USO09592,331 B2
(12) United States Patent (10) Patent No.: US 9,592,331 B2 Kussie et al. (45) Date of Patent: Mar. 14, 2017
(54) METHODS AND SYSTEMIS FOR TREATING 2007/0065425 A1 3/2007 Behrens et al. ECLAMPSA OR PRE-ECLAMPSA 2007/0202113 A1 8/2007 Young et al. 2007/0231.333 A1 10/2007 Boghaert et al. 2008/O177045 A1 7/2008 Lee et al. (75) Inventors: Paul Kussie, New York, NY (US); Woo 2009,0169547 A1 7/2009 Sahin et al. S. Joo, New York, NY (US) 2009,0286271 A1 1 1/2009 Karumanchi et al. (73) Assignee: AGGAMIN LLC, New York, NY (US) FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this WO 2004/0O8946 A2 1, 2004 patent is extended or adjusted under 35 WO 2006/076467 A2 T 2006 U.S.C. 154(b) by 464 days. WO 2010.075475 A1 T 2010 (21) Appl. No.: 13/984,262 OTHER PUBLICATIONS (22) PCT Fed: Feb. 7, 2012 Office Action issued in counterpart Japanese Patent Application No. 2013-552737, dated Feb. 8, 2016. (86) PCT No.: PCT/US2O12/O24,198 Barleon, B. et al., “Mapping of the Sites for Ligand Binding and Receptor Dimerization at the Extracellular Domain of the Vascular S 371 (c)(1), Endothelial Growth Factor Receptor FLT-1’, J. Bio. Chem., (1997), (2), (4) Date: Nov. 15, 2013 vol. 272:16, pp. 10382-10388. Davis-Smyth, T. et al., “The second immunoglobulin-like domain of (87) PCT Pub. No.: WO2O12A1092.82 the VEGF tyrosine kinase receptor Flt-1 determines ligand binding PCT Pub. Date: Aug. 16, 2012 and may initiate a signal transduction cascade', EMBO J. (1996), vol. 15:18, pp. 4919-4927. (65) Prior Publication Data Kendall, R. et al., “Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor'. US 2014/OO6515O A1 Mar. 6, 2014 PNAS (1993), vol. 90, pp. 10705-10709. Thomas, C. et al., “Itronic polyadenylation signal sequences and Related U.S. Application Data alternate splicing generate human soluble Flt 1 variants and regulate Provisional application No. 61/440,169, filed on Feb. the abundance of soluble Flt1 in the placenta'. FASEB J. (2007), (60) vol. 21 (14):3885-3895. 7, 2011. Belgore, F.M. et al., “Measurement of free and complexed soluble vascular endothelial growth factor receptor, Flt-I, in fluid samples: (51) Int. C. development and application of two new immunoassays”. Clinical A 6LX 39/395 (2006.01) Science (2000), vol. 100, pp. 567-575. C07K 6/8 (2006.01) Zhou, C.C. et al., “Angiotensin II Induces Soluble fms-Like Tyro C07K 6/28 (2006.01) sine Kinase-1 Release via Calcineurin Signaling Pathway in Preg A6M I/34 (2006.01) nancy'. Circulation Research (2007), vol. 100, pp. 88-95. Epub C07K I4/7 (2006.01) Dec. 7, 2006. U.S. C. Zhou, C.C. et al., “Angiotensin II Induces Soluble fms-Like Tyro (52) sine Kinase-1 Release via Calcineurin Signaling Pathway in Preg CPC ...... A61M 1/34 (2013.01); A61M 1/3486 nancy'. Circulation Research (2007), vol. 100, pp. 88-95. Online (2014.02); C07K 14/71 (2013.01); C07K Supplemental published Feb. 20, 2007. I6/2863 (2013.01); C07K 2317/24 (2013.01); C07K 2317/41 (2013.01); C07K 2317/52 Primary Examiner — Christine J Saoud (2013.01); C07K 2317/71 (2013.01); C07K Assistant Examiner — Jon M Lockard 2317/734 (2013.01); C07K 2317/92 (2013.01) (74) Attorney, Agent, or Firm — Fox Rothschild LLP (58) Field of Classification Search None (57) ABSTRACT See application file for complete search history. Disclosed are methods and apparatuses for treating a preg (56) References Cited nancy related hypertensive disorder, such as pre-eclampsia and eclampsia, using ex vivo treatment with an anti-SElt-1 U.S. PATENT DOCUMENTS receptor (sElt-1) antibody bound to a solid support in order to reduce blood levels of silt-1. Further disclosed are the 7,335,362 B2 2/2008 Karumanchi et al. 7,407,659 B2 8/2008 Karumanchi et al. sequences of the heavy chain and light chain CDRs of the 7,435,419 B2 10/2008 Karumanchi et al. anti-sfilt-1 antibodies. 2003, OO86924 A1 5, 2003 Sliwkowski 2006, OO67937 A1 3/2006 Karumanchi et al. 19 Claims, 8 Drawing Sheets U.S. Patent Mar. 14, 2017. Sheet 1 of 8 US 9,592,331 B2
PASMA ceil. SEFARAOR
sit-' PEFLETED Plasma
Fig. 1 U.S. Patent Mar. 14, 2017 Sheet 2 of 8 US 9,592,331 B2
A % sFitl depleted
S % SFlt1 depleted
Kid (M-1)
1.
SK 1.
Fig. 2 U.S. Patent Mar. 14, 2017 Sheet 3 of 8 US 9,592,331 B2
*
Fig. 3 U.S. Patent Mar. 14, 2017. Sheet 4 of 8 US 9,592,331 B2
Depletion of sFlt 1 by A G1 OB Waries with Flow Rate 19 ------sc $
------r Š urg ; r. 3. s
------40% ...... & 96 SFIt1 depletion in two
O5 1 O 15 2.5 3. Flow Rate mLimin Fig. 4
Depletion of sFlt1 by A G1 OB Waries with Linear Flow Rate
------&% SFlt1 depletion
Linear Flow Rate cmir Fig. 5 U.S. Patent Mar. 14, 2017. Sheet 5 of 8 US 9,592,331 B2
Depletion of sf till by AG1(B varies with residence tire
$ 8 SFt 1 depletion
Residence Fine in Fig. 6
%sFit-1 Depletion Activity Varies with MAb density % cyc
SO: ------$------. SOE. S.....&.------. % ------. 5%. ------. S& ------4% ------$98 SFlt1 depletion 30% -sc is a ------i ------
Y------,------
MAb density ng MAbfirl beads) Fig. 7 U.S. Patent Mar. 14, 2017. Sheet 6 of 8 US 9,592,331 B2
SEx
s' ...... Š ...... s S S S -ing,10% ...S s.S.1909.1599.Sri 20 –20% i.------MAE : stl Ratio wiy Fig. 8
100% :------s Š 90%04 soar. &Ša...... $ ...... N 80% Š so 70% Š S S. 60% :------59 ------40% ir &% SFlt 1 depletion s s 39 ------1) ------O. : ------O 1OO O 3OO 4. 5OO A GOB:SFlt-1Ratio w w Fig. 9 U.S. Paten Mar. 14, 2017 Sheet 7 of 8
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Fig. 10
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US 9,592,331 B2 1. 2 METHODS AND SYSTEMIS FOR TREATING VEGF-E, VEGF189, VEGF165, or VEGF 121); see Tischer ECLAMPSA OR PRE-ECLAMPSA et al., 1991, J. Biol. Chem. 266, 11947-11954; Neufed et al., 1996, Cancer Metastasis 15:153-158; U.S. Pat. No. 6,447, CROSS-REFERENCE TO RELATED 768: U.S. Pat. No. 5,219,739; and U.S. Pat. No. 5,194,596. APPLICATIONS Also known are mutant forms of VEGF such as the KDR Selective VEGF and Flt-selective VEGF described in Gille This application claims priority to U.S. Application No. et al., 2001, J. Biol. Chem. 276:3222-3230. Modified forms 61/440,169, filed Feb. 7, 2011, which is incorporated herein of VEGF are described in LeCouter et al., 2003, Science by reference in its entirety. 299:890-893. 10 VEGF binds as a homodimer to two homologous mem FIELD OF THE INVENTION brane-spanning tyrosine kinase receptors, the fms-like tyro sine kinase (Flt-1) and the kinase domain receptor (KDR), The present invention relates to methods, systems, which are differentially expressed in endothelial cells devices, and apparatuses for treating pregnancy-related obtained from many different tissues. GenBank accession hypertensive disorders such as pre-eclampsia and eclampsia. 15 number AF063657 provides the nucleotide and amino acid sequences of human Flt-1. Flt-1, but not KDR, is highly BACKGROUND OF THE INVENTION expressed by trophoblast cells which contribute to placental formation. PIGF is a VEGF family member that is also Pre-eclampsia is a syndrome of hypertension, edema, and involved in placental development. PlGF is expressed by proteinuria that affects 5 to 10% of pregnancies and results cytotrophoblasts and syncytiotrophoblasts and is capable of in substantial maternal and fetal morbidity and mortality. inducing proliferation, migration, and activation of endothe Pre-eclampsia accounts for at least 200,000 maternal deaths lial cells. PlGF binds as a homodimer to the Flt-1 receptor, worldwide per year. The symptoms of pre-eclampsia typi but not to the KDR receptor. Both PIGF and VEGF con cally appear after the 20th week of pregnancy and are tribute to the mitogenic activity and angiogenesis that are usually detected by the routine monitoring of blood pressure 25 critical for the developing placenta. and urine. However, these monitoring methods are ineffec SFlt-1, which lacks the transmembrane and cytoplasmic tive for diagnosis of pre-eclampsia at an early stage, which domains of the full-length Flt-1 receptor, was identified in could reduce the risk to the subject or developing fetus, if an the culture medium of human umbilical vein endothelial effective treatment were available. cells and the in vivo expression of sRlt-1 was subsequently Symptoms of pre-eclampsia generally include any of the 30 demonstrated in placental tissue. s.lt-1 binds to VEGF with following: (1) a systolic blood pressure (BP)>140 mmHg high affinity but does not stimulate mitogenesis of endothe and a diastolic BP>90 mmHg after 20 weeks gestation, (2) lial cells. The elevated levels of sRlt-1 found in the serum new onset proteinuria (1 + by dipstik on urinanalysis, >300 samples taken from pregnant women Suffering from, or at mg of protein in a 24 hour urine collection, or random urine risk of developing, a pregnancy-related hypertensive disor protein/creatinine ratio-0.3), or (3) resolution of hyperten 35 der (e.g., pre-eclampsia or eclampsia) indicate that SFlt-1 is sion and proteinuria by 12 weeks postpartum. The symptoms acting as a “physiologic sink to bind to and deplete the of pre-eclampsia can also include renal dysfunction and trophoblast cells and maternal endothelial cells of functional glomerular endotheliosis or hypertrophy. Other symptoms of growth factors required for the proper development and eclampsia may be any of the following symptoms due to angiogenesis of the fetus and/or the placenta. pregnancy or the influence of a recent pregnancy: seizures, 40 coma, thrombocytopenia, liver edema, pulmonary edema, or SUMMARY OF THE INVENTION cerebral edema. Pre-eclampsia can vary in severity from mild to life The present invention provides a method of treating or threatening. A mild form of pre-eclampsia may be treated preventing a disorder associated with SFlt-1. Such as a with bed rest and frequent monitoring. For moderate to 45 pregnancy-related hypertensive disorder in a subject com severe cases, hospitalization is recommended and blood prising providing ex vivo to the Subject anti-SFlt-1 antibod pressure medications or anticonvulsant medications to pre ies, or SFlt-1 binding fragments thereof, or an SFlt-1 ligand, vent seizures are prescribed. If the condition becomes life in an amount Sufficient and for a time Sufficient to decrease threatening to the mother or the fetus, the pregnancy is the subject’s blood levels of slfilt-1 to treat or prevent the terminated and the fetus is delivered pre-term. 50 disorder associated with sElt-1 in the subject. Several factors have been reported to have an association In certain embodiments, the method comprises removing with fetal and placental development and pre-eclampsia. a volume of the subjects blood, bringing the blood or a They include vascular endothelial growth factor (VEGF), component thereof (e.g., plasma) into contact with the soluble Flt-1 receptor (sElt-1), and placental growth factor anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, (PlGF). VEGF is an endothelial cell-specific mitogen, an 55 or sPlt-1 ligands, where the anti-slfilt-1 antibodies, or sPlt-1 angiogenic inducer, and a mediator of vascular permeability. binding fragments thereof, or SFlt-1 ligands, are bound to a VEGF has also been shown to be important for glomerular solid support, to bind sPlt-1 in the subject’s blood or capillary repair. VEGF is disclosed in U.S. Pat. No. 5,332, component thereof to the anti-slfilt-1 antibodies, or sPlt-1 671; U.S. Pat. No. 5,240,848; and U.S. Pat. No. 5,194,596; binding fragments thereof, or SFlt-1 ligands, thereby as well as in Charnock-Jones et al., 1993, Biol. Reproduc 60 decreasing the amount of sRlt-1 in the subjects blood or tion, 48: 1120-1128. VEGF exists as a glycosylated homodi component thereof, and returning the blood or component mer and includes at least four different alternatively spliced thereof to the subject. isoforms. The biological activity of native VEGF includes The invention provides anti-slfilt-1 antibodies and slfilt-1 the promotion of selective growth of vascular endothelial binding fragments thereof. The antibodies are used in the cells or umbilical vein endothelial cells and induction of 65 aforementioned ex vivo methods, and can also be adminis angiogenesis. VEGF includes several family members or tered to a subject. In certain embodiments, the anti-slfilt-1 isoforms (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, antibodies, or SElt-1 binding fragments thereof, comprise US 9,592,331 B2 3 4 one, two, or three heavy chain CDRs having SEQID NO:18, having