NOS: 4, 5 and 6.Iadd., wherein the heavy chain framework
regions are represented by amino acid residues 1 to 30, 36 to 49,
69 to 100 and 108 to 118 of SEQ ID NO: 16, or having 5 or fewer
conservative amino acid substitutions to SEQ ID NO: 16 framework
regions.Iaddend..
13. The antibody of claim 1, dispersed in a pharmaceutically
acceptable carrier.
14. A method of inhibiting activated protein C anticoagulant
activity and/or amidolytic activity in a subject, comprising
administering an effective amount of an antibody according to claim
1 to said subject.
15. A method of treating a subject in need of blood coagulation
comprising administering an effective amount of an antibody
according to claim 1 to said subject.
16. The method of claim 15, wherein said subject is suffering from
hemophilia.
17. A method of promoting hemostasis or thrombosis in a subject,
comprising .[.administrating.]. .Iadd.administering .Iaddend.an
effective amount of an antibody according to claim 1 to said
subject.
18. The method of claim 17, wherein the subject is a trauma
patient.
19. A kit comprising an antibody according to claim 1.
Description
SEQUENCE LISTING
Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted
herewith as an ASCII compliant text file named
"BAYRP0002US_ST25.txt", created on May 1, 2015 and having a size of
.about.25 kilobytes. The content of the aforementioned file is
hereby incorporated by reference in its entirety.
BACKGROUND
1. Introduction
Blood coagulation is a process consisting of a complex interaction
of various blood components, or factors, which eventually give rise
to a fibrin clot. Generally, blood components participating in the
coagulation "cascade" are proenzymes or zymogens--enzymatically
inactive proteins that are converted into an active form by action
of an activator. Regulation of blood coagulation is largely
accomplished enzymatically by proteolytic inactivation of the
pro-coagulation factors Va and VIIIa achieved by activated protein
C (aPC) (Esmon, 1989).
Protein C is the precursor to aPC, a potent natural anticoagulant.
Protein C is activated by thrombin in complex with thrombomodulin
(TM). The activation is augmented by endothelial cell protein C
receptor (EPCR). TM and EPCR can be down-regulated due to
inflammatory mediators, such as tumor necrosis factor, reviewed by
Esmon (1999). TM and EPCR have also been found to be reduced in
some forms of septic shock, meningococcemia in particular. Since
EPCR and TM are expressed on endothelium, it is not possible to
directly determine how well they are functioning without removal of
blood vessels.
aPC functions as an anticoagulant by proteolytically cleaving and
downregulating pro-coagulant factors. aPC also serves important
functions as an anti-apoptosis agent, an anti-inflammatory molecule
and a cytoprotectant. Bleeding disorders where homeostatis is
dysregulated through a loss of a key factor, such as the absence of
Factor VIII in hemophilia, or in trauma patients where the wound
process results in a temporary loss of hemostasis, can be treated
by the removal of aPC. Such treatment, however, could result in
unwanted detrimental consequences of removing the beneficial
functions of aPC in addition to the removal of the anti-coagulant
activity. Therefore it is desirable to have a therapeutic that
selectively targets the anti-coagulant activity of aPC while
leaving other functions of the molecule intact.
SUMMARY
Thus, there is provided an antibody comprising (a) a .[.heavy.].
.Iadd.light .Iaddend.chain comprising .[.heavy.]. .Iadd.light
.Iaddend.chain CDRs represented by SEQ ID NOS: 1, 2 and 3; and (b)
a .[.light.]. .Iadd.heavy .Iaddend.chain comprising .[.light.].
.Iadd.heavy .Iaddend.chain CDRs represented by SEQ ID NOS: 4, 5 and
6. The antibody maybe a humanized antibody, and may have the
following sequence composition:
TABLE-US-00001 TABLE 1 Antibody Sequences FR.sub.1 CDR1 FR.sub.2
CDR2 FR.sub.3 CDR3 FR.sub.4 Light Chain CDR SEQ ID NO: 1 2 3 Heavy
Chain CDR SEQ ID NO: 4 5 6 Light Chain Framework Residues* 1-23 39-
61- 102- 53 92 112 Heavy Chain Framework Residues** 1-30 36- 69-
108- 49 100 118 *Residues in reference to SEQ ID NO: 26 **Residues
in reference to SEQ ID NO: 16
The heavy chain framework regions may be represented by .[.SEQ ID
NOS: 7, 8, 9 and 10,.]. .Iadd.amino acid residues 1 to 30, 36 to
49, 69 to 100, and 108 to 118 of SEQ ID NO: 16 .Iaddend.or having 5
or fewer conservative amino acid substitutions
.Iadd.thereto.Iaddend., and/or the light chain framework regions
may be represented by .[.SEQ ID NOS: 11, 12, 13 and 14.].
.Iadd.amino residues 1 to 23, 39 to 53, 61 to 92 and 102 to 112 of
SEQ ID NO: 26.Iaddend., or having 5 or fewer conservative amino
acid substitutions .Iadd.thereto.Iaddend.. For example residue
.[.14.]. .Iadd.49 .Iaddend.of SEQ ID NO: .[.8.]. .Iadd.16
.Iaddend.may be substituted with Ala, and/or residues .[.11, 13 and
31 of SEQ ID NO: 9.]. .Iadd.79, 81 and 99 of SEQ ID NO: 16
.Iaddend.may be substituted with Serine, Valine and Isoleucine,
respectively; and/or the heavy chain may comprise SEQ ID .[.NOS:
16-24.]. .Iadd.NO: 16, 17, 18, 19, 20, 21 or 22.Iaddend.. Also for
example, residue 4 of SEQ ID NO: .[.11.]. .Iadd.26 .Iaddend.may be
substituted with Leucine; and/or residue .[.12.]. .Iadd.72
.Iaddend.of SEQ ID NO: .[.13.]. .Iadd.26 .Iaddend.may be
substituted with Arginine; and/or the light chain comprises SEQ ID
.[.NOS: 26-30.]. .Iadd.NO: 26, 27, 28 or 29.Iaddend.. The antibody
may be a single-chain antibody or an antibody fragment, such as a
Fab', Fab, F(ab').sub.2, a single domain antibody, Fv, or scFv.
Also provided is a pharmaceutical composition comprising any of the
foregoing embodiments dispersed in a pharmaceutically acceptable
carrier.
The disclosure also provides an expression construct, cell or cell
line comprising a nucleic acid encoding an antibody comprising (a)
a .[.heavy.]. .Iadd.light .Iaddend.chain comprising .[.heavy.].
.Iadd.light .Iaddend.chain CDRs represented by SEQ ID NOS: 1, 2 and
3; and (b) a .[.light.]. .Iadd.heavy .Iaddend.chain comprising
.[.light.]. .Iadd.heavy .Iaddend.chain CDRs represented by SEQ ID
NOS: 4, 5 and 6. The antibody may be a humanized antibody. The
heavy chain framework regions may be represented by .[.SEQ ID NOS:
7, 8, 9 and 10,.]. .Iadd.amino acid residues 1 to 30, 36 to 49, 69
to 100, and 108 to 118 of SEQ ID NO: 16 .Iaddend.or having 5 or
fewer conservative amino acid substitutions .Iadd.thereto.Iaddend.,
and/or the light chain framework regions may be represented by
.[.SEQ ID NOS: 11, 12, 13 and 14, or having 5 or fewer conservative
amino acid substitutions.]. .Iadd.amino residues 1 to 23, 39 to 53,
61 to 92 and 102 to 112 of SEQ ID NO: 26.Iaddend.. For example
residue .[.14.]. .Iadd.49 .Iaddend.of SEQ ID NO: .[.8.]. .Iadd.16
.Iaddend.may be substituted with Ala, and/or residues .[.11, 13 and
31 of SEQ ID NO: 9.]. .Iadd.79, 81 and 99 of SEQ ID NO: 16
.Iaddend.may be substituted with Serine, Valine and Isoleucine,
respectively; and/or the heavy chain may comprise SEQ ID .[.NOS:
16-24.]. .Iadd.NO: 16, 17, 18 , 19, 20, 21 or 22.Iaddend.. Also for
example, residue 4 of SEQ ID NO: .[.11.]. .Iadd.26 .Iaddend.may be
substituted with Leucine; and/or residue .[.12.]. .Iadd.72
.Iaddend.of SEQ ID NO: .[.13.]. .Iadd.26 .Iaddend.may be
substituted with Arginine; and/or the light chain comprises SEQ ID
.[.NOS: 26-30.]. .Iadd.NO: 26, 27, 28 or 29.Iaddend.. The antibody
may be a single-chain antibody or an antibody fragment, such as a
Fab', Fab, F(ab').sub.2, a single domain antibody, Fv, or scFv.
Also provided is method of inhibiting activated protein C
anticoagulant activity in a subject, comprising administering an
effective amount of an antibody according to the description
above.
Also provided is a method of inhibiting activated protein C
amidolytic activity in a subject comprising administering an
effective amount of an antibody according to the description
above.
Also provided is a method of treating a subject in need of blood
coagulation comprising administering an effective amount of an
antibody according to the description above.
Also provided is a method of treating a subject suffering from
sepsis comprising administering an effective amount of an antibody
according to the description above. The method may further comprise
administration of activated protein C.
Also provided is a method of treating a subject suffering from
hemophilia comprising administering an effective amount of an
antibody according to the description above.
Also provided is a method of modulating hemostasis in a subject,
comprising administering an effective amount of an antibody
according to the description above. The subject may be a trauma
patient.
Also provided is a method of modulating thrombosis in a subject,
comprising administering an effective amount of an antibody
according to the description above.
Yet another embodiment includes a kit comprising an antibody
according to the description above. The antibody may be labeled,
such as with a fluorophore, a radiolabel, a chemiluminescent label,
a dye, a quantum dot, a bead or a chromophore. The kit may further
comprise a buffer or diluent, and/or instructions on the use of
said antibody. The antibody may be present in an aqueous
suspension, or be lyophilized.
It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any compound,
method, or composition, and vice versa.
Other objects, features and advantages will become apparent from
the following detailed description. It should be understood,
however, that the detailed description and the specific examples,
while indicating specific embodiments, are given by way of
illustration only, since various changes and modifications within
the spirit and scope will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and
are included to further demonstrate certain aspects of the present
disclosure. The disclosure may be better understood by reference to
one or more of these drawings in combination with the detailed
description of specific embodiments presented herein.
FIG. 1--SEC analysis of 1573 humanized antibodies.
FIG. 2--Immobilization of capture antibody to CM5 chip using amine
coupling method. 9200 RU of anti-mouse FC IgG signal (top figure)
and 6400 RU of anti-human FC IgG (bottom figure) were generated
respectively. The running buffer was HBS-EP running buffer: 10 mM
HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20.
FIG. 3--SPR sensor-grams of binding of human aPC to 1573
antibodies: human aPC was injected over 1573 antibody at
concentration of 0, 1.25, 2.5, 5, 10, 20 nM, respectively, at 30
.mu.l/min for 180 s of association phase and 500 s of dissociation
phase.
FIG. 4--SPR sensor-grams of binding of cyno aPC to 1573 antibodies:
cyno aPC was injected over 1573 antibody at concentration of 0, 5,
10, 20, 40, 80 nM respectively at 30 .mu.l/min for 180 s of
association phase and 420 s of dissociation phase.
FIGS. 5--1573 humanized antibodies binding ELISA of human PC and
aPC.
FIGS. 6--1573 humanized antibodies binding ELISA of monkey PC and
aPC.
DESCRIPTION
The present disclosure relates to the discovery of monoclonal
antibodies that selectively bind to activated protein C, but not
unactivated protein C, and specifically inhibit the
anti-coagulation activity of activated protein C.
Whenever appropriate, terms used in the singular will also include
the plural and vice versa. In the event that any definition set
forth below conflicts with the usage of that word in any other
document, including any document incorporated herein by reference,
the definition set forth below shall always control for purposes of
interpreting this specification and its associated claims unless a
contrary meaning is clearly intended (for example in the document
where the term is originally used). The use of "or" means "and/or"
unless stated otherwise. The use of "a" herein means "one or more"
unless stated otherwise or where the use of "one or more" is
clearly inappropriate. The use of "comprise," "comprises,"
"comprising," "include," "includes," and "including" are
interchangeable and are not limiting. For example, the term
"including" shall mean "including, but not limited to."
The term "Protein C" or "PC" as used herein refers to any variant,
isoform, and/or species homolog of Protein C in its zymogen form
that is naturally expressed by cells and present in plasma and is
distinct from the activated form of Protein C.
The term "activated Protein C" or "aPC" as used herein refers to an
activated form of Protein C that is characterized by the removal
and absence of a 12 amino acid activation peptide present in
Protein C as a result of a thrombin cleavage site.
