U.S. patent application number 17/355530 was filed with the patent office on 2021-12-30 for antibodies to coagulation factor xia and uses thereof.
The applicant listed for this patent is PFIZER INC., THE REGENTS OF THE UNIVERSITY OF CALFORNIA. Invention is credited to Shaun R. COUGHLIN, Tovo DAVID, Lauren K. ELY, Huilan GAO, Yun KIM, Thomas MIKITA, Isaac J. RONDON.
Application Number | 20210403600 17/355530 |
Document ID | / |
Family ID | 1000005830248 |
Filed Date | 2021-12-30 |
United States Patent
Application |
20210403600 |
Kind Code |
A1 |
MIKITA; Thomas ; et
al. |
December 30, 2021 |
ANTIBODIES TO COAGULATION FACTOR XIA AND USES THEREOF
Abstract
In one aspect, antibodies, or antigen-binding fragments thereof,
that specifically bind to activated Factor XI (FXIa) are provided.
Also provided are methods of obtaining such antibodies and nucleic
acids encoding the same. In another aspect, compositions and
therapeutic prevention of thrombotic diseases, disorders or
conditions are provided. In another aspect, anti-idiotype
antibodies that bind anti-FXIa antibodies of the disclosure, as
well as compositions comprising the anti-idiotype antibodies,
methods of obtaining the antibodies and nucleic acids encoding the
same, are also provided.
Inventors: |
MIKITA; Thomas; (Sausalito,
CA) ; ELY; Lauren K.; (Palo Alto, CA) ; GAO;
Huilan; (West Roxbury, MA) ; KIM; Yun; (Walnut
Creek, CA) ; RONDON; Isaac J.; (San Francisco,
CA) ; DAVID; Tovo; (San Francisco, CA) ;
COUGHLIN; Shaun R.; (Tiburon, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALFORNIA
PFIZER INC. |
OAKLAND
NEW YORK |
CA
NY |
US
US |
|
|
Family ID: |
1000005830248 |
Appl. No.: |
17/355530 |
Filed: |
June 23, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15746305 |
Jan 19, 2018 |
11066481 |
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PCT/US2016/043703 |
Jul 22, 2016 |
|
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17355530 |
|
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62196037 |
Jul 23, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 2317/92 20130101; C07K 16/36 20130101; C07K 2299/00 20130101;
C07K 2317/76 20130101; C07K 2317/52 20130101; C07K 2317/622
20130101 |
International
Class: |
C07K 16/36 20060101
C07K016/36 |
Claims
1. An isolated monoclonal antibody, or an antigen-binding portion
thereof, that specifically binds the Factor XIa catalytic domain,
wherein the antibody or antigen-binding portion thereof has at
least one of the following properties: (a) prolongs activated
partial thromboplastin time (APTT) without significantly increasing
prothrombin time (PT); (b) has an increased dissociation rate from
FXIa in the presence of a serine protease inhibitor; (c) has an
increased dissociation rate from FXIa after treatment of the latter
with an agent that chemically modifies the active site serine of a
serine protease; and (d) binds to, and has its anticoagulant
activity decreased by, a recombinant FXIa protease-domain in which
the active site serine is changed to alanine.
2. The isolated monoclonal antibody of claim 1, or an
antigen-binding portion thereof, wherein the antibody comprises: a)
a heavy chain complementarity region (HCDR) 1 comprising the amino
acid sequence of SEQ ID NO: 2 or 5; b) an HCDR2 comprising the
amino acid sequence of SEQ ID NO: 3, 6, 94 or 95; c) an HCDR3
comprising the amino acid sequence of SEQ ID NO: 4; d) a light
chain complementarity region (LCDR)1 comprising the amino acid
sequence of SEQ ID NO: 8, 11, 32, or 33; e) an LCDR2 comprising the
amino acid sequence of SEQ ID NO: 9 or 12; and/or f) an LCDR3
comprising the amino acid sequence of SEQ ID NO: 10 or 13.
3. The antibody or antigen-binding portion of claim 2, wherein the
antibody or antigen-binding portion comprises HCDR1-3 and LCDR1-3
comprising the amino acid sequences of: a) SEQ ID NOs: 2, 3, 4, 8,
9, and 10, respectively; b) SEQ ID NOs: 5, 6, 4, 11, 12, and 13,
respectively; c) SEQ ID NOs: 2, 94, 4, 8, 9, and 10, respectively;
d) SEQ ID NOs: 5, 95, 4, 11, 12, and 13, respectively; e) SEQ ID
NOs: 2, 15, 4, 8, 9, and 10, respectively; f) SEQ ID NOs: 5, 16, 4,
11, 12, and 13, respectively; g) SEQ ID NOs: 2, 66, 4, 8, 9, and
10, respectively; or h) SEQ ID NOs: 5, 67, 4, 11, 12, and 13,
respectively.
4. The isolated monoclonal antibody of claim 2, or an
antigen-binding portion thereof, wherein the antibody comprises: a
heavy chain variable domain (VH) comprising the amino acid sequence
of SEQ ID NO: 1 and/or a light chain variable domain (VL)
comprising the amino acid sequence of SEQ ID NO: 7.
5. The isolated monoclonal antibody of claim 2, or an
antigen-binding portion thereof, wherein the antibody comprises: a
heavy chain variable domain (VH) comprising the amino acid sequence
of SEQ ID NO: 65 and/or a light chain variable domain (VL)
comprising the amino acid sequence of SEQ ID NO: 68.
6. The isolated monoclonal antibody of claim 2, or an
antigen-binding portion thereof, wherein the antibody comprises: a
heavy chain variable domain (VH) comprising the amino acid sequence
of SEQ ID NO: 14 and/or a light chain variable domain (VL)
comprising the amino acid sequence of SEQ ID NO: 17.
7. The isolated monoclonal antibody of claim 2, or an
antigen-binding portion thereof, wherein the antibody comprises: a)
a heavy chain variable domain (VH) sequence comprising the amino
acid sequence of SEQ ID NOs: 1, 14, 18, 22, 24, 26, 28, 34, 38, 40,
43, 47, 51, 55, 59, 63, 65, or 96; and/or b) a light chain variable
domain (VL) sequence comprising the amino acid sequence of SEQ ID
NOs: 7, 17, 21, 23, 25, 27, 31, 37, 39, 42, 46, 50, 54, 58, 62, 64,
68, or 97.
8. The isolated monoclonal antibody of claim 7, or an
antigen-binding portion thereof, wherein the antibody comprises a
heavy chain variable domain and a light chain variable domain
comprising the following amino acid sequences, respectively: SEQ ID
NOs: 18 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 25, SEQ
ID NOs: 26 and 27, SEQ ID NOs: 28 and 31, SEQ ID NOs: 34 and 37,
SEQ ID NOs: 38 and 39, SEQ ID NOs: 40 and 42, SEQ ID NOs: 43 and
46, SEQ ID NOs: 47 and 50, SEQ ID NOs: 51 and 54, SEQ ID NOs: 55
and 58, SEQ ID NOs: 59 and 62, or SEQ ID NOs: 63 and 64.
9. (canceled)
10. The isolated monoclonal antibody or antigen-binding portion of
claim 1, wherein the antibody specifically binds to the active site
of the catalytic domain of Factor XIa.
11. The isolated monoclonal antibody or antigen-binding portion of
claim 1, wherein the antibody is chimeric, humanized, or human.
12. The isolated monoclonal antibody or antigen-binding portion of
claim 11, wherein the antibody comprises a human IgG heavy chain
constant region.
13. The isolated monoclonal antibody or antigen-binding portion of
claim 12, wherein the antibody comprises a human IgG.sub.1 heavy
chain constant region.
14. The isolated monoclonal antibody or antigen-binding portion of
claim 2, wherein the antibody comprises a heavy chain constant
domain comprising the amino acid sequence of SEQ ID NO: 82 and/or a
light chain constant domain comprising the amino acid sequence of
SEQ ID NO: 83.
15. An isolated monoclonal antibody, or an antigen-binding portion
thereof, that specifically binds Factor XIa, wherein the antibody
comprises: a heavy chain variable domain (VH) comprising the amino
acid sequence encoded by the cDNA insert of the plasmid deposited
under ATCC accession number PTA-122090 and/or a light chain
variable domain (VL) comprising the amino acid sequence encoded by
the cDNA insert of the plasmid deposited under ATCC accession
number PTA-122091.
16-17. (canceled)
18. The isolated monoclonal antibody or antigen-binding portion of
claim 2, wherein the agent that chemically modifies the active site
serine of a serine protease is PMSF (phenylmethylsulfonyl
fluoride).
19-21. (canceled)
22. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding the isolated monoclonal antibody or
antigen-binding portion of claim 1.
23. The isolated nucleic acid molecule of claim 22, wherein the
nucleic acid molecule comprises: a) the nucleotide sequence of SEQ
ID NO: 84, 86, or 88; b) the nucleotide sequence of SEQ ID NO: 85,
87, or 89; or c) both a) and b).
24. A vector comprising the nucleic acid molecule of claim 22.
25. An isolated host cell comprising the vector of claim 24.
26. An isolated host cell that produces the antibody or
antigen-binding portion of claim 1.
27. A method of producing an antibody or antigen-binding portion
thereof, comprising culturing the host cell of claim 26 under
conditions that result in production of the antibody, and isolating
the antibody or antigen-binding portion from the host cell or
culture.
28. A method for inhibiting the intrinsic pathway of coagulation in
a subject, comprising administering to said subject the isolated
monoclonal antibody or antigen-binding portion of claim 1.
29. A method for increasing clotting time in a subject, comprising
administering to said subject the isolated monoclonal antibody or
antigen-binding portion of claim 1, wherein the clotting time is
increased compared to the clotting time in the subject prior to
administration of the antibody or antigen-binding portion.
30-33. (canceled)
34. A pharmaceutical composition comprising the isolated monoclonal
antibody or antigen-binding portion of claim 1, and a
pharmaceutically acceptable excipient.
35. An isolated monoclonal anti-idiotype antibody, or an
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-Factor XIa antibody or
antigen-binding portion of claim 1, wherein the antibody comprises:
a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 70 or
73; b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 71
or 74; c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:
72; d) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 76
or 79; e) an LCDR2 comprising the amino acid sequence of SEQ ID NO:
77 or 80; and/or f) an LCDR3 comprising the amino acid sequence of
SEQ ID NO: 78 or 81.
36-38. (canceled)
39. The isolated monoclonal anti-idiotype antibody or
antigen-binding portion of claim 35, wherein the anti-idiotype
antibody comprises: a heavy chain variable domain (VH) comprising
the amino acid sequence of SEQ ID NO: 69 and/or a light chain
variable domain (VL) comprising the amino acid sequence of SEQ ID
NO: 75.
40-44. (canceled)
45. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding the isolated monoclonal anti-idiotype antibody or
antigen-binding portion of claim 35.
46. The isolated nucleic acid molecule of claim 45, wherein the
nucleic acid molecule comprises: (a) the nucleotide sequence of SEQ
ID NO: 90; (b) the nucleotide sequence of SEQ ID NO: 91; or (c)
both (a) and (b).
47-57. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 15/746,305, filing date Jan. 19, 2018, now U.S. Pat. No.
11,066,481, which is a U.S. National Stage Application of
PCT/US2016/043703, International Filing Date Jul. 22, 2016, which
claims the benefit of priority to U.S. Provisional Application No.
62/196,037, filed Jul. 23, 2015, the entire contents of each of
these applications are incorporated by reference herein.
PARTIES TO A JOINT RESEARCH AGREEMENT
[0002] The presently claimed invention was made by or on behalf of
the below listed parties to a joint research agreement. The joint
research agreement was in effect on or before the date the claimed
invention was made and the claimed invention was made as a result
of activities undertaken within the scope of the joint research
agreement. The parties to the joint research agreement are PFIZER
INC. and THE REGENTS OF THE UNIVERSITY OF CALIFORNIA.
REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE
[0003] The Sequence Listing written in file
081906-101657-222210PC_Sequence Listing.txt created on Jul. 21,
2016, containing 80,397 bytes, machine format IBM-PC, MS-Windows
operating system, is hereby incorporated by reference in its
entirety for all purposes.
FIELD OF THE INVENTION
[0004] The present disclosure relates to antibodies, e.g.,
full-length antibodies and antigen-binding fragments thereof that
specifically bind coagulation Factor XIa (FXIa). The disclosure
further relates to compositions comprising antibodies to FXIa, and
methods of using the antibodies as a medicament. The FXIa
antibodies are useful for, for example, inhibiting the intrinsic
pathway of coagulation or increasing clotting time. In addition,
the present disclosure relates to anti-idiotype antibodies e.g.,
full-length antibodies and antigen-binding fragments thereof that
specifically bind to the antigen-binding site of an anti-FXIa
antibody or antigen-binding portion thereof of the disclosure,
compositions comprising such anti-idiotype antibodies, and methods
of using such antibodies as a medicament. The anti-idiotype
antibodies to anti-FXIa antibodies are useful for, for example,
reversing the effects of an anti-FXIa antibody (e.g., decreasing
anticoagulant activity or reducing clotting time).
BACKGROUND OF THE INVENTION
[0005] Two distinct inputs trigger the coagulation cascade that
generates blood clots: (1) the extrinsic pathway comprised of
Tissue Factor (TF)/FVIIa and (2) the intrinsic pathway comprised of
FXII, FXI and other components. Both feed into a common cascade
that triggers conversion of FIX to FIXa, FX to FXa, and prothrombin
to thrombin. Thrombin is the effector protease of the cascade that
activates platelets and cleaves fibrinogen to generate fibrin.
Fibrin actively self-assembles and, in combination with activated
platelets, starts formation of a clot (in the context of
hemostasis) or thrombus (in the context of thrombosis) (Woodruff,
R. S., Sullenger, B., and R. C. Becker. J Thromb Thrombolysis.
2011; 32: 9-20).
[0006] The extrinsic pathway is triggered when blood vessels are
disrupted and FVII and other coagulation factors in plasma reach
tissue factor in the extravascular (or extrinsic) compartment.
TF/VIIa cleaves FX to FXa and, as part of an amplification step,
FIX to FIXa, which also converts FX to Xa. As above, FXa in turn
converts prothrombin to thrombin. All of these extrinsic pathway
and common cascade components are necessary for normal hemostasis,
and all existing anticoagulants target one or more of these
factors.
[0007] In contrast to the extrinsic pathway, all of the components
of the intrinsic pathway are contained in plasma (i.e., intrinsic
to blood). Tissue damage leads to release or exposure of negatively
charged surfaces and polymers, which support assembly of intrinsic
pathway components and activation of Factor XII to FXIIa. FXIIa in
turn converts FXI to FXIa, which connects the intrinsic pathway to
the common cascade by activating FIX to FIXa. Thrombin can also
convert FXI to FXIa in a positive feedback loop that may be
important for thrombus formation in some settings. Unlike the
extrinsic and common pathways, the components of the intrinsic
pathway are unnecessary for hemostasis.
[0008] Recent evidence suggests that intrinsic pathway inhibitors
might provide "next generation" anti-thrombotic drugs. Published
observations suggest that FXIa inhibition may effect thrombosis
while sparing hemostasis. In animal studies, FXI knockout mice show
no apparent bleeding defect, yet they are protected against
thrombosis in the FeCl3-induced arterial injury model (Wang, X.,
Cheng, Q., Xu, L., Feuerstain, G. Z., Hsu, M. Y., Smith, P. L.,
Seiffert, D. A., Schumacher, W. A., Ogletree, M. L., and D.
Gailani. J Thromb Haemost. 2005: 3 (4): 695-702). Other animal
studies have shown that FXI inhibition is protective against
thrombosis in non-human primate models, also with minimal increased
bleeding events (Crosby J R, Marzec U, Revenko A. S., Zhao C, Gao
D, Matafonov A, Gailani D, MacLeod A. R., Tucker E. I., Gruber A,
Hanson S. R., and B. P. Monia. Arterioscler Thromb Vasc Biol. 2013;
33(7):1670-8). In the human population, FXI deficiency is known to
exist, and while some surgeries are associated with a higher
bleeding risk, there is little association with serious spontaneous
bleeding in this population (Seligsohn, U. J. Thromb Haemost. 2009;
7 suppl. 1: 84-87). In addition, case-controlled studies suggest
FXI-deficient humans have less ischemic stroke and venous
thromboembolism (VTE), with the converse being true for individuals
with elevated FXI (He, R., Chen, D., and H. Shilin. Thrombosis
Research. 2012; 129: 541-550). A recent Phase 2 trial in humans
supports the hypothesis that a FXI antisense molecule may be safe
and effective in preventing VTE after total knee replacement
(Buller H. R., Bethune C, Bhanot S, Gailani D, Monia B. P., Raskob
G. E., Segers A., Verhamme P., Weitz J. I.; FXI-ASO TKA
Investigators. N Engl J Med. 2015. 15; 372(3):232-40).
[0009] Efforts to create selective small molecule inhibitors
against FXIa have yet to achieve adequate potency, selectivity, and
pharmacokinetics (Schumacher, W. A., Luettgen, J. M., Quan, M. L.,
and D. A. Seiffert. Arterioscler Thromb Vasc Biol. 2010; 30:
388-392). Limitations also exist, from a treatment standpoint, with
the FXI antisense inhibitor currently in clinical development as
multiple weeks of pre-dosing are required before the treatment
becomes effective.
[0010] Thus, the current state of need for a high affinity, high
potency, high selectivity, and fast acting IgG inhibitor of the
coagulation cascade serine protease FXIa is great. Further, there
is a great and long-felt need for fast-acting reversal agents for
anticoagulants to increase their safety, in this case, an agent to
reverse the action of an inhibitor of FXIa.
BRIEF SUMMARY OF THE INVENTION
[0011] This application discloses isolated antibodies, or
antigen-binding portions thereof, that specifically bind FXIa. This
application also discloses isolated anti-idiotype antibodies, or
antigen-binding portions thereof, that specifically bind to the
antigen-binding site of an anti-FXIa antibody or antigen-binding
portion thereof of the disclosure.
[0012] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds the Factor XIa catalytic domain, wherein the
antibody or antigen-binding portion thereof has at least one of the
following properties: [0013] (a) prolongs activated partial
thromboplastin time (APTT) without significantly increasing
prothrombin time (PT); [0014] (b) has an increased dissociation
rate from FXIa in the presence of a serine protease inhibitor;
[0015] (c) has an increased dissociation rate from FXIa after
treatment of the latter with an agent that chemically modifies the
active site serine of a serine protease (e.g., phenylmethylsulfonyl
fluoride (PMSF)); and [0016] (d) binds to, and has its
anticoagulant activity decreased by, a recombinant FXIa
protease-domain in which the active site serine is changed to
alanine.
[0017] In some embodiments, the antibody or antigen-binding portion
thereof has properties (a) and (b). In some embodiments, the
antibody or antigen-binding portion thereof has properties (a) and
(c). In some embodiments, the antibody or antigen-binding portion
thereof has properties (a) and (d). In some embodiments, the
antibody or antigen-binding portion thereof has properties (b) and
(c). In some embodiments, the antibody or antigen-binding portion
thereof has properties (b) and (d). In some embodiments, the
antibody or antigen-binding portion thereof has properties (c) and
(d). In some embodiments, the antibody or antigen-binding portion
thereof has properties (a), (b), and (c). In some embodiments, the
antibody or antigen-binding portion thereof has properties (a),
(b), and (d). In some embodiments, the antibody or antigen-binding
portion thereof has properties (a), (c), and (d). In some
embodiments, the antibody or antigen-binding portion thereof has
properties (b), (c), and (d). In some embodiments, the antibody or
antigen-binding portion thereof has properties (a), (b), (c), and
(d).
[0018] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, where the antibody comprises: a) a
heavy chain (H) complementarity region (CDR) 1 comprising the amino
acid sequence of SEQ ID NO: 2 or 5; b) an HCDR2 comprising the
amino acid sequence of SEQ ID NO: 3, 6, 94 or 95; c) an HCDR3
comprising the amino acid sequence of SEQ ID NO: 4; d) a light
chain (L) CDR1 comprising the amino acid sequence of SEQ ID NO: 8,
11, 32, or 33; e) an LCDR2 comprising the amino acid sequence of
SEQ ID NO: 9 or 12; and/or f) an LCDR3 comprising the amino acid
sequence of SEQ ID NO: 10 or 13.
[0019] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the antibody, or
antigen-binding portion thereof, comprises HCDR1-3 and LCDR1-3
comprising the amino acid sequences of:
[0020] a) SEQ ID NOs: 2, 3, 4, 8, 9, and 10, respectively;
[0021] b) SEQ ID NOs: 5, 6, 4, 11, 12, and 13, respectively;
[0022] c) SEQ ID NOs: 2, 94, 4, 8, 9, and 10, respectively;
[0023] d) SEQ ID NOs: 5, 95, 4, 11, 12, and 13, respectively;
[0024] e) SEQ ID NOs: 2, 15, 4, 8, 9, and 10, respectively;
[0025] f) SEQ ID NOs: 5, 16, 4, 11, 12, and 13, respectively;
[0026] g) SEQ ID NOs: 2, 66, 4, 8, 9, and 10, respectively; or
[0027] h) SEQ ID NOs: 5, 67, 4, 11, 12, and 13, respectively.
[0028] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the antibody comprises: a
heavy chain variable domain (VH) comprising the amino acid sequence
of SEQ ID NO: 1 and/or a light chain variable domain (VL)
comprising the amino acid sequence of SEQ ID NO: 7.
[0029] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the antibody comprises: a
heavy chain variable domain (VH) comprising the amino acid sequence
of SEQ ID NO: 65 and/or a light chain variable domain (VL)
comprising the amino acid sequence of SEQ ID NO: 68.
[0030] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the antibody comprises: a
heavy chain variable domain (VH) comprising the amino acid sequence
of SEQ ID NO: 14 and/or a light chain variable domain (VL)
comprising the amino acid sequence of SEQ ID NO: 17.
[0031] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the antibody comprises:
[0032] a) a heavy chain variable domain (VH) sequence comprising
the amino acid sequence of SEQ ID NOs: 1, 14, 18, 22, 24, 26, 28,
34, 38, 40, 43, 47, 51, 55, 59, 63, 65, or 96; and/or [0033] b) a
light chain variable domain (VL) sequence comprising the amino acid
sequence of SEQ ID NOs: 7, 17, 21, 23, 27, 31, 37, 39, 42, 46, 50,
54, 58, 62, 64, 68, or 97.
[0034] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the antibody comprises a
heavy chain variable domain and a light chain variable domain
comprising the following amino acid sequences, respectively:
[0035] SEQ ID NOs: 18 and 21,
[0036] SEQ ID NOs: 22 and 23,
[0037] SEQ ID NOs: 24 and 25,
[0038] SEQ ID NOs: 26 and 27,
[0039] SEQ ID NOs: 28 and 31,
[0040] SEQ ID NOs: 34 and 37,
[0041] SEQ ID NOs: 38 and 39,
[0042] SEQ ID NOs: 40 and 42,
[0043] SEQ ID NOs: 43 and 46,
[0044] SEQ ID NOs: 47 and 50,
[0045] SEQ ID NOs: 51 and 54,
[0046] SEQ ID NOs: 55 and 58,
[0047] SEQ ID NOs: 59 and 62, or
[0048] SEQ ID NOs: 63 and 64.
[0049] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the antibody is chimeric,
humanized, or human. In some embodiments, the isolated monoclonal
antibody, or an antigen-binding portion thereof, that specifically
binds Factor XIa comprises a human IgG heavy chain constant region.
In some embodiments, the isolated monoclonal antibody, or an
antigen-binding portion thereof, that specifically binds Factor XIa
comprises a human IgG.sub.1 heavy chain constant region.
[0050] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, comprises a heavy chain constant
domain comprising the amino acid sequence of SEQ ID NO:82 or SEQ ID
NO:103 and/or a light chain constant domain comprising the amino
acid sequence of SEQ ID NO:83.
[0051] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the antibody comprises: a
heavy chain variable domain (VH) comprising the amino acid sequence
encoded by the cDNA insert of the plasmid deposited under ATCC
accession number PTA-122090 and/or a light chain variable domain
(VL) comprising the amino acid sequence encoded by the cDNA insert
of the plasmid deposited under ATCC accession number
PTA-122091.
[0052] In certain aspects, the disclosure relates to an isolated
monoclonal antibody that competes for binding to FXIa and/or binds
the same epitope as an isolated monoclonal antibody, or an
antigen-binding portion thereof, that specifically binds Factor
XIa.
[0053] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the dissociation rate of the
antibody, or antigen-binding portion thereof, from FXIa is
increased in the presence of a serine protease inhibitor. In some
embodiments, the serine protease inhibitor is PMSF
(phenylmethylsulfonyl fluoride).
[0054] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa, wherein the antibody, or
antigen-binding portion thereof, prolongs activated partial
thromboplastin time (APTT). In some embodiments, the antibody, or
antigen-binding portion thereof, does not increase prothrombin time
(PT).
[0055] In certain aspects, the disclosure relates to an isolated
monoclonal antibody that binds an antibody variable region formed
by SEQ ID NO: 69 and SEQ ID NO: 75.
[0056] In certain aspects, the disclosure relates to an isolated
nucleic acid molecule comprising a nucleotide sequence encoding a
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds Factor XIa.
[0057] In certain aspects, the disclosure relates to an isolated
nucleic acid molecule comprising a nucleotide sequence encoding an
antibody, or an antigen-binding portion thereof, that specifically
binds to Factor XIa, wherein the nucleic acid molecule
comprises:
[0058] a) the nucleotide sequence of SEQ ID NO: 84, 86, or 88;
[0059] b) the nucleotide sequence of SEQ ID NO: 85, 87, or 89;
or
[0060] c) both a) and b).
[0061] In certain aspects, the disclosure relates to a vector
comprising an isolated nucleic acid molecule comprising a
nucleotide sequence encoding a monoclonal antibody, or an
antigen-binding portion thereof, that specifically binds Factor
XIa.
[0062] In certain aspects, the disclosure relates to a vector
comprising an isolated nucleic acid molecule comprising a
nucleotide sequence encoding an antibody, or an antigen-binding
portion thereof, that specifically binds to Factor XIa, wherein the
nucleic acid molecule comprises:
[0063] a) the nucleotide sequence of SEQ ID NO: 84, 86, or 88;
[0064] b) the nucleotide sequence of SEQ ID NO: 85, 87, or 89;
or
[0065] c) both a) and b).
[0066] In certain aspects, the disclosure relates to an isolated
host cell comprising a vector comprising an isolated nucleic acid
molecule comprising a nucleotide sequence encoding a monoclonal
antibody, or an antigen-binding portion thereof, that specifically
binds Factor XIa.
[0067] In certain aspects, the disclosure relates to an isolated
host cell comprising a vector comprising an isolated nucleic acid
molecule comprising a nucleotide sequence encoding an antibody, or
an antigen-binding portion thereof, that specifically binds to
Factor XIa, wherein the nucleic acid molecule comprises:
[0068] a) the nucleotide sequence of SEQ ID NO: 84, 86, or 88;
[0069] b) the nucleotide sequence of SEQ ID NO: 85, 87, or 89;
or
[0070] c) both a) and b).
[0071] In certain aspects, the disclosure relates to an isolated
host cell that produces an antibody, or an antigen-binding portion
thereof, that specifically binds to Factor XIa.
[0072] In certain aspects, the disclosure relates to a method of
producing an antibody or antigen-binding portion thereof,
comprising culturing a host cell comprising a vector comprising an
isolated nucleic acid molecule comprising a nucleotide sequence
encoding a monoclonal antibody, or an antigen-binding portion
thereof, that specifically binds Factor XIa, under conditions that
result in production of the antibody, and isolating the antibody,
or antigen-binding portion thereof, from the host cell or
culture.
[0073] In certain aspects, the disclosure relates to a method of
producing an antibody or antigen-binding portion thereof,
comprising culturing a host cell comprising a vector comprising an
isolated nucleic acid molecule comprising a nucleotide sequence
encoding an antibody, or an antigen-binding portion thereof, that
specifically binds to Factor XIa, wherein the nucleic acid molecule
comprises:
[0074] a) the nucleotide sequence of SEQ ID NO: 84, 86, or 88;
[0075] b) the nucleotide sequence of SEQ ID NO: 85, 87, or 89;
or
[0076] c) both a) and b),
under conditions that result in production of the antibody, and
isolating the antibody, or antigen-binding portion thereof, from
the host cell or culture.
[0077] In certain aspects, the disclosure relates to a method of
producing an antibody or antigen-binding portion thereof,
comprising culturing a host cell that produces an antibody, or an
antigen-binding portion thereof, that specifically binds to Factor
XIa under conditions that result in production of the antibody, and
isolating the antibody, or antigen-binding portion thereof, from
the host cell or culture.
[0078] In certain aspects, the disclosure relates to a method for
inhibiting the intrinsic pathway of coagulation in a subject,
comprising administering to said subject an antibody, or an
antigen-binding portion thereof, that specifically binds to Factor
XIa.
[0079] In certain aspects, the disclosure relates to a method for
increasing dotting time in a subject, comprising administering to
said subject an antibody, or an antigen-binding portion thereof,
that specifically binds to Factor XIa, wherein the clotting time is
increased compared to the clotting time in the subject prior to
administration of the antibody, or antigen-binding portion
thereof.
[0080] In certain aspects, the disclosure relates to an antibody,
or an antigen-binding portion thereof, that specifically binds to
Factor XIa for use in inhibiting the intrinsic pathway of
coagulation in a subject.
[0081] In certain aspects, the disclosure relates to an antibody,
or an antigen-binding portion thereof, that specifically binds to
Factor XIa for use in increasing clotting time in a subject.
[0082] In certain aspects, the disclosure relates to use of an
isolated antibody, or an antigen-binding portion thereof, that
specifically binds to Factor XIa in the manufacture of a medicament
for inhibiting the intrinsic pathway of coagulation in a
subject.
[0083] In certain aspects, the disclosure relates to use of an
isolated antibody, or an antigen-binding portion thereof, that
specifically binds to Factor XIa in the manufacture of a medicament
for increasing clotting time in a subject.
[0084] In certain aspects, the disclosure relates to a
pharmaceutical composition comprising an isolated antibody, or an
antigen-binding portion thereof, that specifically binds to Factor
XIa, and a pharmaceutically acceptable excipient.
[0085] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-Factor
XIa antibody, or antigen-binding portion thereof, that specifically
binds to Factor XIa, wherein the antibody comprises: a) an HCDR1
comprising the amino acid sequence of SEQ ID NO: 70 or 73; b) an
HCDR2 comprising the amino acid sequence of SEQ ID NO: 71 or 74; c)
an HCDR3 comprising the amino acid sequence of SEQ ID NO: 72; d) an
LCDR1 comprising the amino acid sequence of SEQ ID NO: 76 or 79; e)
an LCDR2 comprising the amino acid sequence of SEQ ID NO: 77 or 80;
and/or f) an LCDR3 comprising the amino acid sequence of SEQ ID NO:
78 or 81. 37. In some embodiments, the monoclonal antibody
comprises: a heavy chain variable domain (VH) comprising the amino
acid sequence of SEQ ID NO: 69 and/or a light chain variable domain
(VL) comprising the amino acid sequence of SEQ ID NO: 75. In some
embodiments, the isolated monoclonal antibody, or an
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa is chimeric, humanized, or human. In some embodiments, the
isolated monoclonal antibody, or an antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-Factor XIa antibody, or antigen-binding portion thereof, that
specifically binds to Factor XIa comprises a human IgG heavy chain
constant region. In some embodiments, the isolated monoclonal
antibody, or an antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-Factor XIa antibody,
or antigen-binding portion thereof, that specifically binds to
Factor XIa comprises a human IgG.sub.1 heavy chain constant
region.
[0086] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-Factor
XIa antibody, or antigen-binding portion thereof, that specifically
binds to Factor XIa, wherein the antibody comprises three HCDR
sequences from the heavy chain variable domain (VH) comprising the
amino acid sequence of SEQ ID NO: 69 and/or three LCDR sequences
from a light chain variable domain (VL) comprising the amino acid
sequence of SEQ ID NO: 75. In some embodiments, the antibody, or
antigen-binding portion thereof, comprises HCDR1-3 and LCDR1-3
comprising the amino acid sequences of SEQ ID NOs: 70, 71, 72, 76,
77 and 78, respectively. In some embodiments, the antibody or
antigen-binding portion comprises HCDR1-3 and LCDR1-3 comprising
the amino acid sequences of SEQ ID NOs: 73, 74, 72, 79, 80 and 81,
respectively. In some embodiments, the isolated monoclonal
antibody, or an antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-Factor XIa antibody,
or antigen-binding portion thereof, that specifically binds to
Factor XIa is chimeric, humanized, or human. In some embodiments,
the isolated monoclonal antibody, or an antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-Factor XIa antibody, or antigen-binding portion thereof, that
specifically binds to Factor XIa comprises a human IgG heavy chain
constant region. In some embodiments, the isolated monoclonal
antibody, or an antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-Factor XIa antibody,
or antigen-binding portion thereof, that specifically binds to
Factor XIa comprises a human IgG.sub.1 heavy chain constant
region.
[0087] In certain aspects, the disclosure relates to an isolated
monoclonal antibody, or an antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-Factor
XIa antibody, or antigen-binding portion thereof, that specifically
binds to Factor XIa, wherein the antibody comprises a heavy chain
constant domain comprising the amino acid sequence of SEQ ID NO: 82
and/or a light chain constant domain comprising the amino acid
sequence of SEQ ID NO: 83.
[0088] In certain aspects, the disclosure relates to an isolated
monoclonal antibody that competes for binding to FXIa and/or binds
the same epitope as an isolated monoclonal antibody, or an
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa.
[0089] In certain aspects, the disclosure relates to an isolated
nucleic acid molecule comprising a nucleotide sequence encoding an
isolated monoclonal antibody, or an antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-Factor XIa antibody or antigen-binding portion thereof that
specifically binds to Factor XIa.
[0090] In certain aspects, the disclosure relates to an isolated
nucleic acid molecule comprising a nucleotide sequence encoding an
antibody, or an antigen-binding portion thereof, that specifically
binds to an antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa, wherein the nucleic acid molecule comprises:
[0091] a) the nucleotide sequence of SEQ ID NO: 90;
[0092] b) the nucleotide sequence of SEQ ID NO: 91; or
[0093] c) both a) and b).
[0094] In certain aspects, the disclosure relates to a vector
comprising an isolated nucleic acid molecule comprising a
nucleotide sequence encoding an isolated monoclonal antibody, or an
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa.
[0095] In certain aspects, the disclosure relates to a vector
comprising an isolated nucleic acid molecule comprising a
nucleotide sequence encoding an antibody, or an antigen-binding
portion thereof, that specifically binds to an antigen-binding site
of an anti-Factor XIa antibody, or antigen-binding portion thereof,
that specifically binds to Factor XIa, wherein the nucleic acid
molecule comprises:
[0096] a) the nucleotide sequence of SEQ ID NO: 90;
[0097] b) the nucleotide sequence of SEQ ID NO: 91; or
[0098] c) both a) and b).
[0099] In certain aspects, the disclosure relates to a host cell
comprising a vector comprising an isolated nucleic acid molecule
comprising a nucleotide sequence encoding an isolated monoclonal
antibody, or an antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-Factor XIa antibody,
or antigen-binding portion thereof, that specifically binds to
Factor XIa.
[0100] In certain aspects, the disclosure relates to a host cell
comprising a vector comprising an isolated nucleic acid molecule
comprising a nucleotide sequence encoding an antibody, or an
antigen-binding portion thereof, that specifically binds to an
antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa, wherein the nucleic acid molecule comprises:
[0101] a) the nucleotide sequence of SEQ ID NO: 90;
[0102] b) the nucleotide sequence of SEQ ID NO: 91; or
[0103] c) both a) and b).
[0104] In certain aspects, the disclosure relates to an isolated
host cell that produces a monoclonal antibody, or an
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa.
[0105] In certain aspects, the disclosure relates to a method of
producing an antibody, or antigen-binding portion thereof,
comprising culturing a host cell comprising a vector comprising an
isolated nucleic acid molecule comprising a nucleotide sequence
encoding an isolated monoclonal antibody, or an antigen-binding
portion thereof, that specifically binds to the antigen-binding
site of an anti-Factor XIa antibody, or antigen-binding portion
thereof, that specifically binds to Factor XIa, under conditions
that result in production of the antibody, and isolating the
antibody or antigen-binding portion thereof from the host cell or
culture.
[0106] In certain aspects, the disclosure relates to a method of
producing an antibody, or antigen-binding portion thereof,
comprising culturing a host cell comprising a vector comprising an
isolated nucleic acid molecule comprising a nucleotide sequence
encoding an antibody, or an antigen-binding portion thereof, that
specifically binds to an antigen-binding site of an anti-Factor XIa
antibody, or antigen-binding portion thereof, that specifically
binds to Factor XIa, wherein the nucleic acid molecule
comprises:
[0107] a) the nucleotide sequence of SEQ ID NO: 90;
[0108] b) the nucleotide sequence of SEQ ID NO: 91; or
[0109] c) both a) and b),
under conditions that result in production of the antibody, and
isolating the antibody or antigen-binding portion thereof from the
host cell or culture.
[0110] In certain aspects, the disclosure relates to a method of
producing an antibody or antigen-binding portion thereof,
comprising culturing a host cell that produces a monoclonal
antibody, or an antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-Factor XIa antibody,
or antigen-binding portion thereof, that specifically binds to
Factor XIa, under conditions that result in production of the
antibody, and isolating the antibody or antigen-binding portion
thereof from the host cell or culture.
[0111] In certain aspects, the disclosure relates to a method for
decreasing anticoagulant activity in a subject being administered a
first antibody or antigen-binding portion thereof, wherein said
first antibody, or an antigen-binding portion thereof, specifically
binds to Factor XIa, comprising administering to said subject a
second antibody or antigen-binding portion thereof, wherein said
second antibody, or an antigen-binding portion thereof,
specifically binds to the antigen-binding site of an anti-Factor
XIa antibody, or antigen-binding portion thereof, that specifically
binds to Factor XIa, wherein the anticoagulant activity is reduced
compared with the anticoagulant activity in the subject prior to
administration of the second antibody or antigen-binding portion
thereof.
[0112] In certain aspects, the disclosure relates to a method for
reducing clotting time in a subject being administered a first
antibody or antigen-binding portion thereof, wherein said first
antibody, or an antigen-binding portion thereof, specifically binds
to Factor XIa, comprising administering to said subject a second
antibody or antigen-binding portion thereof, wherein said second
antibody, or an antigen-binding portion thereof, specifically binds
to the antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa, wherein the clotting time is reduced compared with the
clotting time in the subject prior to administration of the second
antibody or antigen-binding portion thereof.
[0113] In certain aspects, the disclosure relates to an antibody or
antigen-binding portion thereof, wherein said antibody, or an
antigen-binding portion thereof, specifically binds to the
antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa, for use in decreasing anticoagulant activity in a subject
being administered a first antibody or antigen-binding portion,
wherein said first antibody, or an antigen-binding portion thereof,
specifically binds to Factor XIa, wherein the anticoagulant
activity is reduced compared with the anticoagulant activity in the
subject prior to administration of the antibody or antigen-binding
portion thereof.
[0114] In certain aspects, the disclosure relates to an isolated
antibody or antigen-binding portion thereof, wherein said antibody,
or an antigen-binding portion thereof, specifically binds to the
antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa, for use in reducing clotting time in a subject being
administered a first antibody or antigen-binding portion thereof,
wherein said first antibody, or an antigen-binding portion thereof,
specifically binds to Factor XIa, wherein the clotting time is
reduced compared with the clotting time in the subject prior to
administration of the antibody or antigen-binding portion
thereof.
[0115] In certain aspects, the disclosure relates to use of an
isolated antibody or antigen-binding portion thereof, wherein said
antibody, or an antigen-binding portion thereof, specifically binds
to the antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa, in the manufacture of a medicament for use in decreasing
anticoagulant activity in a subject being administered a first
antibody or antigen-binding portion thereof, wherein said first
antibody, or an antigen-binding portion thereof, specifically binds
to Factor XIa, wherein the anticoagulant activity is reduced
compared with the anticoagulant activity in the subject prior to
administration of the antibody or antigen-binding portion
thereof.
[0116] In certain aspects, the disclosure relates to use of an
isolated antibody or antigen-binding portion thereof, wherein said
antibody, or an antigen-binding portion thereof, specifically binds
to the antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa, in the manufacture of a medicament for use in reducing
clotting time in a subject being administered a first antibody or
antigen-binding portion thereof, wherein said first antibody, or an
antigen-binding portion thereof, specifically binds to Factor XIa,
wherein the clotting time is reduced compared with the clotting
time in the subject prior to administration of the antibody or
antigen-binding portion thereof.
[0117] In certain aspects, the disclosure relates to a
pharmaceutical composition comprising an isolated antibody or
antigen-binding portion, wherein said antibody, or an
antigen-binding portion thereof, specifically binds to the
antigen-binding site of an anti-Factor XIa antibody, or
antigen-binding portion thereof, that specifically binds to Factor
XIa, and a pharmaceutically acceptable excipient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0118] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings. For the purpose of
illustrating the invention there are shown in the drawings
embodiment(s). It should be understood, however, that the invention
is not limited to the precise arrangements and instrumentalities
shown.
[0119] FIG. 1A-1D depict the production and selection of anti-FXIa
mAbs. FIG. 1A and FIG. 1B depict the binding response of positive
scFv clones, after reformatting to IgG, to human FXIa (A) and
cynomolgus (B) FXIa. FIG. 1C and FIG. 1D depict the human (C) and
cynomolgus (D) FXIa inhibitory activity of the positive scFV
clones, after reformatting to IgG, as measured by half-maximal
inhibitory concentration (IC.sub.50) values.
[0120] FIG. 2 depicts the epitope binning data for seven anti-FXIa
mAbs, as determined by measuring the binding of anti-FXIa clones to
FXIa/D4 complexes. Binding of mouse anti-FXI clone AHXI-5061 was
also tested.
[0121] FIG. 3A-3D depict the generation of improved versions of the
D4 anti-FXIa mAb as determined by selective binding to and activity
against FXIa. FIG. 3A shows the binding of D4 variants to hFXI and
hFXIa. FIG. 3B-3D show the inhibitory activity of D4 and its
variants clone 24, DEF, and 24F, against human FXIa (B), cynomolgus
FXIa (C), and rabbit FXIa (D), as measured by IC.sub.50 values.
[0122] FIG. 4A-4B show the FXIa affinity and binding kinetics for
selected mAbs and Fabs. FIG. 4A shows the binding response versus
time for D4 IgG, B11 IgG, clone 24 ("C24") Fab and DEF Fab over a
series of antibody/Fab concentrations. FIG. 4B summarizes the
kinetic rate constant data.
[0123] FIG. 5A-5B show the effect of anti-FXIa mAbs DEF (A) and 24F
(B) on the activity of FXIa and other serine proteases on the
coagulation cascade in an in vitro assay. FIG. 5C-5D show the
effect of anti-FXIa mAbs DEF (C) and 24 (D) on the activity of FXIa
and other serine proteases on the coagulation cascade in an in
vitro assay.
[0124] FIG. 6A-6F depict the dose-dependent inhibition of thrombin
generation by anti-FXIa mAbs in a human plasma assay. FXIIa was
used to trigger the activation of FXI to FXIa to drive the overall
coagulation cascade downstream to the final step of thrombin
activation. FIG. 6A-6D depict the results of the thrombin
generation assay for clone 24 (A), clone B11 (B), clone D4 (C), and
IgG1 control (D). FIG. 6E and FIG. 6F depict the decreases in peak
thrombin activity (E) and lag time to peak thrombin activity
(F).
[0125] FIG. 7 shows the results of a single dose intravenous bolus
pharmacokinetic (PK) study with DEF in New Zealand white
rabbits.
[0126] FIG. 8 shows a plot of activated partial thromboplastin time
(APTT) and prothrombin time (PT) clotting times versus DEF plasma
concentration in New Zealand white rabbits injected with different
doses of DEF.
[0127] FIG. 9A-9F show the dose-dependent inhibition of anti-FXIa
mAb DEF (FIG. 9A-9C) on thrombus weight (A), APTT (B), and PT (C)
in a rabbit venous thromboembolism (VTE) model in comparison to the
effects of rivaroxaban and both IgG and vehicle controls (FIG.
9D-9F) on thrombus weight (D), APTT (E), and PT (F).
[0128] FIG. 10A-10C show the effects of anti-FXIa mAb DEF in a
rabbit cuticle bleeding study. Rivaroxaban, vehicle, and a control
IgG were included for comparison. FIGS. 10A-10C show the pre- and
post-dose bleeding amount (A), pre- and post-dose APTT (B), and
pre- and post-dose PT (C).
[0129] FIG. 11 shows the effect of PMSF modification of FXIa
catalytic serine residue on the binding affinity of anti-FXIa mAbs
DEF and H04 for FXIa.
[0130] FIG. 12 shows the PK of DEF IgG in cynomolgus monkeys.
[0131] FIG. 13A-13B show the effect of high dose DEF exposure on
APTT and PT coagulation time in cynomolgus monkeys. FIG. 13A shows
the mean APTT values while FIG. 13B shows the mean PT values.
[0132] FIG. 14 shows the binding selectivity of C4 mAb, an
anti-idiotype antibody to DEF.
[0133] FIG. 15A-15B show the DEF Fab binding kinetics for C4 mAb.
FIG. 15A shows the binding response versus time for C4 mAb at
various DEF Fab concentrations. FIG. 15B summarizes the kinetic
rate constant data.
[0134] FIG. 16 shows the reversal effect of C4 mAb on the
inhibitory effects of anti-FXIa mAb DEF in an in vitro FXIa
assay.
[0135] FIG. 17 shows the reversal effect of C4 mAb on the
inhibitory effects of anti-FXIa mAb DEF in an in vitro FXIa
assay.
[0136] FIG. 18A, FIG. 18B, and FIG. 18C show the effect of C4 mAb
on the inhibitory effects of anti-FXIa mAb DEF in human plasma in a
FXIIa-triggered thrombin generation assay.
[0137] FIG. 19A-19B show the effect of C4 mAb on anti-FXIa mAb DEF
in an in vivo rabbit dosing experiment followed by ex vivo APTT and
PT assays. FIG. 19A shows the effect of sequential addition of DEF,
ctrl IgG, and C4 mAb on ex vivo APTT coagulation time, with FIG.
19B showing PT coagulation time effects.
[0138] FIG. 20A-20B show the effect on FeCl.sub.3-triggered carotid
artery thrombosis in human FXI-reconstituted FXI-deficient mice.