SEQID NO:8, SEQID NO:10, and SEQID NO:12. SEQ ID NO:20, and SEQ ID NO:22 and one, two, or three In certain embodiments, the anti-sflt-1 antibodies compete light chain CDRs having SEQ ID NO:24, SEQ ID NO:26, for binding with an antibody which comprises one, two, or and SEQ ID NO:28. In certain embodiments, the sRlt-1 three heavy chain CDRs having substantially the same antibodies comprise one, two, or three heavy chain CDRs sequence as SEQ ID NO:2, SEQ ID NO:4, and SEQ ID having substantially the same sequence as SEQ ID NO:18, NO:6 and one, two, or three light chain CDRs having SEQ ID NO:20, and SEQ ID NO:22 and one, two, or three substantially the same sequence as SEQ ID NO:8, SEQ ID light chain CDRs having Substantially the same sequence as NO:10, and SEQ ID NO:12. In some embodiments, the SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28. In anti-SElt-1 antibodies or binding fragments thereof compete some embodiments, the anti-sflt-1 antibodies or binding 10 for binding with an antibody which comprises at least one fragments thereof comprise at least one variable region with variable region with an amino acid sequence selected from an amino acid sequence selected from SEQID NOS: 30 and SEQ ID NOS: 14 and 16, or a sequence at least 85% or at 32, or a sequence at least 85% or at least 90% identical least 90% identical thereto. thereto. In certain embodiments, the pregnancy-related hyperten In certain embodiments, the anti-sfilt-1 antibodies, or 15 sive disorder is eclampsia or pre-eclampsia. In certain SFlt-1 binding fragments thereof, comprise one, two, or embodiments, the pregnancy-related hypertensive disorder three heavy chain CDRs having SEQ ID NO:2, SEQ ID is pre-eclampsia. In certain embodiments, the SFlt-1 related NO:4, and SEQID NO:6 and one, two, or three light chain disorder is kidney disease. CDRs having SEQ ID NO:8, SEQ ID NO:10, and SEQ ID In certain embodiments, the blood or a component thereof NO:12. In certain embodiments, the anti-sfilt-1 antibodies is plasma and the method comprises removing a Volume of comprise one, two, or three heavy chain CDRS having the Subjects blood and separating the blood into plasma and substantially the same sequence as SEQ ID NO:2, SEQ ID cellular components before contacting the plasma with anti NO:4, and SEQID NO:6 and one, two, or three light chain SFlt-1 antibodies, or SFlt-1 antigen binding fragments CDRs having substantially the same sequence as SEQ ID thereof, bound to a solid support. NO:8, SEQ ID NO:10, and SEQ ID NO:12. In some such 25 In certain embodiments, the Subject is a pregnant human, embodiments, the anti-slfilt-1 antibodies or binding frag a post-partum human, or a non-human (e.g., a cow, a horse, ments thereof comprise at least one variable region with an a sheep, a pig, a goat, a dog, or a cat). In certain embodi amino acid sequence selected from SEQID NOS: 14 and 16, ments, the Subject is a pregnant human or a post-partum or a sequence at least 85% or at least 90% identical thereto. human. In certain embodiments, the Subject is a pregnant In certain embodiments of the invention, the anti-sfilt-1 30 human. antibodies do not block ligand binding to sRlt-1. SElt-1 The present invention provides a system comprising anti ligands include PIGF, VEGF, including their isoforms. In sElt-1 antibodies, or sPlt-1 antigen binding fragments certain embodiments, the anti-sfilt-1 antibodies, or sPlt-1 thereof, or sPlt-1 ligands, bound to a solid support, first binding fragments thereof, bind to an epitope in sElt-1 that means for conveying blood from a subject to the anti-slfilt-1 is not present in Flt-1. In certain embodiments, the anti 35 antibodies, or SFlt-1 antigen binding fragments thereof, or sElt-1 antibodies, or sPlt-1 binding fragments thereof, bind SFlt-1 ligands, bound to the Solid Support so as to contact the to an epitope that includes amino acids from the carboxy blood with the anti-slfilt-1 antibodies, or sPlt-1 antigen terminus of an sElt-1 isoform. In certain embodiments, the binding fragments thereof, and thereby remove sRlt-1 from anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, the blood, and second means for conveying the blood to the bind to one or more of domains 1-3 of human sElt-1. 40 subject following contact of the blood with the anti-slfilt-1 It is observed that the ability of an antibody to deplete antibodies, or SFlt-1 antigen binding fragments thereof. sElt-1 from blood or a component thereof is not necessarily In certain embodiments of the present invention, plasma, dependent on binding affinity, and may be influenced by the rather than blood, is contacted with anti-sfilt-1 antibodies, or region of sfilt-1 to which the antibody binds. In certain SFlt-1 antigen binding fragments thereof, or SFlt-1 ligands, embodiments of the invention, the anti-sflt-1 antibodies or 45 bound to a solid Support, in order to treat or prevent a sElt-1 binding fragments thereof compete for binding with pregnancy-related hypertensive disorder. Accordingly, in an antibody which comprises one, two, or three heavy chain certain embodiments, the first means includes a device for CDRs having SEQID NO:18, SEQID NO:20, and SEQID separating the Subject’s blood into plasma and cellular NO:22 and one, two, or three light chain CDRs having SEQ components. ID NO:24, SEQ ID NO:26, and SEQ ID NO:28. In certain 50 In certain embodiments, the first means comprises an embodiments, the anti-slfilt-1 antibodies compete for binding access device. Such as a catheter, needle, cannula, or the like, with an antibody which comprises one, two, or three heavy inserted into a blood vessel of the subject, for accessing the chain CDRs having Substantially the same sequence as SEQ Subject’s blood system, a conduit System, such as tubing, ID NO:18, SEQ ID NO:20, and SEQ ID NO:22 and one, piping, hollow fibers, or the like, which fluidly connects the two, or three light chain CDRs having substantially the same 55 access device to the anti-slfilt-1 antibodies, or sPlt-1 antigen sequence as SEQ ID NO:24, SEQ ID NO:26, and SEQ ID binding fragments thereof, bound to the Solid Support, NO:28. In some embodiments, the anti-sflt-1 antibodies or thereby allowing the subject’s blood to flow to and contact binding fragments thereof compete for binding with an the anti-SFlt-1 antibodies, or SFlt-1 antigen binding frag antibody which comprises at least one variable region with ments thereof, and, optionally, a pump (e.g., a peristaltic an amino acid sequence selected from SEQID NOS: 30 and 60 pump) or the like, for moving blood from the subject 32, or a sequence at least 85% or at least 90% identical through the access device and conduit system to the anti thereto. SFlt-1 antibodies, or SFlt-1 antigen binding fragments In certain embodiments of the invention, the anti-sfilt-1 thereof. antibodies or sPlt-1 binding fragments thereof compete for In certain embodiments, the second means comprises a binding with an antibody which comprises one, two, or three 65 conduit system, such as tubing, piping, hollow fibers, or the heavy chain CDRs having SEQID NO:2, SEQID NO:4, and like, and a return device. Such as a catheter, needle, cannula, SEQ ID NO:6 and one, two, or three light chain CDRs or the like, where the return device is inserted into a blood US 9,592,331 B2 5 6 vessel (e.g., a vein) of the Subject, where the conduit system FIG. 5 shows the effect of linear flow rate on SFlt-1 fluidly connects the blood or plasma in contact with the depletion by antibody AG10B. anti-SElt-1 antibodies, or SElt-1 antigen binding fragments FIG. 6 shows the effect of residence time on sElt-1 thereof, or sPlt-1 ligands, to the return device so as to allow depletion by antibody AG10B. for the return of the blood or plasma to the subject. Option FIG. 7 shows the effect of AG10B density on sElt-1 ally, the second means also comprises a pump (e.g., a depletion. peristaltic pump) or the like, for moving the blood or plasma FIG.8 shows depletion of slfilt-1 from plasma over a range from the anti-slfilt-1 antibodies, or sPlt-1 antigen binding of AG10B:sFlt-1 ratios. A non-specific antibody, Erbitux, fragments thereof, or SElt-1 ligands, through the conduit does not deplete sRlt-1, indicating that the effect of AG10B system to the return device. This pump or the like may be the 10 is specific. same pump or the like that is part of the first means or, FIG. 9 shows depletion of slfilt-1 from serum over a range alternatively, the motive force for the second means for of AG1OB:SFlt-1 ratios. conveying the blood or plasma to the Subject may be a FIG. 10 shows depletion of sRlt-1 is not affected by separate pump or the like, specific to the second means. column bed volume. In certain embodiments, the device for separating a Sub 15 FIG. 11 shows silt-1 depletion by antibody AG10B in the jects blood into plasma and cellular components is a presence of heparin. centrifuge or an apheresis device, e.g., a plasmapheresis FIG. 12 shows binding of antibody AG10B to sRlt-1 does device. not block VEGF binding. In certain embodiments, the first and/or second means FIG. 13 shows AG10B immobilized on Sepharose beads may also comprise one or more sensors for determining the does not activate the complement system. pressure and/or the flow rate of the blood in the conduit system. DETAILED DESCRIPTION OF THE The present invention also provides a column containing INVENTION anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, or SFlt-1 ligands, bound to a solid Support, where the column 25 The present invention provides a method of treating or is suitable for use in treating or preventing a pregnancy preventing an SFlt-1-related disease or disorder comprising related hypertensive disorder Such as eclampsia or pre providing ex vivo to the subject anti-slfilt-1 binding sub eclampsia. stances, including but not limited to SRlt-1 ligands and binding proteins, anti-SFlt-1 antibodies, and SFlt-1 binding BRIEF DESCRIPTION OF THE FIGURES 30 fragments thereof, in an amount Sufficient and for a time sufficient to decrease the subject’s blood levels of sfilt-1. In FIG. 1 shows a schematic depiction of one embodiment of one embodiment, the invention provides a method of treat the present invention where blood from a subject is sepa ing or preventing a pregnancy-related hypertensive disorder rated into plasma and cellular components, the cellular in a Subject having or at risk of developing a pregnancy components are returned to the Subject, the plasma is con 35 related hypertensive disorder and thus in need of treatment veyed to a column filled with SEPHAROSE(R) beads to or prevention for a pregnancy-related hypertensive disorder which anti-sflt-lantibodies have been attached such that comprising providing ex vivo to the Subject anti-SElt-1 contact with the anti-sflt-lantibodies depletes the plasma of binding Substances, including but not limited to SElt-1 sElt-1, and the sRlt-1-depleted plasma is returned to the ligands and binding proteins, anti-SFlt-1 antibodies, and Subject. 40 SFlt-1 binding fragments thereof, in an amount Sufficient and FIG. 2 illustrates the depletion of sRlt-1 from a solution for a time sufficient to decrease the subject’s blood levels of comprising SFlt-1 by the use of SFlt-1 binding compounds, SFlt-1, thereby treating or preventing the pregnancy-related including anti-slfilt-1 antibodies and VEGF bound to a hypertensive disorder in the subject. In another embodiment, Solid Support (panel A) and apparent K, measurements of the invention provides a method of treating pre-term labor. purified monoclonal antibodies and Flt-1 by ForteBio Octet 45 sElt-1 levels are typically elevated during the last several (panel B). weeks of a normal pregnancy, and may not be accompanied FIG. 3 shows one embodiment of a column comprising by a hypertensive disorder. Accordingly, the invention is anti-SElt-1 antibodies, or anti-SFlt-1 antigen binding frag used to treat non-hypertensive sRlt-1-related disorders of ments thereof, bound to a solid Support. The column com late stage pregnancy and labor or prophylactically to avoid prises a cylindrical housing 1 and two connecting caps 2 and 50 Such disorders. In another embodiment, the invention pro 3, where cap 2 is connected to a means for delivering blood vides a method of treating or preventing chronic kidney or plasma from a subject to the anti-slfilt-1 antibodies, or disease. SFlt-1 antigen binding fragments thereof, bound to the solid “Soluble Flt-1 (sElt-1) (also known as sVEGF-R1) refers Support, and cap 3 is connected to a means for returning the to a soluble form of the Flt-1 receptor that is identical or sElt-1-depleted blood or plasma to the subject following 55 homologous to the protein defined by GenBank accession contact of the blood or plasma with the anti-slfilt-1 antibod number AF063657, and has sRlt-1 biological activity. The ies, or SFlt-1 antigenbinding fragments thereof, bound to the biological activity of SFlt-1 may be assayed using any Solid Support. Upper disk 4 is a barrier inserted into cap 2 standard method, for example, by assaying SFlt-1 binding to which keeps the solid support 5 away from the inlet opening. VEGF. SElt-1 lacks the transmembrane domain and the A similar disk is present in lower cap 3 but is not shown. 