As used herein, an "antibody" refers to a whole antibody and any
antigen binding fragment (i.e., "antigen-binding portion") or
single chain thereof. The term includes a full-length
immunoglobulin molecule (e.g., an IgG antibody) that is naturally
occurring or formed by normal immunoglobulin gene fragment
recombinatorial processes, or an immunologically active portion of
an immunoglobulin molecule, such as an antibody fragment, that
retains the specific binding activity. Regardless of structure, an
antibody fragment binds with the same antigen that is recognized by
the full-length antibody. For example, an anti-aPC monoclonal
antibody fragment binds to an epitope of aPC. The antigen-binding
function of an antibody can be performed by fragments of a
full-length antibody. Examples of binding fragments encompassed
within the term "antigen-binding portion" of an antibody include
(i) a Fab fragment, a monovalent fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a V.sub.H domain; (vi) an isolated
complementarity determining region (CDR); (vii) minibodies,
diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g.,
Ill et al., Protein Eng 1997; 10:949-57); (viii) camel IgG; and
(ix) IgNAR. Furthermore, although the two domains of the Fv
fragment, V.sub.L and V.sub.H, are coded for by separate genes,
they can be joined, using recombinant methods, by a synthetic
linker that enables them to be made as a single protein chain in
which the V.sub.L and V.sub.H regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. These antibody fragments are obtained
using conventional techniques known to those with skill in the art,
and the fragments are analyzed for utility in the same manner as
are intact antibodies.
Furthermore, it is contemplated that an antigen binding fragment
can be encompassed in an antibody mimetic. The term "antibody
mimetic" or "mimetic" as used herein is meant a protein that
exhibits binding similar to an antibody but is a smaller
alternative antibody or a non-antibody protein. Such antibody
mimetic can be comprised in a scaffold. The term "scaffold" refers
to a polypeptide platform for the engineering of new products with
tailored functions and characteristics.
As used herein, the term "anti-aPC antibody" refers to an antibody
that specifically binds to an epitope of aPC. When bound in vivo to
an epitope of aPC, the anti-aPC antibodies disclosed herein augment
one or more aspects of the blood clotting cascade.
As used herein, the terms "inhibits binding" and "blocks binding"
(e.g., referring to inhibition/blocking of binding of aPC substrate
to aPC) are used interchangeably and encompass both partial and
complete inhibition or blocking of a protein with its substrate,
such as an inhibition or blocking by at least about 10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, about 96%, about 97%, about 98%, about 99%,
or about 100%. As used herein, "about" means+/-10% of the numerical
value indicated.
In reference to the inhibition and/or blocking of binding of aPC
substrate to aPC, the terms inhibition and blocking also include
any measurable decrease in the binding affinity of aPC to a
physiological substrate when in contact with an anti-aPC antibody
as compared to aPC not in contact with an anti-aPC antibody, e.g.,
the blocking of the interaction of aPC with its substrates,
including Factor Va or with Factor VIIIa, by at least about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80%, about 90%, about 95%, about 96%, about 97%, about 98%,
about 99%, or about 100%.
The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to antibodies displaying a single binding
specificity that have variable and constant regions derived from
human germline immunoglobulin sequences. The human antibodies can
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in
vivo).
An "isolated antibody," as used herein, is intended to refer to an
antibody which is substantially free of other biological molecules,
including antibodies having different antigenic specificities
(e.g., an isolated antibody that binds to aPC is substantially free
of antibodies that bind antigens other than aPC). In some
embodiments, the isolated antibody is at least about 75%, about
80%, about 90%, about 95%, about 97%, about 99%, about 99.9% or
about 100% pure by dry weight. In some embodiments, purity can be
measured by a method such as column chromatography, polyacrylamide
gel electrophoresis, or HPLC analysis. An isolated antibody that
binds to an epitope, isoform or variant of human aPC can, however,
have cross-reactivity to other related antigens, e.g., from other
species (e.g., aPC species homologs). Moreover, an isolated
antibody can be substantially free of other cellular material
and/or chemicals. As used herein, "specific binding" refers to
antibody binding to a predetermined antigen. Typically, an antibody
that exhibits "specific binding" binds to an antigen with an
affinity of at least about 10.sup.5 M.sup.-1 and binds to that
antigen with an affinity that is higher, for example at least
two-fold greater, than its binding affinity for an irrelevant
antigen (e.g., BSA, casein). The phrases "an antibody recognizing
an antigen" and "an antibody specific for an antigen" are used
interchangeably herein with the term "an antibody which binds
specifically to an antigen."
As used herein, the term "minimal binding" refers to an antibody
that does not bind to and/or exhibits low affinity to a specified
antigen. Typically, an antibody having minimal binding to an
antigen binds to that antigen with an affinity that is lower than
about 10.sup.2 M.sup.-1 and does not bind to a predetermined
antigen with higher affinity than it binds to an irrelevant
antigen.
As used herein, the term "high affinity" for an antibody, such as
an IgG antibody refers to a binding affinity of at least about
10.sup.7M.sup.-1, in at least one embodiment at least about
10.sup.8M.sup.-1, in some embodiments at least about
10.sup.9M.sup.-1, 10.sup.10M.sup.-1, 10.sup.11M.sup.-1 or greater,
e.g., up to 10.sup.13M.sup.-1 or greater. However, "high affinity"
binding can vary for other antibody isotypes. For example, "high
affinity" binding for an IgM isotype refers to a binding affinity
of at least about 10.sup.7M.sup.-1. As used herein, "isotype"
refers to the antibody class (e.g., IgM or IgG1) that is encoded by
heavy chain constant region genes.
"Complementarity-determining region" or "CDR" refers to one of
three hypervariable regions within the variable region of the heavy
chain or the variable region of the light chain of an antibody
molecule that form the N-terminal antigen-binding surface that is
complementary to the three-dimensional structure of the bound
antigen. Proceeding from the N-terminus of a heavy or light chain,
these complementarity-determining regions are denoted as "CDR1,"
"CDR2," and "CDR3," respectively [Wu T T, Kabat E A, Bilofsky H,
Proc Natl Acad Sci USA. 1975 December; 72(12):5107 and Wu T T,
Kabat E A, J Exp Med. 1970 Aug. 1; 132(2):211]. CDRs are involved
in antigen-antibody binding, and the CDR3 comprises a unique region
specific for antigen-antibody binding. An antigen-binding site,
therefore, can include six CDRs, comprising the CDR regions from
each of a heavy and a light chain V region.
The term "epitope" refers to the area or region of an antigen to
which an antibody specifically binds or interacts, which in some
embodiments indicates where the antigen is in physical contact with
the antibody. Conversely, the term "paratope" refers to the area or
region of the antibody on which the antigen specifically binds.
Epitopes characterized by competition binding are said to be
overlapping if the binding of the corresponding antibodies are
mutually exclusive, i.e., binding of one antibody excludes
simultaneous binding of another antibody. The epitopes are said to
be separate (unique) if the antigen is able to accommodate binding
of both corresponding antibodies simultaneously.
The term "competing antibodies," as used herein, refers to
antibodies that bind to about, substantially or essentially the
same, or even the same, epitope as an antibody against aPC as
described herein. "Competing antibodies" include antibodies with
overlapping epitope specificities. Competing antibodies are thus
able to effectively compete with an antibody as described herein
for binding to aPC. In some embodiments, the competing antibody can
bind to the same epitope as the antibody described herein.
Alternatively viewed, the competing antibody has the same epitope
specificity as the antibody described herein.
As used herein, "conservative substitutions" refers to
modifications of a polypeptide that involve the substitution of one
or more amino acids for amino acids having similar biochemical
properties that do not result in loss of a biological or
biochemical function of the polypeptide. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
.beta.-branched side chains (e.g., threonine, valine, isoleucine),
and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine). Antibodies of the present disclosure can
have one or more conservative amino acid substitutions yet retain
antigen binding activity.
For nucleic acids and polypeptides, the term "substantial homology"
indicates that two nucleic acids or two polypeptides, or designated
sequences thereof, when optimally aligned and compared, are
identical, with appropriate nucleotide or amino acid insertions or
deletions, in at least about 80% of the nucleotides or amino acids,
usually at least about 85%, in some embodiments about 90%, 91%,
92%, 93%, 94%, or 95%, in at least one embodiment at least about
96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% of the
nucleotides or amino acids. Alternatively, substantial homology for
nucleic acids exists when the segments will hybridize under
selective hybridization conditions to the complement of the strand.
Also included are nucleic acid sequences and polypeptide sequences
having substantial homology to the specific nucleic acid sequences
and amino acid sequences recited herein.
The percent identity between two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
homology=# of identical positions/total # of positions.times.100),
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences. The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm, such as without limitation the AlignX.TM.
module of VectorNTI.TM. (Invitrogen Corp., Carlsbad, Calif.). For
AlignX.TM., the default parameters of multiple alignment are: gap
opening penalty: 10; gap extension penalty: 0.05; gap separation
penalty range: 8; % identity for alignment delay: 40. (further
details found at the world-wide-web at
invitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEA-Communities/Ve-
ctor-NTI-Community/Sequence-analysis-and-data-management-software-for-PCs/-
AlignX-Module-for-Vector-NTI-Advance.reg.us.html).
Another method for determining the best overall match between a
query sequence (a sequence of the present disclosure) and a subject
sequence, also referred to as a global sequence alignment, can be
determined using the CLUSTALW computer program (Thompson et al.,
Nucleic Acids Res, 1994, 2(22): 4673-4680), which is based on the
algorithm of Higgins et al., Computer Applications in the
Biosciences (CABIOS), 1992, 8(2): 189-191). In a sequence alignment
the query and subject sequences are both DNA sequences. The result
of said global sequence alignment is in percent identity.
Parameters that can be used in a CLUSTALW alignment of DNA
sequences to calculate percent identity via pairwise alignments
are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap
Penalty=3, Gap Open Penalty=10, Gap Extension Penalty=0.1. For
multiple alignments, the following CLUSTALW parameters can be used:
Gap Opening Penalty=10, Gap Extension Parameter=0.05; Gap
Separation Penalty Range=8; % Identity for Alignment Delay=40.
The nucleic acids can be present in whole cells, in a cell lysate,
or in a partially purified or substantially pure form. A nucleic
acid is "isolated" or "rendered substantially pure" when purified
away from other cellular components with which it is normally
associated in the natural environment. To isolate a nucleic acid,
standard techniques such as the following can be used: alkaline/SDS
treatment, CsCl banding, column chromatography, agarose gel
electrophoresis and others well known in the art.
I. Activated Protein C (aPC) and Antibodies
A. Activated Protein C
Protein C is activated by thrombin complexed with thrombomodulin on
endothelium. Unlike the few-second transient life of active
thrombin in vivo, human aPC has about a 20 minute half-life in
circulation after its generation (Berg, et al., 2003). Therefore,
one can feasibly measure a level of aPC in plasma to study its
regulation under various pathophysical conditions.
B. Antibodies to aPC
Previously, a murine antibody HAPC1573 was developed which enhanced
FL-aPC binding on the endothelial cells. HAPC1573 facilitated aPC
internalization on endothelium through the interaction of Gla
domain of aPC and EPCR on the cells, and this internalization could
be blocked by either EPCR blocking Ab or Gla domain blocking Ab
(HPC1575). HAPC1573 also dramatically altered the kinetic
parameters of aPC toward its chromogenic substrate, Spectrozyme
PCa. This profound change of aPC toward small peptide substrate in
the presence of HAPC1573 indicated that this mAb recognized an
epitope near active site of aPC and the interaction of Ab and
antigen dramatically increased the affinity of APC toward small
peptide substrate but decreased the off rate of product from aPC
catalytic site. HAPC1573 also almost completely diminished the
prolongation effect of aPC in factor Xa initiated one-stage plasma
clotting assay, suggesting that the interaction of HAPC1573 and aPC
prevents aPC from cleaving factor Va. Surprisingly, HAPC1573 did
not inhibit but actually enhanced aPC cleaving histone H3 and H4.
Consistently, HAPC1573 did not inhibit but slightly enhanced aPC
cytoprotection activity on endothelium against histone H3 and H4.
Finally, their results show that HAPC1573, recognizes aPC, but not
Protein C. See U.S. Pat. No. 8,153,766.
Recent studies have shown that anticoagulant activity of aPC is
dispensable for its cytoprotective function, but aPC cleavage
activity toward PAR1 might be essential for its anti-apoptotic
effect (Mosnier et al., 2004). However, the cytoprotection effect
of aPC has been shown not only in endothelial cells which express
EPCR, but also on other cells such as neuron and keratinocytes
which do not express EPCR on their cell surfaces (Guo et al., 2004;
Berg et al., 2003), indicating other mechanisms than PAR1 mediated
aPC signaling might exist.
C. Applications of the Technology
The ability to distinguish between Protein C and aPC demonstrates
the utility of antibodies in a convenient ELISA method for
measuring aPC level in plasma in vivo. Typically, it takes less
than 4 hours to measure a plasma sample containing 1 ng/ml APC with
this method compared to 19 hours or even weeks with enzyme capture
assays (Gruber and Griffen, 1992; Liaw et al., 2003).