(A) Blood flow after carotid injury with 250 mM v/v FeCl.sub.3 in
FXI-deficient mice injected with vehicle (blue) or human FXI at
0.25 mg/kg (red) or in age-matched wild-type mice from the same
colony (black). % of vessels remaining open as a function of time
after injury is shown. (B) Human FXI-reconstituted FXI-deficient
mice were injected with C24 at 0.5 (blue), 2 (red), 4 (green), 12
(orange) and 35 (gray) mg/kg i.v. or with the same doses of control
IgG1. The % of vessels remaining open as a function of time after
injury was determined as in (A). Carotids in mice injected with all
doses of control IgG1 had median occlusion times similar to
wild-type mice; only 35 mg/kg IgG1 data are shown (black) to avoid
clutter. The rate of occlusion was decreased in human
FXI-reconstituted FXI null mice treated with C24 at 2 mg/kg and
higher doses when compared to the rate in mice treated with control
IgG1 by Log-Rank Analysis (Mantel Cox) (p=0.01).
[0139] FIG. 21A-21D show the Biacore.TM. binding analysis for C24
Fab to FXIa -/+ PMSF (A, B) and FXIa -/+ PPACK (C, D).
[0140] FIG. 22A-22B show the crystal structure of the DEF Fab
binding to the FXIa catalytic domain. (A) The DEF Fab interacts
with the FXIa catalytic domain predominantly via the light chain
CDRs. (B) The DEF Fab light chain makes contacts surrounding the
active site of the FXIa. The catalytic triad residues, Ala
(Ser557Ala mutant), His and Asp, are highlighted.
[0141] FIG. 23A-23B show the overlay of inhibitor bound serine
protease catalytic domains with the FXIa catalytic domain. (A)
Trypsin-PMSF (PDB 1PQA) and (B) thrombin-PPACK (PDB 1Z8I)
superimposed on the FXIa catalytic domain--DEF Fab complex. In the
trypsin-PMSF structure the active site His takes on an alternate
conformation (arrow), which would in turn create a steric clash for
the DEF CDR L1 Gln27.
[0142] FIG. 24A-24D show the effects of anti-FXIa antibodies on
FXIIa-induced thrombin generation, APTT in human plasma, and
intrinsic pathway-triggered clotting in whole blood. (A-B)
FXIIa-triggered thrombin activity as a function of time was
determined in the presence of the indicated concentrations of
anti-FXIa antibodies D4 (blue), B11 (red), C24 (green) or control
IgG1 (black). Peak thrombin activity (A) and lag to onset of
thrombin generation (B) are shown (mean+/-SEM; n=3-5). Note
substantial reduction in peak thrombin generation and prolongation
of time to onset of thrombin generation in samples containing C24
at 4 ug/ml or greater. (C) APTT assay as a function of antibody
concentration (mean+/-SEM; n=2). Control IgG1 had no effect in this
assay. Note prolongation of APTT in samples containing C24 at 10
ug/ml or greater. (D) Effect of C24 or control IgG1 on intrinsic
pathway-triggered clotting of whole blood. Time to clot is shown
(mean+/-SEM; n=3-4). Note prolongation of time to clotting in whole
human blood in samples containing C24.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0143] Current anticoagulants still suffer from efficacy versus
safety limitations (Tahir, F., Riaz, H., Riaz, T., Maaz, Badshah,
B., Riaz, I. R., Hamza, A., and H. Mohiuddin. Thromb J. 2013; 11:
18). If they are under-dosed, the anti-thrombotic effects are not
realized and patients with a wide range of thrombotic disease
complications fail to be adequately managed, resulting in a higher
incidence of dangerous blood clots. If patients are over-treated or
have conditions that predispose them to bleeding, then dangerous
bleeding events result. While the current thrombin and FXa small
molecule inhibitors have shown improved efficacy versus safety
results over older medications like warfarin in many
anti-thrombotic disease indications (for example, atrial
fibrillation (AF) or venous thromboembolism (VTE)), there are other
indications that would benefit from a novel anti-thrombotic
treatment with an improved efficacy versus safety profile, such as
mechanical heart valve replacement, VTE in the medically ill, VTE
prophylaxis in the medically ill, VTE prophylaxis in knee or hip
surgery, Afib in the renal disease population and/or patients
previously identified as bleeders, acute coronary syndromes, use of
extracorporeal circulations, and devices in which blood contacts
artificial surfaces. See, e.g., Ortel, T. L., and G. M. Arepally.
Annu. Rev. Med. 2015; 66: 241-253; Flaumenhaft, R. N Engl J Med
2015. 15; 372(3):277-8.
[0144] Disclosed herein are antibodies that specifically bind to
Factor XIa (e.g., human FXIa). In some embodiments, anti-FXIa
antibodies are useful for inhibiting the intrinsic pathway of
coagulation or increasing clotting time, and preventing or treating
thrombosis with less bleeding risk than existing coagulants, which
inhibit the extrinsic and common pathways of coagulation. Methods
of making FXIa antibodies, compositions comprising these
antibodies, and methods of using these antibodies are also
provided. FXIa antibodies can be used in the prevention, treatment,
and/or amelioration of diseases, disorders or conditions caused by
and/or associated with FXIa activity. Such diseases, disorders or
conditions include, but are not limited to, thrombotic conditions
such as AF, VTE, mechanical heart valve replacement, VTE in the
medically ill, VTE prophylaxis in the medically ill, VTE
prophylaxis in knee or hip surgery, Afib in the renal disease
population and/or patients previously identified as bleeders, acute
coronary syndromes, and use of extracorporeal circulations and
devices in which blood contacts artificial surfaces, as would be
appreciated by one skilled in the art provided with the teachings
disclosed herein.
[0145] Also disclosed herein are anti-idiotype antibodies that
specifically bind to the antigen-binding site of an anti-FXIa
antibody or antigen-binding portion thereof of the disclosure and
that act as reversal agents of the anti-FXIa anticoagulant
antibodies to improve their safety. Methods of making such
anti-idiotype antibodies, compositions comprising these antibodies,
and methods of using these antibodies are provided. The
anti-idiotype antibodies to anti-FXIa antibodies are useful for,
for example, reversing the effects of an anti-FXIa antibody (e.g.,
decreasing anticoagulant activity or reducing clotting time).
II. Definitions
[0146] Unless otherwise defined herein, scientific and technical
terms used herein shall have the meanings that are commonly
understood by those of ordinary skill in the art. Further, unless
otherwise required by context, singular terms shall include
pluralities and plural terms shall include the singular. Generally,
nomenclatures used in connection with, and techniques of, cell and
tissue culture, molecular biology, immunology, microbiology,
genetics and protein and nucleic acid chemistry and hybridization
described herein are those well-known and commonly used in the
art.
[0147] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.
J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press;
Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998)
Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987);
Introduction to Cell and Tissue Culture (J. P. Mather and P. E.
Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,
1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic
Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and
C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells
(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in
Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The
Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current
Protocols in Immunology (J. E. Coligan et al., eds., 1991);
Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd.
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(2001); Ausubel et al., Current Protocols in Molecular Biology,
John Wiley & Sons, N Y (2002); Harlow and Lane Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1998); Coligan et al., Short
Protocols in Protein Science, John Wiley & Sons, N Y (2003);
Short Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.
Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL
Press, 1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
[0148] Enzymatic reactions and purification techniques are
performed according to manufacturer's specifications, as commonly
accomplished in the art or as described herein. The nomenclatures
used in connection with, and the laboratory procedures and
techniques of, analytical chemistry, biochemistry, immunology,
molecular biology, synthetic organic chemistry, and medicinal and
pharmaceutical chemistry described herein are those well known and
commonly used in the art. Standard techniques are used for chemical
syntheses, chemical analyses, pharmaceutical preparation,
formulation, and delivery, and treatment of patients.
[0149] The term "FXIa" refers to Factor XIa, a serine protease that
is activated from a zymogen form (Factor XI, or "FXI") during
coagulation as part of the coagulation cascade. FXI is a homodimer
in which each subunit contains four apple domains (A1-A4) and a
catalytic domain (CD). FXI subunits are activated by cleavage of a
bond between A4 and the catalytic domain. See, Gailani et al., J.
Thromb Haemost., 2009, 7 (Suppl 1):75-78, incorporated by reference
herein. As used herein, "FXIa" refers to any naturally occurring
form of activated Factor XI, whether monomeric or multimeric,
including dimers, trimers, etc., which may be derived from any
suitable organism. In some embodiments, "FXIa" refers to a
mammalian FXIa, such as human, rat or mouse, as well as non-human
primate, bovine, ovine, or porcine FXIa. In some embodiments, the
FXIa is human (see, e.g., Genbank Accession Number M13142, SEQ ID
NO: 98) or from cynomolgus monkey. The term "FXIa" also encompasses
fragments, variants, isoforms, and other homologs of such FXIa
molecules. Variant FXIa molecules will generally be characterized
by having the same type of activity as naturally occurring FXIa,
such as the ability to bind FIX, thrombin or platelets, and the
ability to activate the coagulation cascade.
[0150] As used herein, the term "isolated molecule" (where the
molecule is, for example, a polypeptide, a polynucleotide, or an
antibody or fragment thereof) refers to a molecule that by virtue
of its origin or source of derivation (1) is not associated with
naturally associated components that accompany it in its native
state, (2) is substantially free of other molecules from the same
species (3) is expressed by a cell from a different species, or (4)
does not occur in nature. Thus, for example, a non-naturally
occurring molecule that is chemically synthesized, or expressed in
a cellular system different from the cell from which it naturally
originates, will be "isolated" from its naturally associated
components. A molecule also may be rendered substantially free of
naturally associated components by isolation, using purification
techniques well known in the art. Molecule purity or homogeneity
may be assayed by a number of means well known in the art. For
example, the purity of a polypeptide sample may be assayed using
polyacrylamide gel electrophoresis and staining of the gel to
visualize the polypeptide using techniques well known in the art.
For certain purposes, higher resolution may be provided by using
HPLC or other means well known in the art for purification.
[0151] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the
composition), and preferably a substantially purified fraction is a
composition wherein the object species (e.g., a glycoprotein,
including an antibody or receptor) comprises at least about 50
percent (on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition will have the object
species as at least about 80% of all macromolecular species present
in the composition, more preferably more than about 85%, 90%, 95%,
or 99%. Most preferably, the object species is purified to
essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species. In certain embodiments a substantially pure material is at
least 50% pure (i.e., free from contaminants), more preferably, at
least 90% pure, at least 95% pure, at least 96% pure, at least 97%
pure, least 98% pure, or at least 99% pure.
[0152] As used herein, the term "antibody" refers to an
immunoglobulin molecule capable of specific binding to a target,
such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.,
through at least one antigen recognition site, located in the
variable region of the immunoglobulin molecule. As used herein, the
term encompasses not only intact polyclonal or monoclonal
antibodies, but also, unless otherwise specified, any antigen
binding portion thereof that competes with the intact antibody for
specific binding, fusion proteins comprising an antigen binding
portion, and any other modified configuration of the immunoglobulin
molecule that comprises an antigen recognition site. Antigen
binding portions include, for example, Fab, Fab', F(ab).sub.2, Fd,
Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies),
fragments including complementarity determining regions (CDRs),
single chain variable fragment antibodies (scFv), maxibodies,
minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR
and bis-scFv, and polypeptides that contain at least a portion of
an immunoglobulin that is sufficient to confer specific antigen
binding to the polypeptide. An antibody includes an antibody of any
class, such as IgG, IgA, or IgM (or sub-class thereof), and the
antibody need not be of any particular class. Depending on the
antibody amino acid sequence of the constant region of its heavy
chains, immunoglobulins can be assigned to different classes. There
are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and
IgM, and several of these may be further divided into subclasses
(isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4,
IgA.sub.1 and IgA.sub.2. The heavy-chain constant regions that
correspond to the different classes of immunoglobulins are called
alpha, delta, epsilon, gamma, and mu, respectively. The subunit
structures and three-dimensional configurations of different
classes of immunoglobulins are well known.
[0153] The terms "antigen-binding portion" or "antigen-binding
fragment" of an antibody or "antibody portion," as used
interchangeably herein, refer to one or more fragments of an
antibody that retain the ability to specifically bind to an antigen
(e.g., FXIa). It has been shown that the antigen-binding function
of an antibody can be performed by fragments of a full-length
antibody. Examples of binding fragments encompassed within the term
"antigen-binding portion" of an antibody include (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR),
disulfide-linked Fvs (dsFv), and anti-idiotypic (anti-Id)
antibodies and intrabodies. Furthermore, although the two domains
of the Fv fragment, VL and VH, are coded for by separate genes,
they can be joined, using recombinant methods, by a synthetic
linker that enables them to be made as a single protein chain in
which the VL and VH regions pair to form monovalent molecules
(known as single chain Fv (scFv)); see e.g., Bird et al. Science
242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988)). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. Other forms of single chain antibodies,
such as diabodies are also encompassed. Diabodies are bivalent,
bispecific antibodies in which VH and VL domains are expressed on a
single polypeptide chain, but using a linker that is too short to
allow for pairing between the two domains on the same chain,
thereby forcing the domains to pair with complementary domains of
another chain and creating two antigen binding sites (see e.g.,
Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993);
Poljak et al., 1994, Structure 2:1121-1123).
[0154] Antibodies may be derived from any mammal, including, but
not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats,
mice, etc., or other animals such as birds (e.g. chickens), fish
(e.g., sharks) and camelids (e.g., llamas).
[0155] A "variable region" of an antibody refers to the variable
region of the antibody light chain (VL) or the variable region of
the antibody heavy chain (VH), either alone or in combination. As
known in the art, the variable regions of the heavy and light
chains each consist of four framework regions (FRs) connected by
three complementarity determining regions (CDRs) also known as
hypervariable regions, and contribute to the formation of the
antigen binding site of antibodies. If variants of a subject
variable region are desired, particularly with substitution in
amino acid residues outside of a CDR region (i.e., in the framework
region), appropriate amino acid substitution, preferably,
conservative amino acid substitution, can be identified by
comparing the subject variable region to the variable regions of
other antibodies which contain CDR1 and CDR2 sequences in the same
canonical class as the subject variable region (Chothia and Lesk,
J. Mol. Biol. 196(4): 901-917, 1987).
[0156] In certain embodiments, definitive delineation of a CDR and
identification of residues comprising the binding site of an
antibody is accomplished by solving the structure of the antibody
and/or solving the structure of the antibody-ligand complex. In
certain embodiments, that can be accomplished by any of a variety
of techniques known to those skilled in the art, such as X-ray
crystallography. In certain embodiments, various methods of
analysis can be employed to identify or approximate the CDR
regions. In certain embodiments, various methods of analysis can be
employed to identify or approximate the CDR regions. Examples of
such methods include, but are not limited to, the Kabat definition,
the Chothia definition, the AbM definition, the contact definition,
and the conformational definition.
[0157] The Kabat definition is a standard for numbering the
residues in an antibody and is typically used to identify CDR
regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:
214-8. The Chothia definition is similar to the Kabat definition,
but the Chothia definition takes into account positions of certain
structural loop regions. See, e.g., Chothia et al., 1986, J. Mol.
Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The
AbM definition uses an integrated suite of computer programs
produced by Oxford Molecular Group that model antibody structure.
See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA),
86:9268-9272; "AbM.TM., A Computer Program for Modeling Variable
Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM
definition models the tertiary structure of an antibody from
primary sequence using a combination of knowledge databases and ab
initio methods, such as those described by Samudrala et al., 1999,
"Ab Initio Protein Structure Prediction Using a Combined
Hierarchical Approach," in PROTEINS, Structure, Function and
Genetics Suppl., 3:194-198. The contact definition is based on an
analysis of the available complex crystal structures. See, e.g.,
MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another
approach, referred to herein as the "conformational definition" of
CDRs, the positions of the CDRs may be identified as the residues
that make enthalpic contributions to antigen binding. See, e.g.,
Makabe et al., 2008, Journal of Biological Chemistry,
283:1156-1166. Still other CDR boundary definitions may not
strictly follow one of the above approaches, but will nonetheless
overlap with at least a portion of the Kabat CDRs, although they
may be shortened or lengthened in light of prediction or
experimental findings that particular residues or groups of
residues do not significantly impact antigen binding. As used
herein, a CDR may refer to CDRs defined by any approach known in
the art, including combinations of approaches. The methods used
herein may utilize CDRs defined according to any of these
approaches. For any given embodiment containing more than one CDR,
the CDRs may be defined in accordance with any of Kabat, Chothia,
extended, AbM, contact, and/or conformational definitions.
[0158] The term "contact residue," as used herein with respect to
an antibody or the antigen specifically bound thereby, refers to an
amino acid residue present on an antibody/antigen comprising at
least one heavy atom (i.e., not hydrogen) that is within 4 .ANG. or
less of a heavy atom of an amino acid residue present on the
cognate antibody/antigen.
[0159] As used herein, a "constant region" of an antibody refers to
the constant region of the antibody light chain or the constant
region of the antibody heavy chain, either alone or in
combination.
[0160] As used herein, "monoclonal antibody" refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally-occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present disclosure may be made by
the hybridoma method first described by Kohler and Milstein, 1975,
Nature 256:495, or may be made by recombinant DNA methods such as
described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may
also be isolated from phage libraries generated using the
techniques described in McCafferty et al., 1990, Nature
348:552-554, for example. As used herein, "humanized" antibody
refers to forms of non-human (e.g. murine) antibodies that are
chimeric immunoglobulins, immunoglobulin chains, or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) that contain minimal
sequence derived from non-human immunoglobulin. Preferably,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a CDR of the recipient are replaced by
residues from a CDR of a non-human species (donor antibody) such as
mouse, rat, or rabbit having the desired specificity, affinity, and
capacity. The humanized antibody may comprise residues that are
found neither in the recipient antibody nor in the imported CDR or
framework sequences, but are included to further refine and
optimize antibody performance.
[0161] As used herein, a "human antibody" refers to an antibody
having an amino acid sequence that corresponds to that of an
antibody produced by a human and/or that has been made using any of
the techniques for making human antibodies as disclosed herein.
This definition of a human antibody specifically excludes a
humanized antibody comprising non-human antigen binding
residues.
[0162] As used herein, the term "chimeric antibody" refers to
antibodies in which the variable region sequences are derived from
one species and the constant region sequences are derived from
another species, such as an antibody in which the variable region
sequences are derived from a mouse antibody and the constant region
sequences are derived from a human antibody or vice versa. The term
also encompasses an antibody comprising a V region from one
individual from one species (e.g., a first mouse) and a constant
region from another individual from the same species (e.g., a
second mouse).
[0163] As used herein, the term "antigen" ("Ag") refers to the
molecular entity used for immunization of an immunocompetent
vertebrate to produce the antibody (Ab) that recognizes the Ag or
to screen an expression library (e.g., phage, yeast or ribosome
display library, among others). As used herein, the term "antigen"
or "Ag" includes target molecules that are specifically recognized
by the Ab, thus including fragments or mimics of the molecule used
in an immunization process for raising the Ab or in library
screening for selecting the Ab. Thus, for antibodies of the
disclosure binding to FXIa, full-length FXIa from mammalian species
(e.g., human, monkey, mouse and rat FXIa), including monomers and
multimers, such as dimers, trimers, etc. thereof, as well as
truncated and other variants of FXIa, are referred to as an
antigen.
[0164] As used herein, the term "epitope" refers to the area or
region of an antigen to which an antibody specifically binds, i.e.,
an area or region in physical contact with the antibody. Thus, the
term "epitope" refers to that portion of a molecule capable of
being recognized by and bound by an antibody at one or more of the
antibody's antigen-binding regions. Typically, an epitope is
defined in the context of a molecular interaction between an
"antibody or antigen-binding fragment thereof" ("Ab") and its
corresponding antigen. Epitopes often consist of a surface grouping
of molecules such as amino acids or sugar side chains and have
specific three-dimensional structural characteristics as well as
specific charge characteristics. In some embodiments, the epitope
can be a protein epitope. Protein epitopes can be linear or
conformational. In a linear epitope, all of the points of
interaction between the protein and the interacting molecule (such
as an antibody) occur linearly along the primary amino acid
sequence of the protein. A "nonlinear epitope" or "conformational
epitope" comprises noncontiguous polypeptides (or amino acids)
within the antigenic protein to which an antibody specific to the
epitope binds. The term "antigenic epitope" as used herein, is
defined as a portion of an antigen to which an antibody can
specifically bind as determined by any method well known in the
art, for example, by conventional immunoassays. Alternatively,
during the discovery process, the generation and characterization
of antibodies may elucidate information about desirable epitopes.
From this information, it is then possible to competitively screen
antibodies for binding to the same epitope. An approach to achieve
this is to conduct competition and cross-competition studies to
find antibodies that compete or cross-compete with one another for
binding to FXIa, e.g., the antibodies compete for binding to the
antigen. Similarly, in the case of the anti-idiotype antibodies of
the disclosure, competition and cross-competition studies can be
conducted to find antibodies that compete or cross-compete with one
another for binding to an anti-FXIa antibody, e.g., the antibodies
compete for binding to the antigen-binding site of an anti-FXIa
antibody of the disclosure.
[0165] As used herein, the terms "wild-type amino acid," "wild-type
IgG," "wild-type antibody," or "wild-type mAb," refer to a sequence
of amino or nucleic acids that occurs naturally within a certain
population (e.g., human, mouse, rats, cell, etc.).
[0166] As outlined elsewhere herein, certain positions of the
antibody molecule can be altered. By "position" as used herein is
meant a location in the sequence of a protein. Positions may be
numbered sequentially, or according to an established format, for
example the EU index and Kabat index can be used to number amino
acid residues of an antibody. For example, position 297 is a
position in the human antibody IgG1. Corresponding positions are
determined as outlined above, generally through alignment with
other parent sequences.
[0167] By "residue" as used herein is meant a position in a protein
and its associated amino acid identity. For example, Asparagine 297
(also referred to as Asn297, also referred to as N297) is a residue
in the human antibody IgG1.
[0168] The terms "polynucleotide" or "nucleic acid," as used
interchangeably herein, refer to chains of nucleotides of any
length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or
bases, and/or their analogs, or any substrate that can be
incorporated into a chain by DNA or RNA polymerase. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the chain. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. Other types of modifications include, for
example, "caps," substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, poly-L-lysine, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid supports. The 5' and 3'
terminal OH can be phosphorylated or substituted with amines or
organic capping group moieties of from 1 to 20 carbon atoms. Other
hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or
deoxyribose sugars that are generally known in the art, including,
for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or
2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by P(O)S
("thioate"), P(S)S ("dithioate"), (O)NR.sub.2 ("amidate"), P(O)R,
P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or R' is
independently H or substituted or unsubstituted alkyl (1-20 C)
optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0169] As used herein, a molecule "preferentially binds" or
"specifically binds" (used interchangeably herein) to a cell or to
a substance (e.g., a protein, polypeptide, or antibody, e.g., a
protein, polypeptide, or antibody comprising an epitope) if the
molecule reacts or associates more frequently, more rapidly, with
greater duration and/or with greater affinity with a particular
cell or substance than it does with alternative cells or
substances. An antibody "specifically binds" or "preferentially
binds" to a target if it binds with greater affinity, avidity, more
readily, and/or with greater duration than it binds to other
substances. Also, an antibody "specifically binds" or
"preferentially binds" to a target if it binds with greater
affinity, avidity, more readily, and/or with greater duration to
that target in a sample than it binds to other substances present
in the sample. For example, an antibody that specifically or
preferentially binds to a FXIa epitope is an antibody that binds
this epitope with greater affinity, avidity, more readily, and/or
with greater duration than it binds to other FXIa epitopes or
non-FXIa epitopes. It will be understood by a person of ordinary
skill in the art reading this definition, for example, that an
antibody (or moiety or epitope) that specifically or preferentially
binds to a first target may or may not specifically or
preferentially bind to a second target. As such, "specific binding"
or "preferential binding" does not necessarily require (although it
can include) exclusive binding. Generally, but not necessarily,
reference to binding means preferential binding. "Specific binding"
or "preferential binding" includes a compound, e.g., a protein, a
nucleic acid, an antibody, and the like, which recognizes and binds
to a specific molecule, but does not substantially recognize or
bind other molecules in a sample. For instance, an antibody that
recognizes and binds to a binding partner (e.g., an anti-FXIa
antibody that binds FXIa) in a sample, but does not substantially
recognize or bind other molecules in the sample, specifically binds
to that cognate ligand or binding partner. Thus, under designated
assay conditions, the specified binding moiety (e.g., an antibody
or an antigen-binding portion thereof) binds preferentially to a
particular target molecule and does not bind in a significant
amount to other components present in a test sample.
[0170] A variety of assay formats may be used to select an antibody
or peptide that specifically binds a molecule of interest. For
example, solid-phase ELISA immunoassay, immunoprecipitation,
Biacore.TM. (GE Healthcare, Piscataway, N.J.), kinetic exclusion
assay (KinExA.RTM., Sapidyne Instruments, Inc., Boise, Id.)),
fluorescence-activated cell sorting (FACS), Octet.TM. (ForteBio,
Inc., Menlo Park, Calif.) and Western blot analysis are among many
assays that may be used to identify an antibody that specifically
reacts with an antigen or a receptor, or ligand binding portion
thereof, that specifically binds with a cognate ligand or binding
partner. Typically, a specific or selective reaction will be at
least twice the background signal or noise, more typically more
than 10 times background, even more typically, more than 50 times
background, more typically, more than 100 times background, yet
more typically, more than 500 times background, even more
typically, more than 1000 times background, and even more
typically, more than 10,000 times background. In some embodiments,
an antibody is said to "specifically bind" an antigen when the
equilibrium dissociation constant (K.sub.D) is .ltoreq.7 nM.
[0171] The term "binding affinity" is herein used as a measure of
the strength of a non-covalent interaction between two molecules,
e.g., an antibody, or fragment thereof, and an antigen. The term
"binding affinity" is used to describe monovalent interactions
(intrinsic activity).
[0172] Binding affinity between two molecules, e.g. an antibody, or
fragment thereof, and an antigen, through a monovalent interaction
may be quantified by determination of the dissociation constant
(K.sub.D). In turn, K.sub.D can be determined by measurement of the
kinetics of complex formation and dissociation using, e.g., the
surface plasmon resonance (SPR) method (Biacore.TM.). The rate
constants corresponding to the association and the dissociation of
a monovalent complex are referred to as the association rate
constants k.sub.a (or k.sub.on) and dissociation rate constant
k.sub.d (or k.sub.off), respectively. K.sub.D is related to k.sub.a
and k.sub.d through the equation K.sub.D=k.sub.d/k.sub.a. The value
of the dissociation constant can be determined directly by
well-known methods, and can be computed even for complex mixtures
by methods such as those, for example, set forth in Caceci et al.
(1984, Byte 9: 340-362). For example, the K.sub.D may be
established using a double-filter nitrocellulose filter binding
assay such as that disclosed by Wong & Lohman (1993, Proc.
Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays to
evaluate the binding ability of ligands such as antibodies towards
target antigens are known in the art, including for example,
ELISAs, Western blots, RIAs, and flow cytometry analysis, and other
assays exemplified elsewhere herein. The binding kinetics and
binding affinity of the antibody also can be assessed by standard
assays known in the art, such as Surface Plasmon Resonance (SPR),
e.g. by using a Biacore.TM. system, or KinExA.RTM..
[0173] A competitive binding assay can be conducted in which the
binding of the antibody to the antigen is compared to the binding
of the target by another ligand of that target, such as another
antibody or a soluble receptor that otherwise binds the target. The
concentration at which 50% inhibition occurs is known as the
K.sub.i. Under ideal conditions, the K.sub.i is equivalent to
K.sub.D. The K.sub.i value will never be less than the K.sub.D, so
measurement of K.sub.i can conveniently be substituted to provide
an upper limit for K.sub.D.
[0174] Following the above definition, binding affinities
associated with different molecular interactions, e.g., comparison
of the binding affinity of different antibodies for a given
antigen, may be compared by comparison of the K.sub.D values for
the individual antibody/antigen complexes. K.sub.D values for
antibodies or other binding partners can be determined using
methods well established in the art. One method for determining the
K.sub.D is by using surface plasmon resonance, typically using a
biosensor system such as a Biacore.RTM. system.
[0175] Similarly, the specificity of an interaction may be assessed
by determination and comparison of the K.sub.D value for the
interaction of interest, e.g., a specific interaction between an
antibody and an antigen, with the K.sub.D value of an interaction
not of interest, e.g., a control antibody known not to bind
FXIa.
[0176] An antibody that specifically binds its target may bind its
target with a high affinity, that is, exhibiting a low K.sub.D as
discussed above, and may bind to other, non-target molecules with a
lower affinity. For example, the antibody may bind to non-target
molecules with a K.sub.D of 1.times.10.sup.-6M or more, more
preferably 1.times.10.sup.-5 M or more, more preferably
1.times.10.sup.-4 M or more, more preferably 1.times.10.sup.-3 M or
more, even more preferably 1.times.10.sup.-2 M or more. An
anti-FXIa antibody of the disclosure is preferably capable of
binding to its target with an affinity that is at least two-fold,
10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or
10,000-fold or greater than its affinity for binding to another
non-FXIa molecule. Similarly, an anti-idiotype antibody of the
disclosure is preferably capable of binding to its target with an
affinity that is at least two-fold, 10-fold, 50-fold, 100-fold,
200-fold, 500-fold, 1,000-fold or 10,000-fold or greater than its
affinity for binding to another non-anti-FXIa antibody
molecule.
[0177] A "host cell" includes an individual cell or cell culture
that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected and/or transformed
in vivo with a polynucleotide of this disclosure.
[0178] As used herein, the term "Fc region" refers to a C-terminal
region of an immunoglobulin heavy chain. The "Fc region" may be a
native sequence Fc region or a variant Fc region. Although the
boundaries of the Fc region of an immunoglobulin heavy chain might
vary, the human IgG heavy chain Fc region is usually defined to
stretch from an amino acid residue at position Cys226, or from
Pro230, to the carboxyl-terminus thereof. The numbering of the
residues in the Fc region is that of the EU index as described in
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md., 1991. The Fc region of an immunoglobulin generally comprises
two constant domains, CH2 and CH3. As is known in the art, an Fc
region can be present in dimer or monomeric form.
[0179] As used herein, the term "Fc receptor" or "FcR" refers to a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof. FcRs are
reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92;
Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al.,
1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the
neonatal receptor, FcRn, which is responsible for the transfer of
maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol.,
117:587; and Kim et al., 1994, J. Immunol., 24:249).
[0180] The term "compete," as used herein with regard to an
antibody, means that a first antibody, or an antigen-binding
portion thereof, binds to an epitope in a manner sufficiently
similar to the binding of a second antibody, or an antigen-binding
portion thereof, such that the result of binding of the first
antibody with its cognate epitope is detectably decreased in the
presence of the second antibody compared to the binding of the
first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Both competing and cross-competing
antibodies are encompassed by the present disclosure. Regardless of
the mechanism by which such competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a
common epitope, or portion thereof), the skilled artisan would
appreciate, based upon the teachings provided herein, that such
competing and/or cross-competing antibodies are encompassed and can
be useful for the methods disclosed herein.
[0181] A "functional Fc region," as used herein, possesses at least
one effector function of a native sequence Fc region. Exemplary
"effector functions" include C1q binding; complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity; phagocytosis; down-regulation of cell surface
receptors (e.g. B cell receptor), etc. Such effector functions
generally require the Fc region to be combined with a binding
domain (e.g. an antibody variable domain or antigen-binding portion
thereof) and can be assessed using various assays known in the art
for evaluating such antibody effector functions.
[0182] A "native sequence Fc region," as used herein, comprises an
amino acid sequence identical to the amino acid sequence of an Fc
region found in nature. A "variant Fc region," as used herein,
comprises an amino acid sequence that differs from that of a native
sequence Fc region by virtue of at least one amino acid
modification, yet retains at least one effector function of the
native sequence Fc region. Preferably, the variant Fc region has at
least one amino acid substitution compared to a native sequence Fc
region or to the Fc region of a parent polypeptide, e.g. from about
one to about ten amino acid substitutions, and preferably, from
about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% sequence identity with a native sequence Fc region and/or with
an Fc region of a parent polypeptide, and most preferably, at least
about 90% sequence identity therewith, more preferably, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99% sequence identity therewith.
[0183] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
disclosure, beneficial or desired clinical results include, but are
not limited to, one or more of the following: improved survival
rate (reduced mortality), decrease in the occurrence of disease
(e.g., thrombosis or thromboembolism), decreased extent of damage
from the disease, decreased duration of the disease, and/or
reduction in the number, extent, or duration of symptoms related to
the disease. The term includes the administration of the compounds
or agents of the present disclosure to prevent or delay the onset
of the symptoms, complications, or biochemical indicia of a
disease, alleviating the symptoms or arresting or inhibiting
further development of the disease, condition, or disorder.
Treatment may be prophylactic (to prevent or delay the onset of the
disease, or to prevent the manifestation of clinical or subclinical
symptoms thereof) or therapeutic suppression or alleviation of
symptoms after the manifestation of the disease.
[0184] "Ameliorating," as used with respect to administering an
anti-FXIa antibody as described herein, means a lessening or
improvement of one or more symptoms as compared to not
administering an anti-FXIa antibody. "Ameliorating" also includes
shortening or reduction in duration of a symptom.
[0185] As used herein, an "effective dosage" or "effective amount"
of a drug, compound, or pharmaceutical composition is an amount
sufficient to affect any one or more beneficial or desired results.
In more specific aspects, an effective amount prevents, alleviates
or ameliorates symptoms of disease or infection, and/or prolongs
the survival of the subject being treated. For prophylactic use,
beneficial or desired results include eliminating or reducing the
risk, lessening the severity, or delaying the outset of the
disease, including biochemical, histological and/or behavioral
symptoms of the disease, its complications and intermediate
pathological phenotypes presenting during development of the
disease. For therapeutic use, beneficial or desired results include
clinical results such as reducing one or more symptoms of a
FXIa-mediated disease, disorder or condition, decreasing the dose
of other medications required to treat the disease, enhancing the
effect of another medication, and/or delaying the progression of
the disease of patients. An effective dosage can be administered in
one or more administrations. For purposes of this disclosure, an
effective dosage of drug, compound, or pharmaceutical composition
is an amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0186] An "individual" or a "subject" is a mammal, in some
embodiments, a human. Mammals also include, but are not limited to,
farm animals (e.g., cows, pigs, horses, chickens, etc.), sport
animals, pets, primates, horses, dogs, cats, mice and rats. In some
embodiments, the individual is considered to be at risk for a
disease, disorder or condition mediated by or associated with FXIa.
In certain embodiments, the subject has a thrombotic condition. In
certain embodiments, the subject is being administered an anti-FXIa
antibody and is in need of a reversal agent.
[0187] As used herein, "vector" means a construct, which is capable
of delivering, and, preferably, expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0188] As used herein, "expression control sequence" means a
nucleic acid sequence that directs transcription of a nucleic acid.
An expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0189] As used herein, "pharmaceutically acceptable carrier" or
"pharmaceutical acceptable excipient" includes any material which,
when combined with an active ingredient, allows the ingredient to
retain biological activity and is non-reactive with the subject's
immune system. Examples include, but are not limited to, any of the
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Preferred diluents for aerosol or
parenteral administration are phosphate buffered saline (PBS) or
normal (0.9%) saline. Compositions comprising such carriers are
formulated by well-known conventional methods (see, for example,
Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science
and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
[0190] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X." Numeric ranges are inclusive of the
numbers defining the range.
[0191] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to
encompass any and all subranges subsumed therein. For example, a
stated range of "1 to 10" should be considered to include any and
all subranges between (and inclusive of) the minimum value of 1 and
the maximum value of 10; that is, all subranges beginning with a
minimum value of 1 or more, e.g. 1 to 6.1, and ending with a
maximum value of 10 or less, e.g., 5.5 to 10.
[0192] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the present specification, including definitions, will
control. Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising" will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers. Unless otherwise required by context, singular terms
shall include pluralities and plural terms shall include the
singular. Any example(s) following the term "e.g." or "for example"
is not meant to be exhaustive or limiting.
[0193] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0194] Where aspects or embodiments of the disclosure are described
in terms of a Markush group or other grouping of alternatives, the
present disclosure encompasses not only the entire group listed as
a whole, but each member of the group individually and all possible
subgroups of the main group, but also the main group absent one or
more of the group members. The present invention also envisages the
explicit exclusion of one or more of any of the group members in
the claimed invention.
[0195] Exemplary methods and materials are described herein,
although methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present invention. The materials, methods, and examples are
illustrative only and not intended to be limiting.
III. Antibodies to FXIa and Anti-Idiotype Antibodies that Bind
Anti-FXIa Antibodies
[0196] In one aspect, the present disclosure relates to antibodies,
e.g., full-length antibodies and antigen-binding fragments thereof,
that specifically bind to Factor XIa (FXIa) or a target molecule
comprising an epitope from FXIa. In some embodiments, an anti-FXIa
antibody specifically binds to a mammalian FXIa, such as human, rat
or mouse, as well as non-human primate, bovine, ovine, or porcine
FXIa. In some embodiments, the anti-FXIa antibody specifically
binds a full-length human FXIa (e.g., the human FXIa protein of SEQ
ID NO: 98) or a full-length cynomolgus monkey FXIa. In some
embodiments, the anti-FXIa antibody specifically binds a fragment,
variant, isoform, or homolog of such an FXIa molecule. In some
embodiments, a variant FXIa molecule is characterized by having the
same type of activity as a naturally occurring FXIa, such as the
ability to bind FIX, thrombin or platelets, and the ability to
activate the coagulation cascade.
[0197] In some embodiments, the FXIa variant or fragment may
comprise one or more, two or more, three or more, four or more,
five or more, six or more, seven or more, eight or more, nine or
more, ten or more, twelve or more or fifteen or more solvent
accessible residues of FXIa. Where the FXIa comprises a
homomultimeric form of FXIa, the FXIa variant or fragment may
comprise one or more, two or more, three or more, four or more,
five or more, six or more, seven or more, eight or more, nine or
more, ten or more, twelve or more, or fifteen or more solvent
accessible residues of a first subunit of FXIa, and one or more,
two or more, three or more, four or more, five or more, six or
more, seven or more, eight or more, nine or more, ten or more,
twelve or more, or fifteen or more solvent accessible residues of a
second subunit of FXIa.
[0198] In some embodiments, an antibody or antigen-binding fragment
thereof specifically binds to FXIa or to a target molecule
comprising an epitope from FXIa. In some embodiments, the target
molecule may comprise the catalytic domain of FXIa.
[0199] In another aspect, the present disclosure relates to
anti-idiotype antibodies that specifically bind to anti-FXIa
antibodies as described herein.
[0200] In another aspect, the disclosure also relates to
compositions comprising anti-FXIa antibodies or anti-idiotype
antibodies that specifically bind to anti-FXIa antibodies as
described herein, as well as uses for such antibodies, including
therapeutic and pharmaceutical uses.
[0201] As detailed herein, the antibodies useful in the present
disclosure (e.g. anti-FXIa antibodies and anti-idiotype antibodies
that specifically bind to the antigen-binding site of an anti-FXIa
antibody) can encompass monoclonal antibodies, polyclonal
antibodies, antibody fragments (e.g., Fab, Fab', F(ab').sub.2, Fv,
Fc, etc.), chimeric antibodies, bispecific antibodies,
heteroconjugate antibodies, single chain (ScFv), mutants thereof,
fusion proteins comprising an antibody portion (e.g., a domain
antibody), humanized antibodies, and any other modified
configuration of the immunoglobulin molecule that comprises an
antigen recognition site of the required specificity, including
glycosylation variants of antibodies, amino acid sequence variants
of antibodies, and covalently modified antibodies. The antibodies
may be murine, rat, human, or any other origin (including chimeric
or humanized antibodies). In some embodiments, the FXIa antibody is
a monoclonal antibody. In some embodiments, the FXIa antibody is a
human or humanized antibody. In some embodiments, the anti-idiotype
antibody is a monoclonal antibody. In some embodiments, the
anti-idiotype antibody is a human or humanized antibody.
Anti-FXIa Antibodies
[0202] In one aspect, the present disclosure relates to antibodies
that bind to FXIa. The antibodies preferably specifically bind to
FXIa, i.e., they bind to FXIa but they do not detectably bind, or
bind at a lower affinity, to other molecules. In particular, in
some embodiments, the disclosure relates to antibodies that
specifically bind to FXIa but not the zymogen FXI. In some
embodiments, the anti-FXIa antibodies of the disclosure
specifically bind the FXIa catalytic domain and/or adjacent
residues.
[0203] In some embodiments, the anti-FXIa antibodies of the
disclosure prolong activated partial thromboplastin time (APTT)
without significantly increasing prothrombin time (PT). This
finding reflects inhibition of the intrinsic pathway but not the
extrinsic or common pathways of coagulation by the antibody and was
associated with anti-thrombotic protection without increased
bleeding risk, as shown in the Examples (e.g., FIGS. 9 and 10). As
used herein, the term "prolong activated partial thomboplastin
time" refers to a measurement of the length of time it takes for
plasma to clot after addition of an intrinsic pathway activator
such as ellagic acid or kaolin, and the term "increasing
prothrombin time" refers to a measurement of the length of time it
takes for plasma to clot after addition of an extrinsic pathway
activator such as Tissue Factor or thromboplastin. Thus, APTT is a
measurement of the intrinsic pathway of coagulation, while PT is a
measurement of the extrinsic pathway of coagulation. Both can also
be prolonged by sufficient inhibition of the common pathway.
[0204] In some embodiments, an anti-FXIa antibody prolongs
activated partial thromboplastin time (APTT) if the length of time
it takes for a sample of plasma to clot after addition of an
intrinsic pathway activator (e.g., ellagic acid or kaolin) in the
presence of an anti-FXIa antibody is greater than the length of
time it takes for a sample of plasma to clot after addition of the
intrinsic pathway activator in the absence of the anti-FXIa
antibody. In some embodiments, an anti-FXIa antibody prolongs APTT
by at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 100%, at least 150%, or more. In some embodiments, an
anti-FXIa antibody does not "significantly increase prothrombin
time" if the length of time it takes for a sample of plasma to clot
after addition of an extrinsic pathway activator (e.g., Tissue
Factor or thromboplastin) in the presence of an anti-FXIa antibody
is no more than 20% longer, no more than 15% longer, no more than
10%, no more than 5% longer, or no longer than the length of time
it takes for a sample of plasma to clot after addition of the
extrinsic pathway activator in the absence of the anti-FXIa
antibody. APTT and PT can be measured at a predetermined time after
administration of an anti-FXIa antibody (e.g., 15 mins, 20 mins, 30
mins, 40 mins, 45 mins, 50 mins, 60 mins or more after
administration of an anti-FXIa antibody). Methods of measuring APTT
and PT are known in the art and are also described herein in the
Examples section.
[0205] In some embodiments, the anti-FXIa antibodies of the
disclosure have an increased dissociation rate from FXIa in the
presence of a serine protease inhibitor. In some embodiments, the
anti-FXIa antibodies of the disclosure have an increased
dissociation rate from FXIa after treatment of the latter with an
agent that chemically modifies the active site serine of a serine
protease. In some embodiments, the serine protease inhibitor or
agent is phenylmethylsulfonyl fluoride (PMSF). Methods of measuring
the dissociation rate of an anti-FXIa antibody from FXIa are known
in the art and are also described in the Examples section below. In
some embodiments, dissociate rate is measured using Surface Plasmon
Resonance (SPR), e.g. by using a Biacore.TM. system. In some
embodiments, an anti-FXIa antibody has an increased dissociation
rate from FXIa in the presence of a serine protease inhibitor or
after treatment of FXIa with an agent that chemically modifies the
active site serine of a serine protease if the dissociation rate is
increased at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 100%, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, or more as
compared to the dissociation rate of the anti-FXIa antibody from
FXIa in the absence of the serine protease inhibitor or agent.
[0206] In some embodiments, the anti-FXIa antibodies of the
disclosure bind to, and have their anticoagulant activity decreased
by, a recombinant FXIa protease-domain in which the active site
serine is changed to alanine (e.g., as described in U.S.
Provisional Patent Application "Reversal Agents for FXIa
Inhibitors," No. 62/196,085, incorporated by reference herein).
Methods of measuring specific binding and binding affinity are
known in the art and include, but are not limited to, solid-phase
binding assays, immunoprecipitation, surface plasmon resonance
(e.g., Biacore.TM. (GE Healthcare, Piscataway, N.J.)), kinetic
exclusion assay, fluorescence-activated cell sorting (FACS),
Octet.TM. (ForteBio, Inc., Menlo Park, Calif.), and Western blot
analysis. Methods of measuring anticoagulant activity are known in
the art and include, but are not limited to, measuring clotting
time (e.g., APTT and/or PT) and measuring thrombin production
(e.g., using a thrombin generation assay (TGA)). Methods of
measuring specific binding and binding affinity and anticoagulant
activity are also described in U.S. Provisional Patent Application
No. 62/196,085. In some embodiments, the binding activity of an
anti-FXIa antibody to a recombinant FXIa protease-domain in which
the active site serine is changed to alanine, and the anticoagulant
activity of an anti-FXIa antibody in the presence of a recombinant
FXIa protease-domain in which the active site serine is changed to
alanine, are measured using a recombinant FXIa protease-domain that
is disclosed in U.S. Provisional Patent Application No. 62/196,085
and that has the amino acid sequence of SEQ ID NO:2 in U.S.
Provisional Patent Application No. 62/196,085.
[0207] Preferably, an anti-FXIa antibody of the disclosure has at
least one of these features. In some embodiments, the anti-FXIa
antibody has two or more of these features. In some embodiments,
the anti-FXIa antibody has all of these features.
[0208] In one embodiment, the disclosure provides an antibody
having a light chain sequence, or a portion thereof, and a heavy
chain, or a portion thereof, derived from any of the following
antibodies: D4, DEF, QCA11, B1D2, B10H2, B10E6, B10F6, B10D8,
B10B12, S1D4, S10H9, Clone 8, Clone 16, Clone 20, Clone 22, Clone
32, or Clone 24; or a composition (including pharmaceutical
compositions) comprising such an antibody. The amino acid sequences
of the light chain variable domain (VL) and heavy chain variable
domains (VH) of the anti-FXIa antibodies DEF, D4, QCA11, B1D2,
B10H2, B10E6, B10F6, B10D8, B10B12, S1D4, S10H9, Clone 8, Clone 16,
Clone 20, Clone 22, Clone 32, and Clone 24 are set forth in Table 5
below.