60 cytoplasmic tyrosine kinase domain of the Flt-1 receptor. Solid support 5 is depicted here in the form of beads, but sFlt-1 can bind to VEGF and PIGF with high affinity, but it may be any convenient shape. The anti-SFlt-1 antibodies are cannot induce proliferation orangiogenesis and is therefore not shown, but are bound to solid support 5. 1, 2, 3, and 4 functionally different from the Flt-1 and KDR receptors. are made of blood compatible synthetic materials and are sElt-1 was initially purified from human umbilical endothe interconnected by conventional techniques. 65 lial cells and later shown to be produced by trophoblast cells FIG. 4 shows the effect of flow rate on sElt-1 depletion by in vivo. As used herein, split-1 includes any slflt-1 family antibody AG10B. member or isoform. Non-limiting examples include split-1 US 9,592,331 B2 7 8 isoforms that are recognized to be splice variants. The splice SEQ ID NO:20, and SEQ ID NO:22 and one, two, or three variants have a common transcription start site, but do not light chain CDRs that are substantially identical to SEQ ID contain all 30 spliced exons that encode Flt-1. One isoform NO:24, SEQ ID NO:26, and SEQ ID NO:28. In certain of is encoded by an mRNA having the first 13 exons followed the embodiments, the anti-slfilt-1 antibodies or binding frag by a portion of intron 13 and a poly(A) signal sequence and 5 ments thereof comprise at least one variable region with an contains the first six Ig-like domains, but not the seventh amino acid sequence selected from SEQID NOS:30 and 32, Ig-like domain, transmembrane domain, or intracellular or a sequence at least 85% at least 90%, at least 95%, at least domain. (GenBank Accession No. AF063657; Kendall et al., 97%, at least 98%, or at least 99%, identical thereto. Proc. Natl. Acad. Sci. USA 1993, 90:10705-9). Another The invention further provides an isolated sRlt-1 antibody isoform is encoded by an mRNA having the first 14 exons 10 which comprises one, two, or three heavy chain CDRs followed by a new alternatively spliced terminal exon 15 having SEQID NO:2, SEQID NO:4, and SEQID NO:6 and and a poly(A) signal sequence. The isoform is truncated in one, two, or three light chain CDRs having SEQ ID NO:8, the seventh extracellular Ig-like domain (GenBank Acces SEQ ID NO:10, and SEQ ID NO:12, as well as an sElt-1 sion No. AI188382; Thomas et al., 2007, FASEB J. 21:3885 antibody comprising one, two, or three heavy chain CDRS 3895). Several other alternatively spliced mRNAs and their 15 that are substantially identical to SEQ ID NO:2, SEQ ID translation products have also been reported or predicted. NO:4, and SEQID NO:6 and one, two, or three light chain Each of these proteins contain unique C-terminal sequences CDRs that are substantially identical to SEQID NO:8, SEQ that include amino acids encoded by the alternatively spliced ID NO:10, and SEQ ID NO:12. In certain of the embodi 3' end of the mRNA up to the first translation termination ments, the anti-SFlt-1 antibodies or binding fragments codon. SFlt-1 can also mean degradation products or frag thereof comprise at least one variable region with an amino ments that result from enzymatic cleavage of the Flt-1 acid sequence selected from SEQ ID NOS: 14 and 16, or a receptor where such degradation products or fragments sequence at least 85% at least 90%, at least 95%, at least maintain SFlt-1 biological activity. In one example, specific 97%, at least 98%, or at least 99%, identical thereto. metalloproteinases released from the placenta may cleave “Identity” refers to the number or percentage of identical the extracellular domain of Flt-1 receptor to release the 25 positions shared by two amino acid or nucleic acid N-terminal portion of Flt-1 into circulation. sequences, taking into account the number of gaps, and the “Ex vivo” refers to practicing the methods of treatment or length of each gap, which need to be introduced for optimal prevention disclosed herein outside the body of a subject, alignment of the two sequences. “Substantially identical i.e., extracorporeally, whereby the subject’s blood or blood means an amino acid sequence that which differs only by component (e.g., plasma) is contacted with anti-SFlt-1 anti 30 conservative amino acid substitutions, for example, Substi bodies or sPlt-1 binding fragments thereof outside the body tution of one amino acid for another of the same class (e.g., of the subject. valine for glycine, arginine for lysine, etc.) or by one or more “Anti-slfilt-1 antibody” refers to an antibody that is non-conservative Substitutions, deletions, or insertions capable of binding to sRlt-1. “slfilt-1 binding fragment of an located at positions of the amino acid sequence which do not anti-slfilt-1 antibody refers to a portion of an anti-slfilt-1 35 destroy the function of the protein. Preferably, the amino antibody that retains the ability to bind sPlt-1. acid sequence is at least 80%, more preferably at least about “slfilt-1 ligand refers to a growth factor or derivative 85%, and most preferably at least about 90% similar to thereof that binds to sRlt-1. Naturally occurring sRlt-1 another amino acid sequence. Methods and computer pro ligands include, without limitation, vascular endothelial grams for determining sequence similarity are publically growth factor (VEGF), and placenta growth factor (P1GF). 40 available, including, but not limited to, the GCG program The VEGF is preferably VEGF-A or VEGF-B. VEGF package (Devereux et al., Nucleic Acids Research 12: 387, includes its isoforms, including without limitation, 1984), BLASTP. BLASTN, FASTA (Altschulet al., J. Mol. VEGF, VEGFs, and VEGFs. PlGF includes it iso Biol. 215:403 (1990), and the ALIGN program (version 2.0). forms, including without limitation, PlCF-1, PIGF-2. PlGF The well-known Smith Waterman algorithm may also be 3, and PlGF-4. Derivatives include without limitation VEGF 45 used to determine similarity. The BLAST program is pub and PlGF fusion proteins and sequence variants of VEGF licly available from NCBI and other sources (BLAST and PlGF that bind to SFlt-1. Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. “slfilt-1 binding substances include antibodies, antibody 20894; BLAST 2.0 at http://www.ncbi.nlm.nih.gov/blast/). fragments, ligands, and any other binding molecules (e.g., In comparing sequences, these methods account for various natural or synthetic proteins, polypeptides, and polymers) 50 Substitutions, deletions, and other modifications. Conserva that selectively bind to sRlt-1. tive substitutions typically include substitutions within the The antibodies of the invention are effective to efficiently following groups: glycine, alanine; Valine, isoleucine, leu deplete sRlt-1 in blood or plasma from a subject. The sRlt-1 cine; aspartic acid, glutamic acid, asparagine, glutamine: can be soluble, or in microparticles circulating in the blood serine, threonine; lysine, arginine; and phenylalanine, tyro stream. According to the invention, heparin can be admin 55 S1C. istered to the subject to release tissue-bound sPlt-1, enhanc It is observed herein that the ability of an antibody to ing ex vivo depletion of SFlt-1 and minimizing the pool of deplete sRlt-1 from blood or a component thereof is not non-circulating sRlt-1 left in the subject. necessarily dependent on binding affinity, but also can Non-limiting examples of antibody sequences are pro depends on certain other characteristics, such as the domains vided. The invention provides an isolated sRlt-1 antibody 60 or epitope of silt-1 to which the antibody binds. In certain (including SElt-1 binding fragments thereof) which com embodiments of the invention, the anti-sflt-1 antibodies or prises one, two, or three heavy chain CDRs having SEQID sElt-1 binding fragments of the invention compete for bind NO:18, SEQ ID NO:20, and SEQ ID NO:22 and one, two, ing with an antibody which comprises one, two, or three or three light chain CDRs having SEQ ID NO:24, SEQ ID heavy chain CDRs having SEQID NO:18, SEQID NO:20, NO:26, and SEQID NO:28. The invention also provides an 65 and SEQID NO:22 and one, two, or three light chain CDRs SFlt-1 antibody comprising one, two, or three heavy chain having SEQID NO:24, SEQID NO:26, and SEQID NO:28. CDRs that are substantially identical to SEQ ID NO:18, In certain embodiments, the anti-sflt-1 antibodies compete US 9,592,331 B2 9 10 for binding with an antibody which comprises one, two, or antibody, or sPlt-1 binding fragment thereof, that binds three heavy chain CDRs having substantially the same sElt-1 recognizes the extracellular domain of Flt-1. In certain sequence as SEQ ID NO:18, SEQ ID NO:20, and SEQ ID embodiments, an anti-slfilt-1 antibody, or sPlt-1 binding NO:22 and one, two, or three light chain CDRs having fragment thereof, recognizes an epitope in SFlt-1 that is not substantially the same sequence as SEQID NO:24, SEQID present in Flt-1. In certain embodiments, such an epitope not NO:26, and SEQID NO:28. In some such embodiments, the present in Flt-1 includes amino acids from the carboxy anti-SElt-1 antibodies or binding fragments thereof compete terminus of SFlt-1. In certain embodiments, such an epitope for binding with an antibody which comprises at least one not present in Flt-1 is a discontinuous epitope or a confor variable region with an amino acid sequence selected from mational epitope of slfilt-1. In certain embodiments, the SEQ ID NOS: 30 and 32, or a sequence at least 85% or at 10 anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, least 90% identical thereto. bind to the ligand binding site of Flt-1. In certain embodiments of the invention, the anti-sfilt-1 According to the invention, in certain embodiments, the antibodies or sPlt-1 binding fragments thereof compete for anti-slfilt-1 antibodies, or sPlt-1-binding fragments thereof, binding with an antibody which comprises one, two, or three are particularly suitable for administration to a subject. For heavy chain CDRs having SEQID NO:2, SEQID NO:4, and 15 example, the antibodies can be modified to minimize immu SEQ ID NO:6 and one, two, or three light chain CDRs nogenicity and/or hypersensitivity in a Subject. Such modi having SEQID NO:8, SEQID NO:10, and SEQID NO:12. fications can provide an additional safety factor in the event In certain embodiments, the anti-sflt-1 antibodies compete that antibodies are leached from a column or other solid for binding with an antibody which comprises one, two, or support used for ex vivo the treatment of a subject. Further, three heavy chain CDRs having substantially the same in certain embodiments, the sRlt-1 antibodies, or sPlt-1- sequence as SEQ ID NO:2, SEQ ID NO:4, and SEQ ID binding fragments thereof, can be administered in vivo to NO:6 and one, two, or three light chain CDRs having treat eclampsia or pre-eclampsia. Thus, for both ex vivo and substantially the same sequence as SEQ ID NO:8, SEQ ID in vivo treatment, antibodies used according to the invention NO:10, and SEQID NO:12. In some such embodiments, the include chimeric or humanized antibodies, as well as antigen anti-SElt-1 antibodies or binding fragments thereof compete 25 binding fragments of the anti-slfilt-1 antibodies. Chimeric for binding with an antibody which comprises at least one antibody 10A (V: SEQ ID NO:35; V,: SEQ ID NO:36) variable region with an amino acid sequence selected from comprises the variable region of antibody 102 and a human SEQ ID NOS:14 and 16, or a sequence at least 85% or at IgG1 constant region. The antibodies may also be modified least 90% identical thereto. to minimize or eliminate other effects. For example the The following Table 1 lists the SEQID NOS: correspond 30 constant region of chimeric antibody 10B (V: SEQ ID ing to nucleotide and amino acid sequences of the variable NO:37; V: SEQ ID NO:36), provided herein, includes the domains and CDRs of anti-sPlt-1 antibodies “101' and mutation N298Q, which prevents glycosylation. Antibodies “102 disclosed herein. containing this mutation are deficient in effector functions, Such as complement activation and binding to Fc. Chimeric TABLE 1. 