Also, as discussed above, HAPC1573 altered aPC cleavage activity
toward a chromogenic peptide substrate and also blocked aPC
anticoagulant activity in a plasma clotting assay, suggesting this
mAb recognizes an epitope near the aPC active site and alters its
catalytic activity upon antibody-antigen binding. At the same time,
HAPC1573 actually enhanced aPC cleaving extracellular histones, and
enhanced APC cytoprotection activity on endothelium against
histones. This indicates that APC anticoagulant activity for
cleaving activated factor V and VIII is not required for its
cytoprotection activity by cleaving extracellular histones.
Cleaving extracellular histones independent from its anticoagulant
activity might be one of the molecular mechanisms of aPC regulation
inflammation and cytoprotection.
Thus, such antibodies against aPC can, for example, be used in
treatment of hemophilia A patients. aPC cleaves both factor VIIIa
and factor Va and thus negatively affects blood clotting. In
hemophilia A patients, factor VIII levels are low and the
inactivation of factor Va by aPC is probably a major pathway to
regulate hemostasis and thrombosis in these patients. Recent
clinical reports demonstrated factor V Leiden mutant which is
resistant to aPC cleavage was beneficial to hemophilia A patients
regarding their bleeding symptom (van't Zant et al., 1997).
Blocking aPC anticoagulant activity toward factor Va in vivo with
an antibody is an alternative approach for hemophilia A treatments,
especially for those patients who have high level factor VIII
inhibitors so that the factor VIII replacement therapy would not be
very effective.
In other embodiments, another possible clinical application for
antibodies against aPC is in the treatment of trauma patients
wherein homeostasis is disrupted, excessive bleeding is likely, and
surgical intervention is delayed to regain homeostatis. Treatment
with antibodies can selectively restore the pro-coagulant state
without eliminating the cytoprotective or anti-inflammatory
activities of APC.
Yet another clinical application of antibodies against aPC is in
combination with aPC in sepsis treatment. Its bleeding side effect
in patients is due to aPC anticoagulant activity. Because HAPC1573
blocked aPC anticoagulant activity while maintaining, and even
enhancing, aPC cytoprotective effect, the mAb-aPC complex can be a
better therapeutic than aPC alone regarding its bleeding side
effect.
II. Antibody Structure
Antibodies comprise a large family of glycoproteins with common
structural features. An antibody is comprised of four polypeptides
that form a three dimensional structure. Typically, an antibody is
comprised of two different polypeptides, the heavy chain and the
light chain. An antibody molecule is comprised of one or more of
these units, each unit comprising two heavy chains and two light
chains. An antibody molecule typically consists of three functional
domains: the Fc, Fab, and antigen-binding site.
There are five different types of heavy chain polypeptides
designated as .alpha., .delta., .epsilon., .gamma., and .mu.. There
are two different types of light chain polypeptides designated
.kappa. and .lamda.. An antibody typically contains only one type
of heavy chain and only one type of light chain, although any light
chain can associate with any heavy chain.
The carboxyl terminal of each heavy chain polypeptide is known as
the constant (Fc) region. The amino terminal of each heavy and
light chain polypeptide is known as the variable (V) region. Within
the variable regions of the chains are hypervariable regions known
as complementarity determining regions (CDRs). The variable regions
of one heavy chain and one light chain associate to form an
antigen-binding site. Each heavy chain and each light chain
includes three CDRs. The six CDRs of an antigen-binding site define
the amino acid residues that form the actual binding site for the
antigen. CDR variability accounts for the diversity of antigen
recognition.
Antibodies against aPC may be defined by sequences set forth in the
following table:
TABLE-US-00002 TABLE 1 Antibody Sequences FR.sub.1 CDR1 FR.sub.2
CDR2 FR.sub.3 CDR3 FR.sub.4 Light Chain CDR SEQ ID NO: 1 2 3 Heavy
Chain CDR SEQ ID NO: 4 5 6 Light Chain Framework Residues* 1- 39-
61- 102- 23 53 92 112 Heavy Chain Framework Residues** 1- 36- 69-
108- 30 49 100 118 *Residues in reference to SEQ ID NO: 26
**Residues in reference to SEQ ID NO: 16
III. Antibodies Against aPC
A. Antibody Fragments
Thus, in one embodiment, such molecules will comprise fragments
(such as (F(ab'), F(ab')2) that are produced, for example, by the
proteolytic cleavage of the mAbs, or single-chain immunoglobulins
producible, for example, via recombinant means. Such antibody
derivatives are monovalent. In one embodiment, such fragments can
be combined with one another, or with other antibody fragments or
receptor ligands to form "chimeric" binding molecules.
Significantly, such chimeric molecules can contain substituents
capable of binding to different epitopes of the same molecule, or
they can be capable of binding to an activated protein C epitope
and a "non-activated protein C" epitope.
A single-chain variable fragment (scFv) is another form of antibody
fragment. It comprises a fusion of the variable regions of the
heavy and light chains of immunoglobulins, linked together with a
short (usually serine, glycine) linker. This chimeric molecule
retains the specificity of the original immunoglobulin, despite
removal of the constant regions and the introduction of a linker
peptide. These molecules were created historically to facilitate
phage display where it is highly convenient to express the antigen
binding domain as a single peptide. Alternatively, scFv can be
created directly from subcloned heavy and light chains derived from
a hybridoma. Single chain variable fragments lack the constant Fc
region found in complete antibody molecules, and thus, the common
binding sites (e.g., protein A/G) used to purify antibodies. These
fragments can often be purified/immobilized using Protein L since
Protein L interacts with the variable region of kappa light
chains.
Flexible linkers generally are comprised of helix- and
turn-promoting amino acid residues such as alaine, serine and
glycine. However, other residues can function as well. Tang et al.
(1996) used phage display as a means of rapidly selecting tailored
linkers for single-chain antibodies (scFvs) from protein linker
libraries. A random linker library was constructed in which the
genes for the heavy and light chain variable domains were linked by
a segment encoding an 18-amino acid polypeptide of variable
composition. The scFv repertoire (approx. 5.times.10.sup.6
different members) was displayed on filamentous phage and subjected
to affinity selection with hapten. The population of selected
variants exhibited significant increases in binding activity but
retained considerable sequence diversity. Screening 1054 individual
variants subsequently yielded a catalytically active scFv that was
produced efficiently in soluble form. Sequence analysis revealed a
conserved proline in the linker two residues after the VH C
terminus and an abundance of arginines and prolines at other
positions as the only common features of the selected tethers.
The recombinant antibodies against aPC can also involve sequences
or moieties that permit dimerization or multimerization of the
receptors. Such sequences include those derived from IgA, which
permit formation of multimers in conjunction with the J chain.
Another multimerization domain is the Gal4 dimerization domain. In
other embodiments, the chains can be modified with agents such as
biotin/avidin, which permit the combination of two antibodies.
In a separate embodiment, a single-chain antibody can be created by
joining receptor light and heavy chains using a non-peptide linker
or chemical unit. Generally, the light and heavy chains will be
produced in distinct cells, purified, and subsequently linked
together in an appropriate fashion (i.e., the N-terminus of the
heavy chain being attached to the C-terminus of the light chain via
an appropriate chemical bridge).
Cross-linking reagents are used to form molecular bridges that tie
functional groups of two different molecules, e.g., a stabilizing
and coagulating agent. However, it is contemplated that dimers or
multimers of the same analog or heteromeric complexes comprised of
different analogs can be created. To link two different compounds
in a step-wise manner, heterobifunctional cross-linkers can be used
that eliminate unwanted homopolymer formation. An exemplary
hetero-bifunctional cross-linker contains two reactive groups: one
reacting with primary amine group (e.g., N-hydroxy succinimide) and
the other reacting with a thiol group (e.g., pyridyl disulfide,
maleimides, halogens, etc.). Through the primary amine reactive
group, the cross-linker can react with the lysine residue(s) of one
protein (e.g., the selected antibody or fragment) and through the
thiol reactive group, the cross-linker, already tied up to the
first protein, reacts with the cysteine residue (free sulfhydryl
group) of the other protein (e.g., the selective agent).
A cross-linker having reasonable stability in blood can be
employed. Numerous types of disulfide-bond containing linkers are
known that can be successfully employed to conjugate targeting and
therapeutic/preventative agents. Linkers that contain a disulfide
bond that is sterically hindered can prove to give greater
stability in vivo, preventing release of the targeting peptide
prior to reaching the site of action. These linkers are thus one
group of linking agents.
Another cross-linking reagent is SMPT, which is a bifunctional
cross-linker containing a disulfide bond that is "sterically
hindered" by an adjacent benzene ring and methyl groups. It is
believed that steric hindrance of the disulfide bond serves a
function of protecting the bond from attack by thiolate anions such
as glutathione which can be present in tissues and blood, and
thereby help in preventing decoupling of the conjugate prior to the
delivery of the attached agent to the target site. The SMPT
cross-linking reagent, as with many other known cross-linking
reagents, lends the ability to cross-link functional groups such as
the SH of cysteine or primary amines (e.g., the epsilon amino group
of lysine). Another possible type of cross-linker includes the
heterobifunctional photoreactive phenylazides containing a
cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido
salicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxysuccinimidyl
group reacts with primary amino groups and the phenylazide (upon
photolysis) reacts non-selectively with any amino acid residue.
In addition to hindered cross-linkers, non-hindered linkers also
can be employed in accordance herewith. Other useful cross-linkers,
not considered to contain or generate a protected disulfide,
include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe,
1987). The use of such cross-linkers is well understood in the art.
Another embodiment involves the use of flexible linkers. U.S. Pat.
No. 4,680,338, describes bifunctional linkers useful for producing
conjugates of ligands with amine-containing polymers and/or
proteins, especially for forming antibody conjugates with
chelators, drugs, enzymes, detectable labels and the like. U.S.
Pat. Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates
containing a labile bond that is cleavable under a variety of mild
conditions. This linker is particularly useful in that the agent of
interest can be bonded directly to the linker, with cleavage
resulting in release of the active agent. Particular uses include
adding a free amino or free sulfhydryl group to a protein, such as
an antibody, or a drug.
U.S. Pat. No. 5,856,456 provides peptide linkers for use in
connecting polypeptide constituents to make fusion proteins, e.g.,
single chain antibodies. The linker is up to about 50 amino acids
in length, contains at 5 least one occurrence of a charged amino
acid (e.g., arginine or lysine) followed by a proline, and is
characterized by greater stability and reduced aggregation. U.S.
Pat. No. 5,880,270 discloses aminooxy-containing linkers useful in
a variety of immunodiagnostic and separative techniques.
B. Antibody Conjugates
Further provided are antibody conjugates. For both diagnostic and
therapeutic purposes, one can link or covalently bind or complex an
agent to an antibody. Such a molecule or moiety can be, but is not
limited to, at least one effector or reporter molecule. A reporter
molecule is defined as any moiety which can be detected using an
assay. Non-limiting examples of reporter molecules which have been
conjugated to antibodies include enzymes, radiolabels, haptens,
fluorescent labels, phosphorescent molecules, chemiluminescent
molecules, chromophores, luminescent molecules, photoaffinity
molecules, colored particles or ligands, such as biotin.
Certain examples of antibody conjugates are those conjugates in
which the antibody is linked to a detectable label. "Detectable
labels" are compounds and/or elements that can be detected due to
their specific functional properties, and/or chemical
characteristics, the use of which allows the antibody to which they
are attached to be detected, and/or further quantified if desired.
Another such example is the formation of a conjugate comprising an
antibody linked to a cytotoxic or anti cellular agent, and can be
termed "immunotoxins."
Antibody conjugates are used as diagnostic agents. Antibody
diagnostics generally fall within two classes, those for use in in
vitro diagnostics, such as in a variety of immunoassays, and/or
those for use in vivo diagnostic protocols, generally known as
"antibody-directed imaging."
Many appropriate imaging agents are known in the art, as are
methods for their attachment to antibodies (see, for e.g., U.S.
Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated
herein by reference). The imaging moieties used can be paramagnetic
ions; radioactive isotopes; fluorochromes; NMR-detectable
substances; X-ray imaging.
In the case of paramagnetic ions, one might mention by way of
example ions such as chromium (III), manganese (II), iron (III),
iron (II), cobalt (II), nickel (II), copper (II), neodymium (III),
samarium (III), ytterbium (III), gadolinium (III), vanadium (II),
terbium (III), dysprosium (III), holmium (III) and/or erbium (III).
Ions useful in other contexts, such as X-ray imaging, include but
are not limited to lanthanum (III), gold (III), lead (II), and
especially bismuth (III).