[0209] In some embodiments, an anti-FXIa antibody of the disclosure
comprises both: [0210] a) a VH comprising the amino acid sequence
of SEQ ID NO:1, and a VL comprising the amino acid sequence of SEQ
ID NO:7; [0211] b) a VH comprising the amino acid sequence of SEQ
ID NO:14, and a VL comprising the amino acid sequence of SEQ ID
NO:17; [0212] c) a VH comprising the amino acid sequence of SEQ ID
NO:18, and a VL comprising the amino acid sequence of SEQ ID NO:21;
[0213] d) a VH comprising the amino acid sequence of SEQ ID NO:22,
and a VL comprising the amino acid sequence of SEQ ID NO:23; [0214]
e) a VH comprising the amino acid sequence of SEQ ID NO:24, and a
VL comprising the amino acid sequence of SEQ ID NO:25; [0215] f) a
VH comprising the amino acid sequence of SEQ ID NO:26, and a VL
comprising the amino acid sequence of SEQ ID NO:27; [0216] g) a VH
comprising the amino acid sequence of SEQ ID NO:28, and a VL
comprising the amino acid sequence of SEQ ID NO:31; [0217] h) a VH
comprising the amino acid sequence of SEQ ID NO:34, and a VL
comprising the amino acid sequence of SEQ ID NO:37; [0218] i) a VH
comprising the amino acid sequence of SEQ ID NO:38, and a VL
comprising the amino acid sequence of SEQ ID NO:39; [0219] j) a VH
comprising the amino acid sequence of SEQ ID NO:40, and a VL
comprising the amino acid sequence of SEQ ID NO:42; [0220] k) a VH
comprising the amino acid sequence of SEQ ID NO:43, and a VL
comprising the amino acid sequence of SEQ ID NO:46; [0221] l) a VH
comprising the amino acid sequence of SEQ ID NO:47, and a VL
comprising the amino acid sequence of SEQ ID NO:50; [0222] m) a VH
comprising the amino acid sequence of SEQ ID NO:51, and a VL
comprising the amino acid sequence of SEQ ID NO:54; [0223] n) a VH
comprising the amino acid sequence of SEQ ID NO:55, and a VL
comprising the amino acid sequence of SEQ ID NO:58; [0224] o) a VH
comprising the amino acid sequence of SEQ ID NO:59, and a VL
comprising the amino acid sequence of SEQ ID NO:62; [0225] p) a VH
comprising the amino acid sequence of SEQ ID NO:63, and a VL
comprising the amino acid sequence of SEQ ID NO:64; or [0226] q) a
VH comprising the amino acid sequence of SEQ ID NO:65, and a VL
comprising the amino acid sequence of SEQ ID NO:68.
[0227] In some embodiments, an anti-FXIa antibody of the disclosure
comprises both a VH comprising the consensus amino acid sequence of
SEQ ID NO:96, and a VL comprising the consensus amino acid sequence
of SEQ ID NO:97. The consensus VH sequence of SEQ ID NO:96 and the
consensus VL sequence of SEQ ID NO:97 are described in Table 5
below.
[0228] In another aspect, the antibody comprises a variant of any
one or more of these sequences, wherein such variants can include
both conservative and non-conservative substitutions, deletions,
and/or additions, and typically include peptides that share at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 87%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to any of the specific sequences disclosed herein.
[0229] For example, in one aspect, the disclosure provides an
isolated antibody or antigen-binding portion thereof that comprises
a V.sub.L chain amino acid sequence as set forth in SEQ ID NO: 7,
SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID
NO:27, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:42, SEQ
ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:68, or SEQ ID NO:97 or a variant thereof.
In one aspect, said antibody variant comprises 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative
substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 additions and/or deletions to SEQ ID NO: 7, SEQ ID NO:17,
SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID
NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:46, SEQ
ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:64,
SEQ ID NO:68, or SEQ ID NO:97. In a further aspect, said variant
shares at least 65%, at least 75%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence identity with SEQ ID NO: 7, SEQ ID NO:17, SEQ ID
NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, SEQ
ID NO:37, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50,
SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:64, SEQ ID
NO:68, or SEQ ID NO:97 and wherein said antibody or antigen-binding
portion specifically binds FXIa.
[0230] In a further aspect, the disclosure provides an isolated
antibody or antigen-binding portion thereof that comprises a
V.sub.H chain amino acid sequence as set forth in SEQ ID NO: 1, SEQ
ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID
NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ
ID NO:63, SEQ ID NO:65, or SEQ ID NO:96, or a variant thereof. In
one aspect, said antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative
substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 additions and/or deletions to SEQ ID NO: 1, SEQ ID NO:14,
SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID
NO:28, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ
ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63,
SEQ ID NO:65, or SEQ ID NO:96. In a further aspect, said variant
shares at least 65%, at least 75%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence identity with SEQ ID NO: 1, SEQ ID NO:14, SEQ ID
NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ
ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:47,
SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID
NO:65, or SEQ ID NO:96, and wherein said antibody or
antigen-binding portion specifically binds FXIa.
[0231] An anti-FXIa antibody of the disclosure may comprise a heavy
chain comprising a VH comprising the amino acid sequence of SEQ ID
NO: 1, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40,
SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID
NO:59, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:96, wherein the
antibody further comprises a heavy chain constant domain. As more
fully set forth elsewhere herein, the antibody heavy chain constant
domain can be selected from an IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG.sub.1 or IgG.sub.2 constant region. The IgG
constant region sequence can be any of the various alleles or
allotypes known to occur among different individuals, such as
Gm(1), Gm(2), Gm(3), and Gm(17). For a Fab fragment heavy chain
gene, the VH-encoding DNA can be operatively linked to another DNA
molecule encoding only the heavy chain CH1 constant region. The CH1
heavy chain constant region may be derived from any of the heavy
chain genes.
[0232] In one aspect, the antibody may comprise a heavy chain
comprising a VH selected from a VH comprising the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22,
SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:34, SEQ ID
NO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ
ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID
NO:96, and further comprising the IgG1 constant domain comprising a
triple mutation decreasing or abolishing Fc effector function. In
one aspect, said antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative
substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 additions and/or deletions to the full length heavy
chain. In a further aspect, said variant shares at least 65%, at
least 75%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% sequence identity with
the full length heavy chain, and wherein said antibody or
antigen-binding portion specifically binds FXIa. In some
embodiments, the antibody comprises a heavy chain constant domain
comprising the amino acid sequence of SEQ ID NO:82 or SEQ ID
NO:103.
[0233] An antibody of the disclosure may comprise a light chain
comprising a VL comprising the amino acid sequence of SEQ ID NO: 7,
SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID
NO:27, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:42, SEQ
ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:68, or SEQ ID NO:97, wherein the antibody
further comprises a light chain constant domain. The antibody light
chain constant domain can be selected from a C.kappa. or C.lamda.
constant region, for example the constant region of SEQ ID NO:83.
In one aspect, said antibody variant comprises 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative
substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 additions and/or deletions to the full length light
chain. In a further aspect, said variant shares at least 65%, at
least 75%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% sequence identity with
the full length light chain, and wherein said antibody or
antigen-binding portion specifically binds FXIa. In some
embodiments, the antibody comprises a light chain constant domain
comprising the amino acid sequence of SEQ ID NO:83.
[0234] An antibody of the disclosure may comprise a fragment of one
of the VL or VH amino acid sequences shown in Table 5. For example,
an antibody of the disclosure may comprise a fragment of at least
7, at least 8, at least 9, at least 10, at least 12, at least 15,
at least 18, at least 20 or at least 25 consecutive amino acids
from a VH comprising SEQ ID NO: 1, SEQ ID NO:14, SEQ ID NO:18, SEQ
ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:34,
SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:47, SEQ ID
NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:65, or
SEQ ID NO:96, and/or from a VL comprising SEQ ID NO: 7, SEQ ID
NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ
ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:46,
SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID
NO:64, SEQ ID NO:68, or SEQ ID NO:97. Such a fragment will
preferably retain one or more of the functions discussed above,
such as the ability to bind to FXIa.
[0235] A suitable fragment or variant of any of these VH or VL
sequences will retain the ability to bind to FXIa. In some
embodiments, it will retain the ability to specifically bind to
FXIa. In some embodiments, it will retain the ability to
specifically bind to the same or similar epitope or region of the
FXIa molecule as the antibody from which it is derived.
[0236] An antibody of the disclosure may comprise a CDR region from
the specific antibody identified herein, such as a CDR region from
within SEQ ID NOs: 7, 17, 21, 23, 25, 27, 31, 37, 39, 42, 46, 50,
54, 58, 62, 64, 68, or 97 or within SEQ ID NOs: 1, 14, 18, 22, 24,
26, 28, 34, 38, 40, 43, 47, 51, 55, 59, 63, 65, or 96. Such an
antibody will preferably retain the ability to bind to FXIa as
described herein. For example, the CDR sequences of the antibodies
D4, DEF, QCA11, B1D2, B10H2, B10E6, B10F6, B10D8, B10B12, S1D4,
S10H9, Clone 8, Clone 16, Clone 20, Clone 22, Clone 32, or Clone 24
are shown in Table 5 and in the accompanying Sequence Listing.
[0237] In one aspect, the disclosure provides an antibody variant
comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
conservative or non-conservative substitutions, and/or 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and/or deletions
to a CDR listed herein (e.g., a CDR sequence as shown in Table 5).
In a further aspect, the variant shares at least 65%, at least 75%,
at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% sequence identity with a CDR
sequence listed herein (e.g., a CDR sequence as shown in Table 5),
and wherein the antibody or antigen-binding portion specifically
binds FXIa.
[0238] In some embodiments, an anti-FXIa antibody binds to the
active site of the catalytic domain of FXIa (e.g., human FXIa). In
some embodiments, an anti-FXIa antibody binds to the active site of
the catalytic domain of FXIa near the FXIa catalytic triad (e.g.,
His 431, Asp 480, and Ser 575 in human FXIa). In some embodiments,
the anti-FXIa antibody binds to FXIa at one or more of the
following residues of FXIa: His 414, Tyr 434, Met 474, Ala 475, Ser
477, Asp 480, Tyr 521, Arg 525, Asp 526, Asp 569, Lys 572, Ser 594,
and Gly 598, wherein the FXIa is numbered with reference to the
full-length FXIa sequence of SEQ ID NO:98. In some embodiments, the
anti-FXIa antibody binds two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
10 or more) of the residues His 414, Tyr 434, Met 474, Ala 475, Ser
477, Asp 480, Tyr 521, Arg 525, Asp 526, Asp 569, Lys 572, Ser 594,
and Gly 598 in FXIa, wherein the residues are numbered with
reference to the full-length FXIa sequence of SEQ ID NO:98. In some
embodiments, the anti-FXI antibody binds to all of the residues His
414, Tyr 434, Met 474, Ala 475, Ser 477, Asp 480, Tyr 521, Arg 525,
Asp 526, Asp 569, Lys 572, Ser 594, and Gly 598 of FXIa, wherein
the residues are numbered with reference to the full-length FXIa
sequence of SEQ ID NO:98 (e.g., a full-length FXIa sequence that
includes the native signal peptide). When these residues are
instead numbered with reference to a truncated FXIa catalytic
domain (i.e., Ile370 to Ala606, for example as shown in SEQ ID
NO:100), the residues are numbered His 27, Tyr 47, Met 87, Ala 88,
Ser 90, Asp 93, Tyr 134, Arg 138, Asp 139, Asp 182, Lys 185, Ser
207, and Gly 211, respectively.
[0239] In some embodiments, an anti-FXIA antibody can be defined by
its paratopes. The definition of the term "paratope" is derived
from the above definition of "epitope" by reversing the
perspective. Thus, the term "paratope" refers to the area or region
on the antibody which specifically binds an antigen, i.e., the
amino acid residues on the antibody which make contact with the
antigen (FXIa or anti-FXIa antibody) as "contact" is defined
elsewhere herein.
[0240] In some embodiments, an anti-FXIa antibody comprises a
paratope comprising one or more of the residues Leu 99 or Tyr 33,
wherein the residues are numbered with reference to the sequence of
SEQ ID NO:101; and or comprises a paratope comprising one or more
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) of the residues Ala 25,
Gln 27, Arg 30, Asp 32, Ser 67, Thr 69, His 91, Asp 92, Il3 93, or
Tyr 94, wherein the residues are numbered with reference to the
sequence of SEQ ID NO:102.
Anti-Idiotype Antibodies that Specifically Bind to Anti-FXIa
Antibodies
[0241] In another aspect, the present disclosure relates to
anti-idiotype antibodies that specifically bind to an anti-FXIa
antibody, such as the anti-FXIa antibodies described herein. In
some embodiments, an anti-idiotype antibody specifically binds to
the antigen-binding site of the anti-FXIa antibody.
[0242] In one embodiment, the disclosure provides an anti-idiotype
antibody that binds to the antigen binding site of an anti-FXIa
antibody, wherein the anti-idiotype antibody has a light chain
sequence, or a portion thereof, and a heavy chain, or a portion
thereof, derived from the antibody C4; or a composition (including
pharmaceutical compositions) comprising such an antibody. The amino
acid sequences of the light chain variable domain (VL) and heavy
chain variable domains (VH) of the anti-idiotype antibodies C4 are
set forth in Table 5 below.
[0243] In some embodiments, an anti-idiotype antibody of the
disclosure that specifically binds to the antigen-binding site of
an anti-FXIa antibody or antigen-binding portion thereof of the
disclosure may comprise both a VH comprising the amino acid
sequence of SEQ ID NO:69, and a VL comprising the amino acid
sequence of SEQ ID NO:75.
[0244] In another aspect, the anti-idiotype antibody comprises a
variant of these sequences, wherein such variants can include both
conservative and non-conservative substitutions, deletions, and/or
additions, and typically include peptides that share at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 87%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% sequence identity to
any of the specific sequences disclosed herein.
[0245] For example, in one aspect, the disclosure provides an
isolated antibody or antigen-binding portion thereof that comprises
a V.sub.L chain amino acid sequence as set forth in SEQ ID NO:75 or
a variant thereof. In one aspect, said antibody variant comprises
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative
or non-conservative substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15 additions and/or deletions to SEQ ID
NO:75. In a further aspect, said variant shares at least 65%, at
least 75%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% sequence identity with
SEQ ID NO:75 and wherein said antibody or antigen-binding portion
specifically binds an anti-FXIa antibody of the disclosure (e.g.,
DEF).
[0246] In a further aspect, the disclosure provides an isolated
antibody or antigen-binding portion thereof that comprises a
V.sub.H chain amino acid sequence as set forth in SEQ ID NO:69 or a
variant thereof. In one aspect, said antibody variant comprises 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or
non-conservative substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 additions and/or deletions to SEQ ID
NO:69. In a further aspect, said variant shares at least 65%, at
least 75%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% sequence identity with
SEQ ID NO:69, and wherein said antibody or antigen-binding portion
specifically binds specifically binds an anti-FXIa antibody of the
disclosure.
[0247] An anti-idiotype antibody of the disclosure may comprise a
heavy chain comprising a VH comprising the amino acid sequence of
SEQ ID NO:69, wherein the antibody further comprises a heavy chain
constant domain. The antibody heavy chain constant domain can be
selected from an IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA,
IgE, IgM or IgD constant region, but most preferably is an
IgG.sub.1 or IgG.sub.2 constant region. The IgG constant region
sequence can be any of the various alleles or allotypes known to
occur among different individuals, such as Gm(1), Gm(2), Gm(3), and
Gm(17). For a Fab fragment heavy chain gene, the VH-encoding DNA
can be operatively linked to another DNA molecule encoding only the
heavy chain CH1 constant region. The CH1 heavy chain constant
region may be derived from any of the heavy chain genes.
[0248] In one aspect, the antibody may comprise a heavy chain
comprising a VH comprising the amino acid sequence of SEQ ID NO:69,
and further comprising the IgG1 constant domain comprising a triple
mutation decreasing or abolishing Fc effector function. In one
aspect, said antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 conservative or non-conservative
substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 additions and/or deletions to the full length heavy
chain. In a further aspect, said variant shares at least 65%, at
least 75%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% sequence identity with
the full length heavy chain, and wherein said antibody or
antigen-binding portion specifically binds an anti-FXIa antibody of
the disclosure.
[0249] An antibody of the disclosure may comprise a light chain
comprising a VL comprising the amino acid sequence of SEQ ID NO:75,
wherein the antibody further comprises a light chain constant
domain. The antibody light chain constant domain can be selected
from a C.kappa. or C.lamda. constant region, for example the
constant region of SEQ ID NO:83. In one aspect, said antibody
variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15 conservative or non-conservative substitutions, and/or 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and/or
deletions to the full length light chain. In a further aspect, said
variant shares at least 65%, at least 75%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% sequence identity with the full length light chain, and
wherein said antibody or antigen-binding portion specifically binds
an anti-FXIa antibody of the disclosure.
[0250] An anti-idiotype antibody of the disclosure may comprise a
fragment of SEQ ID NO:69 or SEQ ID NO:75 shown in Table 5. For
example, an antibody of the disclosure may comprise a fragment of
at least 7, at least 8, at least 9, at least 10, at least 12, at
least 15, at least 18, at least 20 or at least 25 consecutive amino
acids from a VH comprising SEQ ID NO:69, or from a VL comprising
SEQ ID NO:75. Such a fragment will preferably retain one or more of
the functions discussed above, such as the ability to bind to an
anti-FXIa antibody of the disclosure.
[0251] A suitable fragment or variant of any of these VH or VL
sequences will retain the ability to bind to an anti-FXIa antibody
of the disclosure. In some embodiments, it will retain the ability
to specifically bind to an anti-FXIa antibody of the disclosure. In
some embodiments, it will retain the ability to specifically bind
to the same or similar epitope or region of the anti-FXIa antibody
(e.g. variable domain) as the antibody from which it is
derived.
[0252] An antibody of the disclosure may comprise a CDR region from
the specific antibody identified herein such as a CDR region from
within SEQ ID NO: 69 or within SEQ ID NO:75. Such an antibody will
preferably retain the ability to bind to an anti-FXIa antibody of
the disclosure as described herein. For example, the CDR sequences
of the anti-idiotype antibody C4 are shown in Table 5 and in the
accompanying Sequence Listing.
[0253] In one aspect, the disclosure provides an antibody variant
comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
conservative or non-conservative substitutions, and/or 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and/or deletions
to the CDRs listed above. In a further aspect, the variant shares
at least 65%, at least 75%, at least 85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%
sequence identity with the CDR sequences listed above, and wherein
the antibody or antigen-binding portion specifically binds an
anti-FXIa antibody of the disclosure.
Identification and Characterization of Anti-FXIa Antibodies and
Anti-Idiotype Antibodies that Specifically Bind to Anti-FXIa
Antibodies
[0254] FXIa antibodies can be identified or characterized using
methods known in the art, whereby binding to FXIa is required for
selection, and reduction, amelioration, or neutralization of FXIa
activity is detected and/or measured, for example, in an in vitro
activity assay with a substrate. In some embodiments, an FXIa
antibody is identified by incubating a candidate agent (e.g., FXIa)
with a substrate and monitoring binding and/or attendant reduction
or inhibition of a biological activity of FXIa (e.g. catalytic
activity). The binding assay may be performed with, e.g., purified
FXIa polypeptide(s) or with human plasma. In one embodiment, the
binding assay is a competitive binding assay, where the ability of
a candidate antibody to compete with a known FXIa antibody for FXIa
binding is evaluated. The assay may be performed in various
formats, including the ELISA format. In some embodiments, a FXIa
antibody is identified by incubating a candidate antibody with FXIa
and monitoring binding.
[0255] Anti-idiotype antibodies that specifically bind to the
antigen-binding site of an anti-FXIa antibody can be identified or
characterized using methods known in the art, whereby reduction,
amelioration, or neutralization of anti-FXIa antibody activity
(e.g. FXIa inhibitory activity) is detected and/or measured.
[0256] Following initial identification, the activity of a
candidate FXIa antibody or anti-idiotype antibody specifically
binds to the antigen-binding site of an anti-FXIa antibody can be
further confirmed and refined by bioassays, known to test the
targeted biological activities. In some embodiments, an in vitro
biochemical assay is used to further characterize a candidate FXIa
antibody or anti-idiotype antibody specifically binds to the
antigen-binding site of an anti-FXIa antibody. For example,
bioassays can be used to screen candidates directly. Some of the
methods for identifying and characterizing FXIa antibody or
anti-idiotype antibody are described in detail in the Examples.
[0257] FXIa antibodies or anti-idiotype antibodies that
specifically bind to the antigen-binding site of an anti-FXIa
antibody may be characterized using methods well known in the art.
For example, one method is to identify the epitope to which it
binds, or "epitope mapping." There are many methods known in the
art for mapping and characterizing the location of epitopes on
proteins, including solving the crystal structure of an
antibody-antigen complex, competition assays, gene fragment
expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. In an additional example,
epitope mapping can be used to determine the sequence to which a
FXIa antibody or anti-idiotype antibody of the disclosure binds.
Epitope mapping is commercially available from various sources, for
example, Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The
Netherlands). The epitope can be a linear epitope, i.e., contained
in a single stretch of amino acids, or a conformational epitope
formed by a three-dimensional interaction of amino acids that may
not necessarily be contained in a single stretch. Peptides of
varying lengths (e.g., at least 4-6 amino acids long) can be
isolated or synthesized (e.g., recombinantly) and used for binding
assays with a FXIa antibody or anti-idiotype antibody of the
disclosure. In another example, the epitope to which the FXIa
antibody binds can be determined in a systematic screening by using
overlapping peptides derived from the FXIa sequence and determining
binding by the antibody. According to the gene fragment expression
assays, the open reading frame encoding FXIa can be fragmented
either randomly or by specific genetic constructions and the
reactivity of the expressed fragments of FXIa with the antibody to
be tested is determined. The gene fragments may, for example, be
produced by PCR and then transcribed and translated into protein in
vitro, in the presence of radioactive amino acids. The binding of
the antibody to the radioactively labeled FXIa fragments is then
determined by immunoprecipitation and gel electrophoresis. Certain
epitopes can also be identified by using large libraries of random
peptide sequences displayed on the surface of phage particles
(phage libraries) or yeast (yeast display). Alternatively, a
defined library of overlapping peptide fragments can be tested for
binding to the test antibody in simple binding assays. In an
additional example, mutagenesis of an antigen, domain swapping
experiments and alanine scanning mutagenesis can be performed to
identify residues required, sufficient, and/or necessary for
epitope binding. For example, alanine scanning mutagenesis
experiments can be performed using a mutant FXIa in which various
residues of the FIXa polypeptide have been replaced with alanine.
By assessing binding of the antibody to the mutant FXIa, the
importance of the particular FXIa residues to antibody binding can
be assessed.
[0258] In another example, the epitope to which the anti-idiotype
antibody binds can be determined in a systematic screening by using
overlapping peptides derived from the anti-FXIa antibody sequence
and determining binding by the anti-idiotype antibody. According to
the gene fragment expression assays, the open reading frame
encoding the anti-FXIa antibody can be fragmented either randomly
or by specific genetic constructions and the reactivity of the
expressed fragments of the anti-FXIa antibody with the antibody to
be tested is determined. The gene fragments may, for example, be
produced by PCR and then transcribed and translated into protein in
vitro, in the presence of radioactive amino acids. The binding of
the antibody to the radioactively labeled the anti-FXIa antibody
fragments is then determined by immunoprecipitation and gel
electrophoresis. Certain epitopes can also be identified by using
large libraries of random peptide sequences displayed on the
surface of phage particles (phage libraries) or yeast (yeast
display). Alternatively, a defined library of overlapping peptide
fragments can be tested for binding to the test antibody in simple
binding assays. In an additional example, mutagenesis of an
antigen, domain swapping experiments and alanine scanning
mutagenesis can be performed to identify residues required,
sufficient, and/or necessary for epitope binding. For example,
alanine scanning mutagenesis experiments can be performed using a
mutant anti-FXIa antibody in which various residues of the
anti-FXIa antibody (e.g. variable domain residues) have been
replaced with alanine. By assessing binding of the anti-idiotype
antibody to the mutant anti-FXIa antibody, the importance of the
particular the anti-FXIa antibody residues to anti-idiotype
antibody binding can be assessed.
[0259] Yet another method that can be used to characterize an
anti-FXIa antibody is to use competition assays with other
antibodies known to bind to the same antigen, i.e., various
fragments on FXIa, to determine if the anti-FXIa antibody binds to
the same epitope as other antibodies. Competition assays are well
known to those of skill in the art. Similarly, another method which
can be used to characterize an anti-idiotype antibody that
specifically binds to the antigen-binding site of an anti-FXIa
antibody is to use competition assays with other antibodies known
to bind to the same antigen, i.e., various fragments of the
anti-FXIa antibody, to determine if the anti-idiotype antibody
binds to the same epitope as other antibodies. Competition assays
are well known to those of skill in the art.
[0260] Further, the epitope for a given antibody/antigen binding
pair can be defined and characterized at different levels of detail
using a variety of experimental and computational epitope mapping
methods. The experimental methods include mutagenesis, X-ray
crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy,
hydrogen/deuterium exchange Mass Spectrometry (H/D-MS) and various
competition binding methods well-known in the art. As each method
relies on a unique principle, the description of an epitope is
intimately linked to the method by which it has been determined.
Thus, the epitope for a given antibody/antigen pair will be defined
differently depending on the epitope mapping method employed.
[0261] At its most detailed level, the epitope for the interaction
between the antigen ("Ag") and the antibody ("Ab") can be defined
by the spatial coordinates defining the atomic contacts present in
the Ag-Ab interaction, as well as information about their relative
contributions to the binding thermodynamics. At a less detailed
level the epitope can be characterized by the spatial coordinates
defining the atomic contacts between the Ag and Ab. At a further
less detailed level the epitope can be characterized by the amino
acid residues that it comprises as defined by a specific criterion,
e.g., by distance between atoms (e.g., heavy, i.e., non-hydrogen
atoms) in the Ab and the Ag. At a further less detailed level the
epitope can be characterized through function, e.g. by competition
binding with other Abs. The epitope can also be defined more
generically as comprising amino acid residues for which
substitution by another amino acid will alter the characteristics
of the interaction between the Ab and Ag (e.g. using alanine
scanning).
[0262] From the fact that descriptions and definitions of epitopes,
dependent on the epitope mapping method used, are obtained at
different levels of detail, it follows that comparison of epitopes
for different Abs on the same Ag can similarly be conducted at
different levels of detail.
[0263] Epitopes described at the amino acid level, e.g., determined
from an X-ray structure, are said to be identical if they contain
the same set of amino acid residues. Epitopes are said to overlap
if at least one amino acid is shared by the epitopes. Epitopes are
said to be separate (unique) if no amino acid residue is shared by
the epitopes.
[0264] 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 or consecutive binding of the other antibody. The
epitopes are said to be separate (unique) if the antigen is able to
accommodate binding of both corresponding antibodies
simultaneously.
[0265] The epitope and paratope for a given antibody/antigen pair
may be identified by routine methods. For example, in the case of
the anti-FXIa antibodies of the disclosure, the general location of
an epitope may be determined by assessing the ability of an
antibody to bind to different fragments or variant FXIa
polypeptides. Similarly, in the case of the anti-idiotype
antibodies of the disclosure, the general location of an epitope
may be determined by assessing the ability of an antibody to bind
to different fragments or variant anti-FXIa antibodies or
antigen-binding fragments thereof. The specific amino acids within
FXIa that make contact with a FXIa antibody (epitope) and the
specific amino acids in an antibody that make contact with FXIa
(paratope) may also be determined using routine methods, such as
those described in the examples. Similarly, the specific amino
acids within the anti-FXIa antibody that make contact with an
anti-idiotype antibody (epitope) and the specific amino acids in an
anti-idiotype antibody that make contact with the anti-FXIa
antibody (paratope) may also be determined using routine methods,
such as those described in the examples. For example, the antibody
and target molecule may be combined and the antibody/antigen
complex may be crystallized. The crystal structure of the complex
may be determined and used to identify specific sites of
interaction between the antibody and its target.
[0266] A FXIa antibody according to the current disclosure may bind
to the same epitope or domain of FXIa as the antibodies of the
disclosure that are specifically disclosed herein. For example,
other as yet unidentified antibodies of the disclosure may be
identified by comparing their binding to FXIa with that of any of
the following monoclonal antibodies: D4, DEF, QCA11, B1D2, B10H2,
B10E6, B10F6, B10D8, B10B12, S1D4, S10H9, Clone 8, Clone 16, Clone
20, Clone 22, Clone 32, Clone 24, and variants thereof; or by
comparing the function of yet unidentified antibodies with that of
the antibodies described herein; and/or by comparing the
epitope/contact residues on FXIa of yet unidentified antibodies
with those of the antibodies of the disclosure. Analyses and assays
that may be used for the purpose of such identification include
assays assessing the competition for binding of FXIa between the
antibody of interest and FXIa substrate, in biological activity
assays as described in Examples 1-13 and 20-22, and in analysis of
the crystal structure of the antibody, such as described in Example
23.
[0267] An anti-idiotype antibody according to the disclosure that
specifically binds to the antigen-binding site of an anti-FXIa
antibody may bind to the same epitope or domain of an anti-FXIa
antibody as the anti-idiotype antibodies of the disclosure that are
specifically disclosed herein. For example, other as yet
unidentified anti-idiotype antibodies of the disclosure may be
identified by comparing their binding to an anti-FXIa antibody with
that of the monoclonal antibody C4, and variants thereof; or by
comparing the function of yet unidentified anti-idiotype antibodies
with that of the anti-idiotype antibodies described herein; and/or
by comparing the epitope/contact residues on the anti-FXIa antibody
of yet unidentified antibodies with those of the anti-idiotype
antibodies of the disclosure. Analyses and assays that may be used
for the purpose of such identification include assays assessing the
competition for binding of anti-FXIa antibody between the
anti-idiotype antibody of interest and FXIa, in biological activity
assays as described in Examples 14-19, and in analysis of the
crystal structure of the antibody.
[0268] An anti-FXIa antibody of the disclosure may have the ability
to compete or cross-compete with another antibody of the disclosure
for binding to FXIa as described herein. For example, an antibody
of the disclosure may compete or cross-compete with antibodies
described herein for binding to FXIa, or to a suitable fragment or
variant of FXIa that is bound by the antibodies disclosed
herein.
[0269] That is, if a first anti-FXIa antibody competes with a
second antibody for binding to FXIa, but it does not compete where
the second antibody is first bound to FXIa, it is deemed to
"compete" with the second antibody (also referred to as
unidirectional competition). Where an antibody competes with
another antibody regardless of which antibody is first bound to
FXIa, then the antibody "cross-competes" for binding to FXIa with
the other antibody. Such competing or cross-competing antibodies
can be identified based on their ability to compete/cross-compete
with a known antibody of the disclosure in standard binding assays.
For example, SPR, e.g. by using a Biacore.TM. system, ELISA assays
or flow cytometry may be used to demonstrate
competition/cross-competition. Such competition/cross-competition
may suggest that the two antibodies bind to identical, overlapping
or similar epitopes.
[0270] An anti-idiotype antibody of the disclosure may have the
ability to compete or cross-compete with another antibody of the
disclosure for binding to an anti-FXIa antibody as described
herein. For example, an anti-idiotype antibody of the disclosure
may compete or cross-compete with anti-idiotype antibodies
described herein for binding to an anti-FXIa antibody, or to a
suitable fragment or variant of an anti-FXIa antibody that is bound
by the anti-idiotype antibodies disclosed herein.
[0271] That is, if a first anti-idiotype antibody competes with a
second anti-idiotype antibody for binding to an anti-FXIa antibody,
but it does not compete where the second anti-idiotype antibody is
first bound to the anti-FXIa antibody, it is deemed to "compete"
with the second anti-idiotype antibody (also referred to as
unidirectional competition). Where an anti-idiotype antibody
competes with another anti-idiotype antibody regardless of which
antibody is first bound to the anti-FXIa antibody, then the
anti-idiotype antibody "cross-competes" for binding to the
anti-FXIa antibody with the other anti-idiotype antibody. Such
competing or cross-competing anti-idiotype antibodies can be
identified based on their ability to compete/cross-compete with a
known anti-idiotype antibody of the disclosure in standard binding
assays. For example, SPR, e.g. by using a Biacore.TM. system, ELISA
assays or flow cytometry may be used to demonstrate
competition/cross-competition. Such competition/cross-competition
may suggest that the two anti-idiotype antibodies bind to
identical, overlapping or similar epitopes.
[0272] An anti-FXIa antibody of the disclosure may therefore be
identified by a method that comprises a binding assay which
assesses whether or not a test antibody is able to
compete/cross-compete with a reference antibody of the disclosure
(e.g., D4, DEF, QCA11, B1D2, B10H2, B10E6, B10F6, B10D8, B10B12,
S1D4, S10H9, Clone 8, Clone 16, Clone 20, Clone 22, Clone 32, Clone
24) for a binding site on the target molecule. Similarly, an
anti-idiotype antibody of the disclosure may be identified by a
method that comprises a binding assay which assesses whether or not
a test antibody is able to compete/cross-compete with a reference
antibody of the disclosure (e.g., C4). Methods for carrying out
competitive binding assays are disclosed herein and/or are well
known in the art. For example they may involve binding a reference
antibody of the disclosure to a target molecule using conditions
under which the antibody can bind to the target molecule. The
antibody/target complex may then be exposed to a test/second
antibody and the extent to which the test antibody is able to
displace the reference antibody of the disclosure from
antibody/target complexes may be assessed. An alternative method
may involve contacting a test antibody with a target molecule under
conditions that allow for antibody binding, then adding a reference
antibody of the disclosure that is capable of binding that target
molecule and assessing the extent to which the reference antibody
of the disclosure is able to displace the test antibody from
antibody/target complexes or to simultaneously bind to the target
(i.e., non-competing antibody).
[0273] The ability of a test antibody to inhibit the binding of a
reference antibody of the disclosure to the target demonstrates
that the test antibody can compete with a reference antibody of the
disclosure for binding to the target and thus that the test
antibody binds to the same, or substantially the same, epitope or
region on the FXIa protein as the reference antibody of the
disclosure. A test antibody that is identified as competing with a
reference antibody of the disclosure in such a method is also an
antibody of the present disclosure. The fact that the test antibody
can bind FXIa in the same region as a reference antibody of the
disclosure and can compete with the reference antibody of the
disclosure suggests that the test antibody may act as an inhibitor
at the same binding site as the antibody of the disclosure and that
the test antibody may therefore mimic the action of the reference
antibody and is, thus, an antibody of the disclosure. This can be
confirmed by comparing the activity of FXIa in the presence of the
test antibody with the activity of FXIa in the presence of the
reference antibody under otherwise identical conditions, using an
assay as more fully described elsewhere herein.
[0274] The reference antibody of the disclosure may be an antibody
as described herein, such as D4, DEF, QCA11, B1D2, B10H2, B10E6,
B10F6, B10D8, B10B12, S1D4, S10H9, Clone 8, Clone 16, Clone 20,
Clone 22, Clone 32, Clone 24, or any variant, or fragment thereof,
as described herein that retains the ability to bind to FXIa. An
antibody of the disclosure may bind to the same epitope as the
reference antibodies described herein or any variant or fragment
thereof as described herein that retains the ability to bind to
FXIa.
[0275] The ability of a test anti-idiotype antibody to inhibit the
binding of a reference anti-idiotype antibody of the disclosure to
the target demonstrates that the test antibody can compete with a
reference antibody of the disclosure for binding to the target and
thus that the test antibody binds to the same, or substantially the
same, epitope or region on the anti-FXIa antibody as the reference
antibody of the disclosure. A test antibody that is identified as
competing with a reference antibody of the disclosure in such a
method is also an antibody of the present disclosure. The fact that
the test antibody can bind an anti-FXIa antibody in the same region
as a reference antibody of the disclosure and can compete with the
reference antibody of the disclosure suggests that the test
antibody may act as an inhibitor at the same binding site as the
antibody of the disclosure and that the test antibody may therefore
mimic the action of the reference antibody and is, thus, an
antibody of the disclosure. This can be confirmed by comparing the
activity of the anti-FXIa antibody in the presence of the test
antibody with the activity of the anti-FXIa antibody in the
presence of the reference antibody under otherwise identical
conditions, using an assay as more fully described elsewhere
herein.
[0276] The reference antibody of the disclosure may be an antibody
as described herein, such as C4, or any variant, or fragment
thereof, as described herein that retains the ability to bind to an
anti-FXIa antibody. An antibody of the disclosure may bind to the
same epitope as the reference antibodies described herein or any
variant or fragment thereof as described herein that retains the
ability to bind to an anti-FXIa antibody.
[0277] As stated previously elsewhere herein, specific binding may
be assessed with reference to binding of the antibody to a molecule
that is not the target. This comparison may be made by comparing
the ability of an antibody to bind to the target and to another
molecule. This comparison may be made as described above in an
assessment of K.sub.D or K.sub.i. The other molecule used in such a
comparison may be any molecule that is not the target molecule.
Preferably, the other molecule is not identical to the target
molecule. Preferably the target molecule is not a fragment of the
target molecule.
[0278] The other molecule used to determine specific binding may be
unrelated in structure or function to the target. For example, the
other molecule may be an unrelated material or accompanying
material in the environment.
[0279] The other molecule used to determine specific binding may be
another molecule involved in the same in vivo pathway as the target
molecule, i.e., FXIa, in the case of an anti-FXIa antibody of the
disclosure. By ensuring that the antibody of the disclosure has
specificity for FXIa over another such molecule, unwanted in vivo
cross-reactivity may be avoided. For example, in some embodiments,
the anti-FXIa antibody of the disclosure fails to inhibit the
ability of human plasma kallikrein, a protease closely related to
human factor XIa, to cleave small fluorogenic substrates.
Similarly, in the case of an anti-idiotype antibody of the
disclosure, the other molecule used to determine specific binding
may be another anti-FXIa antibody. By ensuring that the antibody of
the disclosure has specificity for one anti-FXIa antibody over
another such molecule, unwanted in vivo cross-reactivity may be
avoided.
[0280] In some embodiments, the antibody of the disclosure may
retain the ability to bind to some molecules that are related to
the target molecule.
[0281] Alternatively, the anti-FXIa antibody of the disclosure may
have specificity for a particular target molecule. For example, it
may bind to one target molecule as described herein, but may not
bind, or may bind with significantly reduced affinity to a
different target molecule as described herein. For example, a full
length mature human FXIa may be used as the target, but the
antibody that binds to that target may be unable to bind to or may
bind with lesser affinity to, e.g. other FXIa proteins from other
species, such as other mammalian FXIa. In some embodiments, the
antibody binds to both human and cynomolgus FXIa. In some
embodiments, the antibody binds to one or more of human,
cynomolgus, and rabbit FXIa.
[0282] The anti-idiotype antibody of the disclosure may have
specificity for a particular anti-FXIa antibody (e.g. D4, DEF,
QCA11, B1D2, B10H2, B10E6, B10F6, B10D8, B10B12, S1D4, S10H9, Clone
8, Clone 16, Clone 20, Clone 22, Clone 32, Clone 24). In some
embodiments, the anti-idiotype antibody of the disclosure may have
specificity for DEF.
Antibody Fragments and Variants
[0283] In some embodiments, an antibody comprises an antibody
fragment, e.g., an antigen-binding fragment (Fab) or a single chain
variable fragment (scFv). Polypeptide or antibody "fragments" or
"portions" according to the disclosure may be made by truncation,
e.g. by removal of one or more amino acids from the N and/or
C-terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up
to 40 or more amino acids may be removed from the N and/or C
terminal in this way. Fragments may also be generated by one or
more internal deletions.
[0284] An anti-FXIa antibody of the disclosure may be, or may
comprise, a fragment of, any one of antibodies D4, DEF, QCA11,
B1D2, B10H2, B10E6, B10F6, B10D8, B10B12, S1D4, S10H9, Clone 8,
Clone 16, Clone 20, Clone 22, Clone 32, Clone 24, or a variant
thereof. The FXIa antibody of the disclosure may be or may comprise
an antigen-binding portion of this antibody or a variant thereof.
For example, the antibody of the disclosure may be a Fab fragment
of this antibody or a variant thereof or may be a single chain
antibody derived from this antibody or a variant thereof.
[0285] An anti-idiotype antibody of the disclosure may be, or may
comprise, a fragment of, antibody C4, or a variant thereof. The
anti-idiotype antibody of the disclosure may be or may comprise an
antigen-binding portion of this antibody or a variant thereof. For
example, the antibody of the disclosure may be a Fab fragment of
this antibody or a variant thereof or may be a single chain
antibody derived from this antibody or a variant thereof.
[0286] A variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up
to 20, up to 30 or more amino acid substitutions and/or deletions
and/or insertions from the specific sequences and fragments
discussed above. "Deletion" variants may comprise the deletion of
individual amino acids, deletion of small groups of amino acids
such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid
regions, such as the deletion of specific amino acid domains or
other features. "Insertion" variants may comprise the insertion of
individual amino acids, insertion of small groups of amino acids
such as 2, 3, 4 or 5 amino acids, or insertion of larger amino acid
regions, such as the insertion of specific amino acid domains or
other features. "Substitution" variants preferably involve the
replacement of one or more amino acids with the same number of
amino acids and making conservative amino acid substitutions. For
example, an amino acid may be substituted with an alternative amino
acid having similar properties, for example, another basic amino
acid, another acidic amino acid, another neutral amino acid,
another charged amino acid, another hydrophilic amino acid, another
hydrophobic amino acid, another polar amino acid, another aromatic
amino acid or another aliphatic amino acid. Some properties of the
20 main amino acids which can be used to select suitable
substituents are as described below.
[0287] Substitution variants have at least one amino acid residue
in the antibody molecule removed and a different residue inserted
in its place. The sites of greatest interest for substitutional
mutagenesis include the hypervariable regions, but framework
alterations are also contemplated. Conservative substitutions are
shown in Table 1 under the heading of "conservative substitutions."
If such substitutions result in a change in biological activity,
then more substantial changes, denominated "exemplary
substitutions" shown below, or as further described below in
reference to amino acid classes, may be introduced and the products
screened.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions Original Residue
Conservative Substitutions Exemplary Substitutions Ala (A) Val Val;
Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys;
Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu
Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg
Ile (I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile
Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met
(M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P)
Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr
(Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;
Norleucine
[0288] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a .beta.-sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties: [0289] (1) Non-polar: Norleucine, Met, Ala,
Val, Leu, Ile; [0290] (2) Polar without charge: Cys, Ser, Thr, Asn,
Gln; [0291] (3) Acidic (negatively charged): Asp, Glu; [0292] (4)
Basic (positively charged): Lys, Arg; [0293] (5) Residues that
influence chain orientation: Gly, Pro; and [0294] (6) Aromatic:
Trp, Tyr, Phe, His.
[0295] Non-conservative substitutions are made by exchanging a
member of one of these classes for another class.
[0296] One type of substitution, for example, that may be made is
to change one or more cysteines in the antibody, which may be
chemically reactive, to another residue, such as, without
limitation, alanine or serine. For example, there can be a
substitution of a non-canonical cysteine. The substitution can be
made in a CDR or framework region of a variable domain or in the
constant region of an antibody. In some embodiments, the cysteine
is canonical. Any cysteine residue not involved in maintaining the
proper conformation of the antibody also may be substituted,
generally with serine, to improve the oxidative stability of the
molecule and prevent aberrant cross-linking. Conversely, cysteine
bond(s) may be added to the antibody to improve its stability,
particularly where the antibody is an antibody fragment such as an
Fv fragment.
Generation and Modification of Anti-FXIa Antibodies and
Anti-Idiotype Antibodies
[0297] The disclosure also provides methods of generating,
selecting, and making anti-FXIa antibodies and anti-idiotype
antibodies. The antibodies of this disclosure (e.g. anti-FXIa
antibodies, anti-idiotype antibodies that specifically bind to the
antigen-binding site of an anti-FXIa antibody) can be made by
procedures known in the art. In some embodiments, antibodies may be
made recombinantly and expressed using any method known in the art.
General techniques for production of human and mouse antibodies are
known in the art and/or are described herein.
[0298] In some embodiments, antibodies may be prepared and selected
by phage display technology. See, for example, U.S. Pat. Nos.
5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al.,
Annu. Rev. Immunol. 12:433-455, 1994. Alternatively, the phage
display technology (McCafferty et al., Nature 348:552-553, 1990)
can be used to produce human antibodies and antibody fragments in
vitro, from immunoglobulin variable (V) domain gene repertoires
from unimmunized donors. According to this technique, antibody V
domain genes are cloned in-frame into either a major or minor coat
protein gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B cell. Phage
display can be performed in a variety of formats; for review see,
e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in
Structural Biology 3:564-571, 1993. Several sources of V-gene
segments can be used for phage display. Clackson et al., Nature
352:624-628, 1991, isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from human donors can be constructed and antibodies to a diverse
array of antigens (including self-antigens) can be isolated
essentially following the techniques described by Mark et al.,
1991, J. Mol. Biol. 222:581-597, or Griffith et al., 1993, EMBO J.
12:725-734. In a natural immune response, antibody genes accumulate
mutations at a high rate (somatic hypermutation). Some of the
changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process can be mimicked by employing the technique
known as "chain shuffling." (Marks et al., 1992, Bio/Technol.
10:779-783). In this method, the affinity of "primary" human
antibodies obtained by phage display can be improved by
sequentially replacing the heavy and light chain V region genes
with repertoires of naturally occurring variants (repertoires) of V
domain genes obtained from unimmunized donors. This technique
allows the production of antibodies and antibody fragments with
affinities in the pM-nM range. A strategy for making very large
phage antibody repertoires (also known as "the mother-of-all
libraries") has been described by Waterhouse et al., Nucl. Acids
Res. 21:2265-2266, 1993. Gene shuffling can also be used to derive
human antibodies from rodent antibodies, where the human antibody
has similar affinities and specificities to the starting rodent
antibody. According to this method, which is also referred to as
"epitope imprinting," the heavy or light chain V domain gene of
rodent antibodies obtained by phage display technique is replaced
with a repertoire of human V domain genes, creating rodent-human
chimeras. Selection on antigen results in isolation of human
variable regions capable of restoring a functional antigen-binding
site, i.e., the epitope governs (imprints) the choice of partner.
When the process is repeated in order to replace the remaining
rodent V domain, a human antibody is obtained (see PCT Publication
No. WO 93/06213). Unlike traditional humanization of rodent
antibodies by CDR grafting, this technique provides completely
human antibodies, which have no framework or CDR residues of rodent
origin.
[0299] In some embodiments, antibodies may be made using hybridoma
technology. It is contemplated that any mammalian subject including
humans or antibody producing cells therefrom can be manipulated to
serve as the basis for production of mammalian, including human,
hybridoma cell lines. The route and schedule of immunization of the
host animal are generally in keeping with established and
conventional techniques for antibody stimulation and production, as
further described herein. Typically, the host animal is inoculated
intraperitoneally, intramuscularly, orally, subcutaneously,
intraplantar, and/or intradermally with an amount of immunogen,
including as described herein.
[0300] Hybridomas can be prepared from the lymphocytes and
immortalized myeloma cells using the general somatic cell
hybridization technique of Kohler, B. and Milstein, C., 1975,
Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro,
18:377-381, 1982. Available myeloma lines, including but not
limited to X63-Ag8.653 and those from the Salk Institute, Cell
Distribution Center, San Diego, Calif., USA, may be used in the
hybridization. Generally, the technique involves fusing myeloma
cells and lymphoid cells using a fusogen such as polyethylene
glycol, or by electrical means well known to those skilled in the
art. After the fusion, the cells are separated from the fusion
medium and grown in a selective growth medium, such as
hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate
unhybridized parent cells. Any of the media described herein,
supplemented with or without serum, can be used for culturing
hybridomas that secrete monoclonal antibodies. As another
alternative to the cell fusion technique, EBV immortalized B cells
may be used to produce the anti-FXIa monoclonal antibodies and/or
the anti-idiotype antibodies of the disclosure. The hybridomas or
other immortalized B-cells are expanded and subcloned, if desired,
and supernatants are assayed for anti-immunogen activity by
conventional immunoassay procedures (e.g., radioimmunoassay, enzyme
immunoassay, or fluorescence immunoassay).