35 antibody AG10C (V: SEQID NO:38; V,: SEQID NO:36) includes the mutation 1254A, which disrupts binding of the Antibody SEQ ID NOS antibody to neonatal Fc receptor (FcRn). The FcRn receptor facilitates transport of maternal IgG across the placenta to Antibody Designation the fetus. Accordingly, AG10C would bind sPlt-1 in the 101 102 40 treatment Subject, but not be transported to the growing fetus. In an embodiment of the invention, antibodies for ex nucleotide amino acid nucleotide amino acid Vivo or in vivo administration include both mutations (e.g., Sequence Sequence Sequence Sequence chimeric antibody AG10D; V: SEQ ID NO:39; V: SEQ CDR1EH 1 2 17 18 ID NO:36). CDR2H 3 4 19 2O 45 The anti-slfilt-1 antibodies, or sPlt-1 binding fragments CDR3H 5 6 21 22 CDR1L 7 8 23 24 thereof, or sPlt-1 ligands, are used to neutralize the activity CDR2L 9 10 25 26 of sfilt-1 and one possible mechanism is through direct CDR3L 11 12 27 28 blocking of the binding sites on sElt-1 for growth factors VH 13 14 29 30 such as VEGF or PIGF. However, other mechanisms are also VL 15 16 31 32 50 possible. For example, the anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, may bind to a site on SFlt-1 Such In certain embodiments, the anti-sfilt-1 antibodies, or that binding of VEGF or PIGF to sRlt-1 is not blocked. In SFlt-1 binding fragments thereof, bind to an epitope on either case, the sRlt-1 is removed from the blood or plasma human sElt-1 that is bound by one or more of the antibodies by virtue of being captured by the solid-support bound referred to herein as 101, 102, or AG10A-D. Two antibodies 55 anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, compete (i.e., bind to the same or overlapping epitope) if and is no longer available to bind to, and thus reduce the each competitively inhibits (blocks) binding of the other to concentration of free growth factors such as VEGF or PIGF the antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of in the blood or plasma. Further, when captured by solid one antibody inhibits binding of the other by at least 50%, Support-bound antibodies or binding fragments thereof, preferably 75%, 90%, or even 99% as measured in a 60 sElt-1 is no longer available to form heterodimers with competitive binding assay (see, e.g., Junghans et al., Cancer membrane-bound Flt-1 or KDR. Res. 50:1495, 1990). Additional methods of determining The anti-sfilt-1 antibodies of the invention bind to one or whether one antibody binds to the same or overlapping more extracellular Ig-like domains of Flt-1. In certain epitope as another antibody are well known in the art. embodiments an anti-slfilt-1 antibody, or sPlt-1 binding frag In certain embodiments, an anti-slfilt-1 antibody, or sPlt-1 65 ment thereof, binds to one or more of domains 1-3 of silt-1 binding fragment thereof, binds human sElt-1 but does not and blocks ligand binding. The domain structure of Flt-1 has bind human Flt-1. In certain embodiments, an anti-slit-1 been described. (See, e.g., Davis-Smyth et al., 1996, EMBO US 9,592,331 B2 11 12 Journal, 15(18):4919-27). For example, the first Ig-like variable region sequences and/or CDRs of the anti-slfilt-1 domain extends from about Pro32 to about Ile128. The antibodies disclosed herein. Moreover, it would be a routine second Ig-like domain extends from about Pro 134 to about matter to design additional anti-SFlt-1 antibodies that com Thr226. The third Ig-like domain extends from about prise variable region sequences or CDRS that have certain Va1232 to about Lys331. The fourth Ig-like domain, which specified levels of identity in amino acid sequence to the is thought to be critical for receptor dimer formation, variable region sequences or CDRs of the anti-slfilt-1 anti extends from about Phes33 to about Pro428. The fifth bodies disclosed herein. Ig-like domain extends from about Tyr231 to about Thr553. In designing and producing additional anti-SFlt-1 antibod The sixth Ig-like domain extends from about Gly558 to ies, those skilled in the art may be guided by certain well about Arg656. The seventh Ig-like domain extends from 10 known features of antibodies. The structure of typical natu about Tyró62 to about Thr751. rally occurring antibodies is well known and includes two When such an anti-slfilt-1 antibody, or sPlt-1 binding identical heavy chains and two identical light chains, with fragment thereof, or SFlt-1 ligand, is employed in the ex vivo each light chain covalently linked to a heavy chain by an methods disclosed herein, it binds to sRlt-1 molecules that interchain disulfide bond. The two heavy chains are linked are not bound by sRlt-1 ligand and removes those sRlt-1 15 to one another by additional disulfide bonds. Individual molecules from blood or plasma. In other embodiments, the heavy and light chains can fold into domains having similar anti-slfilt-1 antibody, or sPlt-1 binding fragment thereof, sizes (110-125 amino acids) and structures, but different binds to one or more of domains 1-3 of sRlt-1 and does not functions. Light chains can comprise one variable domain block ligand binding. In certain other embodiments, the (V) and/or one constant domain (C). Heavy chains can anti-slfilt-1 antibody, or sPlt-1 binding fragment thereof, also comprise one variable domain (V) and/or three or four binds to sRlt-1 and bound ligand is displaced. Thus, in constant domains (C1, C2, C3 and C4), depending on certain embodiments, the amount of slfilt-1 in a subject is the class or isotype of antibody. In humans, the isotypes are reduced without a substantial reduction of slflt-1 ligand. IgA, Ig), IgE, IgG, and IgM, with IgA and IgG further In certain embodiments, the anti-sfilt-1 antibodies, or Subdivided into Subclasses or Subtypes (IgA and IgG). sElt-1 binding fragments thereof, or sPlt-1 ligands, bind to 25 As one might expect from their name, variable domains sElt-1 so as to prevent dimerization. Binding of Flt-1 ligand show considerable amino acid sequence variability from one to Flt-1 is understood to be cooperative, such that a stable antibody to the next. This variability is generally greatest at receptor-ligand complex includes a ligand dimer bound to a the location of the antigen-binding sites. Three regions, receptor dimer. Accordingly, blocking receptor dimerization called hyperVariable or complementarity-determining destabilizes receptor-ligand interactions. When anti-slfilt-1 30 regions (CDRs), are found in each of V, and V, which are antibodies, or sPlt-1 binding fragments thereof, or sPlt-1 supported by less variable regions called framework variable ligands, that block dimerization are employed in the ex vivo regions. methods disclosed herein, such antibodies or binding frag It has been found to be convenient to consider certain ments bind to SFlt-1 and reduce the amount of circulating portions of antibody molecules individually. The portion of sElt-1. Thus, the amount of silt-1 in a subject is reduced 35 an antibody consisting of V, and V. domains is designated without a substantial reduction of silt-1 ligand. Since Fv (fragment variable) and constitutes the antigen-binding dimerization of bound sPlt-1 is blocked, the stability of any site. An antibody fragment containing a V, domain and a V SFlt-1 monomer with ligand is reduced. Thus, any reduction domain on one polypeptide chain is referred to as a single of silt-1 ligand in the subject may be insubstantial. chain Fv (scFv) and generally contains the N terminus of one In certain embodiments, the anti-sfilt-1 antibodies, or 40 domain and the C terminus of the other domain joined by a sElt-1 binding fragments thereof, bind to sRlt-1 but do not flexible linker (see, e.g., U.S. Pat. No. 4,946,778 and Inter substantially block or inhibit ligand binding or sPlt-1 national Patent Publication WO 88/09344. dimerization. In certain embodiments, the anti-SFlt-1 anti For certain embodiments disclosed herein, it may be bodies, or sPlt-1 binding fragments thereof, bind to an advantageous to employ Schv fragments because Sclv frag epitope that is present in all isoforms of slfilt-1. 45 ments lack some or all of the constant domains of whole In one embodiment, the anti-slt-1 antibodies, or sPlt-1 antibodies. Therefore, they can overcome some of the side binding fragments thereof, or SFlt-1 ligands, bind to Ig-like effects associated with the use of whole antibodies. For domain 1 of sRlt-1. In another embodiment, the anti-slit-1 example, sclv fragments tend to be free of certain undesired antibodies, or sPlt-1 binding fragments thereof, or sPlt-1 interactions between heavy-chain constant regions and other ligands, bind to Ig-like domain 2 of slfilt-1. In another 50 biological molecules. embodiment, the anti-slfilt-1 antibodies, or sPlt-1 binding In certain embodiments, the solid Support may have fragments thereof, or SFlt-1 ligands, bind to Ig-like domain attached multivalent single chain antibodies, where multiple 3 of sfilt-1. In yet another embodiment, the anti-slfilt-1 single chain antibodies, each single chain having one V and antibodies, or sPlt-1 binding fragments thereof, or sPlt-1 one V, domain covalently linked by a first peptide linker, are ligands, bind to Ig-like domains 1-2 of SFlt-1. In another 55 covalently linked by at least one or more second peptide embodiment, the anti-slfilt-1 antibodies, or sPlt-1 binding linkers to form a multivalent single chain antibody. Each fragments thereof, or SFlt-1 ligands, bind to Ig-like domains chain of a multivalent single chain antibody includes a 2-3 of sRlt-1. In still another embodiment, the anti-slit-1 variable light chain fragment and a variable heavy chain antibodies, or sPlt-1 binding fragments thereof, or sPlt-1 fragment, and is linked by the second peptide linker to at ligands, bind to Ig-like domains 1 and 3 of slfilt-1. 60 least one other chain. The second peptide linker is preferably Disclosed herein are anti-sfilt-1 antibodies suitable for use composed of at least fifteen and fewer than one hundred in the present methods and systems (e.g., 101, 102, AG10A amino acid residues. D). Based on these anti-slfilt-1 antibodies, it would be a In certain embodiments, the solid Support may have routine matter for those skilled in the art to design and attached diabodies, where two single chain antibodies are produce additional anti-slfilt-1 antibodies for use in the 65 combined to form a diabody. Diabodies have two chains and present methods and systems by, e.g., designing and pro two binding sites, each specific for sPlt-1. Each chain of the ducing additional anti-SElt-1 antibodies that comprise the diabody includes a V. domain connected to a V, domain. US 9,592,331 B2 13 14 The domains are connected with linkers that are short individual residues or combinations of residues so that in a enough to prevent pairing between domains on the same population of otherwise identical antigen binding sites, all chain, thus driving the pairing between complementary twenty amino acids are found at particular positions. Alter domains on different chains to recreate the two antigen natively, mutations may be induced over a range of CDR binding sites. residues by error prone PCR methods (see, e.g., Hawkins et In certain embodiments, the Solid Support may have al., J. Mol. Biol., 226: 889-896 (1992)). For example, phage attached triabodies, where three single chain antibodies are display vectors containing heavy and light chain variable combined to form a triabody. In triabodies, the amino acid region genes can be propagated in mutator strains of E. coli terminus of a V, or V. domain is directly fused to the (see, e.g., Low et al., J. Mol. Biol., 250: 359-368 (1996)). carboxyl terminus of a V, or V. domain, i.e., without any 10 These methods of mutagenesis are illustrative of the many linker sequence. The triabody has three Fv