In the case of radioactive isotopes for therapeutic and/or
diagnostic application, one might mention astatine.sup.211,
.sup.14car- bon, .sup.51chromium, .sup.36chlorine, .sup.57cobalt,
.sup.58cobalt, copper.sup.67, .sup.152Eu, gallium.sup.67,
.sup.3hydrogen, iodine .sup.123, iodine.sup.125, iodine .sup.131,
indium.sup.111, .sup.59iron, .sup.32phosphorus, rhenium.sup.186,
rhenium.sup.188, .sup.75selenium, .sup.35sulphur,
technicium.sup.99m and/or yttrium.sup.90. .sup.125I is often being
commonly used in certain embodiments, and technicium.sup.99m and/or
indium.sup.111 are also often used due to their low energy and
suitability for long range detection. Radioactively labeled
monoclonal antibodies can be produced according to well-known
methods in the art. For instance, monoclonal antibodies can be
iodinated by contact with sodium and/or potassium iodide and a
chemical oxidizing agent such as sodium hypochlorite, or an
enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal
antibodies can be labeled with technetium.sup.99m by ligand
exchange process, for example, by reducing pertechnate with
stannous solution, chelating the reduced technetium onto a Sephadex
column and applying the antibody to this column. Alternatively,
direct labeling techniques can be used, e.g., by incubating
pertechnate, a reducing agent such as SNCl.sub.2, a buffer solution
such as sodium-potassium phthalate solution, and the antibody.
Intermediary functional groups which are often used to bind
radioisotopes which exist as metallic ions to antibody are
diethylenetriaminepentaacetic acid (DTPA) or ethylene
diaminetetracetic acid (EDTA).
Among the fluorescent labels contemplated for use as conjugates
include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665,
BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3,
Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green
488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,
Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET,
Tetramethylrhodamine, and/or Texas Red.
Another type of antibody conjugates contemplated are those intended
primarily for use in vitro, where the antibody is linked to a
secondary binding ligand and/or to an enzyme (an enzyme tag) that
will generate a colored product upon contact with a chromogenic
substrate. Examples of suitable enzymes include urease, alkaline
phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.
Secondary binding ligands are biotin and/or avidin and streptavidin
compounds. The use of such labels is well known to those of skill
in the art and are described, for example, in U.S. Pat. Nos.
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149
and 4,366,241; each incorporated herein by reference.
Yet another known method of site-specific attachment of molecules
to antibodies comprises the reaction of antibodies with
hapten-based affinity labels. Essentially, hapten-based affinity
labels react with amino acids in the antigen binding site, thereby
destroying this site and blocking specific antigen reaction.
However, this can not be advantageous since it results in loss of
antigen binding by the antibody conjugate.
Molecules containing azido groups can also be used to form covalent
bonds to proteins through reactive nitrene intermediates that are
generated by low intensity ultraviolet light (Potter & Haley,
1983). In particular, 2- and 8-azido analogues of purine
nucleotides have been used as site-directed photoprobes to identify
nucleotide binding proteins in crude cell extracts (Owens &
Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides
have also been used to map nucleotide binding domains of purified
proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et
al., 1989) and can be used as antibody binding agents.
Several methods are known in the art for the attachment or
conjugation of an antibody to its conjugate moiety. Some attachment
methods involve the use of a metal chelate complex employing, for
example, an organic chelating agent such as described in U.S. Pat.
Nos. 4,472,509 and 4,938,948, each incorporated herein by
reference). Monoclonal antibodies can also be reacted with an
enzyme in the presence of a coupling agent such as glutaraldehyde
or periodate. Conjugates with fluorescein markers are prepared in
the presence of these coupling agents or by reaction with an
isothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast
tumors is achieved using monoclonal antibodies and the detectable
imaging moieties are bound to the antibody using linkers such as
methyl-p-hydroxybenzimidate or
N-succinimidyl-3-(4-hydroxyphenyl)propionate.
In other embodiments, derivatization of immunoglobulins by
selectively introducing sulfhydryl groups in the Fc region of an
immunoglobulin, using reaction conditions that do not alter the
antibody combining site are contemplated. Antibody conjugates
produced according to this methodology are disclosed to exhibit
improved longevity, specificity and sensitivity (U.S. Pat. No.
5,196,066, incorporated herein by reference). Site-specific
attachment of effector or reporter molecules, wherein the reporter
or effector molecule is conjugated to a carbohydrate residue in the
Fc region have also been disclosed in the literature (O'Shannessy
et al., 1987). This approach has been reported to produce
diagnostically and therapeutically promising antibodies which are
currently in clinical evaluation.
In another embodiment, one may choose to modify the immunoglobulins
to improve their stability and half-life in vivo. PEGylation is one
such process that involves covalent attachment of polyethylene
glycol (PEG) polymer chains to the antibody. PEGylation is
routinely achieved by incubation of a reactive derivative of PEG
with the target molecule. The covalent attachment of PEG can "mask"
the antibody from the host's immune system (reduced immunogenicity
and antigenicity), and increase the hydrodynamic size (size in
solution) of the agent which prolongs its circulatory time by
reducing renal clearance. PEGylation can also provide water
solubility. Other polymers used to modify antibodies include
polyethyleneimine and polylysine, often linked through succinic
acid groups.
C. Immunodetection Methods
In still further embodiments, also provided are immunodetection
methods for binding, purifying, removing, quantifying and/or
otherwise generally detecting biological components using
antibodies that react immunologically with such components. Some
immunodetection methods include enzyme linked immunosorbent assay
(ELISA), radioimmunoassay (RIA), immunoradiometric assay,
fluoroimmunoassay, chemiluminescent assay, bioluminescent assay,
and Western blot to mention a few. The steps of various useful
immunodetection methods have been described in the scientific
literature, such as, e.g., Doolittle and Ben-Zeev (1999); Gulbis
and Galand (1993); De Jager et al. (1993); and Nakamura et al.
(1987), each incorporated herein by reference.
In general, the immunobinding methods include obtaining a sample
containing a target of interest, and contacting the sample with a
first antibody that reacts immunologically with the target under
conditions effective to allow the formation of immunocomplexes. The
binding of the antibody to the target can then be assessed using a
variety of different formats.
In one format, the antibody can be linked to a solid support, such
as in the form of a column matrix, and the sample suspected of
containing the target will be applied to the immobilized antibody.
The unwanted components will be washed from the column, leaving the
target immunocomplexed to the immobilized antibody to be
eluted.
The immunobinding methods also include methods for detecting and
quantifying the amount of an target in a sample and the detection
and quantification of any immune complexes formed during the
binding process. Here, one would obtain a sample suspected of
containing a target, and contact the sample with an antibody
against the target, and then detect and quantify the amount of
immune complexes formed under the specific conditions.
In terms of antigen detection, the biological sample analyzed can
be any sample that is suspected of containing a target, such as,
for example, a body fluid like blood, serum, plasma, mucous, urine,
saliva, tears or semen. Alternatively, a tissue can be used.
Contacting the chosen biological sample with the antibody under
effective conditions and for a period of time sufficient to allow
the formation of immune complexes (primary immune complexes) is
generally a matter of simply adding the antibody composition to the
sample and incubating the mixture for a period of time long enough
for the antibodies to form immune complexes with, i.e., to bind to
targets that react immunologically with antibodies present. After
this time, the sample-antibody composition, such as a tissue
section, ELISA plate, dot blot or western blot, will generally be
washed to remove any non-specifically bound species, allowing only
those molecules specifically bound within the primary immune
complexes to be detected.
In general, the detection of immunocomplex formation is well known
in the art and can be achieved through the application of numerous
approaches. These methods are generally based upon the detection of
a label or marker, such as any of those radioactive, fluorescent,
biological and enzymatic tags. U.S. Patents concerning the use of
such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated
herein by reference. Of course, one can find additional advantages
through the use of a secondary binding ligand such as a second
antibody and/or a biotin/avidin ligand binding arrangement, as is
known in the art.
The antibody employed in the detection can itself be linked to a
detectable label, wherein one would then simply detect this label,
thereby allowing the amount of the primary immune complexes in the
composition to be determined. Alternatively, the first antibody
that becomes bound within the primary immune complexes can be
detected by means of a second binding ligand that has binding
affinity for the antibody. In these cases, the second binding
ligand can be linked to a detectable label. The second binding
ligand is itself often an antibody, which can thus be termed a
"secondary" antibody. The primary immune complexes are contacted
with the labeled, secondary binding ligand, or antibody, under
effective conditions and for a period of time sufficient to allow
the formation of secondary immune complexes. The secondary immune
complexes are then generally washed to remove any non-specifically
bound labeled secondary antibodies or ligands, and the remaining
label in the secondary immune complexes is then detected.
Further methods include the detection of primary immune complexes
by a two step approach. A second binding ligand, such as an
antibody, that has binding affinity for the antibody is used to
form secondary immune complexes, as described above. After washing,
the secondary immune complexes are contacted with a third binding
ligand or antibody that has binding affinity for the second
antibody, again under effective conditions and for a period of time
sufficient to allow the formation of immune complexes (tertiary
immune complexes). The third ligand or antibody is linked to a
detectable label, allowing detection of the tertiary immune
complexes thus formed. This system can provide for signal
amplification if this is desired.
One method of immunodetection designed by Charles Cantor uses two
different antibodies. A first step biotinylated, monoclonal or
polyclonal antibody is used to detect the target antigen(s), and a
second step antibody is then used to detect the biotin attached to
the complexed biotin. In that method the sample to be tested is
first incubated in a solution containing the first step antibody.
If the target antigen is present, some of the antibody binds to the
antigen to form a biotinylated antibody/antigen complex. The
antibody/antigen complex is then amplified by incubation in
successive solutions of streptavidin (or avidin), biotinylated DNA,
and/or complementary biotinylated DNA, with each step adding
additional biotin sites to the antibody/antigen complex. The
amplification steps are repeated until a suitable level of
amplification is achieved, at which point the sample is incubated
in a solution containing the second step antibody against biotin.
This second step antibody is labeled, as for example with an enzyme
that can be used to detect the presence of the antibody/antigen
complex by histoenzymology using a chromogen substrate. With
suitable amplification, a conjugate can be produced which is
macroscopically visible.
Another known method of immunodetection takes advantage of the
immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method
is similar to the Cantor method up to the incubation with
biotinylated DNA, however, instead of using multiple rounds of
streptavidin and biotinylated DNA incubation, the
DNA/biotin/streptavidin/antibody complex is washed out with a low
pH or high salt buffer that releases the antibody. The resulting
wash solution is then used to carry out a PCR reaction with
suitable primers with appropriate controls. At least in theory, the
enormous amplification capability and specificity of PCR can be
utilized to detect a single antigen molecule.
Another ELISA in which the antigens are immobilized, involves the
use of antibody competition in the detection. In this ELISA,
labeled antibodies against an antigen are added to the wells,
allowed to bind, and/or detected by means of their label. The
amount of an antigen in an unknown sample is then determined by
mixing the sample with the labeled antibodies against the antigen
during incubation with coated wells. The presence of an antigen in
the sample acts to reduce the amount of antibody against the
antigen available for binding to the well and thus reduces the
ultimate signal. This is also appropriate for detecting antibodies
against an antigen in an unknown sample, where the unlabeled
antibodies bind to the antigen-coated wells and also reduces the
amount of antigen available to bind the labeled antibodies.
As detailed above, immunoassays, in their most simple and/or direct
sense, are binding assays. Certain immunoassays are the various
types of enzyme linked immunosorbent assays (ELISAs) and/or
radioimmunoassays (RIA) known in the art. Immunohistochemical
detection using tissue sections is also particularly useful.
However, it will be readily appreciated that detection is not
limited to such techniques, and/or western blotting, dot blotting,
FACS analyses, and/or the like can also be used. Irrespective of
the format employed, ELISAs have certain features in common, such
as coating, incubating and binding, washing to remove
non-specifically bound species, and detecting the bound immune
complexes. These are described below.
In coating a plate with either antigen or antibody, one will
generally incubate the wells of the plate with a solution of the
antigen or antibody, either overnight or for a specified period of
hours. The wells of the plate will then be washed to remove
incompletely adsorbed material. Any remaining available surfaces of
the wells are then "coated" with a non-specific protein that is
antigenically neutral with regard to the test antisera. These
include bovine serum albumin (BSA), casein or solutions of milk
powder. The coating allows for blocking of nonspecific adsorption
sites on the immobilizing surface and thus reduces the background
caused by nonspecific binding of antisera onto the surface.
In ELISAs, it is probably more customary to use a secondary or
tertiary detection means rather than a direct procedure. Thus,
after binding of a protein or antibody to the well, coating with a
non-reactive material to reduce background, and washing to remove
unbound material, the immobilizing surface is contacted with the
biological sample to be tested under conditions effective to allow
immune complex (antigen/antibody) formation. Detection of the
immune complex then requires a labeled secondary binding ligand or
antibody, and a secondary binding ligand or antibody in conjunction
with a labeled tertiary antibody or a third binding ligand.