[0301] Hybridomas that may be used as source of anti-FXIa
antibodies encompass all derivatives, progeny cells of the parent
hybridomas that produce monoclonal antibodies specific for FXIa, or
a portion thereof. Hybridomas that may be used as source of
anti-idiotype antibodies encompass all derivatives, progeny cells
of the parent hybridomas that produce monoclonal anti-idiotype
antibodies specific for an anti-FXIa antibody, or a portion
thereof.
[0302] Hybridomas that produce such anti-FXIa antibodies or
anti-idiotype antibodies may be grown in vitro or in vivo using
known procedures. The monoclonal antibodies may be isolated from
the culture media or body fluids, by conventional immunoglobulin
purification procedures such as ammonium sulfate precipitation, gel
electrophoresis, dialysis, chromatography, and ultrafiltration, if
desired. Undesired activity, if present, can be removed, for
example, by running the preparation over adsorbents made of the
immunogen attached to a solid phase and eluting or releasing the
desired antibodies off the immunogen. Immunization of a host animal
with FXIa polypeptide, or a fragment containing the target amino
acid sequence conjugated to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups, can yield a population of
antibodies (e.g., monoclonal anti-FXIa antibodies). Immunization of
a host animal with an anti-FXIa polypeptide, or a fragment
containing the target amino acid sequence (e.g. variable domain
sequence) conjugated to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups, can yield a population of
antibodies (e.g., monoclonal anti-idiotype antibodies).
[0303] If desired, the anti-FXIa antibody or anti-idiotype antibody
(monoclonal or polyclonal) of interest may be sequenced and the
polynucleotide sequence may then be cloned into a vector for
expression or propagation. The sequence encoding the antibody of
interest may be maintained in vector in a host cell and the host
cell can then be expanded and frozen for future use. Production of
recombinant monoclonal antibodies in cell culture can be carried
out through cloning of antibody genes from B cells by means known
in the art. See, e.g. Tiller et al., 2008, J. Immunol. Methods 329,
112; U.S. Pat. No. 7,314,622.
[0304] In some embodiments, the polynucleotide sequence may be used
for genetic manipulation to "humanize" the antibody or to improve
the affinity, or other characteristics of the antibody. Antibodies
may also be customized for use, for example, in dogs, cats,
primate, equines and bovines.
[0305] In some embodiments, fully human antibodies may be obtained
by using commercially available mice that have been engineered to
express specific human immunoglobulin proteins. Transgenic animals
that are designed to produce a more desirable (e.g., fully human
antibodies) or more robust immune response may also be used for
generation of humanized or human antibodies. Examples of such
technology are Xenomouse.TM. from Abgenix, Inc. (Fremont, Calif.)
and HuMAb-Mouse.RTM. and TC Mouse.TM. from Medarex, Inc.
(Princeton, N.J.).
[0306] Antibodies may be made recombinantly by first isolating the
antibodies and antibody producing cells from host animals,
obtaining the gene sequence, and using the gene sequence to express
the antibody recombinantly in host cells (e.g., CHO cells). Another
method which may be employed is to express the antibody sequence in
plants (e.g., tobacco) or transgenic milk. Methods for expressing
antibodies recombinantly in plants or milk have been disclosed.
See, for example, Peeters, et al. Vaccine 19:2756, 2001; Lonberg,
N. and D. Huszar Int. Rev. Immunol 13:65, 1995; and Pollock, et
al., J Immunol Methods 231:147, 1999. Methods for making
derivatives of antibodies, e.g., domain, single chain, etc. are
known in the art.
[0307] Immunoassays and flow cytometry sorting techniques such as
fluorescence activated cell sorting (FACS) can also be employed to
isolate antibodies that are specific for FXIa. These assays can
also be employed to isolate antibodies that are specific for an
anti-FXIa antibody (e.g., DEF).
[0308] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors (such as expression vectors disclosed in PCT Publication
No. WO 87/04462), which are then transfected into host cells such
as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)
cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. See, e.g., PCT
Publication No. WO 87/04462. The DNA also may be modified, for
example, by substituting the coding sequence for human heavy and
light chain constant domains in place of the homologous murine
sequences, Morrison et al., Proc. Nat. Acad. Sci. 81:6851, 1984, or
by covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
In that manner, "chimeric" or "hybrid" antibodies are prepared that
have the binding specificity of for example, an anti-FXIa antibody
herein or an anti-idiotype antibody herein.
[0309] Antibody fragments can be produced by proteolytic or other
degradation of the antibodies, by recombinant methods (i.e., single
or fusion polypeptides) as described above or by chemical
synthesis. Polypeptides of the antibodies, especially shorter
polypeptides up to about 50 amino acids, are conveniently made by
chemical synthesis. Methods of chemical synthesis are known in the
art and are commercially available. For example, an antibody could
be produced by an automated polypeptide synthesizer employing the
solid phase method. See also, U.S. Pat. Nos. 5,807,715; 4,816,567;
and 6,331,415.
[0310] In some embodiments, a polynucleotide comprises a sequence
encoding the heavy chain and/or the light chain variable regions of
an anti-FXIa antibody of the present disclosure. The sequence
encoding the antibody of interest may be maintained in a vector in
a host cell and the host cell can then be expanded and frozen for
future use. Vectors (including expression vectors) and host cells
are further described herein.
[0311] In some embodiments, a polynucleotide comprises a sequence
encoding the heavy chain and/or the light chain variable regions of
an anti-idiotype antibody of the present disclosure. The sequence
encoding the antibody of interest may be maintained in a vector in
a host cell and the host cell can then be expanded and frozen for
future use. Vectors (including expression vectors) and host cells
are further described herein.
[0312] The disclosure includes affinity-matured embodiments. For
example, affinity matured antibodies can be produced by procedures
known in the art (Marks et al., 1992, Bio/Technology, 10:779-783;
Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier
et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol.,
155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9;
Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and PCT
Publication No. WO2004/058184).
[0313] The following methods may be used for adjusting the affinity
of an antibody and for characterizing a CDR. One way of
characterizing a CDR of an antibody and/or altering (such as
improving) the binding affinity of a polypeptide, such as an
antibody, termed "library scanning mutagenesis." Generally, library
scanning mutagenesis works as follows. One or more amino acid
positions in the CDR are replaced with two or more (such as 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino
acids using art recognized methods. This generates small libraries
of clones (in some embodiments, one for every amino acid position
that is analyzed), each with a complexity of two or more members
(if two or more amino acids are substituted at every position).
Generally, the library also includes a clone comprising the native
(unsubstituted) amino acid. A small number of clones, e.g., about
20-80 clones (depending on the complexity of the library), from
each library are screened for binding affinity to the target
polypeptide (or other binding target), and candidates with
increased, the same, decreased, or no binding are identified.
Methods for determining binding affinity are well-known in the art.
Binding affinity may be determined using, for example, Biacore.TM.
surface plasmon resonance analysis, which detects differences in
binding affinity of about 2-fold or greater, Kinexa.RTM. Biosensor,
scintillation proximity assays, ELISA, ORIGEN.RTM. immunoassay,
fluorescence quenching, fluorescence transfer, and/or yeast
display. Binding affinity may also be screened using a suitable
bioassay. Biacore.TM. is particularly useful when the starting
antibody already binds with a relatively high affinity, for example
a K.sub.D of about 10 nM or lower.
[0314] In some embodiments, every amino acid position in a CDR is
replaced (in some embodiments, one at a time) with all 20 natural
amino acids using art recognized mutagenesis methods (some of which
are described herein). This generates small libraries of clones (in
some embodiments, one for every amino acid position that is
analyzed), each with a complexity of 20 members (if all 20 amino
acids are substituted at every position).
[0315] In some embodiments, the library to be screened comprises
substitutions in two or more positions, which may be in the same
CDR or in two or more CDRs. Thus, the library may comprise
substitutions in two or more positions in one CDR. The library may
comprise substitution in two or more positions in two or more CDRs.
The library may comprise substitution in 3, 4, 5, or more
positions, said positions found in two, three, four, five or six
CDRs. The substitution may be prepared using low redundancy codons.
See, e.g., Table 2 of Balint et al., 1993, Gene 137(1):109-18.
[0316] The CDR may be heavy chain variable region (VH) CDR3 and/or
light chain variable region (VL) CDR3. The CDR may be one or more
of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3. The
CDR may be a Kabat CDR, a Chothia CDR, an extended CDR, an AbM CDR,
a contact CDR, or a conformational CDR.
[0317] Candidates with improved binding may be sequenced, thereby
identifying a CDR substitution mutant which results in improved
affinity (also termed an "improved" substitution). Candidates that
bind may also be sequenced, thereby identifying a CDR substitution
which retains binding.
[0318] Multiple rounds of screening may be conducted. For example,
candidates (each comprising an amino acid substitution at one or
more position of one or more CDR) with improved binding are also
useful for the design of a second library containing at least the
original and substituted amino acid at each improved CDR position
(i.e., amino acid position in the CDR at which a substitution
mutant showed improved binding). Preparation, and screening or
selection of this library is discussed further below.
[0319] Library scanning mutagenesis also provides a means for
characterizing a CDR, in so far as the frequency of clones with
improved binding, the same binding, decreased binding or no binding
also provide information relating to the importance of each amino
acid position for the stability of the antibody-antigen complex.
For example, if a position of the CDR retains binding when changed
to all 20 amino acids, that position is identified as a position
that is unlikely to be required for antigen binding. Conversely, if
a position of CDR retains binding in only a small percentage of
substitutions, that position is identified as a position that is
important to CDR function. Thus, the library scanning mutagenesis
methods generate information regarding positions in the CDRs that
can be changed to many different amino acids (including all 20
amino acids), and positions in the CDRs which cannot be changed or
which can only be changed to a few amino acids. Candidates with
improved affinity may be combined in a second library, which
includes the improved amino acid, the original amino acid at that
position, and may further include additional substitutions at that
position, depending on the complexity of the library that is
desired, or permitted using the desired screening or selection
method. In addition, if desired, adjacent amino acid position can
be randomized to at least two or more amino acids. Randomization of
adjacent amino acids may permit additional conformational
flexibility in the mutant CDR, which may in turn, permit or
facilitate the introduction of a larger number of improving
mutations. The library may also comprise substitution at positions
that did not show improved affinity in the first round of
screening.
[0320] The second library is screened or selected for library
members with improved and/or altered binding affinity using any
method known in the art, including screening using Biacore.TM.,
Kinexa.TM. biosensor analysis, and selection using any method known
in the art for selection, including phage display, yeast display,
and ribosome display.
[0321] To express the anti-FXIa antibodies of the present
disclosure, DNA fragments encoding VH and VL regions can first be
obtained using any of the methods described above. Various
modifications, e.g. mutations, deletions, and/or additions can also
be introduced into the DNA sequences using standard methods known
to those of skill in the art. For example, mutagenesis can be
carried out using standard methods, such as PCR-mediated
mutagenesis, in which the mutated nucleotides are incorporated into
the PCR primers such that the PCR product contains the desired
mutations or site-directed mutagenesis. In some embodiments, a dNTP
pool biased mutagenesis may be carried out.
[0322] To express the anti-idiotype antibodies of the present
disclosure, DNA fragments encoding VH and VL regions can first be
obtained using any of the methods described above. Various
modifications, e.g. mutations, deletions, and/or additions can also
be introduced into the DNA sequences using standard methods known
to those of skill in the art. For example, mutagenesis can be
carried out using standard methods, such as PCR-mediated
mutagenesis, in which the mutated nucleotides are incorporated into
the PCR primers such that the PCR product contains the desired
mutations or site-directed mutagenesis. In some embodiments, a dNTP
pool biased mutagenesis may be carried out.
[0323] The disclosure encompasses modifications to the variable
regions and the CDRs indicated in Table 3. For example, the
disclosure includes antibodies comprising functionally equivalent
variable regions and CDRs which do not significantly affect their
properties as well as variants which have enhanced or decreased
activity and/or affinity. For example, the amino acid sequence may
be mutated to obtain an antibody with the desired binding affinity
to FXIa. In the case of an anti-idiotype antibody, the amino acid
sequence may be mutated to obtain an antibody with the desired
binding affinity to an anti-FXIa antibody. Examples of modified
polypeptides include polypeptides with conservative substitutions
of amino acid residues, one or more deletions or additions of amino
acids which do not significantly deleteriously change the
functional activity, or which mature (enhance) the affinity of the
polypeptide for its ligand, or use of chemical analogs.
[0324] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the antibody molecule include
the fusion to the N- or C-terminus of the antibody of an enzyme or
a polypeptide that increases the half-life of the antibody in the
blood circulation.
[0325] The antibodies may also be modified, e.g., in the variable
domains of the heavy and/or light chains, e.g., to alter a binding
property of the antibody. Changes in the variable region can alter
binding affinity and/or specificity. In some embodiments, no more
than one to five conservative amino acid substitutions are made
within a CDR domain. In other embodiments, no more than one to
three conservative amino acid substitutions are made within a CDR
domain. For example, a mutation may be made in one or more of the
CDR regions to increase or decrease the K.sub.D of the anti-FXIa
antibody for FXIa, to increase or decrease k.sub.off, or to alter
the binding specificity of the antibody. Similarly, a mutation may
be made in one or more of the CDR regions to increase or decrease
the K.sub.D of the anti-idiotype antibody for an anti-FXIa
antibody, to increase or decrease k.sub.off, or to alter the
binding specificity of the antibody Techniques in site-directed
mutagenesis are well-known in the art. See, e.g., Sambrook et al.
and Ausubel et al., supra.
[0326] A modification or mutation may also be made in a framework
region or constant region to increase the half-life of an an-FXIa
antibody or an anti-idiotype antibody. See, e.g., PCT Publication
No. WO 00/09560. A mutation in a framework region or constant
region can also be made to alter the immunogenicity of the
antibody, to provide a site for covalent or non-covalent binding to
another molecule, or to alter such properties as complement
fixation, FcR binding and antibody-dependent cell-mediated
cytotoxicity. A mutation in a framework region can also be made to
alter the affinity and potency of the antibody. In some
embodiments, the mutation may be a Q->K substitution in the
framework region (e.g., in FR1). According to the disclosure, a
single antibody may have mutations in any one or more of the CDRs
or framework regions of the variable domain or in the constant
region.
[0327] Modifications also include glycosylated and nonglycosylated
polypeptides, as well as polypeptides with other post-translational
modifications, such as, for example, glycosylation with different
sugars, acetylation, and phosphorylation. Antibodies are
glycosylated at conserved positions in their constant regions
(Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and
Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains
of the immunoglobulins affect the protein's function (Boyd et al.,
1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem.
29:4175-4180) and the intramolecular interaction between portions
of the glycoprotein, which can affect the conformation and
presented three-dimensional surface of the glycoprotein (Jefferis
and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.
7:409-416). Oligosaccharides may also serve to target a given
glycoprotein to certain molecules based upon specific recognition
structures. Glycosylation of antibodies has also been reported to
affect antibody-dependent cellular cytotoxicity (ADCC). In
particular, antibodies produced by CHO cells with
tetracycline-regulated expression of
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al., 1999, Nature
Biotech. 17:176-180).
[0328] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine, asparagine-X-threonine, and
asparagine-X-cysteine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0329] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0330] The glycosylation pattern of antibodies may also be altered
without altering the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g. antibodies, as potential therapeutics is rarely
the native cell, variations in the glycosylation pattern of the
antibodies can be expected (see, e.g. Hse et al., 1997, J. Biol.
Chem. 272:9062-9070).
[0331] In addition to the choice of host cells, factors that affect
glycosylation during recombinant production of antibodies include
growth mode, media formulation, culture density, oxygenation, pH,
purification schemes and the like. Various methods have been
proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain
types of glycosylation, can be enzymatically removed from the
glycoprotein, for example, using endoglycosidase H (Endo H),
N-glycosidase F, endoglycosidase F1, endoglycosidase F2,
endoglycosidase F3. In addition, the recombinant host cell can be
genetically engineered to be defective in processing certain types
of polysaccharides. These and similar techniques are well known in
the art.
[0332] Other methods of modification include using coupling
techniques known in the art, including, but not limited to,
enzymatic means, oxidative substitution and chelation.
Modifications can be used, for example, for attachment of labels
for immunoassay. Modified polypeptides are made using established
procedures in the art and can be screened using standard assays
known in the art, some of which are described below and in the
Examples.
[0333] In some embodiments, the antibody comprises a modified
constant region that has increased or decreased binding affinity to
a human Fc gamma receptor, is immunologically inert or partially
inert, e.g., does not trigger complement mediated lysis, does not
stimulate antibody-dependent cell mediated cytotoxicity (ADCC), or
does not activate microglia; or has reduced activities (compared to
the unmodified antibody) in any one or more of the following:
triggering complement mediated lysis, stimulating ADCC, or
activating microglia. Different modifications of the constant
region may be used to achieve optimal level and/or combination of
effector functions. See, for example, Morgan et al., Immunology
86:319-324, 1995; Lund et al., J. Immunology 157:4963-9
157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184,
2000; Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis
et al., Immunological Reviews 163:59-76, 1998. In some embodiments,
the constant region is modified as described in Eur. J. Immunol.,
1999, 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK
Patent Application No. 9809951.8.
[0334] In some embodiments, an antibody constant region can be
modified to avoid interaction with Fc gamma receptor and the
complement and immune systems. The techniques for preparation of
such antibodies are described in WO 99/58572. For example, the
constant region may be engineered to more resemble human constant
regions to avoid immune response if the antibody is used in
clinical trials and treatments in humans. See, e.g., U.S. Pat. Nos.
5,997,867 and 5,866,692.
[0335] In some embodiments, the constant region is modified as
described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Application
No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In
such embodiments, the Fc can be human IgG.sub.2 or human IgG.sub.4.
The Fc can be human IgG.sub.2 containing the mutation A330P331 to
S330S331 (IgG.sub.2.DELTA.a), in which the amino acid residues are
numbered with reference to the wild type IgG.sub.2 sequence. Eur.
J. Immunol., 1999, 29:2613-2624. In some embodiments, the antibody
comprises a constant region of IgG.sub.4 comprising the following
mutations (Armour et al., 2003, Molecular Immunology 40 585-593):
E233F234L235 to P233V234A235 (IgG.sub.4.DELTA.c), in which the
numbering is with reference to wild type IgG.sub.4. In yet another
embodiment, the Fc is human IgG.sub.4 E233F234L235 to P233V234A235
with deletion G236 (IgG.sub.4.DELTA.b). In another embodiment, the
Fc is any human IgG.sub.4 Fc (IgG.sub.4, IgG.sub.4.DELTA.b or
IgG.sub.4.DELTA.c) containing hinge stabilizing mutation 5228 to
P228 (Aalberse et al., 2002, Immunology 105, 9-19).
[0336] In some embodiments, the antibody comprises a human heavy
chain IgG.sub.2 constant region comprising the following mutations:
A330P331 to S330S331 (amino acid numbering with reference to the
wild type IgG.sub.2 sequence). Eur. J. Immunol., 1999,
29:2613-2624. In still other embodiments, the constant region is
aglycosylated for N-linked glycosylation. In some embodiments, the
constant region is aglycosylated for N-linked glycosylation by
mutating the oligosaccharide attachment residue and/or flanking
residues that are part of the N-glycosylation recognition sequence
in the constant region. For example, N-glycosylation site N297 may
be mutated to, e.g., A, Q, K, or H. See, Tao et al., J. Immunology
143: 2595-2601, 1989; and Jefferis et al., Immunological Reviews
163:59-76, 1998. In some embodiments, the constant region is
aglycosylated for N-linked glycosylation. The constant region may
be aglycosylated for N-linked glycosylation enzymatically (such as
removing carbohydrate by enzyme PNGase), or by expression in a
glycosylation deficient host cell.
[0337] Other antibody modifications include antibodies that have
been modified as described in PCT Publication No. WO 99/58572.
These antibodies comprise, in addition to a binding domain directed
at the target molecule, an effector domain having an amino acid
sequence substantially homologous to all or part of a constant
region of a human immunoglobulin heavy chain. These antibodies are
capable of binding the target molecule without triggering
significant complement dependent lysis, or cell-mediated
destruction of the target. In some embodiments, the effector domain
is capable of specifically binding FcRn and/or Fc.gamma.RIIb. These
are typically based on chimeric domains derived from two or more
human immunoglobulin heavy chain CH2 domains. Antibodies modified
in this manner are particularly suitable for use in chronic
antibody therapy, to avoid inflammatory and other adverse reactions
to conventional antibody therapy.
[0338] The disclosure also provides an antibody constant domain
that may be further modified. It is known that variants of the Fc
region, e.g., amino acid substitutions, insertions, and/or
additions and/or deletions, enhance or diminish effector function.
See, e.g., Presta et al, 2002, Biochem. Soc. Trans. 30:487-490;
Strohl, 2009, Curr. Opin. Biotechnol. 20(6):685-691; U.S. Pat. Nos.
5,624,821, 5,648,260, 5,885,573, 6,737,056, 7,317,091; PCT
publication Nos. WO 99/58572, WO 00/42072, WO 04/029207, WO
2006/105338, WO 2008/022152, WO 2008/150494, WO 2010/033736; U.S.
Patent Application Publication Nos. 2004/0132101, 2006/0024298,
2006/0121032, 2006/0235208, 2007/0148170, and 2015/0337053; Armour
et al., 1999, Eur. J. Immunol. 29(8):2613-2624 (reduced ADCC and
CDC); Shields et al., 2001, J. Biol. Chem. 276(9):6591-6604
(reduced ADCC and CDC); Idusogie et al., 2000, J. Immunol.
164(8):4178-4184 (increased ADCC and CDC); Steurer et al., 1995, J.
Immunol. 155(3):1165-1174 (reduced ADCC and CDC); Idusogie et al.,
2001, J. Immunol. 166(4):2571-2575 (increased ADCC and CDC); Lazar
et al., 2006, Proc. Natl. Acad. Sci. USA 103(11): 4005-4010
(increased ADCC); Ryan et al., 2007, Mol. Cancer. Ther., 6:
3009-3018 (increased ADCC); Richards et al., 2008, Mol. Cancer
Ther. 7(8):2517-2527.
[0339] In some embodiments, the antibody comprises a modified
constant region that has increased binding affinity for FcRn and/or
an increased serum half-life as compared with the unmodified
antibody.
[0340] In a process known as "germlining", certain amino acids in
the VH and VL sequences can be mutated to match those found
naturally in germline VH and VL sequences. In particular, the amino
acid sequences of the framework regions in the VH and VL sequences
can be mutated to match the germline sequences to reduce the risk
of immunogenicity when the antibody is administered. Germline DNA
sequences for human VH and VL genes are known in the art (see e.g.,
the "Vbase" human germline sequence database; see also Kabat, E.
A., et al., 1991, Sequences of Proteins of Immunological Interest,
Fifth Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson et al., 1992, J. Mol. Biol.
227:776-798; and Cox et al., 1994, Eur. J. Immunol.
24:827-836).
[0341] Another type of amino acid substitution that may be made is
to remove potential proteolytic sites in the antibody. Such sites
may occur in a CDR or framework region of a variable domain or in
the constant region of an antibody. Substitution of cysteine
residues and removal of proteolytic sites may decrease the risk of
heterogeneity in the antibody product and thus increase its
homogeneity. Another type of amino acid substitution is to
eliminate asparagine-glycine pairs, which form potential
deamidation sites, by altering one or both of the residues. In
another example, the C-terminal lysine of the heavy chain of an
anti-FXI antibody or anti-idiotype antibody of the disclosure can
be cleaved or otherwise removed. In various embodiments of the
disclosure, the heavy and light chains of the antibodies may
optionally include a signal sequence.
[0342] Once DNA fragments encoding the VH and VL segments of the
present disclosure are obtained, these DNA fragments can be further
manipulated by standard recombinant DNA techniques, for example to
convert the variable region genes to full-length antibody chain
genes, to Fab fragment genes, or to a scFv gene. In these
manipulations, a VL- or VH-encoding DNA fragment is operatively
linked to another DNA fragment encoding another protein, such as an
antibody constant region or a flexible linker. The term
"operatively linked", as used in this context, is intended to mean
that the two DNA fragments are joined such that the amino acid
sequences encoded by the two DNA fragments remain in-frame.
[0343] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
E. A., et al., 1991, Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA, IgE, IgM or IgD constant
region, but most preferably is an IgG.sub.1 or IgG.sub.2 constant
region. The IgG constant region sequence can be any of the various
alleles or allotypes known to occur among different individuals,
such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes represent
naturally occurring amino acid substitution in the IgG1 constant
regions. For a Fab fragment heavy chain gene, the VH-encoding DNA
can be operatively linked to another DNA molecule encoding only the
heavy chain CH1 constant region. The CH1 heavy chain constant
region may be derived from any of the heavy chain genes.
[0344] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, E. A., et al., 1991, Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region. The kappa constant region may be any of the
various alleles known to occur among different individuals, such as
Inv(1), Inv(2), and Inv(3). The lambda constant region may be
derived from any of the three lambda genes.
[0345] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker such that the VH and VL sequences can be expressed as a
contiguous single-chain protein, with the VL and VH regions joined
by the flexible linker (See e.g., Bird et al., 1988, Science
242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; McCafferty et al., 1990, Nature 348:552-554. Linkers
of other sequences have been designed and used (Bird et al., 1988,
supra). Linkers can in turn be modified for additional functions,
such as attachment of drugs or attachment to solid supports. The
single chain antibody may be monovalent, if only a single VH and VL
are used, bivalent, if two VH and VL are used, or polyvalent, if
more than two VH and VL are used. Bispecific or polyvalent
antibodies may be generated that bind specifically to FXIa and to
another molecule. In some embodiments, bispecific or polyvalent
anti-idiotype antibodies may be generated that bind specifically to
two or more anti-FXIa antibodies. The single chain variants can be
produced either recombinantly or synthetically. For synthetic
production of scFv, an automated synthesizer can be used. For
recombinant production of scFv, a suitable plasmid containing
polynucleotide that encodes the scFv can be introduced into a
suitable host cell, either eukaryotic, such as yeast, plant, insect
or mammalian cells, or prokaryotic, such as E. coli.
Polynucleotides encoding the scFv of interest can be made by
routine manipulations such as ligation of polynucleotides. The
resultant scFv can be isolated using standard protein purification
techniques known in the art.
[0346] Other forms of single chain antibodies, such as diabodies,
are also encompassed. Diabodies are bivalent, bispecific antibodies
in which VH and VL are expressed on a single polypeptide chain, but
using a linker that is too short to allow for pairing between the
two domains on the same chain, thereby forcing the domains to pair
with complementary domains of another chain and creating two
antigen binding sites (see e.g., Holliger, P., et al., 1993, Proc.
Natl. Acad Sci. USA 90:6444-6448; Poljak, R. J., et al., 1994,
Structure 2:1121-1123).
[0347] Heteroconjugate antibodies, comprising two covalently joined
antibodies, are also provided. Such antibodies have been used to
target immune system cells to unwanted cells (U.S. Pat. No.
4,676,980), and for treatment of HIV infection (PCT Publication
Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate
antibodies may be made using any convenient cross-linking methods.
Suitable cross-linking agents and techniques are well known in the
art, and are described in U.S. Pat. No. 4,676,980.
[0348] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods of synthetic protein chemistry, including those
involving cross-linking agents. For example, immunotoxins may be
constructed using a disulfide exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
[0349] The disclosure also encompasses fusion proteins comprising
one or more fragments or regions from the antibodies disclosed
herein. In some embodiments, a fusion antibody may be made that
comprises all or a portion of an anti-FXIa antibody of the
disclosure linked to another polypeptide. In another embodiment,
only the variable domains of the anti-FXIa antibody are linked to
the polypeptide. In another embodiment, the VH domain of an
anti-FXIa antibody is linked to a first polypeptide, while the VL
domain of an anti-FXIa antibody is linked to a second polypeptide
that associates with the first polypeptide in a manner such that
the VH and VL domains can interact with one another to form an
antigen-binding site. In another preferred embodiment, the VH
domain is separated from the VL domain by a linker such that the VH
and VL domains can interact with one another. The VH-linker-VL
antibody is then linked to the polypeptide of interest. In some
embodiments, a fusion antibody may be made that comprises all or a
portion of an anti-idiotype antibody of the disclosure linked to
another polypeptide. In another embodiment, only the variable
domains of the anti-idiotype antibody are linked to the
polypeptide. In another embodiment, the VH domain of an
anti-idiotype antibody is linked to a first polypeptide, while the
VL domain of an anti-idiotype antibody is linked to a second
polypeptide that associates with the first polypeptide in a manner
such that the VH and VL domains can interact with one another to
form an antigen binding site. In another preferred embodiment, the
VH domain is separated from the VL domain by a linker such that the
VH and VL domains can interact with one another. The VH-linker-VL
antibody is then linked to the polypeptide of interest. In
addition, fusion antibodies can be created in which two (or more)
single-chain antibodies are linked to one another. This is useful
if one wants to create a divalent or polyvalent antibody on a
single polypeptide chain, or if one wants to create a bispecific
antibody.
[0350] In some embodiments, a fusion polypeptide is provided that
comprises at least 10 contiguous amino acids of the variable light
chain region shown in SEQ ID NOs: 7, 17, 21, 23, 25, 27, 31, 37,
39, 42, 46, 50, 54, 58, 62, 64, 68, or 97 and/or at least 10 amino
acids of the variable heavy chain region shown in SEQ ID NOs: 1,
14, 18, 22, 24, 26, 28, 34, 38, 40, 43, 47, 51, 55, 59, 63, 65, or
96. In other embodiments, a fusion polypeptide is provided that
comprises at least about 10, at least about 15, at least about 20,
at least about 25, or at least about 30 contiguous amino acids of
the variable light chain region and/or at least about 10, at least
about 15, at least about 20, at least about 25, or at least about
30 contiguous amino acids of the variable heavy chain region. In
another embodiment, the fusion polypeptide comprises one or more
CDR(s). In still other embodiments, the fusion polypeptide
comprises VH CDR3 and/or VL CDR3. For purposes of this disclosure,
a fusion protein contains one or more antibodies and another amino
acid sequence to which it is not attached in the native molecule,
for example, a heterologous sequence or a homologous sequence from
another region. Exemplary heterologous sequences include, but are
not limited to a "tag" such as a FLAG tag or a 6His tag. Tags are
well known in the art.
[0351] In some embodiments, a fusion polypeptide is provided that
comprises at least 10 contiguous amino acids of the variable light
chain region shown in SEQ ID NO 75 and/or at least 10 amino acids
of the variable heavy chain region shown in SEQ ID NO: 69. In other
embodiments, a fusion polypeptide is provided that comprises at
least about 10, at least about 15, at least about 20, at least
about 25, or at least about 30 contiguous amino acids of the
variable light chain region and/or at least about 10, at least
about 15, at least about 20, at least about 25, or at least about
30 contiguous amino acids of the variable heavy chain region. In
another embodiment, the fusion polypeptide comprises one or more
CDR(s). In still other embodiments, the fusion polypeptide
comprises VH CDR3 and/or VL CDR3. For purposes of this disclosure,
a fusion protein contains one or more antibodies and another amino
acid sequence to which it is not attached in the native molecule,
for example, a heterologous sequence or a homologous sequence from
another region. Exemplary heterologous sequences include, but are
not limited to a "tag" such as a FLAG tag or a 6His tag. Tags are
well known in the art.
[0352] A fusion polypeptide can be created by methods known in the
art, for example, synthetically or recombinantly. Typically, the
fusion proteins of this disclosure are made by preparing and
expressing a polynucleotide encoding them using recombinant methods
described herein, although they may also be prepared by other means
known in the art, including, for example, chemical synthesis.
[0353] In other embodiments, other modified antibodies may be
prepared using nucleic acid molecules encoding an anti-FXIa
antibody. In some embodiments, other modified antibodies may be
prepared using nucleic acid molecules encoding an anti-idiotype
antibody. For instance, "Kappa bodies" (Ill et al., 1997, Protein
Eng. 10:949-57), "Minibodies" (Martin et al., 1994, EMBO J.
13:5303-9), "Diabodies" (Holliger et al., supra), or "Janusins"
(Traunecker et al., 1991, EMBO J. 10:3655-3659 and Traunecker et
al., 1992, Int. J. Cancer (Suppl.) 7:51-52) may be prepared using
standard molecular biological techniques following the teachings of
the specification.
[0354] For example, bispecific antibodies, monoclonal antibodies
that have binding specificities for at least two different
antigens, can be prepared using the antibodies disclosed herein.
Methods for making bispecific antibodies are known in the art (see,
e.g., Suresh et al., 1986, Methods in Enzymology 121:210). For
example, bispecific antibodies or antigen-binding fragments can be
produced by fusion of hybridomas or linking of Fab' fragments. See,
e.g., Songsivilai & Lachmann, 1990, Clin. Exp. Immunol.
79:315-321, Kostelny et al., 1992, J. Immunol. 148:1547-1553.
Traditionally, the recombinant production of bispecific antibodies
was based on the coexpression of two immunoglobulin heavy
chain-light chain pairs, with the two heavy chains having different
specificities (Millstein and Cuello, 1983, Nature 305, 537-539). In
addition, bispecific antibodies may be formed as "diabodies" or
"Janusins." In some embodiments, the bispecific antibody binds to
two different epitopes of FXIa. In some embodiments, the modified
antibodies described above are prepared using one or more of the
variable domains or CDR regions from an anti-FXIa antibody provided
herein. In some embodiments, the bispecific antibody binds to two
different epitopes of an anti-FXIa antibody. In some embodiments, a
bispecific antibody binds two different anti-FXIa antibodies. In
some embodiments, the modified antibodies described above are
prepared using one or more of the variable domains or CDR regions
from an anti-idiotype antibody provided herein.
[0355] According to one approach to making bispecific antibodies,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant region sequences. The fusion preferably is with an
immunoglobulin heavy chain constant region, comprising at least
part of the hinge, CH2 and CH3 regions. It is preferred to have the
first heavy chain constant region (CH1), containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are cotransfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0356] In one approach, the bispecific antibodies are composed of a
hybrid immunoglobulin heavy chain with a first binding specificity
in one arm, and a hybrid immunoglobulin heavy chain-light chain
pair (providing a second binding specificity) in the other arm.
This asymmetric structure, with an immunoglobulin light chain in
only one half of the bispecific molecule, facilitates the
separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations. This approach is described in
PCT Publication No. WO 94/04690.
[0357] This disclosure also provides compositions comprising
anti-FXIa antibodies conjugated (for example, linked) to an agent
that facilitate coupling to a solid support (such as biotin or
avidin). For simplicity, reference will be made generally to
antibodies with the understanding that these methods apply to any
of the FXIa binding embodiments described herein. This disclosure
also provides compositions comprising anti-idiotype antibodies
conjugated (for example, linked) to an agent that facilitate
coupling to a solid support (such as biotin or avidin). For
simplicity, reference will be made generally to antibodies with the
understanding that these methods apply to any of the anti-FXIa
antibody binding embodiments described herein. Conjugation
generally refers to linking these components as described herein.
The linking (which is generally fixing these components in
proximate association at least for administration) can be achieved
in any number of ways. For example, a direct reaction between an
agent and an antibody is possible when each possesses a substituent
capable of reacting with the other. For example, a nucleophilic
group, such as an amino or sulfhydryl group, on one may be capable
of reacting with a carbonyl-containing group, such as an anhydride
or an acid halide, or with an alkyl group containing a good leaving
group (e.g., a halide) on the other.
[0358] The antibodies can be bound to many different carriers.
Carriers can be active and/or inert. Examples of well-known
carriers include polypropylene, polystyrene, polyethylene, dextran,
nylon, amylases, glass, natural and modified celluloses,
polyacrylamides, agaroses and magnetite. The nature of the carrier
can be either soluble or insoluble for purposes of the disclosure.
Those skilled in the art will know of other suitable carriers for
binding antibodies, or will be able to ascertain such, using
routine experimentation.
[0359] An antibody or polypeptide of this disclosure may be linked
to a labeling agent such as a fluorescent molecule, a radioactive
molecule or any others labels known in the art. Labels are known in
the art which generally provide (either directly or indirectly) a
signal.
IV. Polynucleotides, Vectors, and Host Cells
[0360] The disclosure also provides polynucleotides encoding any of
the antibodies, including antibody fragments and modified
antibodies described herein, such as, e.g., antibodies having
impaired effector function. In another aspect, the disclosure
provides a method of making any of the polynucleotides described
herein. Polynucleotides can be made and expressed by procedures
known in the art. Accordingly, the disclosure provides
polynucleotides or compositions, including pharmaceutical
compositions, comprising polynucleotides, encoding any of the
following anti-FXIa antibodies and antigen-binding fragments
thereof: D4 VH (SEQ ID NO:14), DEF VH (SEQ ID NO:1), QCA11 VH (SEQ
ID NO:18), B1D2 VH (SEQ ID NO:22), B10H2 VH (SEQ ID NO:24), B10E6
VH (SEQ ID NO:26), B10F6 VH (SEQ ID NO:28), B10D8 VH (SEQ ID
NO:34), B10B12 VH (SEQ ID NO:38), S1D4 VH (SEQ ID NO:40), S10H9 VH
(SEQ ID NO:43), Clone 8 VH (SEQ ID NO:47), Clone 16 VH (SEQ ID
NO:51), Clone 20 VH (SEQ ID NO:55), Clone 22 VH (SEQ ID NO:59),
Clone 32 VH (SEQ ID NO:63), Clone 24 VH (SEQ ID NO:65), D4 VL (SEQ
ID NO:17), DEF VL (SEQ ID NO:7), QCA11 VL (SEQ ID NO:21), B1D2 VL
(SEQ ID NO:23), B10H2 VL (SEQ ID NO:25), B10E6 VL (SEQ ID NO:27),
B10F6 VL (SEQ ID NO:31), B10D8 VL (SEQ ID NO:37), B10B12 VL (SEQ ID
NO:39), S1D4 VL (SEQ ID NO:42), S10H9 VL (SEQ ID NO:46), Clone 8 VL
(SEQ ID NO:50), Clone 16 VL (SEQ ID NO:54), Clone 20 VL (SEQ ID
NO:58), Clone 22 VL (SEQ ID NO:62), Clone 32 VL (SEQ ID NO:64), or
Clone 24 VL (SEQ ID NO:68), or any fragment or part thereof having
the ability to bind FXIa. The disclosure further provides
polynucleotides or compositions, including pharmaceutical
compositions, comprising polynucleotides, encoding the following
anti-idiotype antibody and antigen-binding fragments thereof: C4 VH
(SEQ ID NO:69), or C4 VL (SEQ ID NO:75), or any fragment or part
thereof having the ability to bind to an anti-FXIa antibody of the
disclosure.
[0361] In one embodiment, the VH and VL domains, or antigen-binding
fragment thereof, or full length HC or LC, are encoded by separate
polynucleotides. Alternatively, both VH and VL, or antigen-binding
fragment thereof, or HC and LC, are encoded by a single
polynucleotide.
[0362] In another aspect, the disclosure provides polynucleotides
and variants thereof encoding an anti-FXIa antibody, wherein such
variant polynucleotides share at least 70%, at least 75%, at least
80%, at least 85%, at least 87%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to any of the specific nucleic acid disclosed herein. In
some embodiments, the polynucleotide has at least 70% sequence
identity to SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87,
SEQ ID NO:88, or SEQ ID NO:89.
[0363] In another aspect, the disclosure provides polynucleotides
and variants thereof encoding an anti-idiotype antibody that
specifically bind to the antigen-binding site of an anti-FXIa
antibody or antigen-binding portion thereof of the disclosure,
wherein such variant polynucleotides share at least 70%, at least
75%, at least 80%, at least 85%, at least 87%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence identity to any of the specific nucleic acid disclosed
herein. In some embodiments, the polynucleotide has at least 70%
sequence identity to SEQ ID NO:90 or SEQ ID NO:91.
[0364] Polynucleotides complementary to any such sequences are also
encompassed by the present disclosure. Polynucleotides may be
single-stranded (coding or antisense) or double-stranded, and may
be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules
include HnRNA molecules, which contain introns and correspond to a
DNA molecule in a one-to-one manner, and mRNA molecules, which do
not contain introns. Additional coding or non-coding sequences may,
but need not, be present within a polynucleotide of the present
disclosure, and a polynucleotide may, but need not, be linked to
other molecules and/or support materials.
[0365] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes an antibody or a fragment thereof)
or may comprise a variant of such a sequence. Polynucleotide
variants contain one or more substitutions, additions, deletions
and/or insertions such that the immunoreactivity of the encoded
polypeptide is not diminished, relative to a native immunoreactive
molecule. The effect on the immunoreactivity of the encoded
polypeptide may generally be assessed as described herein. Variants
preferably exhibit at least about 70% identity, more preferably, at
least about 80% identity, yet more preferably, at least about 90%
identity, and most preferably, at least about 95% identity to a
polynucleotide sequence that encodes a native antibody or a
fragment thereof.
[0366] Two polynucleotide or polypeptide sequences are said to be
"identical" if the sequence of nucleotides or amino acids in the
two sequences is the same when aligned for maximum correspondence
as described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, or 40 to
about 50, in which a sequence may be compared to a reference
sequence of the same number of contiguous positions after the two
sequences are optimally aligned.
[0367] Optimal alignment of sequences for comparison may be
conducted using the MegAlign.RTM. program in the Lasergene.RTM.
suite of bioinformatics software (DNASTAR.RTM., Inc., Madison,
Wis.), using default parameters. This program embodies several
alignment schemes described in the following references: Dayhoff,
M. O., 1978, A model of evolutionary change in proteins--Matrices
for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas
of Protein Sequence and Structure, National Biomedical Research
Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J.,
1990, Unified Approach to Alignment and Phylogenes pp. 626-645
Methods in Enzymology vol. 183, Academic Press, Inc., San Diego,
Calif.; Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153;
Myers, E. W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D.,
1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol.
Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical
Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman
Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J.,
1983, Proc. Natl. Acad. Sci. USA 80:726-730.
[0368] Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polynucleotide or polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid bases or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the reference sequence (i.e., the window size) and
multiplying the results by 100 to yield the percentage of sequence
identity.
[0369] Variants may also, or alternatively, be substantially
homologous to a native gene, or a portion or complement thereof.
Such polynucleotide variants are capable of hybridizing under
moderately stringent conditions to a naturally occurring DNA
sequence encoding a native antibody (or a complementary
sequence).
[0370] Suitable "moderately stringent conditions" include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC,
overnight; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times. and 0.2.times.SSC
containing 0.1% SDS.
[0371] As used herein, "highly stringent conditions" or "high
stringency conditions" are those that: (1) employ low ionic
strength and high temperature for washing, for example 0.015 M
sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate
at 50.degree. C.; (2) employ during hybridization a denaturing
agent, such as formamide, for example, 50% (v/v) formamide with
0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide
at 55.degree. C., followed by a high-stringency wash consisting of
0.1.times.SSC containing EDTA at 55.degree. C. The skilled artisan
will recognize how to adjust the temperature, ionic strength, etc.
as necessary to accommodate factors such as probe length and the
like.
[0372] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Nonetheless,
polynucleotides that vary due to differences in codon usage are
specifically contemplated by the present disclosure. Further,
alleles of the genes comprising the polynucleotide sequences
provided herein are within the scope of the present disclosure.
Alleles are endogenous genes that are altered as a result of one or
more mutations, such as deletions, additions and/or substitutions
of nucleotides. The resulting mRNA and protein may, but need not,
have an altered structure or function. Alleles may be identified
using standard techniques (such as hybridization, amplification
and/or database sequence comparison).
[0373] The polynucleotides of this disclosure can be obtained using
chemical synthesis, recombinant methods, or PCR. Methods of
chemical polynucleotide synthesis are well known in the art and
need not be described in detail herein. One of skill in the art can
use the sequences provided herein and a commercial DNA synthesizer
to produce a desired DNA sequence.
[0374] For preparing polynucleotides using recombinant methods, a
polynucleotide comprising a desired sequence can be inserted into a
suitable vector, and the vector in turn can be introduced into a
suitable host cell for replication and amplification, as further
discussed herein. Polynucleotides may be inserted into host cells
by any means known in the art. Cells are transformed by introducing
an exogenous polynucleotide by direct uptake, endocytosis,
transfection, F-mating or electroporation. Once introduced, the
exogenous polynucleotide can be maintained within the cell as a
non-integrated vector (such as a plasmid) or integrated into the
host cell genome. The polynucleotide so amplified can be isolated
from the host cell by methods well known within the art. See, e.g.,
Sambrook et al., 1989.
[0375] Alternatively, PCR allows reproduction of DNA sequences. PCR
technology is well known in the art and is described in U.S. Pat.
Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR:
The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer
Press, Boston, 1994.
[0376] RNA can be obtained by using the isolated DNA in an
appropriate vector and inserting it into a suitable host cell. When
the cell replicates and the DNA is transcribed into RNA, the RNA
can then be isolated using methods well known to those of skill in
the art, as set forth in Sambrook et al., 1989, supra, for
example.
[0377] Suitable cloning vectors may be constructed according to
standard techniques, or may be selected from a large number of
cloning vectors available in the art. While the cloning vector
selected may vary according to the host cell intended to be used,
useful cloning vectors will generally have the ability to
self-replicate, may possess a single target for a particular
restriction endonuclease, and/or may carry genes for a marker that
can be used in selecting clones containing the vector. Suitable
examples include plasmids and bacterial viruses, e.g., pUC18,
pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,
pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors
such as pSA3 and pAT28. These and many other cloning vectors are
available from commercial vendors such as BioRad, Strategene, and
Invitrogen.
[0378] Expression vectors are further provided. Expression vectors
generally are replicable polynucleotide constructs that contain a
polynucleotide according to the disclosure. It is implied that an
expression vector must be replicable in the host cells either as
episomes or as an integral part of the chromosomal DNA. Suitable
expression vectors include but are not limited to plasmids, viral
vectors, including adenoviruses, adeno-associated viruses,
retroviruses, cosmids, and expression vector(s) disclosed in PCT
Publication No. WO 87/04462. Vector components may generally
include, but are not limited to, one or more of the following: a
signal sequence; an origin of replication; one or more marker
genes; suitable transcriptional controlling elements (such as
promoters, enhancers and terminator). For expression (i.e.,
translation), one or more translational controlling elements are
also usually required, such as ribosome binding sites, translation
initiation sites, and stop codons.
[0379] The vectors containing the polynucleotides of interest
and/or the polynucleotides themselves, can be introduced into the
host cell by any of a number of appropriate means, including
electroporation, transfection employing calcium chloride, rubidium
chloride, calcium phosphate, DEAE-dextran, or other substances;
microprojectile bombardment; lipofection; and infection (e.g.,
where the vector is an infectious agent such as vaccinia virus).