heads with the methods known to one of skill in the art. polypeptides arranged in a cyclic, head-to-tail fashion. Anti-slfilt-1 antibodies can be obtained by standard In certain embodiments, the Solid Support may have hybridoma technology (e.g., Harlow & Lane, ed., Antibod attached Fab fragments. Fab fragments are fragments of an ies: A Laboratory Manual, Cold Spring Harbor, 211-213 antibody consisting of V, C, V, and C1 domains. Those 15 (1998), which is incorporated by reference herein) or by generated following papain digestion simply are referred to using transgenic mice (e.g., KM mice, originally from as Fab and lack the heavy chain hinge region. Following Medarex, San Jose, Calif.) that produce human immuno pepsin digestion, various Fabs retaining the heavy chain globulin gamma heavy and kappa light chains. In certain hinge are generated. Those divalent fragments with the mice known in the art, a Substantial portion of the human interchain disulfide bonds intact are referred to as F(ab'), antibody producing genome is inserted into the genome of while a monovalent Fab' results when the disulfide bonds are the mice, and the mice are rendered deficient in the produc not retained. tion of endogenous murine antibodies. Such mice may be Thus, anti-slfilt-1 antibodies, and sPlt-1 binding fragments immunized with part or all of slfilt-1 (e.g., human sElt-1), thereof, for use in the methods and systems disclosed herein optionally in a suitable adjuvant, e.g., complete or incom include, but are not limited to, naturally occurring antibod 25 plete Freund's adjuvant. ies, bivalent fragments such as (Fab'), monovalent frag Methods for the preparation of antibodies suitable for use ments such as Fab, single chain antibodies, single chain FV in the methods and systems disclosed herein are well known (ScPV), single domain antibodies, multivalent single chain in the art and are described, e.g., in U.S. Pat. No. 6,054,297; antibodies, diabodies, triabodies, and the like that bind U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,365,157; and U.S. SFlt-1. 30 Pat. No. 6,165,464; U.S. Patent Application Publication No. In certain embodiments, specificity of antibodies, or frag 2006/0067937: International Patent Publication WO ments thereof, can be determined based on affinity and/or 06/034507; which are incorporated herein by reference. avidity. Affinity, represented by the equilibrium constant for The anti-sfilt-1 antibodies suitable for use in the methods the dissociation of an antigen with an antibody (K), mea and systems disclosed herein may include polyclonal anti Sures the binding strength between an antigenic determinant 35 bodies, monoclonal antibodies, humanized or chimeric anti and an antibody-binding site. Avidity is the measure of the bodies, FV fragments, single chain FV fragments, Fab frag strength of binding between an antibody with its antigen. ments, or F(ab')2 fragments. In certain embodiments, the Avidity is related to both the affinity between an epitope with antibodies are mouse monoclonal antibodies. The anti-slit-1 its antigen binding site on the antibody, and the Valence of antibodies may include a variety of antibody isotypes, such the antibody, which refers to the number of antigen binding 40 as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, sites of a particular epitope. Antibodies typically bind with IgD, and IgE. a dissociation constant (K) of 10 to 10' liters/mol. Any “Chimericantibody' refers to a polypeptide comprising at K. greater than 10 liters/mol is generally considered to least the antigen-binding portion of an antibody molecule indicate nonspecific binding. The lesser the value of the K, linked to at least part of another protein (typically an the stronger the binding strength between an antigenic 45 immunoglobulin constant domain). determinant and the antibody binding site. “Humanized antibody' refers to an antibody with a frame In certain embodiments, the anti-sfilt-1 antibodies, or work region (FR) having Substantially the amino acid sElt-1 binding fragments, bind slfilt-1 with a dissociation sequence of a human immunoglobulin and a complemen constant(K) of about 10 to 10' liters/mol, about 10 to tarity determining region (CDR) having Substantially the 10' liters/mol, or about 107 to 10 liters/mol. In certain 50 amino acid sequence of a non-human immunoglobulin (the embodiments, anti-slfilt-1 antibodies, or sPlt-1 binding frag “import’ sequences). Generally, a humanized antibody has ments, bind to SFlt-1 with a dissociation constant (K) of at one or more amino acid residues introduced into it from a least about 10 liters/mol, at least 10 liters/mol, at least Source that is non-human. The humanized antibody will 107 liters/mol, at least 10 liters/mol, at least 10 liters/ usually comprise Substantially all of at least one, and typi mol, at least 10" liters/mol, or at least 10' liters/mol. In 55 cally two, variable domains (Fab, Fab'. F(ab'), Fabc. Fv) in certain embodiments, the K is from 10 liters/mol to 10' which all or substantially all of the CDR regions correspond liters/mol. In certain embodiments, embodiments, the K is to those of a non-human immunoglobulin and all or Sub from 10' liters/mol to 10' liters/mol. stantially all of the FR regions are those of a human Anti-sfilt-1 antibodies suitable for use in the methods and immunoglobulin or a human immunoglobulin consensus systems disclosed herein further include those for which 60 sequence. The humanized antibody optimally will comprise binding characteristics have been improved by direct muta at least a portion of an immunoglobulin constant region (Fc), tion, methods of affinity maturation, phage display, or chain typically that of a human immunoglobulin. By "comple shuffling. Affinity and specificity can be modified or mentarity determining region (CDR) is meant the three improved by mutating CDRS and Screening for antigen hyperVariable sequences in the variable regions within each binding sites having the desired characteristics (see, e.g., 65 of the immunoglobulin light and heavy chains. By “frame Yang et al., J. Mol. Biol., 254: 392–403 (1995)). CDRs can work region (FR) is meant the sequences of amino acids be mutated in a variety of ways. One way is to randomize located on either side of the three hypervariable sequences US 9,592,331 B2 15 16 (CDR) of the immunoglobulin light and heavy chains. The suffering from diseases associated with high levels of silt-1 FR and CDR regions of the humanized antibody need not in blood, including preeclampsia. The columns used in correspond precisely to the parental sequences, e.g., the clinical treatments contain anti-SElt-1 antibody-coupled import CDR or the human or consensus human FR may be Sepharose beads at 0.1-5 mg of antibodies per 1 mL of beads mutagenized by Substitution, insertion, or deletion of at least (5-100 mg per 50 mL beads; 0.26-5.2 billion antibody one residue so that the CDR or FR residue at that site does molecules per single bead), at flow rates of 10-100 mL/min, not correspond to either the consensus or the import at linear flow rates of 30-180 cm/hr, and residence times of sequence. Such mutations, however, will not be extensive. 0.5-3 minutes. Patients with average weight will have about Usually, at least 75%, preferably 90%, and most preferably 8 Liters of blood circulating in their body (about 4 Liters of at least 95% of the humanized antibody residues will cor 10 plasma). About 0.5-3 times the total body plasma volume respond to those of the parental FR and CDR sequences. (2-12 Liters of plasma), which corresponds to 40 to 240 The anti-slfilt-1 antibodies may be obtained directly from times the column bed volumes of blood plasma (for a 50 mL hybridomas which express the anti-slfilt-1 antibodies or may column), containing 0.08-0.48 mg of native sRlt-1 (for a be cloned and recombinantly expressed in suitable host cells patient with 40 ng/mL sRlt-1 level in plasma) of various (e.g., CHO cells, NS/0 cells, HEK293 cells). Suitable host 15 forms, are to be applied to the columns containing anti cells include plant cells, mammalian cells, and microorgan sElt-1 antibody-coupled beads at anti-slfilt-1 antibody: silt-1 isms such as E. coli and yeast. Alternatively, anti-SFlt-1 ratios of 50:1 to 2,000:1 (w/w), or molar ratios of 12.5:1 to antibodies may be produced recombinantly in a transgenic 500:1. Under these ranges of conditions, columns containing non-human animal or plant, e.g., a transgenic mouse. Sepharose beads coupled with anti-slfilt-1 antibodies are able In certain embodiments, the anti-slfilt-1 antibodies may be to deplete 50 to 100% of slfilt-1 from plasma of patients with modified prior to, or after, attachment to a solid Support. high sElt-1 levels in their blood. Possible modifications include glycosylation, acetylation, Thus, the invention provides a method treating or pre pegylation, phosphorylation, amidation, derivatization with venting a pregnancy-related hypertensive disorder in a Sub protecting or blocking groups, proteolytic cleavage, or link ject comprising providing ex vivo to the Subject an anti age to a cellular ligand or other protein. In certain embodi 25 sElt-1 antibody, or sPlt-1 binding fragment thereof, wherein ments, the anti-SFlt-1 antibodies may contain one or more the anti-slfilt-1 antibody, or sPlt-1 binding fragment thereof, non-classical amino acids. depletes at least 70%, or at least 80%, or at least 90%, or at The anti-SFlt-1 antibodies, or antigen binding fragments least 95%, or at least 99%, or from 70% to 80%, or from thereofare suitable for ex vivo treatment of an sElt-1-related 80% to 90%, or from 90% to 95%, or from 95% to 99% of disorder. Suitable means that the antibodies effectively 30 SFlt-1 from human plasma in an in vitro analysis, when the reduce the concentration of silt-1 in a subject’s blood or anti-slfilt-1 antibody, or sPlt-1 binding fragment thereof, is plasma when used in a effective amount for an effective attached to a solid support, and the molar antibody:slit-1 time. For example, using a 50 ml/minute flow rate, 5 liters ratio is 500. In another embodiment, the invention provides of plasma (approximately 2.5 human blood Volumes) would a method treating or preventing a pregnancy-related hyper be processed in 100 minutes. As exemplified herein, in one 35 tensive disorder in a Subject comprising providing ex vivo to assay, antibody AG10B depleted 94% of slfilt-1 from a test the subject an anti-slfilt-1 antibody, or sPlt-1 binding frag Solution using a flow rate of 1 ml/min applied to a 1 ml ment thereof, wherein the anti-slfilt-1 antibody, or sPlt-1 column. This is comparable to a 50 ml/min flow rate using binding fragment thereof, depletes at least 70%, or at least a 50 ml column (and comparable to a residence time of 1 80%, or at least 90%, or at least 95%, or at least 99%, or min). Another assay shows that slit-1 depletion in a test 40 from 70% to 80%, or from 80% to 90%, or from 90% to sample was only slightly reduced when the concentration of 95%, or from 95% to 99% of sElt-1 from human plasma in AG1 OB on the solid support was reduced from 0.8 mg/ml of an in vitro analysis, when the anti-slfilt-1 antibody, or sPlt-1 beads to 0.4 mg/ml of beads. binding fragment thereof, is attached to a solid Support, and For research purposes, columns of various dimensions the antibody:sflt-1 ratio is 250. In another embodiment, the containing 0.1-50 mL of Sepharose beads coupled with 45 invention provides a method treating or preventing a preg anti-slfilt-1 antibodies are tested for their ability to deplete nancy-related hypertensive disorder in a Subject comprising recombinant silt-1 spiked into buffered solutions or animal providing ex vivo to the subject an anti-slfilt-1 antibody, or serum or human plasma, or native SElt-1 in amniotic fluid or sElt-1 binding fragment thereof, wherein the anti-slfilt-1 blood plasma of preeclampsia patients. The sRlt-1 depletion antibody, or sPlt-1 binding fragment thereof, depletes at experiments are conducted with columns containing anti 50 least 70%, or at least 80%, or at least 90%, or at least 95%, sElt-1 antibody-coupled Sepharose beads at 0.025-20 mg of or at least 99%, or from 70% to 80%, or from 80% to 90%, antibodies per 1 mL of beads (0.065-52 billion antibody or from 90% to 95%, or from 95% to 99% of SElt-1 from molecules per single bead), at flow rates of 