"Under conditions effective to allow immune complex
(antigen/antibody) formation" means that the conditions can include
diluting the antigens and/or antibodies with solutions such as BSA,
bovine gamma globulin (BGG) or phosphate buffered saline
(PBS)/Tween. These added agents also tend to assist in the
reduction of nonspecific background.
The "suitable" conditions also mean that the incubation is at a
temperature or for a period of time sufficient to allow effective
binding. Incubation steps are typically from about 1 to 2 to 4
hours or so, at temperatures on the order of 25.degree. C. to
27.degree. C., or can be overnight at about 4.degree. C. or so.
D. Purification
In certain embodiments, the antibodies against aPC can be purified.
The term "purified," as used herein, is intended to refer to a
composition, isolatable from other components, wherein the protein
is purified to any degree relative to its naturally-obtainable
state. A purified protein therefore also refers to a protein, free
from the environment in which it can naturally occur. Where the
term "substantially purified" is used, this designation will refer
to a composition in which the protein or peptide forms the major
component of the composition, such as constituting about 50%, about
60%, about 70%, about 80%, about 90%, about 95% or more of the
proteins in the composition.
Protein purification techniques are well known to those of skill in
the art. These techniques involve, at one level, the crude
fractionation of the cellular milieu to polypeptide and
non-polypeptide fractions. Having separated the polypeptide from
other proteins, the polypeptide of interest can be further purified
using chromatographic and electrophoretic techniques to achieve
partial or complete purification (or purification to
homogeneity).
Analytical methods particularly suited to the preparation of a pure
peptide are ion-exchange chromatography, exclusion chromatography;
polyacrylamide gel electrophoresis; isoelectric focusing. Other
methods for protein purification include, precipitation with
ammonium sulfate, PEG, antibodies and the like or by heat
denaturation, followed by centrifugation; gel filtration, reverse
phase, hydroxylapatite and affinity chromatography; and
combinations of such and other techniques.
In purifying an antibody against aPC, it can be desirable to
express the polypeptide in a prokaryotic or eukaryotic expression
system and extract the protein using denaturing conditions. The
polypeptide can be purified from other cellular components using an
affinity column, which binds to a tagged portion of the
polypeptide. As is generally known in the art, it is believed that
the order of conducting the various purification steps can be
changed, or that certain steps can be omitted, and still result in
a suitable method for the preparation of a substantially purified
protein or peptide.
Commonly, complete antibodies are fractionated utilizing agents
(i.e., protein A) that bind the Fc portion of the antibody.
Alternatively, antigens can be used to simultaneously purify and
select appropriate antibodies. Such methods often utilize the
selection agent bound to a support, such as a column, filter or
bead. The antibodies is bound to a support, contaminants removed,
and the antibodies released by applying conditions (salt, heat,
etc.).
Various methods for quantifying the degree of purification of the
protein or peptide will be known to those of skill in the art in
light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. Another method for assessing the purity of a fraction is
to calculate the specific activity of the fraction, to compare it
to the specific activity of the initial extract, and to thus
calculate the degree of purity. The actual units used to represent
the amount of activity will, of course, be dependent upon the
particular assay technique chosen to follow the purification and
whether or not the expressed protein or peptide exhibits a
detectable activity.
It is known that the migration of a polypeptide can vary, sometimes
significantly, with different conditions of SDS/PAGE (Capaldi et
al., 1977). It will therefore be appreciated that under differing
electrophoresis conditions, the apparent molecular weights of
purified or partially purified expression products can vary.
IV. Pharmaceutical Compositions and Uses
A. Compositions
Pharmaceutical compositions can comprise an effective amount of one
or more antibodies, therapeutic agents or additional agent
dissolved or dispersed in a pharmaceutically acceptable carrier.
Aqueous compositions comprise an effective amount of the antibody,
dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous medium. The phrases "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce an adverse, allergic or other untoward reaction when
administered to an animal, or a human, as appropriate.
As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, surfactants,
antioxidants, preservatives (e.g., antibacterial agents, antifungal
agents), isotonic agents, absorption delaying agents, salts,
preservatives, drugs, drug stabilizers, gels, binders, excipients,
disintegration agents, lubricants, sweetening agents, flavoring
agents, dyes, such like materials and combinations thereof, as
would be known to one of ordinary skill in the art (see, for
example, Remington's Pharmaceutical Sciences, 18th Ed. Mack
Printing Company, 1990, pp. 1289-1329, incorporated herein by
reference). The use of such media and agents for pharmaceutical
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. For human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologic
Standards.
The biological material should be extensively dialyzed to remove
undesired small molecular weight molecules and/or lyophilized for
more ready formulation into a desired vehicle, where appropriate.
The active compounds will then generally be formulated for
parenteral administration, e.g., formulated for injection via the
intravenous, intramuscular, sub-cutaneous, intranasal, or
intraperitoneal routes. Typically, such compositions can be
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for using to prepare solutions or suspensions
upon the addition of a liquid prior to injection can also be
prepared; and the preparations can also be emulsified.
The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that easy syringability exists. It must
be stable under the conditions of manufacture and storage and must
be preserved against the contaminating action of microorganisms,
such as bacteria and fungi.
Solutions of the active compounds as free base or pharmacologically
acceptable salts can be prepared in water suitably mixed with a
surfactant, such as hydroxypropylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
The antibodies against aPC can be formulated into a composition in
a free base, in a neutral or salt form. Pharmaceutically acceptable
salts, include the acid addition salts (formed with the free amino
groups of the protein) and which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
The carrier can also be a solvent or dispersion medium containing,
for example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyethylene glycol, and the like),
suitable mixtures thereof, and vegetable oils. The proper fluidity
can be maintained, for example, by the use of a coating, such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. The prevention of
the action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, isotonic agents can be included, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
Sterile injectable solutions are prepared by incorporating the
active compounds in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the methods of
preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
preparation of more, or highly, concentrated solutions for direct
injection is also contemplated, where the use of DMSO as solvent is
envisioned to result in extremely rapid penetration, delivering
high concentrations of the active agents to a small area.
Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
For parenteral administration in an aqueous solution, for example,
the solution should be suitably buffered if necessary and the
liquid diluent first rendered isotonic with sufficient saline or
glucose. These particular aqueous solutions are especially suitable
for intravenous, intramuscular, subcutaneous, intranasal, and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and
1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject.
In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets or
other solids for oral administration; liposomal formulations; time
release capsules; and any other form currently used, including
cremes.
In certain embodiments, the use of liposomes and/or nanoparticles
is contemplated for the formulation and administration of the
antibodies and/or analogs thereof. The formation and use of
liposomes is generally known to those of skill in the art, and is
also described below.
Nanocapsules can generally entrap compounds in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use, and such particles are
easily made.
Liposomes are formed from phospholipids that are dispersed in an
aqueous medium and spontaneously form multilamellar concentric
bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs
generally have diameters of from 25 nm to 4 .mu.m. Sonication of
MLVs results in the formation of small unilamellar vesicles (SUVs)
with diameters in the range of 200-500 .ANG., containing an aqueous
solution in the core.
The following information can also be utilized in generating
liposomal formulations. Phospholipids can form a variety of
structures other than liposomes when dispersed in water, depending
on the molar ratio of lipid to water. At low ratios the liposome is
the recommended structure. The physical characteristics of
liposomes depend on pH, ionic strength and the presence of divalent
cations. Liposomes can show low permeability to ionic and polar
substances, but at elevated temperatures undergo a phase transition
which markedly alters their permeability. The phase transition
involves a change from a closely packed, ordered structure, known
as the gel state, to a loosely packed, less-ordered structure,
known as the fluid state. This occurs at a characteristic
phase-transition temperature and results in an increase in
permeability to ions, sugars and drugs.
Liposomes interact with cells via four different mechanisms:
Endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with cell-surface components;
fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with simultaneous
release of liposomal contents into the cytoplasm; and by transfer
of liposomal lipids to cellular or subcellular membranes, or vice
versa, without any association of the liposome contents. Varying
the liposome formulation can alter which mechanism is operative,
although more than one can operate at the same time.
The therapeutic agent can comprise different types of carriers
depending on whether it is to be administered in solid, liquid or
aerosol form, and whether it needs to be sterile for such routes of
administration as injection. The antibodies against aPC can be
administered intravenously, intradermally, intraarterially,
intraperitoneally, intralesionally, intracranially,
intraarticularly, intraprostaticaly, intrapleurally,
intratracheally, intranasally, intravitreally, intravaginally,
intrarectally, topically, intramuscularly, intraperitoneally,
subcutaneously, subconjunctival, intravesicularlly, mucosally,
intrapericardially, intraumbilically, intraocularally, orally,
topically, locally, by inhalation (e.g., aerosol inhalation), by
injection, by infusion, by continuous infusion, localized perfusion
bathing target cells directly, via a catheter, via a lavage, in
cremes, in lipid compositions (e.g., liposomes), or by other
methods or any combination of the foregoing as would be known to
one of ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
incorporated herein by reference).
The actual dosage amount of a composition administered to an animal
patient can be determined by physical and physiological factors
such as body weight, severity of condition, the type of disease
being treated, previous or concurrent therapeutic interventions,
idiopathy of the patient and the route of administration. The
practitioner responsible for administration will, in any event,
determine the concentration of active ingredient(s) in a
composition and appropriate dose(s) for the individual subject.
In certain embodiments, pharmaceutical compositions can comprise,
for example, at least about 0.1% of an active compound. In other
embodiments, an active compound can comprise between about 2% to
about 75% of the weight of the unit, or between about 25% to about
60%, for example, and any range derivable therein. In other
non-limiting examples, a dose can also comprise from about 1
microgram/kg/body weight, about 5 microgram/kg/body weight, about
10 microgram/kg/body weight, about 50 microgram/kg/body weight,
about 100 microgram/kg/body weight, about 200 microgram/kg/body
weight, about 350 microgram/kg/body weight, about 500
microgram/kg/body weight, about 1 milligram/kg/body weight, about 5
milligram/kg/body weight, about 10 milligram/kg/body weight, about
50 milligram/kg/body weight, about 100 milligram/kg/body weight,
about 200 milligram/kg/body weight, about 350 milligram/kg/body
weight, about 500 milligram/kg/body weight, to about 1000
mg/kg/body weight or more per administration, and any range
derivable therein. In non-limiting examples of a derivable range
from the numbers listed herein, a range of about 5 mg/kg/body
weight to about 100 mg/kg/body weight, about 5 microgram/kg/body
weight to about 500 milligram/kg/body weight, etc., can be
administered, based on the numbers described above.
In any case, the composition can comprise various antioxidants to
retard oxidation of one or more component.
In embodiments where the composition is in a liquid form, a carrier
can be a solvent or dispersion medium comprising but not limited
to, water, ethanol, polyol (e.g., glycerol, propylene glycol,
liquid polyethylene glycol, etc.), lipids (e.g., triglycerides,
vegetable oils, liposomes) and combinations thereof. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin; by the maintenance of the required particle size
by dispersion in carriers such as, for example liquid polyol or
lipids; by the use of surfactants such as, for example
hydroxypropylcellulose; or combinations thereof. In many cases,
isotonic agents can be included, such as, for example, sugars,
sodium chloride or combinations thereof.
In other embodiments, one can use eye drops, nasal solutions or
sprays, aerosols or inhalants. Such compositions are generally
designed to be compatible with the target tissue type. In a
non-limiting example, nasal solutions are usually aqueous solutions
designed to be administered to the nasal passages in drops or
sprays. Nasal solutions are prepared so that they are similar in
many respects to nasal secretions, so that normal ciliary action is
maintained. Thus, in some embodiments the aqueous nasal solutions
usually are isotonic or slightly buffered to maintain a pH of about
5.5 to about 6.5. In addition, antimicrobial preservatives, similar
to those used in ophthalmic preparations, drugs, or appropriate
drug stabilizers, if required, can be included in the formulation.
For example, various commercial nasal preparations are known and
include drugs such as antibiotics or antihistamines.
In certain embodiments the antibodies are prepared for
administration by such routes as oral ingestion. In these
embodiments, the solid composition can comprise, for example,
solutions, suspensions, emulsions, tablets, pills, capsules (e.g.,
hard or soft shelled gelatin capsules), sustained release
formulations, buccal compositions, troches, elixirs, suspensions,
syrups, wafers, or combinations thereof. Oral compositions can be
incorporated directly with the food of the diet. Carriers for oral
administration comprise inert diluents, assimilable edible carriers
or combinations thereof. In other embodiments, the oral composition
can be prepared as a syrup or elixir. A syrup or elixir, can
comprise, for example, at least one active agent, a sweetening
agent, a preservative, a flavoring agent, a dye, a preservative, or
combinations thereof.