The choice of introducing vectors or polynucleotides will often
depend on features of the host cell.
[0380] The disclosure also provides host cells comprising any of
the polynucleotides described herein. Any host cells capable of
over-expressing heterologous DNAs can be used for the purpose of
isolating the genes encoding the antibody, polypeptide or protein
of interest. Non-limiting examples of mammalian host cells include
but not limited to COS, HeLa, and CHO cells. See also PCT
Publication No. WO 87/04462. Suitable non-mammalian host cells
include prokaryotes (such as E. coli or B. subtilis) and yeast
(such as S. cerevisae, S. pombe; or K. lactis). Preferably, the
host cells express the cDNAs at a level of about 5 fold higher,
more preferably, 10 fold higher, even more preferably, 20 fold
higher than that of the corresponding endogenous antibody or
protein of interest, if present, in the host cells. Screening the
host cells for a specific binding to FXIa or anti-FXIa antibody is
effected by an immunoassay or FACS. A cell overexpressing the
antibody or protein of interest can be identified.
[0381] An expression vector can be used to direct expression of an
anti-FXIa antibody or anti-idiotype antibody. One skilled in the
art is familiar with administration of expression vectors to obtain
expression of an exogenous protein in vivo. See, e.g., U.S. Pat.
Nos. 6,436,908; 6,413,942; and 6,376,471. Administration of
expression vectors includes local or systemic administration,
including injection, oral administration, particle gun or
catheterized administration, and topical administration. In another
embodiment, the expression vector is administered directly to the
sympathetic trunk or ganglion, or into a coronary artery, atrium,
ventrical, or pericardium.
[0382] Targeted delivery of therapeutic compositions containing an
expression vector, or subgenomic polynucleotides can also be used.
Receptor-mediated DNA delivery techniques are described in, for
example, Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et
al., Gene Therapeutics: Methods And Applications Of Direct Gene
Transfer, J. A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988,
263:621; Wu et al., J. Biol. Chem., 1994, 269:542; Zenke et al.,
Proc. Natl. Acad. Sci. USA, 1990, 87:3655; Wu et al., J. Biol.
Chem., 1991, 266:338. Therapeutic compositions containing a
polynucleotide are administered in a range of about 100 ng to about
200 mg of DNA for local administration in a gene therapy protocol.
Concentration ranges of about 500 ng to about 50 mg, about 1 .mu.g
to about 2 mg, about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g
to about 100 .mu.g of DNA can also be used during a gene therapy
protocol. The therapeutic polynucleotides and polypeptides can be
delivered using gene delivery vehicles. The gene delivery vehicle
can be of viral or non-viral origin (see generally, Jolly, Cancer
Gene Therapy, 1994, 1:51; Kimura, Human Gene Therapy, 1994, 5:845;
Connelly, Human Gene Therapy, 1995, 1:185; and Kaplitt, Nature
Genetics, 1994, 6:148). Expression of such coding sequences can be
induced using endogenous mammalian or heterologous promoters.
Expression of the coding sequence can be either constitutive or
regulated.
[0383] Viral-based vectors for delivery of a desired polynucleotide
and expression in a desired cell are well known in the art.
Exemplary viral-based vehicles include, but are not limited to,
recombinant retroviruses (see, e.g., PCT Publication Nos. WO
90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO
93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB
Patent No. 2,200,651; and EP Patent No. 0 345 242),
alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki
forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC
VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus
(ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and
adeno-associated virus (AAV) vectors (see, e.g., PCT Publication
Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO
95/11984 and WO 95/00655). Administration of DNA linked to killed
adenovirus as described in Curiel, Hum. Gene Ther., 1992, 3:147 can
also be employed.
[0384] Non-viral delivery vehicles and methods can also be
employed, including, but not limited to, polycationic condensed DNA
linked or unlinked to killed adenovirus alone (see, e.g., Curiel,
Hum. Gene Ther., 1992, 3:147); ligand-linked DNA (see, e.g., Wu, J.
Biol. Chem., 1989, 264:16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO
95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic
charge neutralization or fusion with cell membranes. Naked DNA can
also be employed. Exemplary naked DNA introduction methods are
described in PCT Publication No. WO 90/11092 and U.S. Pat. No.
5,580,859. Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO
95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional
approaches are described in Philip, Mol. Cell Biol., 1994, 14:2411,
and in Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.
V. Therapeutic Methods
[0385] In another aspect, therapeutic methods using the antibodies
or antigen-binding fragments thereof are provided. In some
embodiments, the therapeutic methods comprise the use of isolated
antibodies, or antigen-binding fragments thereof, that specifically
bind FXIa. In some embodiments, the therapeutic methods comprise
the use of isolated antibodies, or antigen-binding fragments
thereof, that specifically bind to the antigen-binding site of an
anti-FXIa antibody or antigen-binding portion thereof (e.g., an
anti-idiotype antibody that specifically binds to an anti-FXIa
antibody described herein).
[0386] Therapeutic methods involve administering to a subject in
need of treatment a therapeutically effective amount, or "effective
amount," of an FXIa antibody, or antigen-binding portion, of the
disclosure or of an anti-idiotype antibody, or antigen-binding
portion thereof, that specifically binds to the antigen-binding
site of an anti-FXIa antibody, or antigen-binding portion, of the
disclosure are contemplated by the present disclosure. As used
herein, a "therapeutically effective", or "effective," amount
refers to an amount of an antibody or portion thereof that is of
sufficient quantity to result in a decrease in severity of disease
symptoms, an increase in frequency and duration of disease
symptom-free periods, or a prevention of impairment or disability
due to the disease affliction--either as a single dose or according
to a multiple dose regimen, alone or in combination with other
agents. Therapeutically effective or effective may also refer to
decreasing an indication of disease that predicts clinically
important events (e.g., for an anti-FXIa antibody, substantially
prolonging APTT or decreasing the occurrence of deep venous
thrombosis in the legs as measured by venography or ultrasound, and
for an anti-idiotype antibody to an anti-FXIa antibody, returning
APTT to normal). One of ordinary skill in the art would be able to
determine such amounts based on such factors as the subject's size,
the severity of the subject's symptoms, laboratory tests that
indicate an effective dosing level (e.g., APTT), and the particular
composition or route of administration selected. The subject may be
a human or non-human animal (e.g., rabbit, rat, mouse, monkey or
other lower-order primate).
[0387] An antibody or antigen-binding portion of the disclosure
might be co-administered with known medicaments, and in some
instances the antibody might itself be modified. Regarding
co-administration with additional therapeutic agents, such agents
can include an anticoagulant agent or a procoagulant agent. The
antibody can be linked to the agent (as an immunocomplex) or can be
administered separately from the agent. In the latter case
(separate administration), the antibody can be administered before,
after or concurrently with the agent or can be co-administered with
other known therapies. Co-administration of the FXIa antibodies, or
antigen binding fragments thereof, of the present disclosure with a
therapeutic agent provides two agents which operate via different
mechanisms may provide a therapeutic and perhaps synergistic effect
to human disease.
[0388] To treat any of the foregoing disorders, pharmaceutical
compositions for use in accordance with the present disclosure may
be formulated in a conventional manner using one or more
pharmaceutically acceptable carriers or excipients and administered
as more fully discussed below.
[0389] Determining a therapeutically effective amount of an
antibody or antigen-binding portion according to the present
disclosure will largely depend on particular patient
characteristics, route of administration, and the nature of the
disorder being treated and is more fully discussed below.
[0390] Administration and dosing of the antibodies are more fully
discussed elsewhere below.
Anti-FXIa Antibodies
[0391] According to the disclosure, an anti-FXIa antibody can be
used to inhibit FXIa-mediated activity. The activity of an
anti-FXIa antibody can be confirmed by bioassays, known to test the
targeted biological activities. Some of the methods for
characterizing an anti-FXIa antibody are described in detail in the
Examples. Non-limiting exemplary tests include a fluorogenic
peptide substrate assay, a thrombin generation assay, and an APTT
test. Other tests are also possible within the knowledge of those
of ordinary skill in the art.
[0392] The disclosure encompasses the use of an anti-FXIa antibody
that binds FXIa as an anticoagulant. In certain embodiments, the
anti-FXIa antibody binds to the catalytic domain of FXIa. In
certain embodiments, the anti-FXIa antibody binds to the active
site of the catalytic domain.
[0393] According to some embodiments, an anti-FXIa antibody reduces
the activity of FXIa in a sample. In some embodiments, treatment
with an anti-FXIa antibody reduces activity of FXIa in a sample at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in
the presence of an anti-FXIa antibody compared to absence of
treatment with an anti-FXIa antibody. In other embodiments,
treatment with an anti-FXIa antibody reduces the activity of FXIa
in a sample about 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%,
30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%,
65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.
These amounts are not meant to be limiting, and increments between
the recited amounts are specifically envisioned as part of the
disclosure.
[0394] According to some embodiments, reversing the effects of FXIa
in a sample by administering an anti-FXIa antibody decreases the
amount of thrombin produced in the sample. In some embodiments,
treatment with an anti-FXIa antibody decreases thrombin production
in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 100%, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,
7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least
50-fold, or more in the presence of an anti-FXIa antibody compared
to the absence of an anti-FXIa antibody. Thrombin production in a
sample can be determined using the thrombin generation assay (TGA)
or other technique familiar to those of ordinary skill in the art.
These amounts are not meant to be limiting, and increments between
the recited amounts are specifically envisioned as part of the
disclosure.
[0395] According to some embodiments, an anti-FXIa antibody
decreases the amount of FXIa enzymatic activity observed in a
fluorogenic substrate assay in a sample. In some embodiments,
treatment with an anti-FXIa antibody decreases enzymatic cleavage
of a fluorogenic substrate in a sample at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 100%, 1.5 fold, 2-fold, 3-fold,
4-fold, 5-fold, 6-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold,
30-fold, at least 50-fold, or more in the presence of an anti-FXIa
antibody compared to the absence of an anti-FXIa antibody. These
amounts are not meant to be limiting, and increments between the
recited amounts are specifically envisioned as part of the
disclosure.
[0396] According to the disclosure, the anti-FXIa antibody or
antibodies selectively bind FXIa over other trypsin-like proteases
by at least 5-fold, at least 6-fold, at least 7-fold, at least
10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at
least 30-fold, at least 50-fold, at least 100-fold, at least,
500-fold, at least 1,000-fold, at least 5,000-fold or at least
10,000-fold. These amounts are not meant to be limiting, and
increments between the recited amounts are specifically envisioned
as part of the disclosure.
[0397] In a clinical setting, anti-FXIa antibody effectiveness can
be detected directly or by measuring the ability of subject blood
to clot and detecting deviations from the expected degree of
anti-coagulation. Blood clotting potential can be measured in ways
familiar to those ordinarily skilled in the art (e.g., APTT).
[0398] In some embodiments, treatment with an anti-FXIa antibody is
monitored using tests or assays performed on blood or plasma from a
subject treated with an anti-FXIa antibody. A blood sample can be
taken from a subject at a predetermined time after treatment with
an anti-FXIa antibody. The blood, or plasma prepared from it, is
then subjected to one or more tests to determine certain hemostatic
pharmacodynamic parameters. Tests for monitoring the effectiveness
of treatment with an anti-FXIa antibody include tests that directly
or indirectly measure the ability to clot or that measure the
activity of an anti-FXIa antibody. Non-limiting exemplary tests
include activated partial thromboplastin time, partial
thromboplastin time, fluorogenic peptide substrate assay,
thromboelastometry, thromboelastography, thrombin generation assay,
level of prothrombin fragment 1+2, or level of
thrombin-antithrombin III complex. Other tests are also possible
within the knowledge of those of ordinary skill in the art.
[0399] According to some embodiments, an anti-FXIa antibody reduces
coagulation in the subject. In some embodiments, treatment with an
anti-FXIa antibody reduces coagulation in a subject at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the presence
of an anti-FXIa antibody compared to absence of an anti-FXIa
antibody. In other embodiments, treatment with an anti-FXIa
antibody reduces coagulation in a subject about 5%-10%, 10%-15%,
15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%,
50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%,
85%-90%, 90%-95%, or 95%-100%. These amounts are not meant to be
limiting, and increments between the recited amounts are
specifically envisioned as part of the disclosure.
[0400] According to some embodiments, an anti-FXIa antibody reduces
the activity of FXIa in the subject. In some embodiments, treatment
with an anti-FXIa antibody reduces activity of FXIa in a subject at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in
the presence of an anti-FXIa antibody compared to absence of an
anti-FXIa antibody. In other embodiments, treatment with an
anti-FXIa antibody reduces the activity of FXIa in a subject about
5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%,
40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%,
75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%. These amounts are
not meant to be limiting, and increments between the recited
amounts are specifically envisioned as part of the disclosure.
[0401] According to some embodiments, an anti-FXIa antibody
decreases the amount of thrombin produced in the blood or plasma of
the subject. In some embodiments, treatment with an anti-FXIa
antibody decreases thrombin production in a subject at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1.5 fold,
2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold, 15-fold,
20-fold, 25-fold, 30-fold, at least 50-fold, or more in the
presence of an anti-FXIa antibody compared to the absence of an
anti-FXIa antibody. Thrombin production in the blood or plasma of a
subject can be determined using the thrombin generation assay (TGA)
or other technique familiar to those of ordinary skill in the art.
These amounts are not meant to be limiting, and increments between
the recited amounts are specifically envisioned as part of the
disclosure.
[0402] According to some embodiments, treatment with an anti-FXIa
antibody decreases the amount of FXIa enzymatic activity observed
in a fluorogenic substrate assay in a sample of blood or plasma of
a subject. In some embodiments, treatment with an anti-FXIa
antibody decreases enzymatic cleavage of a fluorogenic substrate in
a sample at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
100%, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or
more in the presence of an anti-FXIa antibody compared to the
absence of an anti-FXIa antibody. These amounts are not meant to be
limiting, and increments between the recited amounts are
specifically envisioned as part of the disclosure.
[0403] According to some embodiments, an anti-FXIa antibody
decreases clotting by selective triggers in the subject. In some
embodiments, treatment with an anti-FXIa antibody decreases
clotting in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, 100%, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold,
6-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at
least 50-fold, or more in the presence of an anti-FXIa antibody
compared to the absence of an anti-FXIa antibody. These amounts are
not meant to be limiting, and increments between the recited
amounts are specifically envisioned as part of the disclosure.
[0404] According to some embodiments, an anti-FXIa antibody
increases clotting time in the subject. In some embodiments,
treatment with an anti-FXIa antibody increases clotting time in a
subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
100%, 125%, 150% or greater in the presence of an anti-FXIa
antibody compared to absence of treatment with an anti-FXIa
antibody. In other embodiments, treatment with an anti-FXIa
antibody increases clotting time in a subject about 5%-10%,
10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%,
45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%,
80%-85%, 85%-90%, 90%-95%, 95%-100%, 10%-25%, 25%-50%, 50%-75%,
75%-100%, 100%-125%, 125%-150%, or greater than 150%. In some
embodiments, the increase in clotting time is measured by APTT.
These amounts are not meant to be limiting, and increments between
the recited amounts are specifically envisioned as part of the
disclosure.
[0405] In yet other embodiments, the methods of thromboelastometry
or thromboelastography may be used to analyze clot formation or
clotting time.
[0406] According to some embodiments, treatment with an anti-FXIa
antibody decreases the level of prothrombin fragment 1+2 (PF1+2) in
the blood or plasma of the subject. In some embodiments, treatment
with an anti-FXIa antibody decreases PF1+2 in a subject at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1.5-fold,
2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold, 15-fold,
20-fold, 25-fold, 30-fold, at least 50-fold, or more in the
presence of an anti-FXIa antibody compared to the absence of an
anti-FXIa antibody. These amounts are not meant to be limiting, and
increments between the recited amounts are specifically envisioned
as part of the disclosure.
[0407] According to some embodiments, treatment with an anti-FXIa
antibody decreases the level of thrombin-antithrombin III complex
(TAT) in the blood or plasma of the subject. In some embodiments,
treatment with an anti-FXIa antibody decreases TAT in a subject at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%,
1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold,
15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or more in
the presence of an anti-FXIa antibody compared to the absence of an
anti-FXIa antibody. These amounts are not meant to be limiting, and
increments between the recited amounts are specifically envisioned
as part of the disclosure.
[0408] According to some embodiments, treatment with an anti-FXIa
antibody increases activated partial thromboplastin time (APTT) in
the subject. The APTT test measures the time required for clotting
of recalcified human plasma after addition of an intrinsic pathway
activator such as ellagic acid or kaolin. In some embodiments,
treatment with an anti-FXIa antibody increases activated partial
thromboplastin time (APTT) by 20%, 50%, 100%, 150%, 200%, 250% or
more. These amounts are not meant to be limiting, and increments
between the recited amounts are specifically envisioned as part of
the disclosure. In certain embodiments, treatment with an anti-FXIa
antibody increases activated partial thromboplastin time (APTT) in
a subject without prolonging prothrombin time (PT).
[0409] In some embodiments, the disclosed antibodies, or
antigen-binding portions thereof, that specifically bind to FXIa
can be used as anticoagulants. In some embodiments, the disclosed
antibodies, or antigen-binding portions thereof, that specifically
bind to FXIa can be used in the prevention, treatment, and/or
amelioration of diseases, disorders or conditions caused by and/or
associated with FXI activity. Such diseases, disorders or
conditions include, but are not limited to, surgery or other type
of interventional procedure; thrombotic or thromboembolic diseases;
atrial fibrillation (AF); venous thromboembolism (VTE); VTE in the
medically ill; VTE prophylaxis in the medically ill; VTE
prophylaxis in knee or hip surgery; Afib in the renal disease
population and/or patients previously identified as bleeders; acute
coronary syndromes; use of extracorporeal circulations and devices
in which blood contacts artificial surfaces; vascular grafts;
myocardial infarction; acute myocardial infarction; congestive
heart failure; pulmonary embolism; thrombosis; deep vein
thrombosis; renal vein thrombosis; transient ischemic attack;
thrombotic stroke; thromboembolic stroke; cardiogenic
thromboembolism; atherosclerosis; inflammatory diseases; pulmonary
hypertension; pulmonary and/or hepatic fibrosis; and sepsis; among
others, as would be appreciated by one skilled in the art provided
with the teachings disclosed herein. Additional uses include
situations in which blood touches artificial surfaces, including
mechanical heart valves, extracorporeal circulations including but
not limited to extracorporeal membrane oxygenation, cardiopulmonary
bypass, and hemodialysis; vascular grafts; catheters; wires; left
ventricular assist devices (LVAD); transcatheter aortic valve
replacement (TAVR); and and other devices introduced in to the
heart and blood vessels. Examples of diseases and disorders are
provided in WO2013167669, incorporated herein by reference.
[0410] In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof of the disclosure can be used as
an anticoagulant in patients with mechanical heart valves. In some
embodiments, an anti-FXIa antibody or antigen-binding fragment
thereof of the disclosure can be used as an anticoagulant in atrial
fibrillation patients with reduced kidney function or elevated
bleeding risk. In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof of the disclosure can be used for
VTE prophylaxis in the medically ill. In some embodiments, an
anti-FXIa antibody or antigen-binding fragment thereof of the
disclosure can be used for VTE prophylaxis in the surgically ill.
In some embodiments, an anti-FXIa antibody or antigen-binding
fragment thereof of the disclosure can be used as VTE prophylaxis
in patients requiring knee or hip surgery. In some embodiments, an
anti-FXIa antibody or antigen-binding fragment thereof of the
disclosure can be used as an anticoagulant in patients fitted with
an LVAD. In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof of the disclosure can be used as
an anticoagulant in patients on extracorporeal membrane oxygenation
support. In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof of the disclosure can be used as
an anticoagulant in patients with undergoing cardiopulmonary
bypass. In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof of the disclosure can be used as
an anticoagulant in patients fitted with an indwelling catheter. In
some embodiments, an anti-FXIa antibody or antigen-binding fragment
thereof of the disclosure can be used as an anticoagulant in
patients with a vascular graft.
[0411] In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof is used for the prevention of
venous thrombosis and/or venous thromboembolism (VTE) in a patient
undergoing major orthopedic surgery (e.g., total knee replacement,
total hip replacement, or hip fracture surgery); in a patient
hospitalized for medical illness and at increased risk for VTE; in
a patient undergoing abdominal surgery; in a patient with a cancer
associated with increased risk for VTE, such as pancreatic,
gastric, or renal cell carcinoma; in a patient with genetic
thrombophilia or another genetic disorder (e.g., Prader-Willi) that
is associated with increased risk of venous thromboembolism; in a
patient with a history of unprovoked pulmonary embolism or deep
venous thrombosis; in a patient who would typically receive an
inferior vena caval "umbrella" due to high risk for VTE but who is
unable to receive standard anticoagulation therapy; or in a person
with paralytic spinal cord injury or another trauma associated with
elevated VTE risk.
[0412] In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof is used for the prevention of
thromboembolism from the left atrium in a patient with atrial
fibrillation. In some embodiments, the patient is intolerant of or
unlikely to receive standard anticoagulants, including patients on
hemodialysis, patients with end-stage renal disease, patients with
a history of bleeding on standard anticoagulants or having multiple
other risk factors for bleeding.
[0413] In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof is used for the prevention of
arterial thrombi in a patient at elevated risk for myocardial
infarction, including patients with acute coronary syndromes,
patients with diffuse coronary disease and multiple risk factors
(e.g., prior myocardial infarction and diabetes); in a patient with
thromboembolic or thrombo-occlusive stroke (e.g., patients with
severe carotid artery narrowing, transient ischemic attacks); or in
a patient with acute limb ischemia.
[0414] In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof is used for the prevention of
thrombus formation and embolization or device failure in a patient
with an indwelling device that exposes artificial surfaces to
blood, including patients with mechanical heart valves, left
ventricular assist devices, transcatheter aortic valve
replacements, indwelling catheters, vascular grafts, or vascular
stents, including coronary, carotid, or peripheral arterial
stents.
[0415] In some embodiments, an anti-FXIa antibody or
antigen-binding fragment thereof is used for the prevention of
activation of coagulation, consumption of coagulation factors, or
clot formation in a patient connected to extracorporeal
circulation, including hemodialysis machines, cardiopulmonary
bypass, or extracorporeal membrane oxygenation.
[0416] In certain aspects of the disclosure, methods are provided
for increasing anticoagulant activity in a subject comprising
administering to said subject an anti-FXIa antibody as described
herein, wherein the anticoagulant activity is increased compared
with the anticoagulant activity in the subject prior to
administration of the anti-FXIa antibody. In certain aspects of the
disclosure, methods are provided for increasing clotting time in a
subject, comprising administering to said subject an anti-FXIa
antibody as described herein, wherein the clotting time is
increased compared with the clotting time in the subject prior to
administration of the anti-FXIa antibody.
[0417] In some embodiments, anti-FXIa antibodies as described
herein for use in increasing anticoagulant activity in a subject
are provided. In some embodiments, anti-FXIa antibodies as
described herein for use in increasing clotting time in a subject
are provided.
[0418] In some embodiments, use of an anti-FXIa antibody as
described herein in the manufacture of a medicament for increasing
anticoagulant activity in a subject is provided. In some
embodiments, use of an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody as described herein in the manufacture of a
medicament for increasing clotting time in a subject being
administered the anti-FXIa antibody is provided.
Anti-Idiotype Antibodies that Specifically Bind to the
Antigen-Binding Site of an Anti-FXIa Antibody
[0419] According to the disclosure, an antibody, or antigen-binding
portion thereof, that specifically binds to the antigen-binding
site of an anti-FXIa antibody can be used to counteract an
anti-FXIa antibody that binds FXIa. The activity of an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody can be confirmed by
bioassays, known to test the targeted biological activities. Some
of the methods for characterizing antibodies, or antigen-binding
portions thereof, that specifically bind to the antigen-binding
site of an anti-FXIa antibody are described in detail in the
Examples. Non-limiting exemplary tests include a fluorogenic
peptide substrate assay and a thrombin generation assay. Other
tests are also possible within the knowledge of those of ordinary
skill in the art.
[0420] The disclosure encompasses the use of an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody can be used to
counteract an anti-FXIa antibody that binds FXIa. In certain
embodiments, the anti-idiotype antibody is used to counteract an
anti-FXIa antibody that binds to the catalytic domain of FXIa,
e.g., an anti-FXIa antibody binds to the active site of the
catalytic domain.
[0421] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a sample by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces the activity
of an anti-FXIa antibody in the sample. In some embodiments,
treatment with an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody reduces activity of the anti-FXIa antibody in a sample at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in
the presence of an anti-FXIa antibody compared to absence of
treatment with an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody. In other embodiments, treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces the activity
of a an anti-FXIa antibody in a sample about 5%-10%, 10%-15%,
15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%,
50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%,
85%-90%, 90%-95%, or 95%-100%. These amounts are not meant to be
limiting, and increments between the recited amounts are
specifically envisioned as part of the disclosure.
[0422] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a sample by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases the amount
of thrombin produced in the sample. In some embodiments, treatment
with an antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-FXIa
antibody increases thrombin production in a subject at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1.5 fold,
2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold, 15-fold,
20-fold, 25-fold, 30-fold, at least 50-fold, or more in the
presence of an anti-FXIa antibody compared to the absence of an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody.
Thrombin production in a sample can be determined using the
thrombin generation assay (TGA) or other technique familiar to
those of ordinary skill in the art. These amounts are not meant to
be limiting, and increments between the recited amounts are
specifically envisioned as part of the disclosure.
[0423] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a sample by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases the amount
of FXIa enzymatic activity observed in a fluorogenic substrate
assay in the sample. In some embodiments, treatment with an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody
increases enzymatic cleavage of a fluorogenic substrate in a sample
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%,
1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold,
15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or more in
the presence of an anti-FXIa antibody compared to the absence of an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody. These
amounts are not meant to be limiting, and increments between the
recited amounts are specifically envisioned as part of the
disclosure.
[0424] According to the methods of the disclosure, an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody is administered to a
subject whose blood contains an anti-FXIa antibody. In some
embodiments, an antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-FXIa
antibody of the disclosure can be administered to a subject to
reverse the effects of an anti-FXIa antibody where such anti-FXIa
antibody occurs at therapeutic concentrations. In other
embodiments, an antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-FXIa
antibody of the disclosure can be administered to a subject to
reverse the effects of an anti-FXIa antibody where such inhibitor
occurs at supratherapeutic concentrations. A supratherapeutic
concentration is one that is higher than that ordinarily considered
required to safely achieve anti-coagulation in a particular subject
or class of subjects. Supratherapeutic concentrations of an
anti-FXIa antibody can result from accidental or intentional
overdose. Supratherapeutic concentrations of an anti-FXIa antibody
can also result from unexpected effects in particular subjects,
such as an unexpectedly high sensitivity to these drugs, or
unexpectedly slow rate of clearance, due for example to drug
interactions or other factors. Determination of what would be a
therapeutic concentration or supratherapeutic concentration of an
anti-FXIa antibody in a particular subject or class of subjects is
within the knowledge of those ordinarily skilled in the art.
[0425] According to the disclosure, an antibody, or antigen-binding
portion thereof, that specifically binds to the antigen-binding
site of an anti-FXIa antibody is used to counteract an anti-FXIa
antibody or antibodies that selectively bind FXIa over other
trypsin-like proteases by at least 5-fold, at least 6-fold, at
least 7-fold, at least 10-fold, at least 15-fold, at least 20-fold,
at least 25-fold, at least 30-fold, at least 50-fold, at least
100-fold, at least, 500-fold, at least 1,000-fold, at least
5,000-fold or at least 10,000-fold. These amounts are not meant to
be limiting, and increments between the recited amounts are
specifically envisioned as part of the disclosure.
[0426] The anti-FXIa antibody may bind an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody with a K.sub.i of
about 2.times.10.sup.-7M or less. "K.sub.i" refers to the inhibitor
constant of a particular inhibitor-target interaction, which is the
concentration required to produce half maximum inhibition. One can
determine the K.sub.i by using methods known in the art. The
disclosure contemplates, thus, counteracting an anti-FXIa antibody
that binds with an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody with a K.sub.i of about 2.times.10.sup.-8M or less, about
1.times.10.sup.-8 M or less, about 9.times.10.sup.-9M or less,
about 8.times.10.sup.-9M or less, about 7.times.10.sup.-9M or less,
about 6.times.10.sup.-9M or less, about 5.times.10.sup.-9M or less,
about 4.times.10.sup.-9M or less, about 3.times.10.sup.-9M or less,
about 2.times.10.sup.-9M or less, about 1.times.10.sup.-9M or less,
about 9.times.10.sup.-10 M or less, about 8.times.10.sup.-10 M or
less, about 7.times.10.sup.-10 M or less, about 6.times.10.sup.-10
M or less, about 5.times.10.sup.-10 M or less, about
4.times.10.sup.-10 M or less, about 3.times.10.sup.-10 M or less,
about 2.times.10.sup.-10 M or less, about 1.times.10.sup.-10 M or
less, about 9.times.10.sup.-11M or less, about 8.times.10.sup.-11M
or less, about 7.times.10.sup.-11M or less, about
6.times.10.sup.-11M or less, about 5.times.10.sup.-11M or less,
about 4.times.10.sup.-11M or less, about 3.times.10.sup.-11M or
less, about 2.times.10.sup.-11M or less, about 1.times.10.sup.-11M
or less, about 9.times.10.sup.-12M or less, about
8.times.10.sup.-12M or less, about 7.times.10.sup.-12M or less,
about 6.times.10.sup.-12 M or less, about 5.times.10.sup.-12M or
less, about 4.times.10.sup.-12M or less, about 3.times.10.sup.-12M
or less, about 2.times.10.sup.-12M or less, or about
1.times.10.sup.-12M or less, or less. The anti-FXIa antibody to be
counteracted by an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody according to the methods of the disclosure may bind a
wild-type FXIa with a K.sub.i at least 1.5 fold, at least 2-fold,
at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold,
at least 7-fold, at least 10-fold, at least 15-fold, at least
20-fold, at least 25-fold, at least 30-fold, or at least 50-fold
less than it binds the antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody. The anti-FXIa antibody may bind an FXIa dimer
complex comprising a wild-type FXIa with about the same K.sub.i.
These amounts are not meant to be limiting, and increments between
the recited amounts are specifically envisioned as part of the
disclosure.
[0427] In one aspect, the disclosure provides methods for
counteracting the effects of an anti-FXIa antibody in a subject who
is bleeding (internally or externally) or is at risk of bleeding
(e.g., in the course of a planned surgery) by administering
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody. In some
embodiments, the anti-FXIa antibody may be present in the subject
at a therapeutic concentration or a higher concentration (i.e., a
supratherapeutic concentration). In some embodiments, the
therapeutic concentration may be an overdose in sensitive
individuals. The methods of the disclosure, thus, are useful for
providing an antidote to an overdose of an anti-FXIa antibody. In
various embodiments, the subject of treatment may be a human or a
veterinary subject.
[0428] Anti-FXIa antibody overdose can be detected based on
existence of symptoms or signs of excessively reduced clotting
ability. Non-limiting examples include evidence of gastrointestinal
bleeding, including dark tarry stools, bloody stools, and vomiting
of blood. Other examples include nosebleeds, and increased tendency
to, or severity of, bruising or bleeding from minor cuts and
scrapes.
[0429] In a clinical setting, anti-FXIa antibody overdose can be
detected directly or by measuring the ability of subject blood to
clot and detecting deviations from the expected degree of
anti-coagulation. Blood clotting potential can be measured in ways
familiar to those ordinarily skilled in the art. For example,
overdose may be suspected when a subject's activated partial
thromboplastin time is excessively prolonged. In some embodiments,
overdose is confirmed when the activated partial thromboplastin
time is more than 2, 3, 4, or 5 fold, or greater than the activated
partial thromboplastin time of a control sample untreated with an
anti-FXIa antibody.
[0430] The antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-FXIa
antibody may be administered whenever it is desired to counteract
the effects of the anti-FXIa antibody, including but not limited to
before a planned surgery, after an injury resulting in external or
internal bleeding or after an anti-FXIa antibody overdose.
According to the disclosure, the antibody, or antigen-binding
portion thereof, that specifically binds to the antigen-binding
site of an anti-FXIa antibody may be administered at least about 12
hours, at least about 6 hours, at least about 3 hours, at least
about 2 hours, at least about 1 hour, at least about 30 minutes, at
least about 10 minutes, or at least about 5 minutes of when the
desired counteracting effect is needed, such as before a planned
surgery, after an injury resulting in external or internal bleeding
or after an anti-FXIa antibody overdose.
[0431] According to another embodiment, the disclosure provides a
method of administering an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody to effect the urgent reversal of acquired
coagulopathy due to an anti-FXIa antibody therapy in a subject with
acute major bleeding. In some embodiments, subjects are adult human
patients. In other embodiments, subjects are pediatric human
patients.
[0432] In some embodiments, acute major bleeding is caused by
trauma. In other embodiments, acute major bleeding occurs during
surgery or other type of interventional procedure. Exemplary
non-limiting interventional procedures include dental extractions,
incisions, drainage, vascular surgery, appendectomy, herniotomy or
hernioplasty, abdominal surgery, cholecystectomy, trephination
(burr hole), lumbar puncture, cardiac pacemaker insertion, hip
fracture surgery, uterine, kidney, prostate and bladder surgery,
and others. In some embodiments, acute major bleeding may be
menorrhagia. In still other embodiments, acute major bleeding can
be spontaneous bleeding with no apparent cause.
[0433] Without limitation, sites of acute major bleeding include
gastrointestinal bleeding, subcutaneous or intramuscular bleeding,
bladder bleeding, hemarthrosis, subdural hematoma, nasal bleeding,
peritoneal bleeding, uterine bleeding, and other sites of
bleeding.
[0434] Effective treatment with antibodies, or antigen-binding
portions thereof, that specifically bind to the antigen-binding
site of an anti-FXIa antibody of the disclosure can reverse the
effects of an anti-FXIa antibody. Successful reversal of such
effects by an antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-FXIa
antibody can be determined in a variety of ways and be measured or
monitored using different assays, methods, or endpoints.
[0435] In some embodiments, treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody to reverse the
effects of an anti-FXIa antibody is monitored using tests or assays
performed on blood or plasma from a subject treated with an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody. A blood
sample can be taken from a subject at a predetermined time after
treatment with an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody. The blood, or plasma prepared from it, is then subjected
to one or more tests to determine if certain hemostatic
pharmacodynamic parameters have been normalized despite the
presence of an anti-FXIa antibody. If normalization is found then
the subject need not be further treated with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody. If normalization is
not found, however, then further treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody in accordance with
the methods of the disclosure may be required to reverse the effect
of an anti-FXIa antibody. Tests for monitoring the effectiveness of
treatment with an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody include tests that directly or indirectly measure the
ability to clot or that measure the activity of an anti-FXIa
antibody. Non-limiting exemplary tests include fluorogenic peptide
substrate assay, thromboelastometry, thromboelastography, thrombin
generation assay, level of prothrombin fragment 1+2, level of
thrombin-antithrombin III complex, and activated partial
thromboplastin time. Other tests are also possible within the
knowledge of those of ordinary skill in the art.
[0436] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a subject by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces bleeding in
the subject. In some embodiments, treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces bleeding in a
subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
or 99% in the presence of an anti-FXIa antibody compared to absence
of treatment with an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody. In other embodiments, treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces bleeding in a
subject about 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%,
35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%,
70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%. These
amounts are not meant to be limiting, and increments between the
recited amounts are specifically envisioned as part of the
disclosure.
[0437] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a subject by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces the activity
of an anti-FXIa antibody in the subject. In some embodiments,
treatment with an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody reduces activity of the anti-FXIa antibody in a subject at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in
the presence of an anti-FXIa antibody compared to absence of
treatment with an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody. In other embodiments, treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces the activity
of an anti-FXIa antibody in a subject about 5%-10%, 10%-15%,
15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%,
50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%,
85%-90%, 90%-95%, or 95%-100%. These amounts are not meant to be
limiting, and increments between the recited amounts are
specifically envisioned as part of the disclosure.
[0438] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a subject by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases the amount
of thrombin produced in the blood or plasma of the subject. In some
embodiments, treatment with an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody increases thrombin production in a subject at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1.5
fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold,
15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or more in
the presence of an anti-FXIa antibody compared to the absence of an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody.
Thrombin production in the blood or plasma of a subject can be
determined using the thrombin generation assay (TGA) or other
technique familiar to those of ordinary skill in the art. These
amounts are not meant to be limiting, and increments between the
recited amounts are specifically envisioned as part of the
disclosure.
[0439] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a subject by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases the amount
of FXIa enzymatic activity observed in a fluorogenic substrate
assay in a sample of blood or plasma of a subject. In some
embodiments, treatment with an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody increases enzymatic cleavage of a fluorogenic
substrate in a sample at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, 100%, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold,
6-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at
least 50-fold, or more in the presence of an anti-FXIa antibody
compared to the absence of an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody molecule. These amounts are not meant to be
limiting, and increments between the recited amounts are
specifically envisioned as part of the disclosure.
[0440] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a subject by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases clotting in
the subject. In some embodiments, treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases clotting in
a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 100%, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,
7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least
50-fold, or more in the presence of an anti-FXIa antibody compared
to the absence of an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody. These amounts are not meant to be limiting, and
increments between the recited amounts are specifically envisioned
as part of the disclosure.
[0441] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a subject by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces clotting time
in the subject. In some embodiments, treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces clotting time
in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or 99% in the presence of an anti-FXIa antibody compared to
absence of treatment with an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody. In other embodiments, treatment with an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody reduces
clotting time in a subject about 5%-10%, 10%-15%, 15%-20%, 20%-25%,
25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%,
60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or
95%-100%. These amounts are not meant to be limiting, and
increments between the recited amounts are specifically envisioned
as part of the disclosure.
[0442] In yet other embodiments, the methods of thromboelastometry
or thromboelastography may be used to analyze clot formation or
clotting time.
[0443] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a subject by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases the level
of prothrombin fragment 1+2 (PF1+2) in the blood or plasma of the
subject. In some embodiments, treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases PF1+2 in a
subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or
more in the presence of an anti-FXIa antibody compared to the
absence of an antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-FXIa
antibody. These amounts are not meant to be limiting, and
increments between the recited amounts are specifically envisioned
as part of the disclosure.
[0444] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a subject by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases the level
of thrombin-antithrombin III complex (TAT) in the blood or plasma
of the subject. In some embodiments, treatment with an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody increases TAT in a
subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or
more in the presence of an anti-FXIa antibody compared to the
absence of an antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-FXIa
antibody. These amounts are not meant to be limiting, and
increments between the recited amounts are specifically envisioned
as part of the disclosure.
[0445] According to some embodiments, reversing the effects of an
anti-FXIa antibody in a subject by administering an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody reduces activated
partial thromboplastin time (APTT) in the subject. In some
embodiments, treatment with an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody reduces activated partial thromboplastin time
(APTT) in a subject to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 fold or less of the normal
range for the APTT test. In some embodiments, treatment with an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody reduces
activated partial thromboplastin time (APTT) in a subject to less
than 1.2 fold the APTT of a control sample untreated with an
anti-FXIa antibody and an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody. These amounts are not meant to be limiting, and
increments between the recited amounts are specifically envisioned
as part of the disclosure.
[0446] In other embodiments, clinical endpoints can be relied upon
to determine if hemostasis has been adequately restored in a
subject treated with an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody to reverse the effects of an anti-FXIa antibody.
For example, where a subject presents with acute bleeding, clinical
hemostatic efficacy can be scored "very good" where prompt
cessation of existing bleeding occurs after treatment with an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody;
"satisfactory" where there is a 1-2 hr delay in bleeding cessation;
"questionable" where there is a >2 hr delay in bleeding
cessation; and "none" where an effect on bleeding is absent. Where
treatment with an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody is determined to be less than satisfactory, then an
additional dose of an antibody, or antigen-binding portion thereof,
that specifically binds to the antigen-binding site of an anti-FXIa
antibody can be administered to effect adequate hemostasis. In a
further example, where a subject is undergoing an interventional
procedure, clinical hemostatic efficacy can be scored "very good"
where normal hemostasis is attained during the procedure;
"satisfactory" where intraprocedural hemostasis is mildly abnormal
as judged by quantity or quality of blood loss (e.g., slight
oozing); ""questionable" where intraprocedural hemostasis is
moderately abnormal as judged by quantity or quality of blood loss
(e.g., controllable bleeding); and "none" where intraprocedural
hemostasis is severely abnormal as judged by quantity or quality of
blood loss (e.g., severe refractory hemorrhage).
[0447] In some embodiments, the disclosed antibodies, or
antigen-binding portions thereof, that specifically bind to the
antigen-binding site of an anti-FXIa antibody can be used to
decrease anticoagulant activity of an anti-FXIa antibody. In some
embodiments, the disclosed antibodies, or antigen-binding portions
thereof, that specifically bind to the antigen-binding site of an
anti-FXIa antibody can be used in combination with an anti-FXIa
antibody in the prevention, treatment, and/or amelioration of
diseases, disorders or conditions caused by and/or associated with
FXI activity. Such diseases, disorders or conditions include, but
are not limited to, acute major bleeding caused by trauma; acute
major bleeding during surgery or other type of interventional
procedure; thrombotic or thromboembolic diseases; atrial
fibrillation (AF); venous thromboembolism (VTE); VTE in the
medically ill; VTE prophylaxis in the medically ill; VTE
prophylaxis in knee or hip surgery; Afib in the renal disease
population and/or patients previously identified as bleeders; acute
coronary syndromes; use of extracorporeal circulations and devices
in which blood contacts artificial surfaces; vascular grafts;
myocardial infarction; acute myocardial infarction; congestive
heart failure; pulmonary embolism; thrombosis; deep vein
thrombosis; renal vein thrombosis; transient ischemic attack;
thrombotic stroke; thromboembolic stroke; cardiogenic
thromboembolism; atherosclerosis; inflammatory diseases; pulmonary
hypertension; pulmonary and/or hepatic fibrosis; and sepsis; among
others, as would be appreciated by one skilled in the art provided
with the teachings disclosed herein. Additional uses include
situations in which blood touches artificial surfaces, including
mechanical heart valves, extracorporeal circulations,
extracorporeal membrane oxygenation, left ventricular assist
devices, cardiopulmonary bypass, vascular grafts, and catheters,
wires, and other devices introduced in to the heart and blood
vessels. Examples of diseases and disorders are provided in
WO2013167669, incorporated herein by reference.
[0448] In certain aspects of the disclosure, methods are provided
for decreasing anticoagulant activity in a subject being
administered an anti-FXIa antibody, comprising administering to
said subject an antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-FXIa
antibody as described herein, wherein the anticoagulant activity is
reduced compared with the anticoagulant activity in the subject
prior to administration of the anti-FXIa antibody. In certain
aspects of the disclosure, methods are provided for reducing
clotting time in a subject being administered an anti-FXIa
antibody, comprising administering to said subject an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody as described herein,
wherein the clotting time is reduced compared with the clotting
time in the subject prior to administration of the anti-FXIa
antibody.
[0449] In some embodiments, the disclosure provides an antibody, or
antigen-binding portion thereof, that specifically binds to the
antigen-binding site of an anti-FXIa antibody as described herein
for use in decreasing anticoagulant activity in a subject being
administered an anti-FXIa antibody. In some embodiments, the
disclosure provides an antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of an
anti-FXIa antibody as described herein for use in reducing clotting
time in a subject being administered an anti-FXIa antibody.
[0450] In some embodiments, the disclosure provides the use of an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody as
described herein in the manufacture of a medicament for decreasing
anticoagulant activity in a subject being administered an anti-FXIa
antibody. In some embodiments, the disclosure provides the use of
an antibody, or antigen-binding portion thereof, that specifically
binds to the antigen-binding site of an anti-FXIa antibody as
described herein in the manufacture of a medicament for reducing
clotting time in a subject being administered an anti-FXIa
antibody.
VI. Combination Therapies
[0451] Co-administration of an anti-FXIa antibody, or an anti-FXIa
antibody and an anti-idiotype antibody that specifically binds to
the antigen-binding site of the anti-FXIa antibody, or an
antigen-binding portion thereof, as described herein with an
additional therapeutic agent (combination therapy) encompasses
administering a pharmaceutical composition comprising the anti-FXIa
antibody of the disclosure or the anti-idiotype antibody, or
antigen-binding portion thereof, as described herein and the
additional therapeutic agent, as well as administering two or more
separate pharmaceutical compositions, i.e., one comprising the
anti-FXIa antibody of the disclosure or the anti-FXIa antibody and
the anti-idiotype antibody, or an antigen-binding portion thereof,
as described herein and the other(s) comprising the additional
therapeutic agent(s). Co-administration or combination therapy
further includes administering the anti-FXIa antibody of the
disclosure or the anti-FXIa antibody and the anti-idiotype
antibody, or antigen-binding portion thereof, and additional
therapeutic agent(s) simultaneously or sequentially, or both. For
instance, the anti-FXIa antibody of the disclosure or the anti-FXIa
antibody and the anti-idiotype antibody, or antigen-binding portion
thereof, as described herein may be administered once every three
days, while the additional therapeutic agent is administered once
daily at the same time as the anti-FXIa antibody of the disclosure
or the anti-FXIa antibody and the anti-idiotype antibody, or
antigen-binding portion thereof, as described herein, or at a
different time. An anti-FXIa antibody of the disclosure, or
antigen-binding portion thereof, or an anti-FXIa antibody and
anti-idiotype antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of an anti-FXIa
antibody as described herein may be administered prior to or
subsequent to treatment with the additional therapeutic agent.
Similarly, administration of an anti-FXIa antibody of the
disclosure, or antigen-binding portion thereof, or the anti-FXIa
antibody and the anti-idiotype antibody, or antigen-binding portion
thereof, that specifically binds to the antigen-binding site of the
anti-FXIa antibody as described herein may be part of a treatment
regimen that includes other treatment modalities including surgery.
The combination therapy may be administered to prevent recurrence
of the condition. The combination therapy may be administered from
multiple times hourly to weekly. The administrations may be on a
schedule such as every 10 minutes, every 15 minutes, every 20
minutes, every 30 minutes, every hour, every two hours, every three
hours, every four hours, three times daily, twice daily, once
daily, once every two days, once every three days, once weekly, or
may be administered continuously, e.g. via a minipump. The
combination therapy may be administered, for example, via a
parenteral route (e.g., intravenously, subcutaneously,
intraperitoneally, or intramuscularly).
[0452] In another embodiment, the anti-FXIa antibody and the
anti-idiotype antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of the anti-FXIa
antibody as described herein may be co-administered with another
procoagulant including another FXIa decoy molecule, Factor IX,
Factor Xa, Factor XIIa, Factor VIII, Factor VIIa, FEIBA and
prothrombin complex concentrate (PCC).