0.05-50 mL/min, human plasma in an in vitro analysis, when the anti-SElt-1 at linear flow rates of 10-300 cm/hr, and residence times of antibody, or sPlt-1 binding fragment thereof, is attached to 0.25-5 minutes. For these sRlt-1 depletion experiments, 1 to 55 a solid support, and the molar antibody:sFlt-1 ratio is 100. In 400 times the column bed volumes of buffered solutions, still other embodiments, at least 70%, or at least 80%, or at serum or plasma containing SElt-1 are applied to the col least 90%, or at least 95%, or at least 99%, or from 70% to umns at anti-slfilt-1 antibody:sFlt-1 ratios of 5:1 to 5,000:1 80%, or from 80% to 90%, or from 90% to 95%, or from (w/w), or molar ratios of 1.25:1 to 1,250:1. Under these 95% to 99% of slfilt-1 is depleted from human plasma in the ranges of conditions, columns containing Sepharose beads 60 in vitro analysis when the anti-slfilt-1 antibody, or sPlt-1 coupled with anti-slfilt-1 antibodies deplete 50 to 100% of binding fragment thereof, is attached to a solid Support, and sElt-1 in buffered solutions, serum or plasma. the molar antibody: shlt-1 ratio is 50, 25, or 12.5. The For clinical treatments, columns of various dimensions anti-slfilt-1 antibodies and sPlt-1 binding fragments thereof containing 25 to 750 mL of Sepharose beads coupled with include those that bind to Flt-1 Ig-like domains 1-3 in anti-slfilt-1 antibodies are used to deplete native slflt-1 of 65 various combinations, as well as antibodies that bind to various isoforms, alone or in complex with ligands such as Ig-like domains 4, 5, 6, or 7, either alone, or in combination, VEGF or PIGF isoforms from blood plasma of patients or in combination with Ig-like domains 2 and/or 3. US 9,592,331 B2 17 18 According to the analysis method, human serum is spiked ng/ml, less than about 3 ng/ml, less than about 2 ng/ml, less with sElt-1. As exemplified herein, an sElt-1 protein con than about 1 ng/ml, less than about 0.75 ng/ml, or less than sisting of domains 1-3 was used. When slit-1 antibodies about 0.5 ng/ml. against other domains or combinations of domains are In certain embodiments, an sElt-1 molecule is removed tested, an SFlt-1 molecule containing the pertinent domains 5 from blood plasma by immobilization to a solid support, for is used. Non-limiting examples of SFlt-1 molecules contain example, using an anti-SFlt-1 antibody, or SFlt-1 binding domains 1-3, domains 1-4, domains 1-5, domains 1-6. fragment thereof. When slfilt-1 is immobilized to a solid domains 1-7, domains 2-3, domains 2-4, domains 2-5. Support, ligand binding is less favored compared to the case domains 2-6, or domains 2-7 of slfilt-1. (See, e.g., Barleon et where slfilt-1 is free in solution. Accordingly, silt-1 levels al., 1997, J. Biol. Chem. 272:10382-88 for showing expres 10 are reduced in the Subject, and any reduction of circulating sion of various domains of silt-1). In certain embodiments SFlt-1 ligand may be insubstantial. the analysis is performed using Sepharose bead-bound anti In certain embodiments, the anti-sfilt-1 antibodies are sElt-1 antibodies or sPlt-1 binding fragments thereof mixed bound to a solid Support where the solid Support does not in sElt-1-Spiked plasma. In certain embodiments, the analy have anti-endoglin antibodies, or endoglin binding frag sis is performed over a time period that replicates a residence 15 ments thereof, bound to it. In certain embodiments of the time on a clinical column of 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, or methods disclosed herein, the methods do not substantially 5 minutes. Such an analysis can be performed using a decrease the amount of endoglin in the subject’s blood. In solution of bead-bound anti-sfilt-1 antibodies or sPlt-1 bind certain embodiments of the systems disclosed herein, the ing fragments in a column and SFlt-1-Spiked plasma applied systems are not capable of significantly removing endoglin at a flow rate to obtain a desired residence time. Alterna from the subjects blood. tively, the analysis could be performed using slfilt-1 spiked In certain embodiments, the methods of the present inven in amniotic fluid, serum (e.g., horse serum), or a buffer tion comprise: Solution (e.g., PBS), but plasma, particularly human plasma, (a) removing blood from the Subject, is preferred. The analysis can be performed using anti-slfilt-1 (b) passing the blood or a component thereof over a solid antibodies or sPlt-1 binding fragments thereof bound to a 25 support to which are attached anti-slfilt-1 antibodies, or column Support (e.g., Sepharose beads) at various densities sElt-1 binding fragments thereof, or sPlt-1 ligands, to and SElt-1 Spiked in plasma at various concentrations. The decrease the level of slfilt-1 in the blood or component anti-slfilt-1 antibodies or sPlt-1 binding fragments thereof thereof, and can be linked to Sepharose beads in amounts of 0.025, 0.050, (c) returning the blood or component thereof to the 0.1, 0.25, 0.5, 1, or 2 mg/bead. The flow rate can be 0.05, 30 subject’s body. 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, or 100 ml/min, and linear In certain embodiments, the blood is separated into flow rates can be 10, 20, 30, 50, 100, 150, 180, 240, or 300 plasma and cellular components and only the plasma is cm/hr. contacted with the anti-slfilt-1 antibodies, or sPlt-1 binding Antibodies of the invention are effective to efficiently fragments thereof, while the cellular components are deplete sRlt-1 in blood or plasma from a subject. The sRlt-1 35 returned to the Subject without such contact or, in certain can be soluble and/or in microparticles circulating in the embodiments, disposed of rather than returned to the sub bloodstream. In certain embodiments, when an antibody of ject. the invention is attached to a solid Support (e.g., Sepharose Accordingly, in certain embodiments, the method com beads), and contacted with a solution containing SElt-1 Such prises removing a volume of the Subject’s blood, separating that the antibody:SElt-1 ratio is 50, the slit-1 antibody 40 the blood into plasma and cellular components, bringing the depletes (binds to) at least 70%, or at least 80%, or at least plasma into contact with the anti-slfilt-1 antibodies, or sPlt-1 90%, or at least 95% of sRlt-1. In certain embodiments, the binding fragments thereof, to bindsFlt-1 in the plasma to the sFlt-1 antibody depletes from 70% to 80%, of from 80% to anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, 90%, or from 90% to 95%, of from 95 to 99% of sElt-1. The thereby decreasing the amount of slfilt-1 in the subjects Solution can be blood, plasma, serum, or a buffer solution. In 45 plasma, returning the plasma to the Subject, and, optionally, certain embodiments, when an antibody of the invention is returning the cellular components to the Subject. attached to a solid Support (e.g., Sepharose beads), and When practicing the above embodiment, the cellular contacted with a solution containing SElt-1 Such that the components may be returned to the Subject at any time. That antibody:sFlt-1 ratio is 100, the sRlt-1 antibody depletes at is, the cellular components may be returned to the Subject least 70%, or at least 80%, or at least 90%, or at least 95% 50 before the plasma is contacted with the anti-slfilt-1 antibod of slfilt-1. In certain embodiments, the sRlt-1 antibody ies, or sPlt-1 binding fragments thereof, or the cellular depletes from 70% to 80%, of from 80% to 90%, or from components may be returned to the Subject after the plasma 90% to 95%, of from 95 to 99% of SElt-1. In certain is contacted with the anti-slfilt-1 antibodies, or sPlt-1 binding embodiments, when an antibody of the invention is attached fragments thereof. In certain embodiments, the cellular to a solid Support (e.g., Sepharose beads), and contacted 55 components may be combined with the plasma after the with a solution containing slfilt-1 such that the antibody: plasma has been contacted with the anti-slfilt-1 antibodies, or sElt-1 ratio is 250, the sRlt-1 antibody depletes at least 70%, sElt-1 binding fragments thereof, and the combined cellular or at least 80%, or at least 90%, or at least 95% of SElt-1. In components and plasma are returned to the Subject at the certain embodiments, the sRlt-1 antibody depletes from 70% same time, through the same conduit system and/or the same to 80%, of from 80% to 90%, or from 90% to 95%, of from 60 return device. 95 to 99% of SFlt-1. In certain embodiments, the pregnancy-related hyperten In certain embodiments, the anti-slfilt-1 antibody or sPlt-1 sive disorder is eclampsia or pre-eclampsia. In certain binding fragment is capable, under Suitable conditions, of embodiments, the pregnancy-related hypertensive disorder reducing the concentration of slfilt-1 in the subjects blood or is eclampsia. In certain embodiments, the disorder is chronic plasma containing SElt-1 to less than about 50 ng/ml, less 65 kidney disease. than about 40 ng/ml, less than about 25 ng/ml, less than In certain embodiments, the Subject is a pregnant human, about 10 ng/ml, less than about 5 ng/ml, less than about 4 a post-partum human, or a pregnant or post-partum non US 9,592,331 B2 19 20 human (e.g., a cow, a horse, a sheep, a pig, a goat, a dog, or coagulation abnormalities due to pregnancy or the influence a cat). In certain embodiments, the Subject is a pregnant of a recent pregnancy. All forms of pre-eclampsia, Such as human or a post-partum human. In certain embodiments, the premature, mild, moderate, and severe pre-eclampsia are Subject is a pregnant human. included in this definition. Pre-eclampsia generally occurs Optionally, the methods disclosed herein may be prac after the 20th week of gestation. Pre-eclampsia is generally ticed on a subject who is being treated with standard defined as some combination of the following symptoms: (1) pre-eclampsia or eclampsia therapies. Such standard thera a systolic blood pressure (BP)>140 mm Hg and a diastolic pies are known to the skilled artisan and include the methods BP>90 mm Hg after 20 weeks gestation (generally measured described in U.S. Patent Application Publication No. US on two occasions, 4-168 hours apart), (2) new onset pro 2004/0126828; U.S. Patent Application Publication No. US 10 teinuria (1+ by dipstik on urinalysis, >300 mg of protein in 2005/0025762; U.S. Patent Application Publication No. US a 24-hour urine collection, or a single random urine sample 2005/0170444; and U.S. Patent Application Publication No. having a protein creatinine ratio>0.3), and (3) resolution of US 2006/0067937 as well as in International Patent Publi hypertension and proteinuria by 12 weeks postpartum. cation WO 2004/008946; International Patent Publication Severe pre-eclampsia is generally defined as (1) a diastolic WO 2005/077007; and International Patent Publication WO 15 BP>110 mm Hg (generally measured on two occasions, O6/O345O7. 4-168 hours apart) or (2) proteinuria characterized by a The methods disclosed herein may be practiced using a measurement of 3.5 grams or more protein in a 24-hour combination of slfilt-1 binding substances. For example, two urine collection or two random urine specimens with at least or more of anti-slfilt-1 antibodies, slfilt-1 binding fragments 3+ protein by dipstick. In pre-eclampsia, hypertension and thereof, and SFlt-1 ligands may be used. proteinuria generally occur within seven days of each other. The methods disclosed herein may be practiced on a In severe pre-eclampsia, severe hypertension, severe pro Subject who is being treated with chronic hypertension teinuria and HELLP syndrome (hemolysis, elevated liver medications. Medications used for the treatment of hyper enzymes, low platelets) or eclampsia can occur simultane tension during pregnancy include methyldopa, hydralazine ously or only one symptom at a time. HELLP syndrome is hydrochloride, or labetalol. 25 characterized by evidence of thrombocytopenia (<100,000 In certain embodiments, the methods of the present inven cells/ul), increased LDH (>600 IU/L) and increased AST tion can further include the step of administering an anti (>70 IU/L). Occasionally, severe pre-eclampsia can lead to hypertensive compound to the Subject. Such administration the development of seizures. This severe form of the syn may be by conventional means, e.g., administering an oral drome is referred to as "eclampsia.” Eclampsia can also dosage form comprising an anti-hypertensive compound. 