In certain embodiments an oral composition can comprise one or more
binders, excipients, disintegration agents, lubricants, flavoring
agents, and combinations thereof. In certain embodiments, a
composition can comprise one or more of the following: a binder,
such as, for example, gum tragacanth, acacia, cornstarch, gelatin
or combinations thereof; an excipient, such as, for example,
dicalcium phosphate, mannitol, lactose, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate or combinations
thereof; a disintegrating agent, such as, for example, corn starch,
potato starch, alginic acid or combinations thereof; a lubricant,
such as, for example, magnesium stearate; a sweetening agent, such
as, for example, sucrose, lactose, saccharin or combinations
thereof; a flavoring agent, such as, for example peppermint, oil of
wintergreen, cherry flavoring, orange flavoring, etc.; or
combinations of the foregoing. When the dosage unit form is a
capsule, it can contain, in addition to materials of the above
type, carriers such as a liquid carrier. Various other materials
can be present as coatings or to otherwise modify the physical form
of the dosage unit. For instance, tablets, pills, or capsules can
be coated with shellac, sugar or both.
Additional formulations which are suitable for other modes of
administration include suppositories. Suppositories are solid
dosage forms of various weights and shapes, usually medicated, for
insertion into the rectum, vagina or urethra. After insertion,
suppositories soften, melt or dissolve in the cavity fluids. In
general, for suppositories, traditional carriers can include, for
example, polyalkylene glycols, triglycerides or combinations
thereof. In certain embodiments, suppositories can be formed from
mixtures containing, for example, the active ingredient in the
range of about 0.5% to about 10%, and about 1% to about 2%.
The composition must be stable under the conditions of manufacture
and storage, and preserved against the contaminating action of
microorganisms, such as bacteria and fungi. It will be appreciated
that endotoxin contamination should be kept minimally at a safe
level, for example, less that 0.5 ng/mg protein.
In particular embodiments, prolonged absorption of an injectable
composition can be brought about by the use in the compositions of
agents delaying absorption, such as, for example, aluminum
monostearate, gelatin or combinations thereof.
B. Pharmaceutical Uses
The monoclonal antibody can be used for therapeutic purposes for
treating genetic and acquired deficiencies or defects in
coagulation. For example, the monoclonal antibodies in the
embodiments described above can be used to block the interaction of
aPC with its substrate, which can include Factor Va or Factor
VIIIa.
The monoclonal antibodies have therapeutic use in the treatment of
disorders of hemostasis such as thrombocytopenia, platelet
disorders and bleeding disorders (e.g., hemophilia A, hemophilia B
and hemophilia C). Such disorders can be treated by administering a
therapeutically effective amount of the anti-aPC monoclonal
antibody to a patient in need thereof. The monoclonal antibodies
also have therapeutic use in the treatment of uncontrolled bleeds
in indications such as trauma and hemorrhagic stroke. Thus, also
provided is a method for shortening the bleeding time comprising
administering a therapeutically effective amount of an anti-aPC
monoclonal antibody to a patient in need thereof.
In another embodiment, the anti-aPC antibody can be useful as an
antidote for aPC-treated patients, including for example wherein
aPC is used for the treatment of sepsis or bleeding disorder.
The antibodies can be used as monotherapy or in combination with
other therapies to address a hemostatic disorder. For example,
co-administration of one or more antibodies with a clotting factor
such as factor VIIa, factor VIII or factor IX is believed useful
for treating hemophilia. In one embodiment, provided is a method
for treating genetic and acquired deficiencies or defects in
coagulation comprising administering (a) a first amount of a
monoclonal antibody that binds to human tissue factor pathway
inhibitor and (b) a second amount of factor VIII or factor IX,
wherein said first and second amounts together are effective for
treating said deficiencies or defects. In another embodiment,
provided is a method for treating genetic and acquired deficiencies
or defects in coagulation comprising administering (a) a first
amount of a monoclonal antibody that binds to human tissue factor
pathway inhibitor and (b) a second amount of factor VIII or factor
IX, wherein said first and second amounts together are effective
for treating said deficiencies or defects, and further wherein
factor VII is not coadministered. Also included is a pharmaceutical
composition comprising a therapeutically effective amount of the
combination of a monoclonal antibody and factor VIII or factor IX,
wherein the composition does not contain factor VII. "Factor VII"
includes factor VII and factor VIIa. These combination therapies
are likely to reduce the necessary infusion frequency of the
clotting factor. By co-administration or combination therapy is
meant administration of the two therapeutic drugs each formulated
separately or formulated together in one composition, and, when
formulated separately, administered either at approximately the
same time or at different times, but over the same therapeutic
period.
In some embodiments, one or more antibodies described herein can be
used in combination to address a hemostatic disorder. For example,
co-administration of two or more of the antibodies described herein
is believed useful for treating hemophilia or other hemostatic
disorder.
The pharmaceutical compositions can be parenterally administered to
subjects suffering from hemophilia A or B at a dosage and frequency
that can vary with the severity of the bleeding episode or, in the
case of prophylactic therapy, can vary with the severity of the
patient's clotting deficiency.
The compositions can be administered to patients in need as a bolus
or by continuous infusion. For example, a bolus administration of
an inventive antibody present as a Fab fragment can be in an amount
of from 0.0025 to 100 mg/kg body weight, 0.025 to 0.25 mg/kg, 0.010
to 0.10 mg/kg or 0.10-0.50 mg/kg. For continuous infusion, an
inventive antibody present as an Fab fragment can be administered
at 0.001 to 100 mg/kg body weight/minute, 0.0125 to 1.25
mg/kg/min., 0.010 to 0.75 mg/kg/min., 0.010 to 1.0 mg/kg/min. or
0.10-0.50 mg/kg/min. for a period of 1-24 hours, 1-12 hours, 2-12
hours, 6-12 hours, 2-8 hours, or 1-2 hours. For administration of
an inventive antibody present as a full-length antibody (with full
constant regions), dosage amounts can be about 1-10 mg/kg body
weight, 2-8 mg/kg, or 5-6 mg/kg. Such full-length antibodies would
typically be administered by infusion extending for a period of
thirty minutes to three hours. The frequency of the administration
would depend upon the severity of the condition. Frequency could
range from three times per week to once every two weeks to six
months.
Additionally, the compositions can be administered to patients via
subcutaneous injection. For example, a dose of 10 to 100 mg
anti-aPC antibody can be administered to patients via subcutaneous
injection weekly, biweekly or monthly.
As used herein, "therapeutically effective amount" means an amount
of an anti-aPC monoclonal antibody or of a combination of such
antibody and factor VIII or factor IX that is needed to effectively
increase the clotting time in vivo or otherwise cause a measurable
benefit in vivo to a patient in need. The precise amount will
depend upon numerous factors, including, but not limited to the
components and physical characteristics of the therapeutic
composition, intended patient population, individual patient
considerations, and the like, and can readily be determined by one
skilled in the art.
V. Kits
Any of the compositions described herein can be comprised in a kit.
The kits will thus comprise, in suitable container, an antibody
and/or an additional agent. Other components can be included in a
kit. Diagnostic and therapeutic kits comprise in suitable
container, a pharmaceutically acceptable formulation of an antibody
in a pharmaceutically acceptable formulation. The kit can have a
single container, and/or it can have distinct container for each
compound.
When the components of the kit are provided in one and/or more
liquid solutions, the liquid solution is an aqueous solution, with
a sterile aqueous solution being one example of a particular
embodiment. The antibody can also be formulated into a syringeable
composition, in which case, the container can itself be a syringe,
pipette, and/or other such like apparatus, from which the
formulation can be applied to an infected area of the body,
injected into an animal, and/or even applied to and/or mixed with
the other components of the kit.
However, the components of the kit can be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent can also be
provided in another container.
The container will generally include at least one vial, test tube,
flask, bottle, syringe and/or other container, into which the
antibody/antibody formulation is placed, suitably allocated. The
kits can also comprise a second container for containing a sterile,
pharmaceutically acceptable buffer and/or other diluent.
The kits can also include a means for containing the vials in close
confinement for commercial sale, such as, e.g., injection and/or
blow-molded plastic containers into which the desired vials are
retained.
Irrespective of the number and/or type of containers, the kits can
also comprise, and/or be packaged with, an instrument for assisting
with the injection/administration and/or placement of the ultimate
antibody within the body of an animal. Such an instrument can be a
syringe, pipette, forceps, and/or any such medically approved
delivery vehicle.
VI. EXAMPLES
The following examples are included to demonstrate embodiments. It
should be appreciated by those of skill in the art that the
techniques disclosed in the examples which follow represent
techniques discovered by the inventor to function well in the
practice, and thus can be considered to constitute modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments which are disclosed and still obtain a
like or similar result without departing from the spirit and
scope.
Example 1--Materials and Methods
Design of Humanized 1573 VH/VL.
Protein and DNA sequences information of mouse aPC monoclonal
antibody were obtained. The humanization design was done using the
following method: The VH/VL CDR residues were determined and
annotated with Kabat numbering system (world-wide-web at
bioinf.org.uk/abs/#kabatnum). The canonical structures of the VH/VL
CDRs were determined based on reports in literature (1-2). Based on
VH/VL CDR canonical structures, the human germline framework
acceptors with the same canonical structures were selected.
1573 sequence was used to blast search PDB database and obtain
known antibody structures sharing the highest sequence identities
with the target antibody. Based on the output of blast search and
sequence identity ratio, 1M71, 1M7D, and 1M7I were selected as
template for VH modeling, while 1IQW, 11T9 and 2GCY were selected
as template for VL chain modeling. Schrodinger suite software was
used to build homology models for VL and VH chains, with loop
optimization. Then the output models were analyzed with software
"contact" in CCP4 suite to give a list of all residues in framework
regions that interact with residues from CDR regions within 4
.ANG.. Based on the output of software and visual inspection with
the model, the following residues in framework were identified as
residues that contribute to the supporting of CDR loops. Those
were: light chain residues Asp70, Tyr36, Thr69, Phe71, Ile2 and
Tyr49; heavy chain residues Arg94, Arg38, Glu46, Trp47, Asp73,
Arg71, and Trp102. For the design of humanized VH, residues
supporting loop structures and VH/VL interface were identified
(International Application No. WO2008021156). Residues important
for loop conformation and VH/VL interface were to be back-mutated.
Then the VH sequences with the back-mutations were aligned with the
selected germline sub-family. The identities and similarities to
each individual human germline framework sequences within the same
canonical subsets were analyzed and the germline sequence with the
best overall homology to the murine VH sequence was identified. It
was selected as the acceptor human germline framework for grafting
VH CDRs. Additional considerations for mutations included a Q1E
mutation used to eliminate N-terminal pyroglutamate formation.
Mutations also included those to maintain consensus within the
selected VH family, for CDR canonical structures and VH/VL
interface. Mutations might also include those identified as within
4 .ANG. from the CDR binding region according to molecular
modeling. Analysis was performed to make sure no N-linked
glycosylation pattern (N-{P}-S/T) was found in the proposed
humanized construct.
The human JH region was selected based on best sequence homology.
The humanized VL sequences were also designed based on such method
stated above.
Generation of HC and LC Expression Plasmids.
Humanized V-region sequences were built using gene de novo
synthesis approach. The PCR amplified VHs were cloned into pCP-hCgl
expression vector by homologous recombination. Amplified Vks were
cloned into pCP-hck vector using same method.
The variable regions of chimera HC and LC (VH and VL) were
PCR-amplified from 1573. Gel-purified PCR product was cloned into
the same vectors as humanized V-region by homologous
recombination.
Transient Transfection of HEK293 Cells.
Approximately 24 hrs before transfection, pass FreeStyle.TM. 293E
at 0.5.times.10.sup.6 cells/ml and cells were sharked at 120
rpm/min, at 37.degree. C., 8% CO.sub.2. On the day of transfection,
the cell density should be about 1.0-1.2.times.10.sup.6/ml. The
cells were split to 1.times.10.sup.6/ml with growth medium. To
ensure optimal transfection, viability of the cells was determined
to be >95%. DNA was diluted in FreeStyle.TM. 293 expression
medium (293E) in a volume equivalent to one-tenth of culture
transfected. PEI was added into DNA; the mixture was vortex
immediately and incubated for 10 min at room temperature prior to
its addition to the cells. The final concentration of DNA to PEI
ratio was 1:2.
Purification of Humanized 1573 IgG Antibodies.
Conditioned medium above on day 6 was loaded onto a 1 ml Protein A
column, which was pre-equilibrated by 10 ml PBS, pH7.0. The column
was then washed with equilibrating buffer to baseline after sample
loading. After washed, the column was eluted with 100 mM Glycin-HCl
pH3.0, followed with immediate addition of 1M Tris-HCl solution to
adjust pH value to 8.0. The final product was dialyzed against PBS
solution. Protein purity was analyzed by SDS-PAGE, SEC and its
concentration was determined by Bradford method.
Size Exclusion Chromatography Analysis of the Purified
Antibodies.
SEC for analyzing purified antibody was carried out with a Superdex
200 5/150, GL column using a HPLC system (LC-20AD, Shimadzu) at
ambient temperature. PBS buffer pH 7.0, at a flow rate 0.3 mL/min
was used as the mobile phase. The protein detection was under 280
nm.