[0453] In some embodiments, the antibodies of the disclosure are
given in combination with lipid lowering compounds; compounds
suitable for the treatment of coronary diseases and/or compounds
exhibiting vasodilatative activities; diuretics; inhibitors of
calcium channels; inhibitors of the coagulation cascade; and
anticoagulants like non-fractionated heparins, low molecular weight
heparins, hirudin, bivalirudin and/or argatroban. Examples of
suitable combination therapeutics are provided in WO2013167669,
incorporated herein by reference.
[0454] In another embodiment, the anti-FXIa antibody and the
anti-idiotype antibody, or antigen-binding portion thereof, that
specifically binds to the antigen-binding site of the anti-FXIa
antibody as described herein may be co-administered with another
procoagulant including another FXIa decoy molecule, Factor IX,
Factor Xa, Factor XIIa, Factor VIII, Factor VIIa, FEIBA and
prothrombin complex concentrate (PCC).
[0455] Co-administration of an antibody of the disclosure with an
additional therapeutic agent (combination therapy) encompasses
administering a pharmaceutical composition comprising the antibody
of the disclosure and the additional therapeutic agent, as well as
administering two or more separate pharmaceutical compositions,
i.e., one comprising the antibody and the other(s) comprising the
additional therapeutic agent(s). Co-administration or combination
therapy further includes administering the antibody of the
disclosure and additional therapeutic agent(s) simultaneously or
sequentially, or both. For instance, the antibody may be
administered once every three days, while the additional
therapeutic agent is administered once daily at the same as the
antibody, or at a different time. An antibody of the disclosure may
be administered prior to or subsequent to treatment with the
additional therapeutic agent. Similarly, administration of an
antibody of the disclosure may be part of a treatment regimen that
includes other treatment modalities including surgery. The
combination therapy may be administered to prevent recurrence of
the condition. The combination therapy may be administered from
multiple times hourly to weekly. The administrations may be on a
schedule such as every 10 minutes, every 15 minutes, every 20
minutes, every 30 minutes, every hour, every two hours, every three
hours, every four hours, three times daily, twice daily, once
daily, once every two days, once every three days, once weekly, or
may be administered continuously, e.g. via a minipump. The
combination therapy may be administered, for example, via a
parenteral route (e.g., intravenously, subcutaneously,
intraperitoneally, or intramuscularly).
VII. Compositions
[0456] In one aspect, the disclosure provides pharmaceutical
compositions comprising an effective amount of an anti-FXIa
antibody described herein. Examples of such compositions, as well
as how to formulate, are also described herein. In some
embodiments, the composition comprises one or more anti-FXIa
antibodies. In other embodiments, the anti-FXIa antibody recognizes
FXIa. In other embodiments, the anti-FXIa antibody is a human
antibody. In other embodiments, the anti-FXIa antibody is a
humanized antibody. In some embodiments, the anti-FXIa antibody
comprises a constant region that is capable of triggering a desired
immune response, such as antibody-mediated lysis or ADCC. In other
embodiments, the anti-FXIa antibody comprises a constant region
that does not trigger an unwanted or undesirable immune response,
such as antibody-mediated lysis or ADCC. In other embodiments, the
anti-FXIa antibody comprises one or more CDR(s) of the antibody
(such as one, two, three, four, five, or, in some embodiments, all
six CDRs).
[0457] It is understood that the compositions can comprise more
than one anti-FXIa antibody (e.g., a mixture of anti-FXIa
antibodies that recognize different epitopes of FXIa). Other
exemplary compositions comprise more than one anti-FXIa antibody
that recognize the same epitope(s), or different species of
anti-FXIa antibodies that bind to different epitopes of anti-FXIa.
In some embodiments, the compositions comprise a mixture of
anti-FXIa antibodies that recognize different variants of
anti-FXIa.
[0458] The disclosure also provides pharmaceutical compositions
comprising an effective amount of an anti-idiotype antibody that
specifically binds to the antigen-binding site of an anti-FXIa
antibody or antigen-binding portion thereof, as described herein.
Examples of such compositions, as well as how to formulate, are
also described herein. In some embodiments, the composition
comprises one or more anti-idiotype antibodies. In other
embodiments, the anti-idiotype antibody recognizes an anti-FXIa
antibody of the disclosure. In other embodiments, the anti-FXIa
antibody is a human antibody. In other embodiments, the anti-FXIa
antibody is a humanized antibody. In some embodiments, the
anti-idiotype antibody comprises a constant region that is capable
of triggering a desired immune response, such as antibody-mediated
lysis or ADCC. In other embodiments, the anti-FXIa antibody
comprises a constant region that does not trigger an unwanted or
undesirable immune response, such as antibody-mediated lysis or
ADCC. In other embodiments, the anti-idiotype antibody comprises
one or more CDR(s) of the antibody (such as one, two, three, four,
five, or, in some embodiments, all six CDRs).
[0459] It is understood that the compositions can comprise more
than one anti-idiotype antibody (e.g., a mixture of anti-idiotype
antibodies that recognize different anti-FXIa antibodies or
different epitopes on the same anti-FXIa antibody). Other exemplary
compositions comprise more than one anti-idiotype antibody that
recognize the same epitope(s), or different species of
anti-idiotype antibodies that bind to different epitopes of an
anti-FXIa antibody. In some embodiments, the compositions comprise
a mixture of anti-idiotype antibodies that recognize different
variants of an anti-FXIa antibody.
[0460] The compositions of the present disclosure can further
comprise pharmaceutically acceptable carriers, excipients, or
stabilizers (Remington: The Science and practice of Pharmacy 20th
Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations, and may comprise
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrans; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
Pharmaceutically acceptable excipients are further described
herein.
[0461] The anti-FXIa antibody and compositions thereof or the
anti-idiotype antibody and compositions thereof can also be used in
conjunction with other agents that serve to enhance and/or
complement the effectiveness of the agents.
[0462] The disclosure also provides compositions, including
pharmaceutical compositions, comprising any of the polynucleotides
of the disclosure. In some embodiments, the composition comprises
an expression vector comprising a polynucleotide encoding the
antibody as described herein. In other embodiments, the composition
comprises an expression vector comprising a polynucleotide encoding
any of the antibodies described herein. In still other embodiments,
the composition comprises either or both of the polynucleotides
comprising the sequence shown in SEQ ID NO: 84 and SEQ ID NO: 85,
either or both of the polynucleotides shown in SEQ ID NO: 86 and
SEQ ID NO: 87, or either or both of the polynucleotides shown in
SEQ ID NO:88 and SEQ ID NO:89. In still other embodiments, the
composition comprises either or both of the polynucleotides
comprising the sequence shown in SEQ ID NO: 90 and SEQ ID NO:
91.
[0463] In another aspect, the polynucleotide can encode the VH, VL
and/or both VH and VL of the anti-FXIa antibody of the disclosure.
In another aspect, the polynucleotide can encode the VH, VL and/or
both VH and VL of an anti-idiotype antibody of the disclosure. That
is, the composition comprises a single polynucleotide or more than
one polynucleotide encoding the antibody, or antigen-binding
portion thereof, or the disclosure.
[0464] Pharmaceutical compositions of the disclosure also can be
administered in combination therapy, such as, combined with other
agents. For example, the combination therapy can include an
anti-FXIa antibody, or antigen binding fragment thereof, of the
present disclosure combined with at least one other therapy wherein
the therapy may be surgery, immunotherapy, or drug therapy.
[0465] The pharmaceutical compounds of the disclosure may include
one or more pharmaceutically acceptable salts. Examples of such
salts include acid addition salts and base addition salts. Acid
addition salts include those derived from nontoxic inorganic acids,
such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,
hydroiodic, phosphorous and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic
acids, aliphatic and aromatic sulfonic acids and the like. Base
addition salts include those derived from alkaline earth metals,
such as sodium, potassium, magnesium, calcium and the like, as well
as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0466] A pharmaceutical composition of the disclosure also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0467] Examples of suitable aqueous and non-aqueous carriers that
may be employed in the pharmaceutical compositions of the
disclosure include water, ethanol, polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like), and suitable
mixtures thereof, vegetable oils, such as olive oil, and injectable
organic esters, such as ethyl oleate. Proper fluidity can be
maintained, for example, by the use of coating materials, such as
lecithin, by the maintenance of the required particle size in the
case of dispersions, and by the use of surfactants.
[0468] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0469] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures 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 in the case of
dispersion and by the use of surfactants. In many cases, it will be
suitable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0470] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration.
[0471] Generally, dispersions are prepared by incorporating the
active compound into a sterile vehicle that contains a 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 preferred methods
of preparation are vacuum drying and freeze-drying (lyophilization)
that yield a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution
thereof.
[0472] A pharmaceutical composition of the present disclosure may
be prepared, packaged, or sold in a formulation suitable for
ophthalmic administration. Such formulations may, for example, be
in the form of eye drops including, for example, a 0.1 1.0% (w/w)
solution or suspension of the active ingredient in an aqueous or
oily liquid carrier. Such drops may further comprise buffering
agents, salts, or one or more other of the additional ingredients
described herein. Other ophthalmically-administrable formulations
which are useful include those which comprise the active ingredient
in microcrystalline form or in a liposomal preparation.
[0473] As used herein, "additional ingredients" include, but are
not limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the disclosure are
known in the art and described, for example in Remington's
Pharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton,
Pa. (1985), which is incorporated herein by reference.
[0474] In one embodiment, the anti-FXIa antibody, or antigen
binding fragment thereof, is administered in an intravenous
formulation as a sterile aqueous solution containing 5 mg/ml, or
more preferably, about 10 mg/ml, or yet more preferably, about 15
mg/ml, or even more preferably, about 20 mg/ml of antibody, with
sodium acetate, polysorbate 80, and sodium chloride at a pH ranging
from about 5 to 6. Preferably, the intravenous formulation is a
sterile aqueous solution containing 5 or 10 mg/ml of antibody, with
20 mM sodium acetate, 0.2 mg/ml polysorbate 80, and 140 mM sodium
chloride at pH 5.5. Further, a solution comprising an antibody, or
antigen binding fragment thereof, can comprise, among many other
compounds, histidine, mannitol, sucrose, trehalose, glycine,
poly(ethylene) glycol, EDTA, methionine, and any combination
thereof, and many other compounds known in the relevant art.
[0475] In one embodiment, a pharmaceutical composition of the
present disclosure comprises the following components: 100 mg
anti-FXIa antibody or antigen binding fragment of the present
disclosure, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80
at pH 5.8. This composition may be provided as a lyophilized
powder. When the powder is reconstituted at full volume, the
composition retains the same formulation. Alternatively, the powder
may be reconstituted at half volume, in which case the composition
comprises 100 mg FXIa antibody or antigen binding fragment thereof
of the present disclosure, 20 mM histidine, 10% sucrose, and 0.02%
polysorbate 80 at pH 5.8.
[0476] In one embodiment, part of the dose is administered by an
intravenous bolus and the rest by infusion of the antibody
formulation. For example, a 0.01 mg/kg intravenous injection of the
anti-FXIa antibody, or antigen binding fragment thereof, may be
given as a bolus, and the rest of the antibody dose may be
administered by intravenous injection. A predetermined dose of the
anti-FXIa antibody, or antigen binding fragment thereof, may be
administered, for example, over a period of an hour and a half to
two hours to five hours.
[0477] In one embodiment, the anti-idiotype antibody, or antigen
binding fragment thereof, is administered in an intravenous
formulation as a sterile aqueous solution containing 5 mg/ml, or
more preferably, about 10 mg/ml, or yet more preferably, about 15
mg/ml, or even more preferably, about 20 mg/ml of antibody, with
sodium acetate, polysorbate 80, and sodium chloride at a pH ranging
from about 5 to 6. Preferably, the intravenous formulation is a
sterile aqueous solution containing 5 or 10 mg/ml of antibody, with
20 mM sodium acetate, 0.2 mg/ml polysorbate 80, and 140 mM sodium
chloride at pH 5.5. Further, a solution comprising an antibody, or
antigen binding fragment thereof, can comprise, among many other
compounds, histidine, mannitol, sucrose, trehalose, glycine,
poly(ethylene) glycol, EDTA, methionine, and any combination
thereof, and many other compounds known in the relevant art.
[0478] In one embodiment, a pharmaceutical composition of the
present disclosure comprises the following components: 100 mg
anti-idiotype antibody or antigen binding fragment of the present
disclosure, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80
at pH 5.8. This composition may be provided as a lyophilized
powder. When the powder is reconstituted at full volume, the
composition retains the same formulation. Alternatively, the powder
may be reconstituted at half volume, in which case the composition
comprises 100 mg anti-idiotype antibody or antigen binding fragment
thereof of the present disclosure, 20 mM histidine, 10% sucrose,
and 0.02% polysorbate 80 at pH 5.8.
[0479] In one embodiment, part of the dose is administered by an
intravenous bolus and the rest by infusion of the antibody
formulation. For example, a 0.01 mg/kg intravenous injection of the
anti-idiotype antibody, or antigen binding fragment thereof, may be
given as a bolus, and the rest of the antibody dose may be
administered by intravenous injection. A predetermined dose of the
anti-FXIa antibody, or antigen binding fragment thereof, may be
administered, for example, over a period of an hour and a half to
two hours to five hours.
[0480] With regard to a therapeutic agent, where the agent is,
e.g., a small molecule, it can be present in a pharmaceutical
composition in the form of a physiologically acceptable ester or
salt, such as in combination with a physiologically acceptable
cation or anion, as is well known in the art.
[0481] The formulations of the pharmaceutical compositions
described herein may be prepared by any method known or hereafter
developed in the art of pharmacology. In general, such preparatory
methods include the step of bringing the active ingredient into
association with a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or
packaging the product into a desired single- or multi-dose
unit.
[0482] In one embodiment the compositions of the disclosure are
pyrogen-free formulations which are substantially free of
endotoxins and/or related pyrogenic substances. Endotoxins include
toxins that are confined inside a microorganism and are released
when the microorganisms are broken down or die. Pyrogenic
substances also include fever-inducing, thermostable substances
(glycoproteins) from the outer membrane of bacteria and other
microorganisms. Both of these substances can cause fever,
hypotension and shock if administered to humans. Due to the
potential harmful effects, it is advantageous to remove even low
amounts of endotoxins from intravenously administered
pharmaceutical drug solutions. The Food and Drug Administration
("FDA") has set an upper limit of 5 endotoxin units (EU) per dose
per kilogram body weight in a single one hour period for
intravenous drug applications (The United States Pharmacopeial
Convention, Pharmacopeial Forum 26 (1):223 (2000)). When
therapeutic proteins are administered in amounts of several hundred
or thousand milligrams per kilogram body weight it is advantageous
to remove even trace amounts of endotoxin. In one embodiment,
endotoxin and pyrogen levels in the composition are less than 10
EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1
EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg. In
another embodiment, endotoxin and pyrogen levels in the composition
are less than about 10 EU/mg, or less than about 5 EU/mg, or less
than about 1 EU/mg, or less than about 0.1 EU/mg, or less than
about 0.01 EU/mg, or less than about 0.001 EU/mg.
[0483] In one embodiment, the disclosure comprises administering a
composition wherein said administration is oral, parenteral,
intramuscular, intranasal, vaginal, rectal, lingual, sublingual,
buccal, intrabuccal, intravenous, cutaneous, subcutaneous or
transdermal.
[0484] In another embodiment the disclosure further comprises
administering a composition in combination with other therapies,
such as surgery, chemotherapy, hormonal therapy, biological
therapy, immunotherapy or radiation therapy.
VIII. Dosing and Administration
[0485] To prepare pharmaceutical or sterile compositions including
an anti-FXIa antibody or antigen binding fragment thereof of the
disclosure, or an anti-idiotype antibody or antigen binding
fragment thereof of the disclosure, the antibody is mixed with a
pharmaceutically acceptable carrier or excipient. Formulations of
therapeutic and diagnostic agents can be prepared by mixing with
physiologically acceptable carriers, excipients, or stabilizers in
the form of, e.g., lyophilized powders, slurries, aqueous
solutions, lotions, or suspensions (see, e.g., Hardman, et al.
(2001) Goodman and Gilman's The Pharmacological Basis of
Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000)
Remington: The Science and Practice of Pharmacy, Lippincott,
Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker,
NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY;
Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel
Dekker, Inc., New York, N.Y.).
[0486] Selecting an administration regimen for a therapeutic
depends on several factors, including the serum or tissue turnover
rate of the entity, the level of symptoms, the immunogenicity of
the entity, and the accessibility of the target cells in the
biological matrix. In certain embodiments, an administration
regimen maximizes the amount of therapeutic delivered to the
patient consistent with an acceptable level of side effects.
Accordingly, the amount of biologic delivered depends in part on
the particular entity and the severity of the condition being
treated. Guidance in selecting appropriate doses of antibodies,
cytokines, and small molecules are available (see, e.g.,
Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.),
1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert, et al., 2003, New
Engl. J. Med. 348:601-608; Milgrom, et al., 1999, New Engl. J. Med.
341:1966-1973; Slamon, et al., 2001, New Engl. J. Med. 344:783-792;
Beniaminovitz, et al., 2000, New Engl. J. Med. 342:613-619; Ghosh,
et al., 2003, New Engl. J. Med. 348:24-32; Lipsky, et al., 2000,
New Engl. J. Med. 343:1594-1602).
[0487] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced.
[0488] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present disclosure may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present disclosure
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0489] In some embodiments, an anti-idiotype antibody or antigen
binding fragment thereof of the disclosure is administered to a
subject who is being administered an anti-FXIa antibody of the
disclosure. In some embodiments, the anti-FXIa antibody is selected
from: D4, DEF, QCA11, B1D2, B10H2, B10E6, B10F6, B10D8, B10B12,
S1D4, S10H9, Clone 8, Clone 16, Clone 20, Clone 22, Clone 32, or
Clone 24. In some embodiments, the anti-FXIa antibody is DEF. In
some embodiments, the anti-FXIa antibody is DEF and the
anti-idiotype antibody is C4.
[0490] Compositions comprising anti-FXIa antibodies or antigen
binding fragments thereof of the disclosure, or anti-idiotype
antibodies or antigen binding fragments thereof of the disclosure,
can be provided by continuous infusion, or by doses at intervals
of, e.g., one day, one week, or 1-7 times per week. Doses may be
provided intravenously, subcutaneously, topically, orally, nasally,
rectally, intramuscular, intracerebrally, or by inhalation. A
specific dose protocol is one involving the maximal dose or dose
frequency that avoids significant undesirable side effects. A total
weekly dose may be at least 0.05 .mu.g/kg body weight, at least 0.2
.mu.g/kg, at least 0.5 .mu.g/kg, at least 1 .mu.g/kg, at least 10
.mu.g/kg, at least 100 .mu.g/kg, at least 0.2 mg/kg, at least 1.0
mg/kg, at least 2.0 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at
least 20 mg/kg, at least 25 mg/kg, or at least 50 mg/kg (see, e.g.,
Yang, et al., 2003, New Engl. J. Med. 349:427-434; Herold, et al.,
2002, New Engl. J. Med. 346:1692-1698; Liu, et al., 1999, J.
Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al., 2003,
Cancer. Immunol. Immunother. 52: 133-144). The dose may be at least
15 .mu.g, at least 20 .mu.g, at least 25 .mu.g, at least 30 .mu.g,
at least 35 .mu.g, at least 40 .mu.g, at least 45 .mu.g, at least
50 .mu.g, at least 55 .mu.g, at least 60 .mu.g, at least 65 .mu.g,
at least 70 .mu.g, at least 75 .mu.g, at least 80 .mu.g, at least
85 .mu.g, at least 90 .mu.g, at least 95 .mu.g, or at least 100
.mu.g. The doses administered to a subject may number at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.
[0491] For anti-FXIa antibodies or antigen binding fragments
thereof of the disclosure, or anti-idiotype antibodies or antigen
binding fragments thereof of the disclosure, the dosage
administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the
patient's body weight. The dosage may be between 0.0001 mg/kg and
20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg,
0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75
mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg,
0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg,
0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body
weight.
[0492] The dosage of the anti-FXIa antibody, or antigen binding
fragment thereof, or the anti-idiotype antibody, or antigen binding
fragment thereof, may be calculated using the patient's weight in
kilograms (kg) multiplied by the dose to be administered in mg/kg.
The dosage of the antibodies of the disclosure may be 150 .mu.g/kg
or less, 125 .mu.g/kg or less, 100 .mu.g/kg or less, 95 .mu.g/kg or
less, 90 .mu.g/kg or less, 85.mu./kg or less, 80.mu./kg or less,
75.mu./kg or less, 70.mu./kg or less, 65.mu./kg or less, 60.mu./kg
or less, 55.mu./kg or less, 50.mu./kg or less, 45.mu./kg or less,
40.mu./kg or less, 35.mu./kg or less, 30.mu./kg or less, 25.mu./kg
or less, 20.mu./kg or less, 15.mu./kg or less, 10.mu./kg or less,
5.mu./kg or less, 2.5.mu./kg or less, 2.mu./kg or less, 1.5.mu./kg
or less, 1.mu./kg or less, 0.5.mu./kg or less, or 0.1.mu./kg or
less of a patient's body weight.
[0493] A unit dose of the anti-FXIa antibodies or antigen binding
fragments thereof of the disclosure, or the anti-idiotype
antibodies or antigen binding fragments thereof of the disclosure,
may be 0.1 mg to 200 mg, 0.1 mg to 175 mg, 0.1 mg to 150 mg, 0.1 mg
to 125 mg, 0.1 mg to 100 mg, 0.1 mg to 75 mg, 0.1 mg to 50 mg, 0.1
mg to 30 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1
mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to
2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10
mg, 0.25 to 8 mg, 0.25 mg to 7 m g, 0.25 mg to 5 mg, 0.5 mg to 2.5
mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1
mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.
[0494] The dosage of the anti-FXIa antibodies or antigen binding
fragments thereof of the disclosure, or the anti-idiotype
antibodies or antigen binding fragments thereof of the disclosure,
may achieve a serum titer of at least 0.1 .mu.g/ml, at least 0.5
.mu.g/ml, at least 1 .mu.g/ml, at least 2 .mu.g/ml, at least 5
.mu.g/ml, at least 6 .mu.g/ml, at least 10 .mu.g/ml, at least 15
.mu.g/ml, at least 20 .mu.g/ml, at least 25 .mu.g/ml, at least 50
.mu.g/ml, at least 100 .mu.g/ml, at least 125 .mu.g/ml, at least
150 v, at least 175 .mu.g/ml, at least 200 .mu.g/ml, at least 225
.mu.g/ml, at least 250 .mu.g/ml, at least 275 .mu.g/ml, at least
300 .mu.g/ml, at least 325 .mu.g/ml, at least 350 .mu.g/ml, at
least 375 .mu.g/ml/ml, or at least 400 .mu.g/ml/ml in a subject.
Alternatively, the dosage of the antibodies of the disclosure may
achieve a serum titer of at least 0.1 .mu.g/ml, at least 0.5
.mu.g/ml, at least 1 .mu.g/ml, at least, 2 .mu.g/ml, at least 5
.mu.g/ml, at least 6 .mu.g/ml, at least 10 .mu.g/ml, at least 15
.mu.g/ml, at least 20 .mu.g/ml, at least 25 .mu.g/ml, at least 50
.mu.g/ml, at least 100 .mu.g/ml, at least 125 .mu.g/ml, at least
150 .mu.g/ml, at least 175 .mu.g/ml, at least 200 .mu.g/ml, at
least 225 .mu.g/ml, at least 250 .mu.g/ml, at least 275 .mu.g/ml,
at least 300 .mu.g/ml, at least 325 .mu.g/ml, at least 350
.mu.g/ml, at least 375 .mu.g/ml, or at least 400 .mu.g/ml in the
subject.
[0495] Doses of anti-FXIa antibodies or antigen binding fragments
thereof of the disclosure, or anti-idiotype antibodies or antigen
binding fragments thereof of the disclosure, may be repeated and
the administrations may be separated by at least 1 day, 2 days, 3
days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75
days, 3 months, or at least 6 months.
[0496] An effective amount for a particular patient may vary
depending on factors such as the condition being treated, the
overall health of the patient, the method route and dose of
administration and the severity of side effects (see, e.g.,
Maynard, et al., 1996, A Handbook of SOPs for Good Clinical
Practice, Interpharm Press, Boca Raton, FIa.; Dent, 2001, Good
Laboratory and Good Clinical Practice, Urch Publ, London, UK).
[0497] The route of administration may be by, e.g., topical or
cutaneous application, injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intracerebrospinal, intralesional, or by sustained
release systems or an implant (see, e.g., Sidman et al., 1983,
Biopolymers 22:547-556; Langer, et al., 1981, J. Biomed. Mater.
Res. 15: 167-277; Langer, 1982, Chem. Tech. 12:98-105; Epstein, et
al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al.,
1980, Proc. Natl. Acad. Sci. USA 77:4030-4034; U.S. Pat. Nos.
6,350,466 and 6,316,024). Where necessary, the composition may also
include a solubilizing agent and a local anesthetic such as
lidocaine to ease pain at the site of the injection. In addition,
pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309,
5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT
Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO
98/31346, and WO 99/66903, each of which is incorporated herein by
reference their entirety. In one embodiment, the anti-FXIa antibody
or antigen binding fragment thereof, or a composition of the
disclosure is administered using Alkermes AIR.TM. pulmonary drug
delivery technology (Alkermes, Inc., Cambridge, Mass.). In one
embodiment, the anti-idiotype antibody or antigen binding fragment
thereof, or a composition of the disclosure is administered using
Alkermes AIR.TM. pulmonary drug delivery technology (Alkermes,
Inc., Cambridge, Mass.).
[0498] A composition of the present disclosure may also be
administered via one or more routes of administration using one or
more of a variety of methods known in the art. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
Selected routes of administration for antibodies of the disclosure
include intravenous, intramuscular, intradermal, intraperitoneal,
subcutaneous, spinal or other parenteral routes of administration,
for example by injection or infusion. Parenteral administration may
represent modes of administration other than enteral and topical
administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion. Alternatively, a
composition of the disclosure can be administered via a
non-parenteral route, such as a topical, epidermal or mucosal route
of administration, for example, intranasally, orally, vaginally,
rectally, sublingually or topically.
[0499] If the anti-FXIa antibodies or antigen binding fragments
thereof of the disclosure, or anti-idiotype antibodies or antigen
binding fragments thereof of the disclosure, are administered in a
controlled release or sustained release system, a pump may be used
to achieve controlled or sustained release (see Langer, supra;
Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al.,
1980, Surgery 88:501; Saudek et al., 1989, N. Engl. J. Med.
321:514).
[0500] Polymeric materials can be used to achieve controlled or
sustained release of the therapies of the disclosure (see e.g.,
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, FIa. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,
Macromol. ScL Rev. Macromol. Chem. 23:61; see also Levy et al,
1985, Science 11 225:190; During et al., 19Z9, Ann. Neurol. 25:351;
Howard et al, 1989, J. Neurosurg. 71: 105); U.S. Pat. Nos.
5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT
Publication No. WO 99/15154; and PCT Publication No. WO 99/20253.
Examples of polymers used in sustained release formulations
include, but are not limited to, poly(2-hydroxy ethyl
methacrylate), poly(methyl methacrylate), poly(acrylic acid),
poly(ethylene-co-vinyl acetate), poly(methacrylic acid),
polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),
polyvinyl alcohol), polyacrylamide, polyethylene glycol),
polylactides (PLA), polyoeactide-co-glycolides) (PLGA), and
polyorthoesters. In one embodiment, the polymer used in a sustained
release formulation is inert, free of leachable impurities, stable
on storage, sterile, and biodegradable. A controlled or sustained
release system can be placed in proximity of the prophylactic or
therapeutic target, thus requiring only a fraction of the systemic
dose (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra, vol. 2, pp. 115-138 (1984)).
[0501] Controlled release systems are discussed in the review by
Langer, 1990, Science 249:1527-1533. Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more antibodies of the disclosure or
conjugates thereof. See, e.g., U.S. Pat. No. 4,526,938,
International Patent Publication Nos. WO 91/05548, WO 96/20698,
Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human
Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy
and Oncology 59:179-189, Song et al., 1995, "Antibody Mediated Lung
Targeting of Long-Circulating Emulsions," PDA Journal of
Pharmaceutical Science and Technology 50:372-397, Cleek et ah,
1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for
Cardiovascular Application," Pro. Ml. Symp. Control. Rel. Bioact.
Mater. 24:853-854, and Lam et al., 1997, "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Ml. Symp. Control Rel. Bioact. Mater. 24:759-160, each of
which is incorporated herein by reference in their entirety.
[0502] If the anti-FXIa antibody or antigen binding fragment
thereof of the disclosure, or the anti-idiotype antibody or antigen
binding fragment thereof of the disclosure, is administered
topically, it can be formulated in the form of an ointment, cream,
transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,
emulsion, or other form well-known to one of skill in the art. See,
e.g., Remington's Pharmaceutical Sciences and Introduction to
Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.
(1995). For non-sprayable topical dosage forms, viscous to
semi-solid or solid forms comprising a carrier or one or more
excipients compatible with topical application and having a dynamic
viscosity, in some instances, greater than water are typically
employed. Suitable formulations include, without limitation,
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, and the like, which are, if desired, sterilized
or mixed with auxiliary agents (e.g., preservatives, stabilizers,
wetting agents, buffers, or salts) for influencing various
properties, such as, for example, osmotic pressure. Other suitable
topical dosage forms include sprayable aerosol preparations wherein
the active ingredient, in some instances, in combination with a
solid or liquid inert carrier, is packaged in a mixture with a
pressurized volatile (e.g., a gaseous propellant, such as freon) or
in a squeeze bottle. Moisturizers or humectants can also be added
to pharmaceutical compositions and dosage forms if desired.
Examples of such additional ingredients are well-known in the
art.
[0503] If the composition comprising the anti-FXIa antibody or
antigen binding fragment thereof of the disclosure, or the
anti-idiotype antibody or antigen binding fragment thereof of the
disclosure, is administered intranasally, it can be formulated in
an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to
the present disclosure can be conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0504] Methods for co-administration or treatment with a second
therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic
agent, antibiotic, or radiation, are well known in the art (see,
e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10 th ed., McGraw-Hill, New
York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics
for Advanced Practice: A Practical Approach, Lippincott, Williams
and Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer
Chemotherapy and Biotherapy, Lippincott, Williams and Wilkins,
Phila., Pa.). An effective amount of therapeutic may decrease the
symptoms by at least 10 percent; by at least 20 percent; at least
about 30 percent; at least 40 percent, or at least 50 percent.
[0505] In certain embodiments, the anti-FXIa antibodies or antigen
binding fragments thereof of the disclosure, or the anti-idiotype
antibodies or antigen binding fragments thereof of the disclosure,
can be formulated to ensure proper distribution in vivo. For
example, the blood-brain barrier (BBB) excludes many highly
hydrophilic compounds. To ensure that the therapeutic compounds of
the disclosure cross the BBB (if desired), they can be formulated,
for example, in liposomes. For methods of manufacturing liposomes,
see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V. V. Ranade, 1989, J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al.,
Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G.
Bloeman et al., 1995, FEBSLett. 357: 140; M. Owais et al., 1995,
Antimicrob. Agents Chemother. 39: 180); surfactant protein A
receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); and
p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.
Keinanen; M. L. Laukkanen, 1994, FEBSLett. 346:123; Killion;
Fidler, 1994; Immunomethods 4:273.
[0506] The disclosure provides protocols for the administration of
pharmaceutical composition comprising anti-FXIa antibodies or
antigen binding fragments thereof of the disclosure, alone or in
combination with other therapies to a subject in need thereof. The
disclosure provides protocols for the administration of
pharmaceutical composition comprising anti-idiotype antibodies or
antigen binding fragments thereof of the disclosure, alone or in
combination with other therapies to a subject in need thereof. The
therapies (e.g., prophylactic or therapeutic agents) of the
combination therapies of the present disclosure can be administered
concomitantly or sequentially to a subject. The therapy (e.g.,
prophylactic or therapeutic agents) of the combination therapies of
the present disclosure can also be cyclically administered. Cycling
therapy involves the administration of a first therapy (e.g., a
first prophylactic or therapeutic agent) for a period of time,
followed by the administration of a second therapy (e.g., a second
prophylactic or therapeutic agent) for a period of time and
repeating this sequential administration, i.e., the cycle, in order
to reduce the development of resistance to one of the therapies
(e.g., agents) to avoid or reduce the side effects of one of the
therapies (e.g., agents), and/or to improve, the efficacy of the
therapies.
[0507] The therapies (e.g., prophylactic or therapeutic agents) of
the combination therapies of the disclosure can be administered to
a subject concurrently. The term "concurrently" is not limited to
the administration of therapies (e.g., prophylactic or therapeutic
agents) at exactly the same time, but rather it is meant that a
pharmaceutical composition comprising anti-FXIa antibodies or
antigen binding fragments thereof of the disclosure, or
anti-idiotype antibodies or antigen binding fragments thereof of
the disclosure, are administered to a subject in a sequence and
within a time interval such that the antibodies of the disclosure
or conjugates thereof can act together with the other therapy(ies)
to provide an increased benefit than if they were administered
otherwise. For example, each therapy may be administered to a
subject at the same time or sequentially in any order at different
points in time; however, if not administered at the same time, they
should be administered sufficiently close in time so as to provide
the desired therapeutic or prophylactic effect. Each therapy can be
administered to a subject separately, in any appropriate form and
by any suitable route. In various embodiments, the therapies (e.g.,
prophylactic or therapeutic agents) are administered to a subject
less than 15 minutes, less than 30 minutes, less than 1 hour apart,
at about 1 hour apart, at about 1 hour to about 2 hours apart, at
about 2 hours to about 3 hours apart, at about 3 hours to about 4
hours apart, at about 4 hours to about 5 hours apart, at about 5
hours to about 6 hours apart, at about 6 hours to about 7 hours
apart, at about 7 hours to about 8 hours apart, at about 8 hours to
about 9 hours apart, at about 9 hours to about 10 hours apart, at
about 10 hours to about 11 hours apart, at about 11 hours to about
12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or
1 week apart. In other embodiments, two or more therapies (e.g.,
prophylactic or therapeutic agents) are administered to a within
the same patient visit.
[0508] The prophylactic or therapeutic agents of the combination
therapies can be administered to a subject in the same
pharmaceutical composition. Alternatively, the prophylactic or
therapeutic agents of the combination therapies can be administered
concurrently to a subject in separate pharmaceutical compositions.
The prophylactic or therapeutic agents may be administered to a
subject by the same or different routes of administration.
IX. Kits
[0509] In another aspect, kits comprising any or all of the
antibodies described herein are provided. In some embodiments, kits
of the disclosure include one or more containers comprising an
anti-FXIa antibody described herein and instructions for use in
accordance with any of the methods of the disclosure described
herein. In some embodiments, kits of the disclosure include one or
more containers comprising an anti-idiotype antibody described
herein and instructions for use in accordance with any of the
methods of the disclosure described herein. In some embodiments,
kits of the disclosure include one or more containers comprising an
anti-FXIa antibody described herein and one or more containers
comprising an anti-idiotype antibody described herein and
instructions for use in accordance with any of the methods of the
disclosure described herein. Generally, these instructions comprise
a description of administration of the antibody for the above
described therapeutic treatments. In some embodiments, kits are
provided for producing a single-dose administration unit. In
certain embodiments, the kit can contain both a first container
having a dried protein and a second container having an aqueous
formulation. In certain embodiments, kits containing an applicator,
e.g., single and multi-chambered pre-filled syringes (e.g., liquid
syringes and lyosyringes), are included.
[0510] The instructions relating to the use of an anti-FXIa
antibody and/or anti-idiotype antibody generally include
information as to dosage, dosing schedule, and route of
administration for the intended treatment. The containers may be
unit doses, bulk packages (e.g., multi-dose packages) or sub-unit
doses. Instructions supplied in the kits of the disclosure are
typically written instructions on a label or package insert (e.g.,
a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable.
[0511] The kits of this disclosure are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an anti-FXIa antibody of the disclosure
or an anti-idiotype antibody of the disclosure. The container may
further comprise a second pharmaceutically active agent.
[0512] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container.
[0513] In some embodiments, a kit comprising one or more anti-FXIa
antibodies and/or anti-idiotype antibodies is used in a therapeutic
method as described herein (e.g., in Section V above). In some
embodiments, a kit comprising an anti-FXIa antibody and/or an
anti-idiotype antibody against an anti-FXIa antibody as described
herein is used in a method for inhibiting the intrinsic pathway of
coagulation in a subject. In some embodiments, a kit comprising an
anti-FXIa antibody and/or an anti-idiotype antibody against an
anti-FXIa antibody as described herein is used in a method for
increasing clotting time in a subject.
[0514] The disclosure also provides diagnostic kits comprising any
or all of the antibodies described herein. The diagnostic kits
comprising anti-FXIa antibodies are useful for, for example,
detecting the presence of FXIa in a sample. In some embodiments, a
diagnostic kit can be used to identify an individual with a latent
disease, disorder or condition that may put them at risk of
developing FXIa-mediated disease, disorder or condition. In some
embodiments, a diagnostic kit can be used to detect the presence
and/or level of FXIa in an individual suspected of having a FXIa
mediated disease. The diagnostic kits comprising anti-idiotype
antibodies are useful for, for example, detecting the presence of
an anti-FXIa antibody in a sample. In some embodiments, a
diagnostic kit can be used to identify an individual with who is at
risk for bleeding disorders. In some embodiments, a diagnostic kit
can be used to detect the presence and/or level of anti-FXIa
antibody in an individual being administered the anti-FXIa
antibody.
[0515] Diagnostic kits of the disclosure include one or more
containers comprising an anti-FXIa antibody described herein and
instructions for use in accordance with any of the methods of the
disclosure described herein. Generally, these instructions comprise
a description of use of the anti-FXIa antibody to detect the
presence of FXIa in individuals at risk for, or suspected of
having, an FXIa mediated disease. In some embodiments, an exemplary
diagnostic kit can be configured to contain reagents such as, for
example, an anti-FXIa antibody, a negative control sample, a
positive control sample, and directions for using the kit.
[0516] In some embodiments, diagnostic kits of the disclosure
include one or more containers comprising an anti-idiotype antibody
described herein and instructions for use in accordance with any of
the methods of the disclosure described herein. Generally, these
instructions comprise a description of use of the anti-idiotype
antibody to detect the presence of an anti-FXIa antibody in
individuals at risk for developing a bleeding disorder. In some
embodiments, an exemplary diagnostic kit can be configured to
contain reagents such as, for example, an anti-idiotype antibody, a
negative control sample, a positive control sample, and directions
for using the kit.
X. Examples
[0517] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
Example 1. Production and Selection of Anti-FXIa mAbs
[0518] Anti-FXIa scFvs were selected from an antibody phage display
library. The antigen used to screen the library, human FXIa
(Haematologic Technologies Inc.), was biotinylated with
Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer's
protocol. This biotinylated FXIa was immobilized on
streptavidin-coated magnetic Dynabeads M-280 (Invitrogen) and used
to select binders from a scFv antibody phage display library, using
standard methods. Four rounds of selection were performed with
decreasing concentrations of the target (FXIa) as follows, 150 nM
(1.sup.st round), 75 nM (2.sup.nd round), 30 nM (3.sup.rd round)
and 5 nM (4.sup.th round). To obtain antibodies specific to FXIa
that did not substantially bind the zymogen, all selections were
performed in the presence of 300 nM human FXI. A total of 6000
clones were screened by FXI/FXIa ELISA from the 3.sup.rd and
4.sup.th round outputs, resulting in 166 FXIa specific hits that
exhibited binding to FXIa, but did not detectably bind FXI.
[0519] After sequencing, 13 unique clones specific to human factor
XIa were identified. These clones bound FXIa and also inhibited
FXIa in an in vitro activity assay. After testing these scFv clones
for cross-reactivity to cynomolgus monkey ("cyno") FXIa by ELISA,
11 clones moved forward. After reformatting, 7 reformatted IgGs
retained binding selectivity to FXIa and exhibited cyno
cross-reactivity. In this ELISA binding assay, a series of wells
were coated with either 1 .mu.g of FXIa or 1 .mu.g of FXI. After
the necessary incubation time, the wells were washed, blocked, and
then the anti-FXIa mAbs were added at various concentrations. After
a series of washes, an anti-human IgG HRP secondary (Southern
Biotech) was added for the standard incubation period, followed by
additional washes, addition of developing solutions, with the
specific binding signal then measured on a spectrophotometer at 450
nM OD. To measure the inhibitory activity of the mAbs against FXIa,
an in vitro assay was run that involved a 5 minute pre-incubation
of the mAbs at various concentrations with either 200 pM human FXI
or 200 pM cyno FXIa in the standard assay buffer (50 mM Tris-Hcl,
pH 7.4, 250 mM NaCl, 1 mM EDTA). This was followed by addition of
100 .mu.M of a fluorogenic peptide (SN-59, Haematological
Technologies) to start the reaction. The plate was then read in a
SpectraMax.RTM. plate reader at 37.degree. C. for 30 minutes.
Settings: excitation 353 nM, emission 470 nM. Data was collected in
one minute intervals. Instrument determined V max values taken from
the linear part of each reaction curve were then plotted for the
determination of IC.sub.50 values.
Results
[0520] After reformatting to IgG, seven out of the original eleven
positive scFv clones retained binding selectivity to human (FIG.
1A) and cyno FXIa (FIG. 1B). These seven anti-FXIa mAbs also
inhibit human FXIa (FIG. 1C) and cyno FXIa (FIG. 1D) activity with
a range of IC.sub.50 values in an in vitro assay involving a
fluorogenic peptide substrate that is cleaved by FXIa.
Example 2. Binding of Anti-FXIa Clones to FXIa/D4 mAb Complexes
[0521] Antibody epitope binning data was collected using an Octet
QK384 instrument (ForteBio). Biotinylated blood-derived FXIa
(Haematologic Technologies Inc.) was diluted in phosphate-buffered
saline with 0.1% bovine serum albumin (PBS-BSA) and loaded onto
streptavidin-conjugated Octet.RTM. biosensors (ForteBio).
Biosensors were then washed in PBS to remove unbound FXIa and
loaded to saturation with 500 nM D4 IgG in PBS-BSA. Each sensor was
subsequently dipped into a second antibody (500 nM IgG in PBS-BSA)
including the commercially available mouse anti-FXI clone AHXI-5061
(Haematologic Technologies Inc.) to assess if concurrent binding to
the FXIa-D4 complex is possible. Data is reported as the change in
response (nm) for the second antibody clone binding to the FXIa-D4
complex.
Results
[0522] All 7 anti-FXIa mAbs tested bind the same or an overlapping
epitope on FXIa, as indicated by the lack of increased response
signal when D4 anti-FXIa mAb is bound first (FIG. 2). Binding
signal with mouse anti-FXI clone AHXI-5061, which binds a different
FXI/FXIa epitope, illustrates an increased signal seen when an
additional IgG binding event occurs at the same time that D4 mAb is
bound.
Example 3. Generating Improved Versions of the D4 Anti-FXIa mAb
[0523] Error prone PCR (ep-PCR) based random mutagenesis was
performed on the D4 scFv gene in order to optimize this antibody.
The amplified D4 scFv DNA from ep-PCR was cloned into a proprietary
parental vector and generated a 2e10 scFv phage library. After
rescuing the library, 3 rounds of selection were performed. In the
1.sup.st round, 900 pM of human FXIa (hu-FXIa) on streptavidin
magnetic beads was used to capture binding phage for 1 hour at room
temperature. In the 2.sup.nd round, 90 pM of antigen was reacted
with output phage from the 1.sup.st round in solution for 1 hour
followed by streptavidin magnetic beads capture. In the final
round, 9 pM of hu-FXIa on streptavidin magnetic beads was reacted
with output phage from the 2.sup.nd round for 5 minutes and the
washed beads were incubated with soluble FXIa overnight. A total of
500 colonies from the 3.sup.rd round were picked and tested in the
following assays: 1) direct binding ELISA, 2) competition ELISA in
the presence of excess parental D4 scFv and 3) Homogeneous
Time-Resolved Fluorescence (HTRF) assay in the scFv format. Of the
total clones screened, 367 were ELISA positive and 87 clones were
identified as likely higher affinity clones than parental D4 based
on competition ELISA and HTRF assay using D4 scFv as the
competitor. Of these 87 clones, 70 clones were identified as unique
and all of these unique clones were reformatted into full length
human IgG followed by HTRF assay in order to assess its affinity by
competition with D4 IgG. All 70 clones were also tested as
inhibitors in the human FXIa fluorogenic peptide assay. Nine clones
(QCA11, B1D2, B10H2, B10E6, B10F6, B10D8, B10B12, S1D4 and S10H9)
were selected based on their HTRF assay result, ELISA signal and
their IC.sub.50 in the fluorogenic peptide assay. Sequence
alignment of these 9 clones identified several position hot spots:
W50R, N52D, N54D and G56D in the heavy chain CDR2 and Q6K in light
chain framework 1. These hot spots were then used to generate 32
IgGs (clone 1 through clone 32 including clone 8, 16, 20, 22, 24
and 32 as shown in the amino acid alignment table) having different
combinations of these 5 mutations, followed by ELISA, Biacore.TM.
and in vitro FXIa activity assays run as previously described.
Clone 24 containing N51D, N53D and G55D mutations in heavy chain
CDR2 and Q6K in light chain framework 1 was the IgG with the
highest affinity of about 10 pM. Clone 24 had a possible
deamination site in the CDRH2, thus 54S was changed to 54E and this
new clone was named "DEF." DEF showed the same affinity and potency
in in vitro assays as clone 24.
Results
[0524] Increased binding affinity and potency was achieved via
selective substitutions in original D4 anti-FXIa mAb. Affinity for
human FXIa increases as follows: clone 24>B11>D4 mAb (FIG.
3A). Anti-FXIa potency against the human (FIG. 3B), cyno (FIG. 3C),
and rabbit (FIG. 3D) FXIa enzymes increases with a similar trend
seen across all three species tested: DEF=24F=clone 24>>D4
mAb. Clone 24F is an effector null version of clone 24 that differs
from Clone 24 at 3 residues in the Fc region (SEQ ID NO: 82).
[0525] A human DNA Insert encoding the IgG heavy chain of DEF was
deposited under ATCC accession number PTA-122090. A human DNA
Insert encoding the IgG light chain of DEF was deposited under ATCC
accession number PTA-122091. The deposits were made under terms in
accordance with the Budapest Treaty with the American Type Culture
Collection (ATCC), 10801 University Blvd., Manassas, Va.
20110-2209.
Example 4. Anti-FXIa mAb Binding Kinetics
[0526] Biotinylated FXIa was captured on a streptavidin-coated
Biacore.TM. chip and the binding response versus time for D4 IgG,
B11 IgG, C24 Fab and DEF Fab measured over a series of antibody/Fab
concentrations. Representative background subtracted Biacore.TM.
sensorgrams overlaid with the kinetic curve fits are shown. Basic
methods in brief: blood-derived FXIa and FXI (Haematologic
Technologies Inc.) were biotin labeled via primary amines and
immobilized on a CAP chip using a Biacore.TM. T200 instrument (GE
Healthcare). IgG binding experiments were performed at 25.degree.