30 include dysfunction or damage to several organs or tissues In certain embodiments, the method of the present inven Such as the liver (e.g., hepatocellular damage, periportal tion can further include administering a growth factor or necrosis) and the central nervous system (e.g., cerebral cytokine, such as, without limitation, a VEGFR ligand, to edema and cerebral hemorrhage). The etiology of the Sei the subject. In one embodiment, the growth factor is VEGF. Zures is thought to be secondary to the development of In another embodiment, the growth factor is PlGF. 35 cerebral edema and focal spasm of small blood vessels in the The methods disclosed herein may be practiced during kidney. pregnancy for the treatment or prevention of pre-eclampsia “Subject” refers to a mammal, including, but not limited or eclampsia or after pregnancy to treat post-partum pre to, a human or non-human mammal Such as a cow, a horse, eclampsia or eclampsia. a sheep, a pig, a goat, a dog, or a cat. “Treating refers to practicing the ex vivo methods dis 40 “At risk of developing a pregnancy-related hypertensive closed herein for therapeutic purposes. To “treat' or to use disorder Such as pre-eclampsia or eclampsia refers to a for “therapy” refers to administering treatment to a subject Subject who does not currently have, but has a greater than already diagnosed as having or Suffering from a pregnancy average chance of developing, a pregnancy-related hyper related hypertensive disorder to improve the subject’s con tensive disorder. Such at risk Subjects include pregnant dition. For example, the Subject may be diagnosed as having 45 women with an sElt-1 blood concentration of greater than or suffering from pre-eclampsia or eclampsia, based on about 3 ng/ml, greater than about 4 ng/ml, greater than about identification of any of the characteristic symptoms 5 ng/ml, greater than about 6 ng/ml, greater than about 7 described herein or based on measurement of the concen ng/ml, greater than about 8 ng/ml, greater than about 9 tration of slflt-1 in the subject’s blood, as described herein. ng/ml, greater than about 10 ng/ml, greater than about 15 “Prevent refers to prophylactic treatment of a subject 50 ng/ml, greater than about 20 ng/ml, greater than about 25 who is not yet ill, but who is susceptible to, or otherwise at ng/ml, greater than about 30 ng/ml, greater than about 40 risk for, developing a pregnancy-related hypertensive disor ng/ml, or greater than about 45 ng/ml, but who show no der, e.g., a Subject who is determined to be at risk for other signs of a pregnancy-related hypertensive disorder developing pre-eclampsia or eclampsia. Such as pre-eclampsia. “Pregnancy-related hypertensive disorder” refers to any 55 The stage of pregnancy at which the methods described condition or disease during pregnancy that is associated with herein may be practiced depends on various clinical factors or characterized by an increase in blood pressure. Included including the overall health of the subject and the severity of among these conditions and diseases are pre-eclampsia the symptoms of pre-eclampsia. In general, once pre-ec (including premature pre-eclampsia, severe pre-eclampsia), lampsia or a predisposition to pre-eclampsia is detected, the eclampsia, gestational hypertension, HELLP syndrome, (he 60 methods may be employed. Treatment can be continued for molysis, elevated liver enzymes, low platelets), abruption a period of time ranging from 1 to 100 days, more preferably placenta, chronic hypertension during pregnancy, pregnancy 1 to 60 days, 1 to 10 days, or 1 to 5 days, and most preferably with intra uterine growth restriction, and pregnancy with a 1 to 20 days. Small for gestational age (SGA) infant. In certain embodiments, the method is carried out on a “Pre-eclampsia' refers to a multi-system disorder that is 65 subject on or after the 14th week of pregnancy, the 16th characterized by hypertension with proteinuria or edema, or week of pregnancy, the 18th week of pregnancy, the 20th both, glomerular dysfunction, brain edema, liver edema, or week of pregnancy, the 22nd week of pregnancy, the 24th US 9,592,331 B2 21 22 week of pregnancy, the 26th week of pregnancy, the 28th without interposing a cell removal step, e.g., a centrifugation week of pregnancy, the 30th week of pregnancy, the 32nd step, prior to contact of the blood or plasma with the week of pregnancy, the 34th week of pregnancy, or the 36th anti-sfilt-1 antibodies. week of pregnancy. In certain embodiments, the method is Solid supports for use in the methods described herein carried out on a subject between the 14th and 16th weeks of preferably should be non-toxic and stable when exposed to pregnancy, the 16th and 18th weeks of pregnancy, the 18th blood or blood components. The solid supports may be and 20th weeks of pregnancy, the 20th and 22nd weeks of chosen from among those well known in the art. For pregnancy, the 22nd and 24th weeks of pregnancy, the 24th example, any suitable porous material may be used as the and 26th weeks of pregnancy, the 26th and 28th weeks of Solid Support. Examples of Suitable solid Supports include, pregnancy, the 28th and 30th weeks of pregnancy, the 30th 10 e.g., carbohydrate-based materials such as the various types and 32nd weeks of pregnancy, the 32nd and 34th weeks of of SEPHAROSE(R) (a crosslinked, beaded-form of agarose), pregnancy, or the 34th and 36th weeks of pregnancy. e.g., SEPHAROSE 4B(R), 4FFR, CL-4B(R) and CL-6B. In certain embodiments, the Subject’s blood or plasma is The solid Support may be comprised of organic or inor contacted with anti-sfilt-1 antibodies or ligands only to the ganic molecules, or a combination of organic and inorganic extent necessary to reduce sRlt-1 to a desired level. A desired 15 molecules, and may be comprised of one or more functional level can be, for example, a level of sRlt-1 characteristic of groups, e.g., hydroxyl groups, Suitable for forming covalent a normal pregnancy. It has been observed that in normal bonds with activating agents. The Solid Support may be pregnancy, the serum concentration of SElt-1 decreases from comprised of a hydrophilic compound, a hydrophobic com 8-12 weeks to 16-20 weeks, gradually increases at 26-30 pound, or any combination thereof. The Solid Support may weeks, rapidly elevates at 35-39 weeks, and returns to be comprised of a polymer or a copolymer. normal level after delivery. Accordingly, in one embodi Examples of Suitable materials for use in Solid Supports ment, the desired level is the normal level for the subjects include, but are not limited to, agarose, cellulose, polyether stage of pregnancy. In another embodiment, the level is Sulfones, polyamides, polysaccharides, polytetrafluoroeth higher or lower that the normal level for the subject’s stage ylene, polyesters, polyurethanes, polyvinylidene fluoride, of pregnancy. One of ordinary skill in the art would be able 25 polypropylene, fluorocarbons, e.g., poly(tetrafluoroethyl to determine a desired level, depending for example on the ene-co-perfluoro(alkyl vinyl ether)), polyethylene, glass, patient and the frequency with which the ex vivo procedure polycarbonates, polyacrylate, polyacrylamide, poly(aZolac is to be performed. tone), polystyrene, ceramics, and nylon. The desired sRlt-1 level can be achieved by controlling, The Solid Support need not be in any particular shape. For for example, the length of time a subject is treated (i.e., the 30 example, the Solid Support may be in the form of beads, volume of blood or plasma treated for a particular flow rate), membranes, gels, columns, chips, plates, tubes, sheets, the flow rate over the immobilized antibody or ligand, and/or fibers, or hollow fibers. The solid support can also be in the the binding capacity of the solid Support bearing the anti form of a coating on the interior of one or more lengths of body or ligand that binds to sfilt-1. In one embodiment, a tubing, piping, or hollow fibers through which blood or diagnostic is used to measure sRlt-1 levels at the time of 35 plasma flows. In Such embodiments, the tubing, piping, or treatment. In another embodiment, the diagnostic provides a hollow fibers are preferably coiled or otherwise convoluted real-time measure of slfilt-1 level and treatment is stopped or bent, in order to maximize the amount of Solid Support when the desired sPlt-1 level is reached. In another embodi contacted by the blood or plasma flowing through the tubing, ment, the time, flow rate, and/or capacity is predetermined piping, or hollow fibers. based on the slit-1 level diagnosed in the subject at the start 40 Methods of attaching antibodies and ligands to a solid of the procedure and the sRlt-1 level desired to be reached. Support are well known in the art and may be used to attach In certain embodiments, the method decreases blood the anti-sflt-1 antibodies, or sPlt-1 binding fragments levels of sRlt-1 in the subject by 10%-90%. 20%–80%, or thereof, used in the methods described herein to a solid 30%-50%, as compared to the blood levels of sRlt-1 in the Support. Such methods include, without limitation, the use subject before the method is practiced on the subject. In 45 of cyanogen bromide, 1,1'-carbonyldiimidazole (CDI), or certain embodiments, the method decreases blood levels of triethylamine. sFlt-1 in the subject by 10%-20%, 20%-30%, 30%-40%, In general, Solid Supports may be activated for the attach 40%-50%, 50%-60%, 60%–70%, 70%–80%, 80%-90%, or ment of anti-slfilt-1 antibodies, or sPlt-1 binding fragments 90%-100% as compared to the blood levels of sElt-1 in the thereof, by contacting the Solid Supports with an activating subject before the method is practiced on the subject. 50 agent Such as an aldehyde, an epoxide, a cyanogen, or an The anti-slfilt-1 antibodies, or sPlt-1 binding fragments activated carboxylic acid. thereof, attached to a solid Support, can be used to remove Methods of attaching antibodies to solid supports are well sElt-1 from the body fluids of subjects suffering from, or at known in the art. See, e.g., Hermanson et al. 1992, Immo risk of developing, pre-eclampsia or eclampsia. In certain bilized Affinity Ligand Techniques, Academic Press; U.S. embodiments, the anti-slfilt-1 antibodies, or sPlt-1 binding 55 Pat. No. 5,874,165; U.S. Pat. No. 3,932,557; U.S. Pat. No. fragments thereof, attached to a solid Support, are used to 4,772,635: U.S. Pat. No. 4,210,723; U.S. Pat. No. 52,506, remove sRlt-1 from blood or blood plasma. In certain 123; European Patent Application EP 1 352 957 A1, and embodiments, the anti-slfilt-1 antibodies, or sPlt-1 binding International Patent Publication WO 2004/074471. Typi fragments thereof, attached to a solid Support are used in cally, the Solid Support is activated with a reactive functional extracorporeal immunoadsorbent devices, which are known 60 group Such as an epoxide (e.g., by the use of epichlorohy in the art. Blood or plasma is exposed to the attached drin), cyanogens (e.g., cyanogen bromide (CNBr)), N.N- support-bound anti-slfilt-1 antibodies, or sPlt-1 binding frag disuccinimidylcarbonate (DSC), aldehydes, or an activated ments thereof, resulting in partial or complete removal of carboxylic acid (e.g., N-hydroxysuccinimide (NHS) esters, circulating slflt-1 (free or in complexes with other blood or carbonyldiimidazole (CDI) activated esters). Activated proteins), following which the blood or plasma is returned to 65 groups may be attached directly to the solid Support, as is the subject’s body. The methods disclosed herein may be generally the case for CNBr, or the activated groups may be implemented in a continuous flow arrangement, with or part of a linker or spacer molecule, which is typically a linear US 9,592,331 B2 23 24 chain of carbon, optionally Substituted with oxygen and/or above about 7.5 ng/ml, above about 10 ng/ml, above about nitrogen atoms. A typical example of Such a linker is the ten 20 ng/ml, above about 30 ng/ml, above about 40 ng/ml, or membered chain of carbon and oxygen found in the linker above about 50 ng/ml. butanediol digycidyl ether (a common epoxide coupling In certain embodiments where the subject’s blood levels agent). The activated Solid Support is then contacted with the of sRlt-1 are determined and/or monitored, the methods antibody under coupling conditions. described herein may