Immobilization of Anti-Mouse FC Antibody onto CM5 Chip.
A CM5 sensor chip was activated in FC2 by 6-min injection (10
.mu.l/min) of freshly prepared 1:1 50 mM NHS: 200 mM EDC. Then anti
mouse FC antibody in 10 mM sodium acetate buffer PH 5.0 (1.4 .mu.l
diluted in 90 .mu.l NaAc, pH 5.0) was injected onto the activated
chip at 5 .mu.l/min (HBS-EP running buffer: 10 mM HEPES, pH 7.4,
150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20). The remaining
active coupling sites were blocked with 7 min injection of 1M
ethanolamine at 10 .mu.l/min. About 9200 RU was produced.
Immobilization of Anti-Human FC Antibody onto CM5 Chip.
A CM5 sensor chip was activated in FC2 by 7-min injection (10
.mu.l/min) of freshly prepared 1:1 50 mM NHS: 200 mM EDC. Then anti
human FC antibody in 10 mM sodium acetate buffer PH 5.0 (2.5 .mu.l
diluted in 90 .mu.l NaAc, pH 5.0) was injected onto the activated
chip at 5 .mu.l/min (HBS-EP running buffer: 10 mM HEPES, pH 7.4,
150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20). The remaining
active coupling sites were blocked with 7 min injection of 1M
ethanolamine at 10 .mu.l/min. About 6400 RU was produced.
Biacore Analysis of Human aPC Binding to 1573 Antibodies.
1573 antibody was first captured on the anti-human FC IgG coated
CM5 chip, followed by injection of antigen human aPC at
concentration of 0, 1.25, 2.5, 5, 10 and 20 nM. Cycle conditions
were as follows: 30 .mu.l/min for 180 s of association phase and
500 s of dissociation phase. The surface was regenerated with a 30
s injection of Gly pH1.5 at 10 .mu.l/min. HBS-EP running buffer: 10
mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20.
Kinetics was calculated with Biacore X100 evaluation software
ver2.0.
Biacore Analysis of Cyno aPC Binding to 1573 Antibodies.
1573 antibody was first captured on the anti-human FC IgG-coated
CM5 chip, followed by injection of antigen cyno aPC at
concentration of 0, 5, 10, 20, 40, 80 nM. Cycle conditions were as
follows: 30 .mu.l/min for 180 s of association phase and 420 s of
dissociation phase. The surface was regenerated with a 45 s
injection of Gly pH1.5 at 10 .mu.l/min. Kinetics was calculated
with Biacore X100 evaluation software ver2.0.
Binding ELISA of Purified Antibodies.
Plates (Nunc, cat#442404) were coated with 100 .mu.l of human aPC
(1 ng/ml), hPC (2 ng/ml), monkey aPC (2 ng/ml), or monkey PC (2
ng/ml) diluted in DPBS (Gibco, cat#14040) overnight (o/n) at
4.degree. C. After washing, the ELISA plate was blocked with 200
.mu.l MPBST for 1 hr at RT, and tapped dry on a stack of paper
towels. To each well 100 .mu.l of IgG to be tested was added, and
incubated 1 hr at RT (for EC.sub.50 determination start at 20 nM
and do 1:3 dilutions).
Plates were washed 5.times. with PBST. 100 .mu.l of anti-hIgG
Fc-HRP (Sigma, cat#A0170) diluted 1:10000 in PBST was added to each
well. Plates were washed and 100 .mu.l/well of TMB substrate was
added and incubated at room temperature for 5 min. 100 .mu.l/well
of 1N HCl was added to terminate reaction. The plate was read with
an ELISA plate reader (Biotek, Elx405) at 450 nm wavelength.
Example 2--Results
Design and Sequence Analysis of Chimera and Humanized 1573
Antibody.
Variable region sequence annotations with Kabat numbering
(world-wide-web at bioinf.org.uk/abs/#kabatnum) CDR residues are
underlined:
TABLE-US-00003 VH: (SEQ ID NO: 31; DNA is SEQ ID NO: 33)
EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYYLNWVRQSPEKGLEWVAD
IRLKSNNYEKHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCIR
EGDYFDYWGQGTTLTVSS VL: (SEQ ID NO: 32; DNA is SEQ ID NO: 34)
NIVLTQSPASLAVSLGQRATISCRASESVDSFGATFMHWYQQKPGQPPKL
LIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPY TFGGGTKLEIKR
1573 VH canonical structure: 1-4-based on H1 and H2 canonical
structures, the VH can be humanized to subset of the VH3 germline
framework sequences. 1573 Vk canonical structure: 4-1-1-based on a
4-1-1 Vk CDR canonical structure, the Vk can be humanized to subset
of the VKII germline framework sequences.
1573 VH Humanization Design.
Germline VH3-72 had the best overall homology with the 1573 VH
sequence. It was selected as the acceptor human germline framework
for grafting 1573 VH CDR sequences. hJH6 will be used for 1573
humanization due to best homology. All possible back-mutations are
listed below. G49A--Vernier Zone residue, canonical residue (effect
CDR H2, score 3), human residue. N76S--Canonical residue (effect
CDRH1, score 3), human residue L78V--Vernier Zone residue,
canonical residue (effect CDRH1, score 3), human residue
A93I--Vernier zone residue. Canonical residue (effect CDR H3, score
3), human residue. Humanized 1573 VH design with CDRs underlined
and back-mutations double-underlined:
TABLE-US-00004 1573 VH (SEQ ID NO: 15)
EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYYLNWVRQSPEKGLEWVAD
IRLKSNNYEKHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCIR
EGDYFDYWGQGTTLTVSS H1573 VH.1 (SEQ ID NO: 16)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGD
IRLKSNNYEKHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR
EGDYFDYWGQGTTVTVSS H1573 VH.1A (SEQ ID NO: 17)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGD
IRLKSNNYEKHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC R
EGDYFDYWGQGTTVTVSS H1573 VH.1B (SEQ ID NO: 18)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGD
IRLKSNNYEKHYAESVKGRFTISRDDSKNS YLQMNSLKTEDTAVYYC R
EGDYFDYWGQGTTVTVSS H1573 VH.1C (SEQ ID NO: 19)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWV D
IRLKSNNYEKHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC R
EGDYFDYWGQGTTVTVSS H1573 VH.1D (SEQ ID NO: 20)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGD
IRLKSNNYEKHYAESVKGRFTISRDDSK SLYLQMNSLKTEDTAVYYC R
EGDYFDYWGQGTTVTVSS H1573 VH.1E (SEQ ID NO: 21)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVAD
IRLKSNNYEKHYAESVKGRFTISRDDSKNS YLQMNSLKTEDTAVYYC R
EGDYFDYWGQGTTVTVSS H1573 VH.1F (SEQ ID NO: 22)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVAD
IRLKSNNYEKHYAESVKGRFTISRDDSK S YLQMNSLKTEDTAVYYC R
EGDYFDYWGQGTTVTVSS VH3-72 (SEQ ID NO: 23)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVGR
TRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR VH3-72/JH6 (SEQ
ID NO: 24) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVGR
TRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR
-------WGQGTTVTVSS
1573 Vk Humanization Design.
The Vk germline A2 had the best overall homology to the 1573 Vk
sequence. It was selected as the acceptor human germline framework
for grafting 1573 Vk CDR sequences. hJk2 will be used for 1573
humanization due to best homology. Residues important for loop
conformation and VH/VL interface are highlighted in yellow and the
CDRs with the Kabat numbers in red.
Possible back-mutations in 1573Vk humanization design:
M4L--Vernier zone residue (effect CDRL1, 3, score 3), human
residue
G68R--Vernier zone residue
Humanized 1573 Vk design with CDR's underlined and back-mutations
in double-underline:
TABLE-US-00005 1573 Vk (SEQ ID NO: 25)
NIVLTQSPASLAVSLGQRATISCRASESVDSFGATFMH-
WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAA
TYYCQQNNEDPYTFGGGTKLEIKR H1573Vk.2 (SEQ ID NO: 26)
DIVMTQTPLSLSVTPGQPASISCRASESVDSFGATFMH-
WYLQKPGQPPQLLIYLASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVG
VYYCQQNNEDPYTFGQGTKLEIKR H1573Vk.2A (SEQ ID NO: 27) DIV
TQTPLSLSVTPGQPASISCRASESVDSFGATFMH-
WYLQKPGQPPQLLIYLASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVG
VYYCQQNNEDPYTFGQGTKLEIKR H1573Vk.2B (SEQ ID NO: 28)
DIVMTQTPLSLSVTPGQPASISCRASESVDSFGATFMH-
WYLQKPGQPPQLLIYLASNLESGVPDRFSGSGS TDFTLKISRVEAEDVG
VYYCQQNNEDPYTFGQGTKLEIKR H1573Vk.2C (SEQ ID NO: 29) DIV
TQTPLSLSVTPGQPASISCRASESVDSFGATFMH-
WYLQKPGQPPQLLIYLASNLESGVPDRFSGSGS TDFTLKISRVEAEDVG
VYYCQQNNEDPYTFGQGTKLEIKR A2 (SEQ ID NO: 30)
DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQ
LLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLP
No N-linked glycosylation pattern (N-{P}-S/T) was found in the
proposed humanized construct in both VH and VL.
Humanized VH and VL Cloning.
All humanized VH and VL were built by gene de novo synthesis
approach and clone them into 293 expression vector pCp. Constructs
for humanized antibody are summarized in Table 2:
TABLE-US-00006 TABLE 2 Construct summary for 1573 humanization VH
(VH3-72) + Construction VL (A2) + Construction JH6/FW4 status
JK2/FW4 status H1573VH.1 H1573Vk.2 H1573VH.1A H1573Vk.2A H1573VH.1B
H1573Vk.2B H1573VH.1C H1573Vk.2C H1573VH.1D 1573Vk (chimeric)
H1573VH.1E H1573VH.1F 1573VH (chimeric)
Expression and Purification of Chimera and Humanized 1573
Antibody.
293 transfection designs were summarized in Table 3. Conditioned
medium (Method 3.3) on day 6 was loaded onto a 1 ml Protein A
column. Recombinant 1573 antibody was collected, purified. The
chimera and humanized 1573 IgG proteins all showed good expression
levels.
TABLE-US-00007 TABLE 3 HEK293 co-transfection design VH 1573VH VK
H1573VH.1 H1573VH.1A H1573VH.1B H1573VH.1C H1573VH.1D H1573VH.1E
H1573V- H.1F chimeric H1573Vk.2 Hu1573-3 Hu1573-6 Hu1573-9
Hu1573-12 Hu1573-15 H1573Vk.2A Hu1573-4 Hu1573-7 Hu1573-10
Hu1573-13 Hu1573-16 H1573Vk.2B Hu1573-5 Hu1573-8 Hu1573-11
Hu1573-14 Hu1573-17 H1573Vk.2C Hu1573-1 Hu1573-2 1573Vk
chimetric
SEC Characterization of Purified Chimera and Humanized 1573
Antibody.
To evaluate the purity and percentage of aggregation of the
purified chimera 1573 and humanized antibodies, the samples were
loaded onto SEC respectively (FIG. 1). All CP generated antibodies
was eluted as a single sharp peak around 5.75 min with PBS pH 7.0
buffer. The elution time is very close to that of one of the
protein markers (158 kDa, elution time is 5.85 min), suggesting
most of them show more than 90% of monomer, two antibodies show
more than 80% monomer.
Biacore Analysis of 1573 Binding to the Humanized Antibodies.
aPC was immobilized directly onto a new CM5 chip using amine
coupling method. After overnight equilibrium, 100 nM 1573
antibodies were injected over the surface and strong signal was
detected. Therefore, the affinity determination was done using this
chip. Each of antibody was injected over the chip surfaces
respectively at 30 .mu.l/min for 180 s of association phase and 300
s of dissociation phase.