C. using a 50 .mu.l/min flow rate in 0.01 M HEPES pH 7.4, 0.15 M
NaCl and 0.005% v/v surfactant P20 (HBS-P) buffer. After each
antibody injection, the chip surface was regenerated with a mixture
of 6 M guanidine HCl and 0.25 M NaOH, and new FXIa/FXI was
captured. Fab binding experiments were performed at 37.degree. C.
using a 50 .mu.l/min flow rate in HBS-P. All data was analyzed
using the Biacore.TM. T200 Evaluation software. Kinetic constants
for at least three experiments were obtained and reported as the
mean. The table of values for affinity measurements for IgGs and
Fabs shows overall affinity of Fab or IgG for human FXIa target, as
well as rate constants (FIG. 4B). No binding was seen with
immobilized human FXI in similar experiments (data not shown)
consistent with FXIA/FXI ELISA data (see Example 2).
Results
[0527] Significant increases in overall affinity to human FXIa were
achieved by making selected amino acid substitutions to original D4
mAb molecule. Greater than 25-fold affinity (KD) increase was seen
with 24 and DEF Fabs over that of original D4 mAb (FIG. 4A and FIG.
4B).
Example 5. Anti-FXIa mAbs do not Inhibit Other Serine Proteases on
the Coagulation Cascade
[0528] The effects of anti-FXIa mAbs DEF and 24F on serine
proteases of the coagulation cascade were tested in an assay as
shown in FIGS. 5A-B. The following standard conditions were used
for all reactions regardless of enzyme tested: 50 .mu.l diluted
enzyme (as indicated below), 8 .mu.l SN59 peptide (final reaction
concentration is 100 .mu.M), 92 .mu.l Standard Assay buffer, 50
.mu.l test IgG (DEF or clone 24F) or buffer (no IgG wells). Final
IgG concentration of first dilution is 25.6 .mu.g/ml, seven 3-fold
serial dilutions were tested). Set up and order of addition:
Pre-incubate the DEF or 24 IgG at concentrations shown (in FIG. 5A
and FIG. 5B) for 5 minutes with the various enzymes in standard
assay buffer prior to the addition of the SN-59 peptide substrate
which starts the reaction. The plate was then read in a fluorescent
SpectraMax.RTM. plate reader at 37.degree. C. for 30 minutes.
Settings: excitation 353 nM, emission 470 nM. Data was collected in
one minute intervals. Instrument determined V max values taken from
the linear part of each reaction curve were then plotted as shown.
Hematological Technologies Inc. (HTI) was the supplier for majority
of human enzymes used in this screen. Final enzyme concentrations
were as follows: FXa (HCXA-0060, HTI) was 2 .mu.g/ml. Thrombin
(HCT-0020, HTI) was 5 .mu.g/ml. FVIIa (HCVIIA-0031, HTI) was 5
.mu.g/ml with Tissue Factor (RTF-0300, HTI) was 0.5 .mu.g/ml and
added phospholipid (PC) at 12 .mu.M. FXIIa (HFXIIa1212a, Enzyme
Research) was 0.9 .mu.g/ml. FXIa (HCXIA-0160, HTI) was 0.2
.mu.g/ml. APC (HCAPC-0080, HTI) was 1.2 .mu.g/ml. Kallikrein-1
(KLK1) (JNV-367, Reagent Protein) was 2.5 .mu.g/ml. All enzymes
were of human origin. As shown in FIG. 5A-B, anti-FXIa mAbs did not
inhibit other serine proteases on the coagulation cascade or other
related proteases tested. V max data plots show the high
selectivity of anti-FXIa mAb DEF (FIG. 5A) and clone 24F (FIG. 5B)
for inhibition of FXIa only in these in vitro assay
comparisons.
[0529] The effects of anti-FXIa mAbs DEF and clone 24 on serine
proteases of the coagulation cascade were tested in an assay as
shown in FIGS. 5C-D. The following standard conditions were used
for all reactions regardless of enzyme tested: 50 .mu.l diluted
enzyme (as indicated below), 8 .mu.l fluorogenic peptide (final
reaction concentration is 100 .mu.M), 92 .mu.l Standard Assay
buffer, 50 .mu.l test IgG (DEF or clone 24) or buffer (no IgG
wells). Hematological Technologies Inc. (HTI) was the supplier of
human enzymes and fluorogenic substrates used in this screen,
unless otherwise indicated. Final IgG concentration of first
dilution is 25.6 .mu.g/ml, with seven 3-fold serial dilutions
tested. Set up and order of addition: Pre-incubate the DEF or clone
24 IgG at concentrations shown (in FIG. 5A and FIG. 5B) for 5
minutes with the various enzymes in standard assay buffer. The
following fluorogenic substrates were then added at a final
concentration of 100 .mu.M to start the reaction: SN-59 for FXIIa,
FXIa, APC and PK; SN-17A for Thrombin and FVIIa; and SN-7 for FXa.
The plate was then read in a fluorescent SpectraMax.RTM. plate
reader at 37.degree. C. for 30 minutes. Settings: excitation 353
nM, emission 470 nM. Data was collected in one minute intervals.
Instrument determined V max values taken from the linear part of
each reaction curve were then plotted as shown. Final enzyme
concentrations were as follows: FXa (HCXA-0060, HTI) was 2
.mu.g/ml. Thrombin (HCT-0020, HTI) was 5 .mu.g/ml. FVIIa
(HCVIIA-0031, HTI) was 5 .mu.g/ml with Tissue Factor (RTF-0300,
HTI) was 0.5 .mu.g/ml and added phospholipid (PC) at 12 .mu.M.
FXIIa (HFXIIa1212a, Enzyme Research) was 0.9 .mu.g/ml. FXIa
(HCXIA-0160, HTI) was 0.2 .mu.g/ml. APC (HCAPC-0080) at 1.2
.mu.g/ml; and plasma kallikrein (PK; KLKB1; 2497-SE, R&D
Systems) at 4 .mu.g/ml. The latter was purchased as zymogen and
activated with thermolysin according to R&D's instructions.
Thermolysin alone had no detectable activity in the SN-59
hydrolysis assay. Due to low efficiency of small peptide substrate
cleavage by FIXa, the activity against FIXa was assessed by using a
coupled assay involving FIXa activation of FX in the presence of
FVIIIa and phospholipid and the FXa chromogenic substrate SXa-11
(Biophen FIXa, Ref A221812, Aniara; used according to supplier
protocol). All enzymes were of human origin. As shown in FIG. 5C-D,
anti-FXIa mAbs did not inhibit other serine proteases on the
coagulation cascade or other related proteases tested. V max data
plots show the high selectivity of anti-FXIa mAb DEF (FIG. 5C) and
clone 24 (FIG. 5D) for inhibition of FXIa only in these in vitro
assay comparisons.
Example 6. Effects of Anti FXIa mAbs in FXIIa-Triggered Human
Plasma Reactions that Measure Thrombin Generation Readout
[0530] Thrombin generation was measured using a fluorogenic
thrombin substrate on a multi-well automated fluorescent plate
reader (ThrombinoSCOPE, Maastricht, the Netherlands) according to
the manufacturer's protocol. Briefly, 5 .mu.L of anti-FXIa antibody
or IgG ctrl (from 5 .mu.g/mL to 443 .mu.g/mL of D4, B11, 24, 32,
and IgG1 ctrl) was mixed with 20 .mu.L PBS-60 nM human Factor XIIa
(Enzyme Research Laboratories, South Bend, Ind., USA)-PC/PS
(Phospholipid-TGT, DiaPharma, West Chester, Ohio, USA) in a 96-well
plate. Finally, 75 .mu.L human Plasma (Triclinical Reference
Plasma, TCoag, Wicklow, Ireland) was added. Due to lot to lot
variability for the PC/PS reagent, for each lot the concentration
was adjusted to achieve a .about.10 min Lag Time and .about.125 nM
Thrombin Peak. Finally, clotting was triggered with the addition of
calcium chloride buffer and a fluorogenic thrombin substrate. The
amount of thrombin generated in the reaction was measured over time
and plotted as to peak activity, and time to peak.
Results
[0531] Anti-FXIa mAbs inhibit FXIa in the more complex setting of
human plasma. In this assay, FXIIa was used to trigger the start of
the coagulation process, and readout was at the level of thrombin
generation, which was the final activation step on the coagulation
cascade. Higher affinity clones (as previously determined by ELISA
and Biacore.TM. measurements and shown above in FIGS. 2 and 3) were
more potent in this assay (Clone 24>B11>D4; FIGS. 6A, 6B, 6C,
6E and 6F). Potency was measured by decreases in peak thrombin
activity (FIG. 6E) and in delays (lag time) to peak thrombin
activity (FIG. 6F). These results indicate that anti-FXIa mAbs are
active against FXIa in human plasma, even when, under these
conditions, it is being continuously generated by FXIIa-mediated
conversion of FXI to FXIa. The ctrl IgG has no effect in this assay
(FIGS. 6D, 6E and 6F).
Example 7. Single Dose i.v. Bolus Pharmacokinetic (PK) Study with
DEF in New Zealand White Rabbits
[0532] All procedures performed on these animals were in accordance
with regulations and established guidelines and were reviewed and
approved by an Institutional Animal Care and Use Committee or
through an ethical review process. Three animals in each dosing
group received an intravenous (i.v) bolus injection of either 10,
1, 0.1 or 0.03 mg/kg of the DEF IgG, or with 1 or 0.1 mg/kg of the
Ctrl IgG. The following sampling times of 0.02, 1, 2, 4, 8 24, 48
120 and 168 hours were used to collect 0.5 ml of blood, half of
which was processed to serum (for PK determination) and half for
plasma (for pharmacodynamics (PD) markers). Serum IgG levels were
determined by ELISA using standard protocols for detecting human
IgG. The concentration of DEF measured in serum for all doses and
times was plotted to evaluate the PK.
Results
[0533] DEF exhibits normal human IgG PK in rabbits, with typical
exposure seen at all concentrations tested over time (FIG. 7).
Values were near to, or identical with, that measured with the
negative control IgG (data not shown).
Example 8. Injection of Different Doses of DEF in New Zealand White
Rabbits Reveals Concentration Dependent and Selective Prolongation
of APTT Clotting Times with No Effect on PT Clotting Times
[0534] Three animals in each dosing group received an i.v bolus
injection of either 10, 1, 0.1 or 0.03 mg/kg DEF IgG, or with 1 or
0.1 mg/kg Ctrl IgG. The following sampling times of 0.02, 1, 2, 4,
8, 24, 48, 120 and 168 hours were used to collect 0.5 ml of blood,
half of which was processed to serum (for PK determination) and
half for plasma (for PD markers). Serum samples were analyzed for
DEF concentration (plotted on X-axis in mg/L) and plasma samples
(pre-dose, 30 min, 24 hr, 7 d and 14 d post-dose) were analyzed for
drug effects on time to clotting (plotted on the Y-axis in seconds)
for both the APTT and the PT coagulation assays (FIG. 8). The PT
and APTT assays were run on the plasma by CRO (Covance) using
standard assay formats. The PK was determined on serum by standard
ELISA format.
Results
[0535] Rabbit PK/PD (APTT, PT) summary data shows that DEF IgG
prolongs time to clotting in a dose dependent fashion in the APTT
coagulation assay, but has no effect on clotting time in the PT
coagulation assay (FIG. 8). Negative control IgG had no effect on
either APTT or PT readouts over time or dose tested (data not
shown).
Example 9. Effects of Anti-FXIa mAb DEF in Rabbit Model of Venous
Thromboembolism (VTE) with Effects Monitored at Level of Thrombus
Formation (Thrombus Weight) and at Level of Plasma PD Readout (APTT
and PT Assays to Measure Clotting Time Effects)
[0536] All procedures performed on these animals were in accordance
with regulations and established guidelines and were reviewed and
approved by an Institutional Animal Care and Use Committee or
through an ethical review process. Rabbits were anesthetized
according to established protocols. Six animals in each dosing
group received an i.v bolus injection of either 10, 3, 1, 0.1 or
0.03 mg/kg of DEF IgG, 10 mg/kg of the negative control IgG, 1.5
mg/kg Rivaroxaban, 0.045 mg/kg Rivaroxaban, or a 10% DMA/30%
PEG400/60% Water vehicle control. Furthermore, for the Rivaroxaban
treated animals, the i.v. bolus injection was supplemented by a
continuous i.v. infusion of Rivaroxaban to maintain proper dosing
throughout the duration of the study due to short half-life
concerns. For all treatment groups, 30 minutes after bolus
injection, a sheath of the appropriate size was placed in the left
femoral vein. Then a wire of approximately 14 cm with 8 strands of
cotton thread attached to it (each 3 cm long) was inserted into the
femoral vein. Fluoroscopy was used for guidance of the wire with
the threads towards the target area in the vena cava. The device
remained in the inferior vena cava for 90 minutes during which
clots formed on the cotton threads. After 90 minutes the device was
removed by surgical dissection, cut from the wire, and the
clot-containing cotton threads were weighed after having been
blotted dry. Prior to IgG dosing, and also prior to surgical
removal of threads, a blood sample was drawn for preparation of
serum (PK) and plasma (for PD biomarkers APTT and PT). Data is
plotted to show dose-dependent effects of DEF on clot weight
reduction, APTT prolongation, and PT prolongation.
Results
[0537] The anti-FXIa mAb DEF dose dependently decreased
thrombus/clot weight (FIG. 9A), dose dependently prolonged time to
clotting in the APTT assay (FIG. 9B), but had no effect at any dose
on clotting times measured in the PT assay of coagulation (FIG.
9C). In contrast, the 1.5 mg/kg dose of Rivaroxaban decreased
thrombus/clot weight (FIG. 9D), but prolonged time to clotting in
both the APTT (FIG. 9E) and PT (FIG. 9F) assays of coagulation. DEF
treatment is thus distinguished by having no effects on PT values,
but can confer equivalent thrombus/clot weight reduction to that
seen with Rivaroxaban at the 1.5 mg/kg dose. No effects were seen
for the 0.045 mg/kg Rivaroxaban dose on any read outs measured.
Both the IgG control (FIGS. 9A, 9B and 9C) and the DMA/PEG400/water
vehicle control for rivaroxaban (FIGS. 9D, 9E and 9F) had no
effects on any readouts measured (all panels).
Example 10. Rabbit Cuticle Bleeding Study Comparing Effects of DEF
mAb to Rivaroxaban and Controls
[0538] All procedures performed on these animals were in accordance
with regulations and established guidelines and were reviewed and
approved by an Institutional Animal Care and Use Committee or
through an ethical review process. Rabbits were anesthetized
according to established protocols. After anesthesia and
cannulation etc., both front paws were shaved to remove fur that
would otherwise contaminate saline and wick it out of the tube. Pre
dosing bleeds were performed on the middle digit of the left front
paw. Post dosing bleeds were performed on the same digit on the
right front paw. There were four dosing groups of 10 animals each.
Ten animals in each dosing group received either an i.v bolus
injection of either 10 mg/kg DEF IgG, 10 mg/kg control IgG, 1.5
mg/kg of Rivaroxaban, or a 10% DMA/30% PEG400/60% water vehicle
control. Furthermore, for the Rivaroxaban treated animals, the i.v.
bolus injection was supplemented by a continuous i.v. infusion of
Rivaroxaban to maintain proper dosing throughout the duration of
the study due to short half-life concerns.
[0539] Nails were transilluminated with white light to visualize
the quick and cut with a razor blade so as to transect the cuticle
approximately 1 mm proximal to the apex of the quick. The nail was
then immediately immersed in a saline solution in 10.times.75 mm 3
ml polystyrene (clear) tube. The tube with saline was stored at
37.degree. C. before the cut. The nail was kept in the solution and
time to cessation of flow was measured. If bleeding did not stop,
the procedure was stopped at 20 min and logged as >20 min. At
the end of the blood collection the tube is capped and inverted 5
times and centrifuged at 200 to 250.times.g for 15 min. The blood
cell pellet was resuspended in 3 mL erythrocyte lysis buffer (8.3
g/L NH4Cl, 1.0 g/LKHCO3, and 0.037 g/L EDTA in water). After at
least 15 min of lysis time, hemoglobin (Hb) concentration was
measured at OD 575 nM using a Spectrophotometer) and expressed as
arbitrary absorbance units.
[0540] As indicated above, baseline cuticle bleeding times were
performed prior to dosing. After cessation of bleeding, animals
were then dosed i.v. at t=0 min with DEF IgG, inactive control IgG,
Rivaroxaban, or vehicle. 30 minutes later a second cuticle cut was
made and bleeding times recorded and blood loss measured. In
addition, blood samples obtained before dosing and at the end of
the study were used for serum PK determinations of Ab and
Rivaroxaban levels and plasma was used to determine PD markers (via
standard APTT and PT coagulation assays). Data is plotted to show
effects of dosing with DEF IgG, ctrl IgG, Rivaroxaban, and vehicle
on total blood loss, APTT prolongation, and PT prolongation.
Results
[0541] The anti-FXIa mAb DEF at 10 mg/kg had no effect on total
blood loss following cuticle cutting (FIG. 10A), induced a
significant prolongation in time to clotting as measured in the
APTT coagulation assay (FIG. 10B), but had no effect on time to
clotting as measured in the PT assay of coagulation (FIG. 10C). In
contrast, Rivaroxaban at 1.5 mg/kg significantly increased total
blood loss following cuticle cutting (FIG. 10A), and induced a
significant prolongation in time to clotting in the both the APTT
(FIG. 10B) and PT (FIG. 10C) coagulation assays. Both the IgG
control and the DMA/PEG400/water vehicle control had no effects on
any readouts measured (FIGS. 10A-10C). Combining the results from
FIG. 8 and FIG. 9, we conclude that the DEF IgG can reduce
thrombus/clot weight (anti-thrombotic effect) while at the same
time have little or no effect on injury-induced blood loss
(hemostatic effect) as measured in these rabbit studies.
Example 11. Anti-FXIa mAb has an Increased Off-Rate when Bound to
FXIa Inhibited with PMSF
[0542] Biotinylated FXIa was incubated with 1 mM PMSF prior to
capture on a streptavidin-coated Biacore.TM. chip. The binding of
100 nM DEF IgG or H04 IgG (e.g., as described in WO 2013/167669 A1)
was observed over 120 sec followed by 120 sec dissociation phase.
Blood-derived FXIa (Haematologic Technologies Inc.) was biotin
labeled via primary amines and immobilized on a CAP chip using a
Biacore.TM. T200 instrument (GE Healthcare). The binding experiment
was performed at 25.degree. C. using a 50 .mu.l/min flow rate in
0.01 M HEPES pH 7.4, 0.15 M NaCl and 0.005% v/v surfactant P20
(HBS-P) buffer. After the antibody injection the chip surface was
regenerated with a mixture of 6 M guanidine HCl and 0.25 M NaOH,
and new FXIa captured. The data was background subtracted using the
signal from the adjacent streptavidin coupled flow cell and
buffer-only injections over the FXIa surface using Biacore.TM. T200
Evaluation software (GE Healthcare).
Results
[0543] A significant increase in the DEF mAb dissociation rate but
not that of the anti-FXIa H04 mAb (described in WO 2013/167669,
incorporated herein by reference) was observed when the FXIa was
pre-bound to the serine protease inhibitor PMSF compared to FXIa
alone (FIG. 11). PMSF covalently binds to the active site serine,
irreversibly inhibiting the protease. The ability of PMSF to
partially disrupt DEF mAb binding to FXIa indicates that the
binding epitope overlaps with the catalytic serine and/or adjacent
residues within the active site cleft. This was not observed for
the H04 mAb, which binds to both FXIa and FXIa-PMSF with similar
kinetics, suggesting that the H04 mAb binding epitope is distinct
from that of the DEF mAb.
Example 12. Multiple Dose PK Study with DEF in Cynomolgus
Monkeys
[0544] As part of an exploratory toxicology and PK study in
cynomolgus monkeys, two animals (one female, one male) in each dose
group were dosed with either 20, 75, or 266.5 mg/kg DEF i.v. or
266.4 mg/kg DEF s.c. on days 1, 8 and 15 with necropsy occurring on
day 15. Daily observations were made over the course of this study,
and no test article-related mortality, clinical signs, effects on
body weight or food consumption, hematology or clinical chemistry
parameters, or microscopic observations were observed.
Additionally, over the course of this study multiple blood samples
were drawn for preparation of serum (PK) and plasma (PD makers).
Serum IgG levels were determined by ELISA using standard protocols
for detecting human IgG. Sampling times were at 0.08, 6, 24, 168,
168.08, 174, 192, 240, 280, 288 and 336 hours. The concentration of
DEF measured in serum for all doses and times was plotted to
evaluate the PK.
Results
[0545] DEF exhibits normal human IgG pharmacokinetics (PK) in
cynomolgus monkeys, with typical exposure seen at all
concentrations tested over time (FIG. 12). Antibody accumulation
was observed after day 8 in all DEF dose groups. The exposure
ratios of Day 8 vs Day 1 ranged from 1.36.times.-1.64.times.. Given
the apparent typical antibody pharmacokinetics observed in this
study, ADA measurements were not performed. Due to the small sample
size, comparisons of DEF antibody exposure between males and
females were not performed. Following i.v. administration of DEF,
systemic exposures (assessed by C.sub.max and AUC) on Day 1 and Day
8 increased proportionally as dose increased from 20 to 266.5
mg/kg. Comparison of AUC.sub.(0-168h) in the i.v. and s.c. 266.5
mg/kg dose groups shows that subcutaneously administered DEF is 61%
bioavailable, and reached T.sub.max 72 hours after the first dose.
Overall exposure after subcutaneous dosing, expressed as
AUC.sub.(0-168h), was 74% of that seen via the intravenous route.
Due to uncertainties arising from the short study duration and
dosing regimen, antibody half-life was not calculated.
Example 13. Prolongation of APTT, but not PT, Coagulation Times
Seen in 15 Day Study of Cynomolgus Monkeys Treated with High Doses
of DEF IgG
[0546] As part of an exploratory toxicology and PK study in
cynomolgus monkeys, two animals (one female, one male) in each
group were dosed with either 20, 75, or 266.5 mk/kg DEF i.v or
266.4 mg/kg DEF s.c. on days 1, 8 and 15 with necropsy occurring on
day 16. Daily observations were made over the course of this study,
and no test article-related mortality, clinical signs, effects on
body weight or food consumption, hematology or clinical chemistry
parameters, or microscopic observations were observed.
Additionally, over the course of this study, multiple blood samples
were drawn for plasma preparation and assessment of coagulation
parameters. These samples were collected on Day -9 and then just
before dosing and 1 h after dosing on days 1, 8, and 15, and then
just prior to necropsy on Day 16. The Day -9 and just prior to
dosing on Day 1 are used to determine a base line value. Plasma
samples were analyzed for drug effects on time to clotting (plotted
on the Y-axis in seconds) in both the APTT and the PT coagulation
assays. The APTT and PT assays were performed using standard assay
formats.
Results
[0547] DEF IgG prolongs clotting time in APTT coagulation assay
(FIG. 13A) but has no effects on clotting time in PT coagulation
assay (FIG. 13B) in cynomolgus monkeys treated with high doses of
DEF IgG over course of 15 days. Even at the high concentrations of
DEF IgG dosing used in this toxicology study, the only evident sign
of treatment in these animals was the expected pharmacological
change of APTT prolongation. This result is significant as it has
previously been shown in earlier rabbit studies (see, e.g., as
described in Examples 8-9) that APTT prolongation was associated
with anti-thrombotic protection but not with increased bleeding
risk. Rather, PT prolongation was associated with bleeding risk.
Thus, the essential mechanism of DEF action (intrinsic pathway
inhibition reflected as selective APTT prolongation) is the same in
rabbits and in this non-human primate model, and thus likely to
translate to humans as well.
Example 14. Identification of an Anti-Idiotype Antibody to DEF
[0548] The antigen DEF is an IgG1 human antibody specific for FXIa.
DEF was chemically biotinylated using biotin-LC-NHS (Pierce; Cat.
NO: 21347), according to the manufacturer's protocol. This
biotinylated DEF was immobilized on streptavidin-coated magnetic
Dynabeads M-280 (Invitrogen, Cat. No: 11206D) and used to select
binders from a scFv antibody phage display library (WyHN5 kappa and
lambda), using standard methods. Prior to each round of selection,
phage antibody library was absorbed to streptavidin-coated
Dynabeads M-280 in order to deplete streptavidin binders. The phage
selection was performed in the presence of 500 nM of human serum
IgG, using decreasing concentrations of the antigen (DEF) and
increasing number of washes with PBS containing 0.1% Tween20
(PBST), as follows: 1.sup.st round 100 nM/5.times.PBST/2.times.PBS,
2.sup.nd round 10 nM/10.times.PBST/5.times.PBS and 3.sup.rd round 1
nM/15.times.PBST/5.times.PBS. A total of 3,000 clones were picked
from the 3.sup.rd round output colonies and tested in ELISA assay
for DEF, Human-IgG and streptavidin binding. This selection and
screening gave a single specific anti-DEF hit. After sequencing,
the unique clone (C4) specific to DEF was reformatted into a fully
human IgG1. In order to evaluate the specificity of full length C4
IgG against DEF, ELISA assay was performed as follows. 1 .mu.g/ml
of biotinylated control antibody, human serum IgG or DEF were added
on to ELISA plate on which 10 mg/ml of streptavidin was previously
coated overnight. After blocking and washing, 100 ng/ml of C4 IgG
was added to each well and incubated at room temperature for 1
hour. After washing and treating with anti-human IgG-HRPO, enzyme
substrate (TMB) was added to develop the color. The signal was read
at 450 nm after stopping the reaction by adding 0.16M sulfuric
acid.
Results
[0549] The reformatted C4 mAb binds selectivity to DEF. ELISA data
shows no binding to a negative control IgG, no binding to human
serum IgG, but strong binding to DEF IgG (FIG. 14).
Example 15. Binding Kinetics for the Anti DEF mAb C4
[0550] For C4-DEF binding experiments, C4 IgG was captured by
anti-human IgG (Fc) antibody amine coupled to a CMS chip using a
Biacore.TM. T200 instrument (GE Healthcare). The anti-human IgG
capture chip surface was prepared using a Biacore.TM. Human
Antibody Capture Kit according to the manufactures directions (GE
Healthcare). DEF Fab binding experiments were performed at
25.degree. C. using a 30 .mu.l/min flow rate in 0.01 M HEPES pH
7.4, 0.15 M NaCl and 0.005% v/v surfactant P20 (HBS-P) buffer.
After each cycle, the chip surface was regenerated with 3 M MgCl2
and new C4 antibody captured. DEF Fab samples ranging from 2-100 nM
were injected over the surface for 3 minutes and the dissociation
monitored for a further 20 minutes. Data was analyzed using the
Biacore.TM. T200 Evaluation software and the results reported as
the mean of two experiments.
Results
[0551] The affinity of the C4 mAb for the DEF mAb has a Kd of 3 nM.
Representative background subtracted Biacore.TM. sensorgrams
overlaid with the kinetic curve fits are shown (FIG. 15A) along
with the measured kinetic rate constants in the Table (FIG.
15B).
Example 16. Effects of the C4 mAb to Reverse Inhibitory Effects of
Anti-FXIa mAb DEF in an In Vitro FXIa Assay
[0552] DEF IgG and anti-DEF C4 IgG (4/20/100/500 nM) were premixed
and incubated for 20 min in the FXIa assay buffer. Following this
time 0.7 nM FXIa was incubated for further 5 min. The reaction was
then initiated by adding the fluorogenic peptide substrate which
starts the reaction. The plate was immediately inserted into and
read on a fluorescent plate reader at 37.degree. C. for 30 minutes.
Excitation setting was 352 nM, emission setting was 470 nM. Data
were collected every 1 minute on a SpectraMax.RTM. 3 instrument.
The V max data was then plotted for the individual conditions
tested.
Results
[0553] Excess C4 mAb (at approximately 2-10-fold that of DEF
concentration) can reverse the inhibitory effects of anti-FXIa mAb
DEF in a human FXIa activity assay in vitro (FIG. 16). It is
predicted that even only partial restoration of FXIa function will
probably be sufficient to reverse anticoagulant effects.
Example 17. Effects of the C4 mAb to Reverse Inhibitory Effects of
Anti-FXIa mAb DEF in an In Vitro FXIa Assay
[0554] 10 nM DEF antibody was premixed and incubated with the
indicated concentrations of either the C4 IgG or C4 Fab for 10 min
in the FXIa assay buffer. Following this time 0.5 nM FXIa was added
and incubated for an additional 5 min. The reaction was then
initiated by adding the fluorogenic peptide substrate which starts
the reaction. The plate was immediately inserted into and read on a
fluorescent plate reader at 37 C for 30 minutes. Excitation setting
was 352 nM, emission setting was 470 nM. Data were collected every
1 minute on a Spectramax 3 instrument. The V max (% FXIa alone)
data was then plotted for the individual conditions tested. Control
FXIa assay reactions containing no DEF or with only C4 mAb or C4
Fab are also shown.
Results
[0555] Excess C4 Fab (at approximately 5-20-fold that of DEF
concentration) can reverse the inhibitory effects of anti-FXIa mAb
DEF in a human FXIa activity assay in vitro (FIG. 17). The greater
activity of C4 IgG compared to C4 Fab may be a result of their
bivalent and monovalent structures, respectively. It is predicted
that even only partial restoration of FXIa function will probably
be sufficient to reverse anticoagulant effects.
Example 18. Reversal Effects of the Anti-DEF mAb C4 on the
Anti-FXIa mAb DEF in a FXIIa-Triggered Human Plasma Reaction that
Measures Downstream Thrombin Generation as a Readout
[0556] Thrombin generation was measured using a fluorogenic
thrombin substrate on a multiwell automated fluorescent plate
reader (ThrombinoSCOPE, Maastricht, the Netherlands) according to
the manufacturer's protocol. Briefly, 5 .mu.L of anti-FXIa DEF
antibody (16 .mu.g/ml) was added in combination with different
ratios of the anti DEF C4 IgG (1, 5, 10, 20, 40, 60, 80 .mu.g/ml)
and mixed with 20 .mu.L PBS-60 nM human Factor XIIa (Enzyme
Research Laboratories, South Bend, Ind., USA)-PC/PS
(Phospholipid-TGT, DiaPharma, West Chester, Ohio, USA) in a 96-well
plate. Finally, 75 .mu.L human Plasma (Triclinical Reference
Plasma, TCoag, Wicklow, Ireland) was added. Due to lot-to-lot
variability for the PC/PS reagent, for each lot, the concentration
was adjusted to achieve a .about.10 min Lag Time and .about.125 nM
Thrombin Peak. Finally, clotting was triggered with the addition of
calcium chloride buffer and a fluorogenic thrombin substrate. The
amount of thrombin generated in the reaction was measured over
time.
Results
[0557] The C4 mAb reverses the inhibitory effects of the anti-FXIa
mAb DEF on FXIIa-triggered thrombin generation in a human plasma
assay. Reversal of DEF effects are seen for all doses at or above
20 .mu.g/ml of the C4 mAb, indicating that the reversal properties
are first evident in this assay at an .about.1:1 ratio with the DEF
mAb (FIGS. 18A-C).
Example 19. C4 mAb Reverses Effects of Anti-FXIa mAb DEF in In Vivo
Rabbit Dosing Experiment
[0558] All procedures performed on these animals were in accordance
with regulations and established guidelines and were reviewed and
approved by an Institutional Animal Care and Use Committee or
through an ethical review process. Rabbits were anesthetized
according to established protocols. A 90-minute in-life rabbit
study was then carried out involving the following procedures. Five
rabbits were treated, with all animals being treated the same. At
time 0 each animal received a 1 mg/kg bolus injection of the DEF
IgG, 30 minutes later each animal then received a bolus injection
of 3 mg/kg ctrl IgG, 30 minutes after that each animal then
received a bolus injection of 3 mg/kg ctrl C4 IgG. Just prior to
each bolus injection, and 30 minutes after the final (C4 IgG)
injection, blood was drawn to make plasma for APTT and PT clotting
time assays. Data is plotted to show the sequential effects of DEF,
ctrl IgG, and C4 IgG injection on APTT and PT coagulation
times.
Results
[0559] The results of this sequential dosing experiment in the live
rabbit show that the C4 mAb reverses the effects of the DEF mAb, as
measured in the ex vivo APTT assay, 30 minutes after dosing with C4
(FIG. 19A). This result provides further evidence that dosing of
the C4 mAb could quickly reverse the effects of DEF dosing in vivo
were any DEF-related adverse bleeding events to occur. No effects
on PT coagulation times were seen between pre- and post-dose
samples, as expected (FIG. 19B).
Example 20. Effects of Anti-FXIa Antibody C24 on FeCl3-Triggered
Carotid Artery Thrombosis in Human FXI-Reconstituted FXI-Deficient
Mice
[0560] A mouse model known to be FXI-dependent was utilized for an
initial determination of whether Clone 24 ("C24") antibody could
alter thrombosis in vivo. FXI-deficient mice are protected against
FeCl.sub.3-induced carotid artery occlusion, a commonly used assay
of injury-induced arterial thrombosis. See, Rosen et al.,
Thrombosis and haemostasis 87, 774-776 (2002); Wang et al., Journal
of thrombosis and haemostasis: JTH 3, 695-702 (2005). Because our
antibodies did not cross react with mouse FXIa, a FXI-humanized
mouse analogous to that reported by Geng et al. (Blood 121,
3962-3969 (2013)) was established, but by administration of human
FXI protein rather than hydrodynamic transduction. Administration
of human FXI (0.25 mg/kg i.v.) to FXI-deficient mice rescued
FXIIa-driven thrombin generation in plasma to wild-type values and
provided a concentration of human FXI in plasma as measured by
ELISA of .about.1.5 .mu.g/ml, .about.30% of the level in human
plasma, for the duration of the thrombosis protocol.
[0561] FXI-knockout mice received an i.v. bolus injection via tail
vein of 0.25 mg/kg purified human FXI and 0.5, 2, 4, 12, 35 mg/kg
C24 (a fully human IgG1 monoclonal antibody) or control IgG1 at the
same concentrations. Fifteen minutes later, the mice were
anesthetized. The left common carotid artery was exposed and a flow
probe (model MA0.5PSB, Transonic Systems Inc., Ithaca, N.Y.) was
placed around the artery proximal to the bifurcation (Wang et al.,
Journal of thrombosis and haemostasis: JTH 3, 695-702 (2005);
Cornelissen et al., Proceedings of the National Academy of Sciences
of the United States of America 107, 18605-18610 (2010)). Filter
papers soaked in a 250 mM ferric chloride (FeCl.sub.3) solution
were placed above and below the artery for 3 minutes then removed.
Arterial flow was measured continuously using a TS420 flow meter
(Transonic Systems inc., Ithaca, N.Y.) connected to an
ADinstruments Powerlab 4/30 and Chart software. Monitoring was
continued until the artery was occluded (defined as no flow for
.gtoreq.1 minutes) or for 20 minutes if no occlusion occurred. Data
are plotted as % of vessels remaining open as a function of time
after injury. Human FXI preparations had no detectable (<1%)
FXIa activity in the SN-59 hydrolysis assay.
Results
[0562] Reconstitution of FXI-deficient mice with human FXI restored
carotid occlusion after application of FeCl.sub.3 (4% w/v; 250 mM)
(FIG. 20A). 4/4, 0/4, and 4/4 carotids were occluded by the end of
the protocol in wild-type, FXI-deficient, and FXI-humanized mice,
respectively. Median time to occlusion was similar in wild-type and
FXI-humanized mice (850 vs. 740 sec). In FXI-humanized mice that
received control IgG1 at 35 mg/kg i.v., the highest dose tested,
7/8 carotids were occluded by the end of the protocol and median
time to occlusion was 750 sec, a rate indistinguishable from that
seen in wild-type or FXI-humanized mice. By contrast, only 3/19
carotids occluded in FXI-humanized mice that received C24 at 2
mg/kg i.v. or above (FIG. 20B). At 0.5 mg/kg, C24 prolonged median
time to occlusion to 1100 sec, and at 2 mg/kg and above, median
times to occlusion were greater than 1380 sec, the end of the
protocol. Achieving substantial inhibition of carotid occlusion at
C24 dose of 2 mg/kg (FIG. 20B) supported further exploration of its
activity in vivo. In summary, inhibition of human FXIa function by
the active-site directed FXIa antibody C24 decreased or prevented
arterial thrombosis in this mouse model.
Example 21. IgG Potency is Improved in Human FXIa Activity Assay by
Addition of Q.fwdarw.K FW1 Substitution into Related IgG
Sequences
[0563] Table 2 below illustrates how IgG potency is improved in
human FXIa activity assay by the addition of a Q.fwdarw.K
substitution in framework 1 of the D4 IgG, and others, to generate
related sequences.
TABLE-US-00002 TABLE 2 in vitro CDR2 other IC50 IgG subs FW1 Sub Fc
sub (nM) D4 WNNG DIVMTQSPSSLSAS wt S54 5.4 VGDRVTITC (wt) B10B12
WNNG Q .fwdarw. K wt S54 0.74 clonel6 RDDD wt wt S54 0.57 clone32
RDDD Q .fwdarw. K wt S54 0.28 B10D8 WDND wt wt S54 1.4 clone22 WDND
Q .fwdarw. K wt S54 0.38 DEW WDDD wt Fc- S54E 0.36 DEF WDDD Q
.fwdarw. K Fc- S54E 0.21 clone8 WDDD wt wt S54 0.73 clone24 WDDD Q
.fwdarw. K wt S54 0.22
Example 22. Anti-FXIa Binding to FXIa is Inhibited by the Serine
Protease Inhibitors PMSF and PPACK
[0564] The binding of the anti-FXIa C24 Fab to FXIa in the presence
of the serine protease inhibitors PMSF and PPACK (also known as
FPRCK) (Haematologic Technologies Inc.) was evaluated by surface
plasmon resonance as described in Example 4. Binding analysis was
performed at 37.degree. C. with concentrations of C24 Fab ranging
from 0.1-5 nM. Biotinylated FXIa protein (Haematologic Technologies
Inc.) was diluted into the HBS-P buffer and incubated at room
temperature with or without 1 mM PMSF or 0.2 mM PPACK for at least
30 minutes. For each experiment, equivalent amounts of FXIa+/-
inhibitor was captured on two different Biacore chip flow cells,
and the C24 Fab passed over for 3 minutes and allowed to dissociate
for a further 20 minutes. The reported response is the C24 Fab
binding to FXIa +/- inhibitor with dual reference subtraction to
remove any background signal from the adjacent streptavidin-only
control flow cell and buffer only injections.
Results
[0565] A significant loss of C24 Fab binding was observed when FXIa
is inhibited with either PMSF (FIG. 21A-B) or PPACK (FIG. 21C-D).
For the FXIa-PMSF complex, a small amount of C24 DEF binding was
observed which may be reflective of incomplete inhibition of FXIa
by PMSF. No binding was observed at concentrations up to 5 nM C24
Fab for the FXIa-PPACK complex. These results suggest that the C24
Fab binding epitope may be in close proximity to the active site
and thus sterically blocked by these active site inhibitors.
Example 23. Crystal Structure of the DEF Fab Bound to the Human
FXIa Catalytic Domain
Complex Formation and Crystallization
[0566] The FXIa catalytic domain was produced using a gene fragment
synthesized to encode a mammalian signal peptide derived from mouse
IgG followed by the catalytic domain of human FXI (i.e., Ile370 to
Ala606), and a C-terminal 6-His tag. The catalytic domain active
site Ser was substituted with an Ala (Ser557A1a), the unpaired Cys
with a Ser (Cys482Ser), and the Asn residues of the two predicted
N-linked glycosylation sites, identified by the consensus sequence
Asn-X-Ser, were substituted with Gln residues to inhibit N-linked
glycosylation ("glyco-"). This gene was subcloned into a mammalian
expression vector and transiently expressed by Expi293F cells (Life
Technologies, CA, USA). The DEF Fab was expressed by
co-transfecting two mammalian expression plasmids, one encoding the
Fab heavy chain with a C-terminal 6-His tag and the second the
light chain, into Expi293F cells. Supernatants were harvested 3
days post transfection and the recombinant proteins captured on
HisTrap columns (GE Healthcare Bio-Sciences, PA, USA). Purified
FXIa protein was mixed with DEF Fab in a 1.5-fold molar excess and
concentrated in a centrifugal filter unit at 4.degree. C. The
complex was purified using a Superdex 200 gel filtration column (GE
Healthcare Bio-Sciences) equilibrated in 10 mM Tris (pH8.0), 150 mM
sodium chloride buffer (TBS) on an AKTA Avant FPLC instrument (GE
Healthcare Bio-Sciences). Fractions corresponding to peaks in 280
nm absorbance were run on SDS-PAGE under reducing and non-reducing
conditions. Fractions containing the DEF Fab-FXIa catalytic domain
complex were pooled and concentrated for crystallization
trials.
[0567] Purified DEF Fab-FXIa catalytic domain complex was
concentrated to 25.7 mg/ml. A 0.25 .mu.l drop of protein sample was
mixed with 0.25 .mu.l of reservoir solution (1.86 M ammonium
sulfate, 8 mM CoCl.sub.2, 30 mM K/Na phosphate, pH 6.5) and
crystallized in hanging-drop configuration over 100 .mu.l of
reservoir solution at 20.degree. C. Crystals appeared overnight and
grew to full size in 1 week. Crystals were harvested and
cryoprotected with a saturated ammonium sulfate solution containing
8 mM CoCl.sub.2, 30 mM K/Na phosphate, pH 6.5 and 3% glycerol and
then flash frozen in liquid nitrogen.
Data Collection and Processing
[0568] Two datasets were collected from a single crystal at 100 K
using synchrotron radiation (APS GM/CAT beamline 23-IDB, Chicago,
Ill.). A native dataset was collected at .lamda.=1.033 .ANG. with
180.degree. rotation. An additional dataset was collected near the
cobalt K-edge (.lamda.=1.606 .ANG.) in order to maximize the cobalt
anomalous signal present in the crystallization solution. The two
datasets were processed with XDS, scaled and merged with Aimless
and a resolution cutoff of 1.8 .ANG. and 2.5 .ANG. for the native
and derivative datasets respectively, was applied accordingly to
the CC.sub.1/2 criterion (Diederichs and Karplus, 2013; Karplus and
Diederichs, 2012). Crystals belong to the 1422 space group.
Structure Determination and Refinement
[0569] The complex structure of the DEF Fab-FXIa catalytic domain
complex was solved by molecular replacement using the Fab fragment
of the human germline antibody 5-51/012 (PDB code: 4KMT) as search
model. Once the Fab fragment was placed, the electron density for
the missing FXIa catalytic domain was clearly visible. It was
finally placed with a second MR round using the PDB code 3SOS as
search model. Iterative rounds of model building using, COOT
(Emsley and Cowtan, 2004), and refinement using REFMAC5
(Collaborative Computational Project, 1994) and PHENIX (Adams et
al., 2010) were carried out to improve the electron density
map.
[0570] To help assign the residual positive peaks, an anomalous map
was generated using the dataset collected at cobalt K-edge. The
wavelength used was sufficient to get a strong signal from the
sulfur ions, helping us to model 4 sulfate anions and 2 cobalt
cations. Data set and refinement statistics are summarized in Table
3.
X-Ray Structure-Based Epitope Mapping
[0571] The complex of DEF Fab and FXIa crystallized as 1 copy of
the complex per asymmetric unit. Residues of the DEF Fab (paratope)
in contact with FXIa (epitope) were determined with PISA (Protein
Interactions, Surfaces and Assemblies) (E. Krissinel and K.
Henrick, J. Mol. Biol., 2007, 372, 774-797) and are listed in Table
4 below.
Results
[0572] The FXIa catalytic domain-DEF Fab complex crystal structure
was determined to 1.8 .ANG. (Table 3). The total interface area
between FXIa and DEF Fab light chain is 976.4 .ANG..sup.2 and the
total interface between FXIa and DEF Fab heavy chain is 276.6
.ANG..sup.2. The DEF Fab acts as a direct competitive inhibitor
blocking the active site of FXIa. The DEF Fab recognizes FXIa
predominantly via the light chain CDR L1 and CDR L3, additional
hydrogen bonds are made by the light chain variable region, the CDR
H1 and CDR H3 (Table 4, FIG. 22). The interaction with the FXIa
catalytic domain is focused around the active site. Specifically
the FXIa epitope is formed by the following residues: His 27, Tyr
47, Met 87, Ala 88, Ser 90, Asp 93, Tyr 134, Arg 138, Asp 182, Asp
139, Lys 185, Ser 207, and Gly 211, with the numbering convention
starting with Ile1 at the NH2-terminal (i.e., numbered with respect
to SEQ ID NO:100). CDR L1 is in close proximately to Ala 188,
corresponding to Ser 188 in catalytic triad of the active site in
wild-type human Factor XIa. Such proximity is consistent with our
experimental results showing that the C24 Fab loses binding
affinity for FXIa when it is bound to the serine protease
inhibitors PMSF and PPACK (Example 22). A superimposition of
published serine protease catalytic domain structures bound to PMSF
(PDB 1PQA) or PPACK (PDB 1Z8I) on the FXIa catalytic domain
illustrates that the inhibitors may sterically impede the Fab from
binding to FXIa through blocking the CDRL1 loop interactions (FIG.
23).
TABLE-US-00003 TABLE 3 Data set and refinement statistics for DEF
Fab - FXIa complex FXIa cat. domain - DEF Fab FXIa cat. domain -
DEF Fab complex (native) complex (derivative) Data Collection Space
group I422 I422 Cell dimensions a/b/c (.ANG.) 136.09/136.09/175.99
136.61/136.61/176.31 .alpha./.beta./.gamma. (.degree.)