TABLE-US-00008 TABLE 4 Kinetic parameters for interaction between
1573 antibodies and antigen hAPC cyno APC determined by surface
plasmon resonance (data fit using 1:1 binding model) Binding to
human APC Binding to cyno APC Overall Overall Antibody affinity
affinity Code ka (1/Ms) kd (1/s) KD (M) ka (1/Ms) kd (1/s) KD (M)
1573 Chimeric 2.32E+06 0.008112 3.49E-09 5.55E+05 0.007664 1.38E-08
1573 mouse 9.48E+05 0.005199 5.49E-09 3.44E+05 0.007254 2.11E-08 Hu
1573-1 1.46E+06 0.005538 3.80E-09 2.40E+05 0.004016 1.67E-08 Hu
1573-2 1.35E+06 0.00624 4.61E-09 2.35E+05 0.003843 1.64E-08 Hu
1573-3 1.44E+06 0.008182 5.70E-09 7.43E+05 0.01053 1.42E-08 Hu
1573-4 1.52E+06 0.007723 5.10E-09 7.43E+05 0.01053 1.42E-08 Hu
1573-5 1.54E+06 0.006397 4.16E-09 2.02E+05 0.002926 1.45E-08 Hu
1573-6 1.43E+06 0.008477 5.94E-09 6.27E+05 0.007074 1.13E-08 Hu
1573-7 1.36E+06 0.008574 6.33E-09 2.55E+05 0.004707 1.85E-08 Hu
1573-8 1.49E+06 0.005995 4.03E-09 3.63E+05 0.004466 1.23E-08 Hu
1573-9 1.45E+06 0.008152 5.63E-09 4.75E+05 0.007239 1.52E-08 Hu
1573-10 1.45E+06 0.006364 4.38E-09 4.34E+05 0.004786 1.10E-08 Hu
1573-11 1.38E+06 0.008958 6.49E-09 2.69E+05 0.004199 1.56E-08 Hu
1573-12 1.40E+06 0.008443 6.03E-09 3.37E+05 0.005404 1.61E-08 Hu
1573-13 1.40E+06 0.006553 4.69E-09 2.29E+05 0.00343 1.50E-08 Hu
1573-14 1.40E+06 0.008559 6.13E-09 2.99E+05 0.005241 1.76E-08 Hu
1573-15 1.37E+06 0.008448 6.15E-09 2.25E+05 0.004524 2.02E-08 Hu
1573-16 1.42E+06 0.006434 4.52E-09 2.36E+05 0.003725 1.58E-08 Hu
1573-17 1.45E+06 0.008329 5.75E-09 2.69E+05 0.004199 1.56E-08
Binding ELISA of Purified Antibodies.
For human PC and monkey PC as negative controls, humanized
antibodies at different concentrations showed very weak response.
They have no binding to the human PC and monkey PC antigen. The
results were showed in FIG. 5 and FIG. 6. For human aPC and monkey
aPC, all the humanized antibodies showed good binding affinity, it
confirmed the Biacore analysis (Table 4). With the dilution curve
of humanized antibodies, the inventors also calculated the
EC.sub.50 of each antibody, which are shown in Table 5.
Chimera and humanized 1573 are cloned in CP's expression vector and
generated in CP. The interaction between aPC and antibodies
including chimera and humanized are characterized as fast
association and fast dissociation by Biacore assay. Furthermore,
all the humanized 1573 variants fulfilled affinity criteria.
Hu1573-1, 2, 5, 8, 10 could be good candidates, as they have
affinity higher than 5 nM and with only 2-3 back-mutations.
TABLE-US-00009 TABLE 5 EC.sub.50 of 1573 humanized antibodies
Cyno-APC Cyno-PC Human-APC Human-PC Humanized Ab EC50 (nM) EC50
(nM) EC50 (nM) EC50 (nM) 1573 chimeric 0.8302 No binding 0.3337 No
binding Hu1573-1 0.8647 No binding 0.3693 No binding Hu1573-2
0.7745 No binding 0.2652 No binding Hu1573-3 0.8723 No binding
0.5915 No binding Hu1573-4 0.9041 No binding 0.5834 No binding
Hu1573-5 1.0350 No binding 0.5520 No binding Hu1573-6 0.8996 No
binding 0.2741 No binding Hu1573-7 0.8525 No binding 0.4446 No
binding Hu1573-8 0.7129 No binding 0.6972 No binding Hu1573-9
0.9857 No binding 0.3134 No binding Hu1573-10 0.6800 No binding
0.5752 No binding Hu1573-11 1.466 No binding 0.6868 No binding
Hu1573-12 0.6418 No binding 0.3394 No binding Hu1573-13 0.8045 No
binding 0.2356 No binding Hu1573-14 0.9838 No binding 0.2247 No
binding Hu1573-15 0.8891 No binding 0.2880 No binding Hu1573-16
1.2290 No binding 0.2328 No binding Hu1573-17 1.2100 No binding
0.6267 No binding
All of the compositions and/or methods disclosed and claimed herein
can be made and executed without undue experimentation in light of
the present disclosure. While the compositions and methods have
been described in terms of specific embodiments, it will be
apparent to those of skill in the art that variations can be
applied to the compositions and/or methods and in the steps or in
the sequence of steps of the method described herein without
departing from the concept, spirit and scope of the disclosure.
More specifically, it will be apparent that certain agents which
are both chemically and physiologically related can be substituted
for the agents described herein while the same or similar results
would be achieved. All such similar substitutes and modifications
apparent to those skilled in the art are deemed to be within the
spirit, scope and concept as defined by the appended claims.
SEQUENCE LISTINGS
1
34115PRTArtificial SequenceSyntethic peptide 1Arg Ala Ser Glu Ser
Val Asp Ser Phe Gly Ala Thr Phe Met His1 5 10 1527PRTArtificial
SequenceSynthetic peptide 2Leu Ala Ser Asn Leu Glu Ser1
539PRTArtificial SequenceSynthetic peptide 3Gln Gln Asn Asn Glu Asp
Pro Tyr Thr1 545PRTArtificial SequenceSynthetic peptide 4Asn Tyr
Tyr Leu Asn1 5519PRTArtificial SequenceSynthetic peptide 5Asp Ile
Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu Ser1 5 10 15Val
Lys Gly67PRTArtificial SequenceSynthetic peptide 6Glu Gly Asp Tyr
Phe Asp Tyr1 5723PRTArtificial SequenceSynthetic peptide 7Asn Ile
Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln
Arg Ala Thr Ile Ser Cys 20815PRTArtificial SequenceSyntehtic
peptide 8Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
Tyr1 5 10 15932PRTArtificial SequenceSynthetic peptide 9Gly Val Pro
Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr1 5 10 15Leu Thr
Ile Asp Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys 20 25
301011PRTArtificial SequenceSynthetic peptide 10Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg1 5 101130PRTArtificial SequenceSynthetic
peptide 11Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe
Ser 20 25 301214PRTArtificial SequenceSynthetic peptide 12Trp Val
Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala1 5
101332PRTArtificial SequenceSynthetic peptide 13Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln1 5 10 15Met Asn Asn Leu
Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Ile Arg 20 25
301411PRTArtificial SequenceSynthetic peptide 14Trp Gly Gln Gly Thr
Thr Leu Thr Val Ser Ser1 5 1015118PRTArtificial SequenceSynthetic
polypeptide 15Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr
Phe Ser Asn Tyr 20 25 30Tyr Leu Asn Trp Val Arg Gln Ser Pro Glu Lys
Gly Leu Glu Trp Val 35 40 45Ala Asp Ile Arg Leu Lys Ser Asn Asn Tyr
Glu Lys His Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln Met Asn Asn
Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95Tyr Cys Ile Arg Glu Gly
Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val
Ser Ser 11516118PRTArtificial SequenceSyntethic polypeptide 16Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30Tyr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr
Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu
Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Glu Gly Asp Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11517118PRTArtificial SequenceSynthetic polypeptide 17Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Tyr
Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu
50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Ile Arg Glu Gly Asp Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11518118PRTArtificial SequenceSynthetic polypeptide 18Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Tyr
Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu
50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Ser65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Ile Arg Glu Gly Asp Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11519118PRTArtificial SequenceSynthetic polypeptide 19Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Tyr
Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu
50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Ile Arg Glu Gly Asp Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11520118PRTArtificial SequenceSynthetic polypeptide 20Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Tyr
Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Gly Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu
50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Ile Arg Glu Gly Asp Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11521118PRTArtificial SequenceSynthetic polypeptide 21Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Tyr
Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu
50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Ser65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Ile Arg Glu Gly Asp Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11522118PRTArtificial SequenceSynthetic polypeptide 22Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Tyr
Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr Ala Glu
50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
Ser65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Ile Arg Glu Gly Asp Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11523100PRTArtificial SequenceSynthetic peptide 23Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30Tyr Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly
Arg Thr Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65
70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val
Tyr 85 90 95Tyr Cys Ala Arg 10024111PRTArtificial SequenceSynthetic
peptide 24Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asp His 20 25 30Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Gly Arg Thr Arg Asn Lys Ala Asn Ser Tyr Thr
Thr Glu Tyr Ala Ala 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu
Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser 100 105 11025112PRTArtificial
SequenceSynthetic polypeptide 25Asn Ile Val Leu Thr Gln Ser Pro Ala
Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg
Ala Ser Glu Ser Val Asp Ser Phe 20 25 30Gly Ala Thr Phe Met His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu
Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser
Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp65 70 75 80Pro Val Glu
Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
11026112PRTArtificial SequenceSynthetic polypeptide 26Asp Ile Val
Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro
Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30Gly
Ala Thr Phe Met His Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro 35 40
45Gln Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp
50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
Ser65 70 75 80Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln
Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 11027112PRTArtificial SequenceSynthetic
polypeptide 27Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val
Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser
Val Asp Ser Phe 20 25 30Gly Ala Thr Phe Met His Trp Tyr Leu Gln Lys
Pro Gly Gln Pro Pro 35 40 45Gln Leu Leu Ile Tyr Leu Ala Ser Asn Leu
Glu Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile Ser65 70 75 80Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
11028112PRTArtificial SequenceSynthetic polypeptide 28Asp Ile Val
Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro
Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30Gly
Ala Thr Phe Met His Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro 35 40
45Gln Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp
50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Lys Ile
Ser65 70 75 80Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln
Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 11029112PRTArtificial SequenceSynthetic
polypeptide 29Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val
Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser
Val Asp Ser Phe 20 25 30Gly Ala Thr Phe Met His Trp Tyr Leu Gln Lys
Pro Gly Gln Pro Pro 35 40 45Gln Leu Leu Ile Tyr Leu Ala Ser Asn Leu
Glu Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr
Asp Phe Thr Leu Lys Ile Ser65 70 75 80Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
11030100PRTArtificial SequenceSynthetic polypeptide 30Asp Ile Val
Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro
Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asp
Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro 35 40
45Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Met Gln Ser 85 90 95Ile Gln Leu Pro 10031118PRTMus musculus 31Glu
Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30Tyr Leu Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp
Val 35 40 45Ala Asp Ile Arg Leu Lys Ser Asn Asn Tyr Glu Lys His Tyr
Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Ser Ser65 70 75 80Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu
Asp Thr Gly Ile Tyr 85 90 95Tyr Cys Ile Arg Glu Gly Asp Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser
11532112PRTMus musculus 32Asn Ile Val Leu Thr Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala
Ser Glu Ser Val Asp Ser Phe 20 25 30Gly Ala Thr Phe Met His Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Ala
Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp65 70 75 80Pro Val Glu Ala
Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
11033657DNAMus musculus
33tacattgttc tgacccagag tccggcaagc ctggcagtga gtctgggtca gcgtgcaacg
60atcagctgcc gtgcatctga aagtgttgat agctttggtg caaccttcat gcattggtat
120cagcagaaac cgggccagcc gccgaaactg ctgatttacc tggcgtctaa
tctggaaagt 180ggtgtgccgg cccgttttag cggttctggc agtcgcaccg
atttcaccct gacgatcgat 240ccggttgaag ccgatgatgc ggccacctat
tactgccagc agaacaatga agatccgtat 300acgtttggcg gtggcaccaa
actggaaatt aaacgtgcag atgcagcacc gaccgtgagc 360atcttcccgc
cgagctctga acagctgacc agcggtggcg cgtctgtggt ttgttttctg
420aacaacttct acccgaaaga tatcaacgtg aaatggaaaa tcgatggttc
tgaacgccag 480aacggcgttc tgaatagttg gacggatcag gattctaaag
atagtaccta cagcatgagt 540agcaccctga cgctgaccaa agatgaatat
gaacgtcata atagctacac gtgcgaagcg 600acccacaaaa cgagcacctc
tccgattgtt aaatctttca accgcaatga atgttaa 65734660DNAMus musculus
34gaagttaaac tggaagaaag cggcggtggc ctggtgcagc cgggtggcag catgaaactg
60tcttgcgttg caagtggttt taccttctct aactattacc tgaattgggt gcgtcagagt
120ccggaaaaag gcctggaatg ggttgcagat attcgcctga aaagcaacaa
ctacgaaaaa 180cattacgcgg aatctgtgaa aggtcgtttt accatcagcc
gcgatgattc taaaagctct 240gtgtatctgc agatgaacaa tctgcgtgcg
gaagatacgg gtatttatta ctgcatccgc 300gaaggcgatt atttcgatta
ctggggtcag ggcaccacgc tgaccgtgag ctcagcgaaa 360accacgccgc
cgagcgttta tccgctggca ccgggtagtg cagcacagac caacagcatg
420gtgacgctgg gttgtctggt taaaggctac tttccggaac cggtgaccgt
tacgtggaat 480tctggtagtc tgtctagtgg cgtgcatacc ttcccggcgg
ttctgcagag tgatctgtat 540acgctgtcta gcagtgtgac cgttccgagc
tctacgtggc cgtctgaaac cgtgacgtgc 600aacgttgccc acccggcaag
tagcaccaaa gtggataaga aaattgttcc gcgtgattgt 660
* * * * *
References