90.0/90.0/90.0 90.0/90.0/90.0 Resolution (.ANG.) 64.94-1.80
(1.86-1.80) 107.99-2.50 (2.59-2.50) Rsym (%) 5.4 3.4 I/.sigma. I
12.99 (0.83) 25.77 (3.30) Correlation Coefficient 0.99 (0.28) 0.99
(0.92) Completeness (%) 100 (98.0) 100 (100) Redundancy 5.7 (5.4)
15.9 (14.6) Unique reflections 75924 (7371) 28898 (2817) Wilson
B-factor 34.35 47.30 Refinement Rwork/Rfree (%) 18.8/22.8 No. of
chains in AU 3 No. of protein residues 653 No. of ligand atoms 19
No. of water atoms 618 RMSD bond lengths (.ANG.) 0.008 RMSD angles
(.degree.) 1.19 Ramachandran 98.0/0.0 best/disallowed regions
(%)
TABLE-US-00004 TABLE 4 Contact residues at the FXIa - DEF Fab
interface DEF FAB DEF FAB Type (H-bond/ Light chain Heavy chain
FXIa Salt bridge) Tyr 94 His 27 H-bond Ala 25 Tyr 47 H-bond Thr 69
Met 87 H-bond Ser 67 Ala 88 H-bond Gln 27 Ser 90 H-bond Gln 27 Asp
93 H-bond Asp 92 Tyr 134 H-bond Ile 93 Asp 32 Leu 99 Arg 138 Salt
bridge Tyr 33 H-bond H-bond Arg 30 Asp 182 Salt bridge Tyr 33 Asp
139 H-bond His 91 Lys 185 H-bond Asp 32 Salt bridge Gln 27 Ser 207
H-bond Arg 30 Gly 211 H-bond
Example 24. Effect of Anti-FXIa Antibodies on FXIIa-Induced
Thrombin Generation, APTT in Human Plasma and Intrinsic
Pathway-Triggered Clotting in Whole Human Blood
[0573] Thrombin generation in platelet-poor plasma was measured
using a fluorogenic thrombin substrate and a multi-well automated
fluorescent plate reader (ThrombinoSCOPE, Maastricht, the
Netherlands). 5 .mu.L of anti-FXIa or IgG1 control (Mab 8.8)
antibody solution was mixed with 20 .mu.L phosphate-buffered saline
containing 60 nM human FXIIa (Enzyme Research Laboratories, South
Bend, Ind., USA) and PC/PS (Phospholipid-TGT, DiaPharma, West
Chester, Ohio, USA) in a 96-well plate. 75 .mu.L citrated
platelet-poor human plasma (Triclinical Reference Plasma, TCoag,
Wicklow, Ireland) was added, and coagulation was triggered with the
addition of 20 .mu.L calcium chloride buffer and fluorogenic
thrombin substrate according to the ThrombinoSCOPE manufacturer's
protocol. Thrombin activity was measured as velocity of fluorogenic
peptide hydrolysis as a function of time. Total thrombin activity
generated (commonly called endogenous thrombin potential, ETP),
peak activity, and time to onset of thrombin generation (lag time)
and time to peak activity were measured. The final concentrations
of the antibodies tested (D4, B11, 24, DEF, and IgG1 ctrl) ranged
from 5 .mu.g/mL to 443 .mu.g/mL. Due to lot-to-lot variability, the
concentration of each lot of PC/PS reagent was adjusted to achieve
a .about.15 min lag to onset and .about.150 nM peak thrombin
activity.
[0574] APTT was measured using standard pooled human platelet-poor
plasma and a Triniclot aPTT kit (Parsippany, N.J.) according to
manufacturer instructions.
[0575] For whole blood clotting, whole blood was collected in 3.8%
citrate a blood:citrate ratio of 9:1 (v/v). 5 .mu.l of C24, control
IgG1, or saline control was added to 300 .mu.l of citrated whole
blood. Samples were preincubated at 37.degree. C. for 5 minutes,
and clotting was initiated by addition of 7 .mu.l of INTEM reagent
(ellagic acid/phospholipid; TEM systems, Inc., Durham, N.C.) and 20
.mu.l STARTEM reagent (0.2M CaCl.sub.2) in Hepes buffer, pH 7.4).
Time to clotting was measured using a semi-automated coagulation
analyzer (KC4 Delta; Tcoag, Wicklow, Ireland).
Results
[0576] As shown in FIG. 24A and FIG. 24B, FXIIa-triggered thrombin
activity as a function of time was determined in the presence of
the indicated concentrations of anti-FXIa antibodies D4 (blue), B11
(red), C24 (green) or control IgG1 (black). Peak thrombin activity
(A) and lag to onset of thrombin generation (B) are shown
(mean+/-SEM; n=3-5). There was a substantial reduction in peak
thrombin generation and prolongation of time to onset of thrombin
generation in samples containing C24 at 4 ug/ml or greater. FIG.
24C shows an APTT assay as a function of antibody concentration
(mean+/-SEM; n=2). Control IgG1 had no effect in this assay. There
was a prolongation of APTT in samples containing C24 at 10 ug/ml or
greater. FIG. 24D shows the effect of C24 or control IgG1 on
intrinsic pathway-triggered clotting of whole blood. Time to clot
is shown (mean+/-SEM; n=3-4). There was a prolongation of time to
clotting in whole human blood in samples containing C24.
TABLE-US-00005 TABLE 5 Sequence Listing Table CDR amino acid
sequences are underlined (Chothia) or in bold (Kabat). DEF_VH AA
SEQ ID NO: EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 1
VRQAPGQGLEWMGWIDPDEGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS DEF_HCDR1 SEQ ID NO:
GYYMH Kabat AA 2 DEF_HCDR2 SEQ ID NO: WIDPDEGDTNYAQKFQG Kabat AA 3
DEF_HCDR3 SEQ ID NO: LASGFRDY Kabat/ 4 Chothia AA DEF_HCDR1 SEQ ID
NO: GYTFTGYYMH Chothia AA 5 DEF_HCDR2 SEQ ID NO: WIDPDEGD Chothia
AA 6 DEF_VL AA SEQ ID NO: DIVMTKSPSSLSASVGDRVTITCRASQGIRNDLGWY 7
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR DEF_LCDR1 SEQ ID NO:
RASQGIRNDLG Kabat AA 8 DEF_LCDR2 SEQ ID NO: YAASSLQS Kabat AA 9
DEF_LCDR3 SEQ ID NO: LQHDIYAST Kabat AA 10 DEF_LCDR1 SEQ ID NO:
ASQGIRNDL Chothia AA 11 DEF_LCDR2 SEQ ID NO: YAASS Chothia AA 12
DEF_LCDR3 SEQ ID NO: QHDIYAST Chothia AA 13 D4_VH AA SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 14
VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS D4_HCDR2 SEQ ID NO:
WINPNSGGTNYAQKFQG Kabat AA 15 D4_HCDR2 SEQ ID NO: WINPNSGG Chothia
AA 16 D4_VL AA SEQ ID NO: DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY 17
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR QCA11_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 18
VRQAPGQGLEWMGRINPNSGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS QCA11_HCDR2 SEQ ID
NO: RINPNSGDTNYAQKFQG Kabat AA 19 QCA11_HCDR2 SEQ ID NO: RINPNSGD
Chothia 20 AA QCA11_VL SEQ ID NO:
DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 21
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR B1D2_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 22
VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTR
DTSISTAYMELSRLSSDDTAVYYCARLASGFRDYWG QGTLVTVSS B1D2_VL AA SEQ ID
NO: DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY 23
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR B10H2_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 24
VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS B10H2_VL SEQ ID NO:
DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 25
QQKPGKAPKRLIYAASSLQSGVPSRFSGSVSGTEFT
LTISSLQPEDLATYYCLQHDIYASTFGPGTKVDIKR B10E6_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 26
VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS B10E6_VL SEQ ID NO:
DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 27
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR B10F6_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 28
VRQAPGQGLEWMGRINPNSGGTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS B10F6_HCDR2 SEQ ID
NO: RINPNSGGTNYAQKFQG Kabat AA 29 B10F6_HCDR2 SEQ ID NO: RINPNSGG
Chothia 30 AA B10F6_VL SEQ ID NO:
DIVMTQSPSSLSASVGDRVTITCRASLGIRNDLGWY AA 31
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFS
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR B10F6_LCDR1 SEQ ID NO:
RASLGIRNDLG Kabat AA 32 B10F6_LCDR1 SEQ ID NO: ASLGIRNDL Chothia AA
33 B10D8_VH SEQ ID NO: EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 34
VRQAPGQGLEWMGWIDPNSGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS B10D8_HCDR2 SEQ ID
NO: WIDPNSGDTNYAQKFQG Kabat AA 35 B10D8_HCDR2 SEQ ID NO: WIDPNSGD
Chothia 36 AA B10D8_VL SEQ ID NO:
DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 37
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR B10B12_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 38
VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS B10B12_VL SEQ ID NO:
DIVMTKSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 39
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR S1D4_VH AA SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 40
VRQAPGQGLEWMGWINPNSGGTNYAPKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS S1D4_HCDR2 SEQ ID
NO: WINPNSGGTNYAPKFQG Kabat AA 41 S1D4_VL AA SEQ ID NO:
DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY 42
QQKPGKAPKRLIYAASSLQSGVPSRFSGSASGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR S10H9_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 43
VRQAPGQGLEWMGWIDPDSGGTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG HGTLVTVSS S10H9_HCDR2 SEQ ID
NO: WIDPDSGGTNYAQKFQG Kabat AA 44 S10H9_HCDR2 SEQ ID NO: WIDPDSGG
Chothia 45 AA S10H9_VL SEQ ID NO:
DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 46
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR Clone 8_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 47
VRQAPGQGLEWMGWIDPDSGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS Clone SEQ ID NO:
WIDPDSGDTNYAQKFQG 8_HCDR2 48 Kabat AA Clone SEQ ID NO: WIDPDSG
8_HCDR2 49 Chothia AA Clone 8_VL SEQ ID NO:
DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 50
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR Clone 16_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 51
VRQAPGQGLEWMGRIDPDSGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS Clone SEQ ID NO:
RIDPDSGDTNYAQKFQG 16_HCDR2 52 Kabat AA Clone SEQ ID NO: RIDPDSGD
16_HCDR2 53 Chothia AA Clone 16_VL SEQ ID NO:
DIVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 54
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR Clone 20_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 55
VRQAPGQGLEWMGWINPDSGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS Clone SEQ ID NO:
WINPDSGDTNYAQKFQG 20_HCDR2 56 Kabat AA Clone SEQ ID NO: WINPDSGD
20_HCDR2 57 Chothia AA Clone 20_VL SEQ ID NO:
DIVMTKSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 58
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR Clone 22_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 59
VRQAPGQGLEWMGWIDPNSGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS Clone SEQ ID NO:
WIDPNSGDTNYAQKFQG 22_HCDR2 60 Kabat AA Clone SEQ ID NO: WIDPNSGD
22_HCDR2 61 Chothia AA Clone 22_VL SEQ ID NO:
DIVMTKSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 62
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR
Clone 32_VH SEQ ID NO: EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 63
VRQAPGQGLEWMGRIDPDSGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS Clone 32_VL SEQ ID
NO: DIVMTKSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 64
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR Clone 24_VH SEQ ID NO:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW AA 65
VRQAPGQGLEWMGWIDPDSGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG QGTLVTVSS Clone SEQ ID NO:
WIDPDSGDTNYAQKFQG 24_HCDR2 66 Kabat AA Clone SEQ ID NO: WIDPDSGD
24_HCDR2 67 Chothia AA Clone 24_VL SEQ ID NO:
DIVMTKSPSSLSASVGDRVTITCRASQGIRNDLGWY AA 68
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKR C4_VH AA SEQ ID NO:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHW 69
VRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCARDTIPGIAVAG TDYWGQGTLVTVSS C4_HCDR1 SEQ ID
NO: SYYMH Kabat AA 70 C4_HCDR2 SEQ ID NO: IINPSGGSTSYAQKFQG Kabat
AA 71 C4_HCDR3 SEQ ID NO: DTIPGIAVAGTDY Kabat/ 72 Chothia AA
C4_HCDR1 SEQ ID NO: GYTFTSYYMH Chothia AA 73 C4_HCDR2 SEQ ID NO:
IINPSGGS Chothia AA 74 C4_VL AA SEQ ID NO:
QSVLTQPPSVSAAPGQKVTISGSGSTSNIGNNYVSW 75
YQQVPGTPPKLLIYDNDKRPSGIPDRFSGSKSGTSA
TLDITGLQTGDEADYYCGTWHSGLYVVVFGGGTKLT VL C4_LCDR1 SEQ ID NO:
SGSTSNIGNNYVS Kabat AA 76 C4_LCDR2 SEQ ID NO: YDNDKRPS Kabat AA 77
C4_LCDR3 SEQ ID NO: GTWHSGLYVVV Kabat AA 78 C4_LCDR1 SEQ ID NO:
GSTSNIGNNYV Chothia AA 79 C4_LCDR2 SEQ ID NO: YDNDK Chothia AA 80
C4_LCDR3 SEQ ID NO: TWHSGLYVVV Chothia AA 81 HC Constant SEQ ID NO:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP Region AA 82
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK LC Constant SEQ ID NO:
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA Region AA 83
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC DEF_VH NT SEQ ID NO:
GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAG 84
AAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCT
TCTGGATACACCTTCACCGGCTACTATATGCACTGG
GTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG
GGATGGATCgACCCTgACgaaGGTGaCACAAACTAT
GCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGG
GACACGTCCATCAGCACAGCCTACATGGAGCTGAGC
AGGCTGAGATCTGACGACACGGCCGTGTATTACTGT
GCGAGATTAGCTAGTGGCTTTCGTGACTACTGGGGC CAGGGAACCCTGGTCACCGTCTCGAGC
DEF_VL NT SEQ ID NO: GACATCGTGATGACCaAGTCTCCATCCTCCCTGTCT 85
GCtTCTGTAGGAGACAGAGTCACCATCACTTGCCGG
GCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTAT
CAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTCATC
TATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCA
AGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACT
CTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCA
ACTTATTACTGTCTACAGCATGATATTTACGCTAGC
ACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGT D4_VH NT SEQ ID NO:
GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAG 86
AAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCT
TCTGGATACACCTTCACCGGCTACTATATGCACTGG
GTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG
GGATGGATCAACCCTAACAGTGGTGGCACAAACTAT
GCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGG
GACACGTCCATCAGCACAGCCTACATGGAGCTGAGC
AGGCTGAGATCTGACGACACGGCCGTGTATTACTGT
GCGAGATTAGCTAGTGGCTTTCGTGACTACTGGGGC CAGGGAACCCTGGTCACCGTCTCGAGC
D4_VL NT SEQ ID NO: GACATCGTGATGACCCAGTCTCCATCCTCCCTGTCT 87
GCATCTGTAGGAGACAGAGTCACCATCACTTGCCGG
GCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTAT
CAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTCATC
TATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCA
AGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACT
CTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCA
ACTTATTACTGTCTACAGCATGATATTTACGCTAGC
ACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGT Clone 24_VH SEQ ID NO:
GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAG NT 88
AAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCT
TCTGGATACACCTTCACCGGCTACTATATGCACTGG
GTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG
GGATGGATCgACCCTgACAGTGGTGaCACAAACTAT
GCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGG
GACACGTCCATCAGCACAGCCTACATGGAGCTGAGC
AGGCTGAGATCTGACGACACGGCCGTGTATTACTGT
GCGAGATTAGCTAGTGGCTTTCGTGACTACTGGGGC CAGGGAACCCTGGTCACCGTCTCGAGC
Clone 24_VL SEQ ID NO: GACATCGTGATGACCaAGTCTCCATCCTCCCTGTCT NT 89
GCtTCTGTAGGAGACAGAGTCACCATCACTTGCCGG
GCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTAT
CAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTCATC
TATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCA
AGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACT
CTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCA
ACTTATTACTGTCTACAGCATGATATTTACGCTAGC
ACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGT C4_VH NT SEQ ID NO:
CAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAG 90
AAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCA
TCTGGATACACCTTCACCAGCTACTATATGCACTGG
GTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG
GGAATAATCAACCCTAGTGGTGGTAGCACAAGCTAC
GCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGG
GACACGTCCACGAGCACAGTCTACATGGAGCTGAGC
AGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT
GCGAGAGACACTATTCCGGGTATAGCAGTGGCTGGT
ACGGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCGAGC C4_VL NT SEQ ID NO:
CAGTCTGTCTTGACGCAGCCGCCCTCAGTGTCTGCG 91
GCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGA
AGCACCTCCAACATTGGCAATAATTATGTATCCTGG
TACCAGCAGGTCCCAGGAACACCCCCCAAACTCCTC
ATTTATGACAATGATAAGCGACCCTCAGGGATTCCT
GACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCC
ACCCTGGACATCACCGGACTCCAGACTGGGGACGAG
GCCGATTATTACTGCGGAACATGGCATAGTGGCCTG
TATGTCGTGGTGTTCGGCGGAGGGACCAAGCTGACC GTCCTA HC Constant SEQ ID NO:
GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCA Region NT 92
CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC
CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG
GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA
GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAAC
GTGAATCACAAGCCCAGCAACACCAAGGTGGACAAG
AAAGTTGAGCCCAAATCTTGTGACAAAACTCACACA
TGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCA
CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC
ACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG
CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG
ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC
AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC
ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC
CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGG
CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA LC Constant
SEQ ID NO: ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA Region NT 93
TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT
GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC
AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG
GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC
AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG
CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC
GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC GTCACAAAGAGCTTCAACAGGGGAGAGTGT
HCDR2 Kabat SEQ ID NO:
X.sub.1IX.sub.2PX.sub.2X.sub.3GX.sub.4TNYAX.sub.5KFQG consensus AA
94 X.sub.1 = W or R X.sub.2 = N or D X.sub.3 = s or e X.sub.4 = g
or d X.sub.5 = q or p HCDR2 SEQ ID NO:
X.sub.1IX.sub.2PX.sub.2X.sub.3GX.sub.4 Chothia 95 X.sub.1 = W or R
consensus AA X.sub.2 = N or D X.sub.3 = s or e X.sub.4 = g or d VH
SEQ ID NO: EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW Consensus aa 96
VRQAPGQGLEWMGX.sub.1IX.sub.2PX.sub.2X.sub.3GX.sub.4TNYAX.sub.5KFQGRV
TMTRDTSISTAYMELSRLX.sub.6SDDTAVYYCARLASGF RDYWGX.sub.7GTLVTVSS
X.sub.1 = WorR X.sub.2 = N or D X.sub.3 = s or e X.sub.4 = g or d
X.sub.5 = q or p X.sub.6 = R or S X.sub.7 = Q or H VL consensus SEQ
ID NO: DIVMTX.sub.1SPSSLSASVGDRVTITCRASX.sub.2GIRNDLG aa 97
WYQQKPGKAPKRLIYAASSLQSGVPSRFSGSX.sub.3SGT
EFX.sub.4LTISSLQPEDX.sub.5ATYYCLQHDIYASTFGPGTK VDIKR X.sub.1 = Q or
K X.sub.2 = Q or L X.sub.3 = G or V OR A X.sub.4 = T or S X.sub.5 =
F or L Human Factor SEQ ID NO: MIFLYQVVHFILFTSVSGECVTQLLKDTCFEGGDIT
XIa 98 TVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTR
WFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQIS
ACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDV
HCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITK
LDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNID
SVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQR
NLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIP
VFCHSSFYHDTDFLGEELDIVAAKSHEACQKLCTNA
VRCQFFTYTPAQASCNEGKGKCYLKLSSNGSPTKIL
HGRGGISGYTLRLCKMDNECTTKIKPRIVGGTASVR
GEWPWQVTLHTTSPTQRHLCGGSIIGNQWILTAAHC
FYGVESPKILRVYSGILNQSEIKEDTSFFGVQEIII
HDQYKMAESGYDIALLKLETTVNYTDSQRPICLPSK
GDRNVIYTDCWVTGWGYRKLRDKIQNTLQKAKIPLV
TNEECQKRYRGHKITHKMICAGYREGGKDACKGDSG
GPLSCKHNEVWHLVGITSWGEGCAQRERPGVYTNVV EYVDWILEKTQAV Human FXI SEQ ID
NO: MGWSCIILFLVATATGVHSIVGGTASVRGEWPWQVT catalytic 99
LHTTSPTQRHLCGGSIIGNQWILTAAHCFYGVESPK domain
ILRVYSGILQQSEIKEDTSFFGVQEIIIHDQYKMAE S557A glyco-
SGYDIALLKLETTVQYTDSQRPISLPSKGDRNVIYT
DCWVTGWGYRKLRDKIQNTLQKAKIPLVTNEECQKR
YRGHKITHKMICAGYREGGKDACKGDAGGPLSCKHN
EVWHLVGITSWGEGCAQRERPGVYTNVVEYVDWILE KTQAHHHHHH Human FXI SEQ ID
NO: IVGGTASVRGEWPWQVTLHTTSPTQRHLCGGSIIGN catalytic 100
QWILTAAHCFYGVESPKILRVYSGILQQSEIKEDTS domain
FFGVQEIIIHDQYKMAESGYDIALLKLETTVQYTDS S557A glyco-
QRPISLPSKGDRNVIYTDCWVTGWGYRKLRDKIQNT mature
LQKAKIPLVTNEECQKRYRGHKITHKMICAGYREGG peptide
KDACKGDAGGPLSCKHNEVWHLVGITSWGEGCAQRE RPGVYTNVVEYVDWILEKTQAHHHHHH
DEF_FAB_HEAVY SEQ ID NO: EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHW 101
VRQAPGQGLEWMGWIDPDEGDTNYAQKFQGRVTMTR
DTSISTAYMELSRLRSDDTAVYYCARLASGFRDYWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCGGSHHHHHH DEF_FAB_LIGHT
SEQ ID NO: DIVMTKSPSSLSASVGDRVTITCRASQGIRNDLGWY 102
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT
LTISSLQPEDFATYYCLQHDIYASTFGPGTKVDIKT
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC Constant SEQ ID NO:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP Region AA 103
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK
[0577] Although the disclosed teachings have been described with
reference to various applications, methods, kits, and compositions,
it will be appreciated that various changes and modifications can
be made without departing from the teachings herein and the claimed
invention below. The foregoing examples are provided to better
illustrate the disclosed teachings and are not intended to limit
the scope of the teachings presented herein. While the present
teachings have been described in terms of these exemplary
embodiments, the skilled artisan will readily understand that
numerous variations and modifications of these exemplary
embodiments are possible without undue experimentation. All such
variations and modifications are within the scope of the current
teachings.
[0578] All publications, patents, patent applications or other
documents cited herein are hereby incorporated by reference in
their entirety for all purposes to the same extent as if each
individual publication, patent, patent application, or other
document was individually indicated to be incorporated by reference
for all purposes.
Sequence CWU 1
1
1031117PRTArtificial SequenceSynthetic construct 1Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asp Pro Asp Glu Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11525PRTArtificial
SequenceSynthetic Construct 2Gly Tyr Tyr Met His1 5317PRTArtificial
SequenceSynthetic Construct 3Trp Ile Asp Pro Asp Glu Gly Asp Thr
Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly48PRTArtificial
SequenceSynthetic Construct 4Leu Ala Ser Gly Phe Arg Asp Tyr1
5510PRTArtificial SequenceSynthetic Construct 5Gly Tyr Thr Phe Thr
Gly Tyr Tyr Met His1 5 1068PRTArtificial SequenceSynthetic
Construct 6Trp Ile Asp Pro Asp Glu Gly Asp1 57108PRTArtificial
SequenceSynthetic Construct 7Asp Ile Val Met Thr Lys Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala Ser 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105811PRTArtificial
SequenceSynthetic Construct 8Arg Ala Ser Gln Gly Ile Arg Asn Asp
Leu Gly1 5 1098PRTArtificial SequenceSynthetic Construct 9Tyr Ala
Ala Ser Ser Leu Gln Ser1 5109PRTArtificial SequenceSynthetic
Construct 10Leu Gln His Asp Ile Tyr Ala Ser Thr1 5119PRTArtificial
SequenceSynthetic Construct 11Ala Ser Gln Gly Ile Arg Asn Asp Leu1
5125PRTArtificial SequenceSynthetic Construct 12Tyr Ala Ala Ser
Ser1 5138PRTArtificial SequenceSynthetic Construct 13Gln His Asp
Ile Tyr Ala Ser Thr1 514117PRTArtificial SequenceSynthetic
Construct 14Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Gly Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr
Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe
Arg Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
1151517PRTArtificial SequenceSynthetic Construct 15Trp Ile Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10
15Gly168PRTArtificial SequenceSynthetic Construct 16Trp Ile Asn Pro
Asn Ser Gly Gly1 517108PRTArtificial SequenceSynthetic Construct
17Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn
Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
His Asp Ile Tyr Ala Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys Arg 100 10518117PRTArtificial SequenceSynthetic Construct
18Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Asn Pro Asn Ser Gly Asp Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
1151917PRTArtificial SequenceSynthetic Construct 19Arg Ile Asn Pro
Asn Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10
15Gly208PRTArtificial SequenceSynthetic Construct 20Arg Ile Asn Pro
Asn Ser Gly Asp1 521108PRTArtificial SequenceSynthetic Construct
21Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn
Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
His Asp Ile Tyr Ala Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys Arg 100 10522117PRTArtificial SequenceSynthetic Construct
22Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Ser Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11523108PRTArtificial SequenceSynthetic Construct 23Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala
Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100
10524117PRTArtificial SequenceSynthetic Construct 24Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11525108PRTArtificial
SequenceSynthetic Construct 25Asp Ile Val Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Val Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Leu
Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala Ser 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 10526117PRTArtificial
SequenceSynthetic Construct 26Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn
Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser 11527108PRTArtificial SequenceSynthetic
Construct 27Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Leu Gln His Asp Ile Tyr Ala Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys Arg 100 10528117PRTArtificial SequenceSynthetic
Construct 28Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Gly Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr
Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe
Arg Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
1152917PRTArtificial SequenceSynthetic Construct 29Arg Ile Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10
15Gly308PRTArtificial SequenceSynthetic Construct 30Arg Ile Asn Pro
Asn Ser Gly Gly1 531108PRTArtificial SequenceSynthetic Construct
31Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Gly Ile Arg Asn
Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
His Asp Ile Tyr Ala Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys Arg 100 1053211PRTArtificial SequenceSynthetic Construct
32Arg Ala Ser Leu Gly Ile Arg Asn Asp Leu Gly1 5 10339PRTArtificial
SequenceSynthetic Construct 33Ala Ser Leu Gly Ile Arg Asn Asp Leu1
534117PRTArtificial SequenceSynthetic Construct 34Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asp Pro Asn Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 1153517PRTArtificial
SequenceSynthetic Construct 35Trp Ile Asp Pro Asn Ser Gly Asp Thr
Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly368PRTArtificial
SequenceSynthetic Construct 36Trp Ile Asp Pro Asn Ser Gly Asp1
537108PRTArtificial SequenceSynthetic Construct 37Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala
Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100
10538117PRTArtificial SequenceSynthetic Construct 38Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11539108PRTArtificial
SequenceSynthetic Construct 39Asp Ile Val Met Thr Lys Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala Ser 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys
Arg 100 10540117PRTArtificial SequenceSynthetic Construct 40Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Pro Lys
Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
1154117PRTArtificial SequenceSynthetic Construct 41Trp Ile Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Pro Lys Phe Gln1 5 10
15Gly42108PRTArtificial SequenceSynthetic Construct 42Asp Ile Val
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu
Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Ala Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile
Tyr Ala Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg
100 10543117PRTArtificial SequenceSynthetic Construct 43Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Trp Ile Asp Pro Asp Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly
His Gly Thr Leu 100 105 110Val Thr Val Ser Ser 1154417PRTArtificial
SequenceSynthetic Construct 44Trp Ile Asp Pro Asp Ser Gly Gly Thr
Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly458PRTArtificial
SequenceSynthetic Construct 45Trp Ile Asp Pro Asp Ser Gly Gly1
546108PRTArtificial SequenceSynthetic Construct 46Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala
Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100
10547117PRTArtificial SequenceSynthetic Construct 47Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asp Pro Asp Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 1154817PRTArtificial
SequenceSynthetic Construct 48Trp Ile Asp Pro Asp Ser Gly Asp Thr
Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly497PRTArtificial
SequenceSynthetic Construct 49Trp Ile Asp Pro Asp Ser Gly1
550108PRTArtificial SequenceSynthetic Construct 50Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala
Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100
10551117PRTArtificial SequenceSynthetic Construct 51Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Arg Ile Asp Pro Asp Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 1155217PRTArtificial
SequenceSynthetic Construct 52Arg Ile Asp Pro Asp Ser Gly Asp Thr
Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly538PRTArtificial
SequenceSynthetic Construct 53Arg Ile Asp Pro Asp Ser Gly Asp1
554108PRTArtificial SequenceSynthetic Construct 54Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala
Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100
10555117PRTArtificial SequenceSynthetic Construct 55Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asn Pro Asp Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 1155617PRTArtificial
SequenceSynthetic Construct 56Trp Ile Asn Pro Asp Ser Gly Asp Thr
Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly578PRTArtificial
SequenceSynthetic Construct 57Trp Ile Asn Pro Asp Ser Gly Asp1
558108PRTArtificial SequenceSynthetic Construct 58Asp Ile Val Met
Thr Lys Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala
Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100
10559117PRTArtificial SequenceSynthetic Construct 59Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asp Pro Asn Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 1156017PRTArtificial
SequenceSynthetic Construct 60Trp Ile Asp Pro Asn Ser Gly Asp Thr
Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly618PRTArtificial
SequenceSynthetic Construct 61Trp Ile Asp Pro Asn Ser Gly Asp1
562108PRTArtificial SequenceSynthetic Construct 62Asp Ile Val Met
Thr Lys Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala
Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100
10563117PRTArtificial SequenceSynthetic Construct 63Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Arg Ile Asp Pro Asp Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11564108PRTArtificial
SequenceSynthetic Construct 64Asp Ile Val Met Thr Lys Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala Ser 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 10565117PRTArtificial
SequenceSynthetic Construct 65Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Asp
Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser 1156617PRTArtificial SequenceSynthetic
Construct 66Trp Ile Asp Pro Asp Ser Gly Asp Thr Asn Tyr Ala Gln Lys
Phe Gln1 5 10 15Gly678PRTArtificial SequenceSynthetic Construct
67Trp Ile Asp Pro Asp Ser Gly Asp1 568108PRTArtificial
SequenceSynthetic Construct 68Asp Ile Val Met Thr Lys Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala Ser 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 10569122PRTArtificial
SequenceSynthetic Construct 69Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser
Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Asp Thr Ile Pro Gly Ile Ala Val Ala Gly Thr Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120705PRTArtificial
SequenceSynthetic Construct 70Ser Tyr Tyr Met His1
57117PRTArtificial SequenceSynthetic Construct 71Ile Ile Asn Pro
Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln1 5 10
15Gly7213PRTArtificial SequenceSynthetic Construct 72Asp Thr Ile
Pro Gly Ile Ala Val Ala Gly Thr Asp Tyr1 5 107310PRTArtificial
SequenceSynthetic Construct 73Gly Tyr Thr Phe Thr Ser Tyr Tyr Met
His1 5 10748PRTArtificial SequenceSynthetic Construct 74Ile Ile Asn
Pro Ser Gly Gly Ser1 575110PRTArtificial SequenceSynthetic
Construct 75Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro
Gly Gln1 5 10 15Lys Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile
Gly Asn Asn 20 25 30Tyr Val Ser Trp Tyr Gln Gln Val Pro Gly Thr Pro
Pro Lys Leu Leu 35 40 45Ile Tyr Asp Asn Asp Lys Arg Pro Ser Gly Ile
Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu
Asp Ile Thr Gly Leu Gln65 70 75 80Thr Gly Asp Glu Ala Asp Tyr Tyr
Cys Gly Thr Trp His Ser Gly Leu 85 90 95Tyr Val
Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
1107613PRTArtificial SequenceSynthetic Construct 76Ser Gly Ser Thr
Ser Asn Ile Gly Asn Asn Tyr Val Ser1 5 10778PRTArtificial
SequenceSynthetic Construct 77Tyr Asp Asn Asp Lys Arg Pro Ser1
57811PRTArtificial SequenceSynthetic Construct 78Gly Thr Trp His
Ser Gly Leu Tyr Val Val Val1 5 107911PRTArtificial
SequenceSynthetic Construct 79Gly Ser Thr Ser Asn Ile Gly Asn Asn
Tyr Val1 5 10805PRTArtificial SequenceSynthetic Construct 80Tyr Asp
Asn Asp Lys1 58110PRTArtificial SequenceSynthetic Construct 81Thr
Trp His Ser Gly Leu Tyr Val Val Val1 5 1082330PRTArtificial
SequenceSynthetic Construct 82Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230
235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 33083106PRTArtificial SequenceSynthetic
Construct 83Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln1 5 10 15Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr 20 25 30Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser 35 40 45Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 50 55 60Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys65 70 75 80His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 85 90 95Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 100 10584351DNAArtificial SequenceSynthetic Construct
84gaggtccagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc
60tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcgaccctg acgaaggtga
cacaaactat 180gcacagaagt ttcagggcag ggtcaccatg accagggaca
cgtccatcag cacagcctac 240atggagctga gcaggctgag atctgacgac
acggccgtgt attactgtgc gagattagct 300agtggctttc gtgactactg
gggccaggga accctggtca ccgtctcgag c 35185324DNAArtificial
SequenceSynthetic Construct 85gacatcgtga tgaccaagtc tccatcctcc
ctgtctgctt ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattaga
aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct
catctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct
240gaagattttg caacttatta ctgtctacag catgatattt acgctagcac
tttcggccct 300gggaccaaag tggatatcaa acgt 32486351DNAArtificial
SequenceSynthetic Construct 86gaggtccagc tggtgcagtc tggggctgag
gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc
ggctactata tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggatgg atcaacccta acagtggtgg cacaaactat 180gcacagaagt
ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac
240atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc
gagattagct 300agtggctttc gtgactactg gggccaggga accctggtca
ccgtctcgag c 35187324DNAArtificial SequenceSynthetic Construct
87gacatcgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca
120gggaaagccc ctaagcgcct catctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca
caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag
catgatattt acgctagcac tttcggccct 300gggaccaaag tggatatcaa acgt
32488351DNAArtificial SequenceSynthetic Construct 88gaggtccagc
tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcgaccctg acagtggtga
cacaaactat 180gcacagaagt ttcagggcag ggtcaccatg accagggaca
cgtccatcag cacagcctac 240atggagctga gcaggctgag atctgacgac
acggccgtgt attactgtgc gagattagct 300agtggctttc gtgactactg
gggccaggga accctggtca ccgtctcgag c 35189324DNAArtificial
SequenceSynthetic Construct 89gacatcgtga tgaccaagtc tccatcctcc
ctgtctgctt ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattaga
aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct
catctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct
240gaagattttg caacttatta ctgtctacag catgatattt acgctagcac
tttcggccct 300gggaccaaag tggatatcaa acgt 32490366DNAArtificial
SequenceSynthetic Construct 90caggtccagc tggtacagtc tggggctgag
gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg catctggata caccttcacc
agctactata tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggaata atcaacccta gtggtggtag cacaagctac 180gcacagaagt
tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac
240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc
gagagacact 300attccgggta tagcagtggc tggtacggac tactggggcc
agggaaccct ggtcaccgtc 360tcgagc 36691330DNAArtificial
SequenceSynthetic Construct 91cagtctgtct tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcacctc caacattggc
aataattatg tatcctggta ccagcaggtc 120ccaggaacac cccccaaact
cctcatttat gacaatgata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tggacatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatggcata gtggcctgta
tgtcgtggtg 300ttcggcggag ggaccaagct gaccgtccta
33092990DNAArtificial SequenceSynthetic Construct 92gcctccacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
120tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 180ggactctact ccctcagcag cgtggtgacc gtgccctcca
gcagcttggg cacccagacc 240tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 300aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaagc cgctggggca 360ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
420gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 600gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
720atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 780gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 840ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc
tctccctgtc tccgggtaaa 99093318DNAArtificial SequenceSynthetic
Construct 93actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt
gaaatctgga 60actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa
agtacagtgg 120aaggtggata acgccctcca atcgggtaac tcccaggaga
gtgtcacaga gcaggacagc 180aaggacagca cctacagcct cagcagcacc
ctgacgctga gcaaagcaga ctacgagaaa 240cacaaagtct acgcctgcga
agtcacccat cagggcctga gctcgcccgt cacaaagagc 300ttcaacaggg gagagtgt
3189417PRTArtificial SequenceSynthetic ConstructVARIANT(1)..(1)Xaa
= Trp or ArgVARIANT(3)..(3)Xaa = Asn or AspVARIANT(5)..(5)Xaa = Asn
or AspVARIANT(6)..(6)Xaa = Ser or GluVARIANT(8)..(8)Xaa = Gly or
AspVARIANT(13)..(13)Xaa = Gln or Pro 94Xaa Ile Xaa Pro Xaa Xaa Gly
Xaa Thr Asn Tyr Ala Xaa Lys Phe Gln1 5 10 15Gly958PRTArtificial
SequenceSynthetic ConstructVARIANT(1)..(1)Xaa = Trp or
ArgVARIANT(3)..(3)Xaa = Asn or AspVARIANT(5)..(5)Xaa = Asn or
AspVARIANT(6)..(6)Xaa = Ser or GluVARIANT(8)..(8)Xaa = Gly or Asp
95Xaa Ile Xaa Pro Xaa Xaa Gly Xaa1 596117PRTArtificial
SequenceSynthetic ConstructVARIANT(50)..(50)Xaa = Trp or
ArgVARIANT(52)..(52)Xaa = Asn or AspVARIANT(54)..(54)Xaa = Asn or
AspVARIANT(55)..(55)Xaa = Ser or GluVARIANT(57)..(57)Xaa = Gly or
AspVARIANT(62)..(62)Xaa = Gln or ProVARIANT(87)..(87)Xaa = Arg or
SerVARIANT(109)..(109)Xaa = Gln or His 96Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Xaa Ile
Xaa Pro Xaa Xaa Gly Xaa Thr Asn Tyr Ala Xaa Lys Phe 50 55 60Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Arg Leu Xaa Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly Xaa Gly Thr
Leu 100 105 110Val Thr Val Ser Ser 11597108PRTArtificial
SequenceSynthetic ConstructVARIANT(6)..(6)Xaa = Gln or
LysVARIANT(27)..(27)Xaa = Gln or LeuVARIANT(66)..(66)Xaa = Gly, Val
or AlaVARIANT(72)..(72)Xaa = Thr or SerVARIANT(83)..(83)Xaa = Phe
or Leu 97Asp Ile Val Met Thr Xaa Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Xaa Gly Ile
Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Xaa Ser Gly Thr Glu Phe Xaa Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Xaa Ala Thr Tyr Tyr Cys
Leu Gln His Asp Ile Tyr Ala Ser 85 90 95Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys Arg 100 10598625PRTHomo sapiens 98Met Ile Phe Leu
Tyr Gln Val Val His Phe Ile Leu Phe Thr Ser Val1 5 10 15Ser Gly Glu
Cys Val Thr Gln Leu Leu Lys Asp Thr Cys Phe Glu Gly 20 25 30Gly Asp
Ile Thr Thr Val Phe Thr Pro Ser Ala Lys Tyr Cys Gln Val 35 40 45Val
Cys Thr Tyr His Pro Arg Cys Leu Leu Phe Thr Phe Thr Ala Glu 50 55
60Ser Pro Ser Glu Asp Pro Thr Arg Trp Phe Thr Cys Val Leu Lys Asp65
70 75 80Ser Val Thr Glu Thr Leu Pro Arg Val Asn Arg Thr Ala Ala Ile
Ser 85 90 95Gly Tyr Ser Phe Lys Gln Cys Ser His Gln Ile Ser Ala Cys
Asn Lys 100 105 110Asp Ile Tyr Val Asp Leu Asp Met Lys Gly Ile Asn
Tyr Asn Ser Ser 115 120 125Val Ala Lys Ser Ala Gln Glu Cys Gln Glu
Arg Cys Thr Asp Asp Val 130 135 140His Cys His Phe Phe Thr Tyr Ala
Thr Arg Gln Phe Pro Ser Leu Glu145 150 155 160His Arg Asn Ile Cys
Leu Leu Lys His Thr Gln Thr Gly Thr Pro Thr 165 170 175Arg Ile Thr
Lys Leu Asp Lys Val Val Ser Gly Phe Ser Leu Lys Ser 180 185 190Cys
Ala Leu Ser Asn Leu Ala Cys Ile Arg Asp Ile Phe Pro Asn Thr 195 200
205Val Phe Ala Asp Ser Asn Ile Asp Ser Val Met Ala Pro Asp Ala Phe
210 215 220Val Cys Gly Arg Ile Cys Thr His His Pro Gly Cys Leu Phe
Phe Thr225 230 235 240Phe Phe Ser Gln Glu Trp Pro Lys Glu Ser Gln
Arg Asn Leu Cys Leu 245 250 255Leu Lys Thr Ser Glu Ser Gly Leu Pro
Ser Thr Arg Ile Lys Lys Ser 260 265 270Lys Ala Leu Ser Gly Phe Ser
Leu Gln Ser Cys Arg His Ser Ile Pro 275 280 285Val Phe Cys His Ser
Ser Phe Tyr His Asp Thr Asp Phe Leu Gly Glu 290 295 300Glu Leu Asp
Ile Val Ala Ala Lys Ser His Glu Ala Cys Gln Lys Leu305 310 315
320Cys Thr Asn Ala Val Arg Cys Gln Phe Phe Thr Tyr Thr Pro Ala Gln
325 330 335Ala Ser Cys Asn Glu Gly Lys Gly Lys Cys Tyr Leu Lys Leu
Ser Ser 340 345 350Asn Gly Ser Pro Thr Lys Ile Leu His Gly Arg Gly
Gly Ile Ser Gly 355 360 365Tyr Thr Leu Arg Leu Cys Lys Met Asp Asn
Glu Cys Thr Thr Lys Ile 370 375 380Lys Pro Arg Ile Val Gly Gly Thr
Ala Ser Val Arg Gly Glu Trp Pro385 390 395 400Trp Gln Val Thr Leu
His Thr Thr Ser Pro Thr Gln Arg His Leu Cys 405 410 415Gly Gly Ser
Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala Ala His Cys 420 425 430Phe
Tyr Gly Val Glu Ser Pro Lys Ile Leu Arg Val Tyr Ser Gly Ile 435 440
445Leu Asn Gln Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe Gly Val Gln
450 455 460Glu Ile Ile Ile His Asp Gln Tyr Lys Met Ala Glu Ser Gly
Tyr Asp465 470 475 480Ile Ala Leu Leu Lys Leu Glu Thr Thr Val Asn
Tyr Thr Asp Ser Gln 485 490 495Arg Pro Ile Cys Leu Pro Ser Lys Gly
Asp Arg Asn Val Ile Tyr Thr 500 505 510Asp Cys Trp Val Thr Gly Trp
Gly Tyr Arg Lys Leu Arg Asp Lys Ile 515 520 525Gln Asn Thr Leu Gln
Lys Ala Lys Ile Pro Leu Val Thr Asn Glu Glu 530 535 540Cys Gln Lys
Arg Tyr Arg Gly His Lys Ile Thr His Lys Met Ile Cys545 550 555
560Ala Gly Tyr Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly
565 570 575Gly Pro Leu Ser Cys Lys His Asn Glu Val Trp His Leu Val
Gly Ile 580 585 590Thr Ser Trp Gly Glu Gly Cys Ala Gln Arg Glu Arg
Pro Gly Val Tyr 595 600 605Thr Asn Val Val Glu Tyr Val Asp Trp Ile
Leu Glu Lys Thr Gln Ala 610 615 620Val62599262PRTArtificial
SequenceSynthetic Construct 99Met Gly Trp Ser Cys Ile Ile Leu Phe
Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ile Val Gly Gly Thr
Ala Ser Val Arg Gly Glu Trp Pro 20 25 30Trp Gln Val Thr Leu His Thr
Thr Ser Pro Thr Gln Arg His Leu Cys 35 40 45Gly Gly Ser Ile Ile Gly
Asn Gln Trp Ile Leu Thr Ala Ala His Cys 50 55 60Phe Tyr Gly Val Glu
Ser Pro Lys Ile Leu Arg Val Tyr Ser Gly Ile65 70 75 80Leu Gln Gln
Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe Gly Val Gln 85 90 95Glu Ile
Ile Ile His Asp Gln Tyr Lys Met Ala
Glu Ser Gly Tyr Asp 100 105 110Ile Ala Leu Leu Lys Leu Glu Thr Thr
Val Gln Tyr Thr Asp Ser Gln 115 120 125Arg Pro Ile Ser Leu Pro Ser
Lys Gly Asp Arg Asn Val Ile Tyr Thr 130 135 140Asp Cys Trp Val Thr
Gly Trp Gly Tyr Arg Lys Leu Arg Asp Lys Ile145 150 155 160Gln Asn
Thr Leu Gln Lys Ala Lys Ile Pro Leu Val Thr Asn Glu Glu 165 170
175Cys Gln Lys Arg Tyr Arg Gly His Lys Ile Thr His Lys Met Ile Cys
180 185 190Ala Gly Tyr Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp
Ala Gly 195 200 205Gly Pro Leu Ser Cys Lys His Asn Glu Val Trp His
Leu Val Gly Ile 210 215 220Thr Ser Trp Gly Glu Gly Cys Ala Gln Arg
Glu Arg Pro Gly Val Tyr225 230 235 240Thr Asn Val Val Glu Tyr Val
Asp Trp Ile Leu Glu Lys Thr Gln Ala 245 250 255His His His His His
His 260100243PRTArtificial SequenceSynthetic Construct 100Ile Val
Gly Gly Thr Ala Ser Val Arg Gly Glu Trp Pro Trp Gln Val1 5 10 15Thr
Leu His Thr Thr Ser Pro Thr Gln Arg His Leu Cys Gly Gly Ser 20 25
30Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala Ala His Cys Phe Tyr Gly
35 40 45Val Glu Ser Pro Lys Ile Leu Arg Val Tyr Ser Gly Ile Leu Gln
Gln 50 55 60Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe Gly Val Gln Glu
Ile Ile65 70 75 80Ile His Asp Gln Tyr Lys Met Ala Glu Ser Gly Tyr
Asp Ile Ala Leu 85 90 95Leu Lys Leu Glu Thr Thr Val Gln Tyr Thr Asp
Ser Gln Arg Pro Ile 100 105 110Ser Leu Pro Ser Lys Gly Asp Arg Asn
Val Ile Tyr Thr Asp Cys Trp 115 120 125Val Thr Gly Trp Gly Tyr Arg
Lys Leu Arg Asp Lys Ile Gln Asn Thr 130 135 140Leu Gln Lys Ala Lys
Ile Pro Leu Val Thr Asn Glu Glu Cys Gln Lys145 150 155 160Arg Tyr
Arg Gly His Lys Ile Thr His Lys Met Ile Cys Ala Gly Tyr 165 170
175Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ala Gly Gly Pro Leu
180 185 190Ser Cys Lys His Asn Glu Val Trp His Leu Val Gly Ile Thr
Ser Trp 195 200 205Gly Glu Gly Cys Ala Gln Arg Glu Arg Pro Gly Val
Tyr Thr Asn Val 210 215 220Val Glu Tyr Val Asp Trp Ile Leu Glu Lys
Thr Gln Ala His His His225 230 235 240His His
His101229PRTArtificial SequenceSynthetic Construct 101Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Trp Ile Asp Pro Asp Glu Gly Asp Thr Asn Tyr Ala Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Leu Ala Ser Gly Phe Arg Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185
190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly
Ser His 210 215 220His His His His His225102213PRTArtificial
SequenceSynthetic Construct 102Asp Ile Val Met Thr Lys Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln His Asp Ile Tyr Ala Ser 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys Thr Val Ala Ala Pro 100 105
110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly
Glu Cys 210103330PRTArtificial SequenceSynthetic Construct 103Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330
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