be employed until the concentration of Other linkers may include a branched, unbranched, or sElt-1 in the subjects blood is less than about 50 ng/ml, less cyclic carbon chain comprising from 1 to 30 carbon atoms. than about 45 ng/ml, less than about 40 ng/ml, less than In certain embodiments, the linker may be comprised of about 35 ng/ml, less than about 30 ng/ml, less than about 25 more than 30 carbonatoms. The linker may comprise at least 10 ng/ml, less than about 20 ng/ml, less than about 15 ng/ml. one hetero-atom Such as nitrogen, oxygen, or Sulfur. less than about 10 ng/ml, less than about 7.5 ng/ml, less than The commercial product AFFI-GEL 150 (BioRad, Her about 5 ng/ml, less than about 4 ng/ml, less than about 3 cules, Calif.) may be used for linker-assisted coupling. ng/ml, less than about 2 ng/ml, less than about 1.5 ng/ml, or AFFI-GEL 150 is an agarose support derivatized with an 15 less than about 1 ng/ml. NHS activated carboxylic acid as part of a linker arm In certain embodiments, the methods disclosed herein containing a positively charged secondary amine. Another may be employed until an improvement is detected in the charged linker is disclosed in U.S. Pat. No. 5,260.373. A symptoms of a pregnancy-related hypertensive disorder. In shorter linker arm comprised of arginine may be used to certain embodiments, the pregnancy-related hypertensive facilitate coupling to an agarose Support. The arginine linker disorder is pre-eclampsia and the improvement is a decrease is activated with NHS and carries a positive charge. in blood pressure to a value of less than 140 mmHg Anti-SFlt-1 antibodies, binding fragments thereof, and (systolic) and/or less than 90 mmHg (diastolic). SFlt-1 specific polypeptides and ligands can be covalently The present invention provides a housing or chamber Such coupled to a solid Support in a manner that provides more as a column containing anti-SFlt-1 antibodies, or SElt-1 uniform orientation and efficient slflt-1 binding. Most meth 25 binding fragments thereof, bound to a Solid Support, where ods involve modifying a protein with a unique chemical the housing or chamber is suitable for use in treating or group at a predefined position, and reacting that group with preventing a pregnancy-related hypertensive disorder Such a complementary group on the Solid Support. In another as eclampsia or pre-eclampsia. embodiment, anti-SElt-1 antibodies, antibody fragments, and In certain embodiments, the housing or chamber is a ligands are produced with N- or C-terminal linkers capable 30 column. “Column” refers to a container, chamber, or hous of being coupled to a solid Support. In certain embodiments, ing, generally cylindrical in shape, containing a solid Sup polypeptides and ligands are synthesized directly on a solid port to which anti-slfilt-1 antibodies, or sPlt-1 binding frag Support. ments thereof, or sPlt-1 ligands, can be or have been Diagnostic methods known in the art can be used to attached. monitor a subject’s pre-eclampsia or eclampsia during 35 In certain embodiments, the column contains a volume of therapy to determine the effectiveness of therapy according about 5 ml to 2000 ml, about 10 ml to about 1000 ml, about to the methods disclosed herein. Suitable diagnostic meth 50 ml to about 500 ml, or about 200 ml to about 400 ml of ods are disclosed in, e.g., U.S. Pat. No. 7,335.362; U.S. Pat. anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, No. 7,435,419; and U.S. Pat. No. 7,407,659. bound to a solid Support. In certain embodiments, the In certain embodiments, diagnostic methods are 40 column contains a volume of about 5 ml, about 10 ml, about employed that determine and/or monitor the concentration 25 ml, about 50 ml, about 100 ml, about 200 ml, about 300 of sRlt-1 in a subject’s blood in order to identify subjects ml, about 500 ml, about 750 ml, about 1000 ml, about 1500 Suitable for treatment or prevention using the methods ml, or about 2000 ml of anti-sfilt-1 antibodies, or sPlt-1 disclosed herein. In certain embodiments, diagnostic meth binding fragments thereof, bound to a solid Support. In ods are employed to identify Subjects at risk of developing 45 certain embodiments, the column contains one or more a pregnancy-related hypertensive disorder Such as pre-ec anti-coagulant Substances, e.g., heparin. In certain embodi lampsia or eclampsia where the Subjects are pregnant ments, the interior of the column has been treated in a women with an sElt-1 blood concentration of greater than manner intended to reduce the amount of bacteria, myco about 5 ng/ml, greater than about 6 ng/ml, greater than about plasma and/or viruses in the interior of the column. In 7 ng/ml, greater than about 8 ng/ml, greater than about 9 50 certain embodiments, the interior of the column is sterile. ng/ml, greater than about 10 ng/ml, greater than about 15 In certain embodiments, the column contains Sufficient ng/ml, greater than about 20 ng/ml, greater than about 25 anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, ng/ml, greater than about 30 ng/ml, greater than about 40 bound to a solid Support, to remove at least 10 ug, at least ng/ml, or greater than about 45 ng/ml, but who show no 25 ug, at least 50 ug, at least 75 ug, at least 100 ug, at least other signs of a pregnancy-related hypertensive disorder 55 150 ug, at least 200 ug, at least 300 ug, at least 400 ug, at Such as pre-eclampsia. least 500 ug, at least 600 ug, at least 700 ug, at least 800 ug, Accordingly, the present invention provides a method of at least 900 ug, at least 1000 ug, at least 1500 ug, or at least identifying a subject having, or at risk of developing, a 2000 g of silt-1 from human blood or plasma. In certain pregnancy-related hypertensive disorder and then practicing embodiments, the column contains sufficient anti-SElt-1 the ex vivo methods disclosed herein on the subject so 60 antibodies, or sPlt-1 binding fragments thereof, bound to a identified, thereby treating or preventing the pregnancy Solid Support, to remove at least 10 ug to 2000 ug, at least related hypertensive disorder. In certain embodiments, a 20 ug to 1000 ug, at least 50 lug to 500 ug, or at least 100 g pregnant human is identified as a Subject Suitable for treat to 200 ug of slfilt-1 from human blood or plasma. ment or prevention by the methods disclosed herein if the The present invention provides methods of making a concentration of slfilt-1 in the subject’s blood during the 65 device for treating or preventing a pregnancy-related hyper second trimester of pregnancy is determined to be above tensive disorder Such as eclampsia or pre-eclampsia com about 3.5 ng/ml, above about 4 ng/ml, above about 5 ng/ml. prising: US 9,592,331 B2 25 26 (a) attaching anti-SFlt-1 antibodies, or SFlt-1 binding thereof, and returned to the subjects body following fragments thereof, to a solid Support to produce anti-SFlt-1 removal of slfilt-1 from the blood by the anti-sflt-1 antibod antibodies, or sPlt-1 binding fragments thereof, bound to a ies, or SFlt-1 binding fragments thereof. In some embodi Solid Support, ments, the apheresis system is a plasmapheresis system and (b) introducing the anti-slfilt-1 antibodies, or sPlt-1 bind plasma rather than blood is passed over a solid Support ing fragments thereof, bound to the Solid Support into a containing bound anti-slfilt-1 antibodies, or sPlt-1 binding housing or chamber Such as a column to produce a housing fragments thereof, and returned to the subject’s body fol or chamber containing the anti-slfilt-1 antibodies, or sPlt-1 lowing removal of silt-1 from the plasma by the anti-slfilt-1 binding fragments thereof, bound to the Solid Support, antibodies, or sPlt-1 binding fragments thereof. (c) fluidly connecting the housing or chamber containing 10 the anti-slfilt-1 antibodies, or sPlt-1 binding fragments In certain embodiments, the methods disclosed herein thereof, bound to the Solid Support, to a means for conveying may be carried out using a modified version of a device blood or plasma from a subject to the anti-slfilt-1 antibodies, known in the art that enables removal and extracorporeal or anti-SFlt-1 antigen binding fragments thereof, bound to treatment of a body fluid such as whole blood or plasma. the Solid Support, 15 One Such device is a dialysis machine. Dialysis machines are (d) fluidly connecting the housing or chamber containing in routine use and methods to control blood flow, remove air the anti-slfilt-1 antibodies, or sPlt-1 binding fragments bubbles, and maintain proper electrolyte balance, blood thereof, bound to the Solid Support, to a means for conveying Sugar, oxygenation, temperature, sterility, and other vital the blood or plasma from the anti-slfilt-1 antibodies, or sPlt-1 factors during dialysis, are well known and established in the binding fragments thereof, bound to the Solid Support, to the art. In certain embodiments, the methods disclosed herein Subject, where the means are connected to the housing or may be carried out using existing dialysis systems where the chamber so as to allow for contact of the blood or plasma dialyzer is replaced by a housing or chamber, such as a from the subject with the anti-slfilt-1 antibodies, or anti column, containing a solid Support to which anti-SElt-1 SFlt-1 antigen binding fragments thereof, bound to the solid antibodies, or SFlt-1 binding fragments thereof, are attached. support, and thereby remove sRlt-1 from the blood or 25 When blood flows through the housing or chamber, the plasma. anti-slfilt-1 antibodies, or sPlt-1 binding fragments thereof, The present invention provides methods of making a remove slfilt-1 from the blood, thereby lowering the concen device for treating or preventing a pregnancy-related hyper tration of sRlt-1 in the blood and treating or preventing a tensive disorder Such as eclampsia or pre-eclampsia com pregnancy-related hypertensive disorder Such as pre-ec prising modifying a dialysis or apheresis device or system so 30 lampsia or eclampsia. as to provide the dialysis or apheresis device or system with Another well known device that can be used to practice a housing or chamber Such as a column containing anti the methods described herein is an apheresis system, e.g., a sElt-1 antibodies, or sPlt-1 binding fragments thereof, bound plasmapheresis system. Plasmapheresis involves the extra to a solid Support, so as to allow the dialysis or apheresis corporeal manipulation and removal of certain cellular com device or system to provide for the contact of blood or 35 ponents of the blood, after which the blood is reinfused into plasma from a subject with the anti-slfilt-1 antibodies, or the subject to induce a desired clinical effect. During plas anti-SElt-1 antigen binding fragments thereof, bound to the mapheresis, blood is initially taken out of the body through solid support, and thereby remove sRlt-1 from the blood or an access device such as a needle or catheter. Plasma is then plasma to produce silt-1-depleted blood or plasma. removed from the blood by a cell separator. Three proce In certain embodiments, the present invention provides 40 dures are commonly used to separate the plasma from blood methods of identifying an anti-slfilt-1 antibody suitable for cells: (1) Discontinuous flow centrifugation, where, typi use in ex vivo methods of treating or preventing a preg cally, a 300 ml volume of blood is removed at a time and nancy-related hypertensive disorder Such as eclampsia or centrifuged to separate plasma from blood cells. (2) Con pre-eclampsia comprising: tinuous flow centrifugation, where centrifugation is used to (a) obtaining an antibody that binds to sRlt-1; 45 continuously spin out plasma. (3) Plasma filtration, where (b) attaching the antibody that binds to sRlt-1 to a solid the plasma is filtered using standard hemodialysis equip Support to produce a solid Support comprising bound anti ment. sElt-1 antibody; Apheresis devices suitable for modification for use in the (c) determining if the Solid Support comprising bound methods disclosed herein are described, e.g., in U.S. Pat. No. anti-slfilt-1 antibody can bindsElt-1 in a fluid sample from a 50 5,098,372; U.S. Pat. No. 5,112,298; and U.S. Pat. No. subject and thereby remove silt-1 from the fluid sample: 6,319,471. Other suitable devices include the LIFE-18R) where if the solid support comprising bound anti-slfilt-1 plasma therapy device from PlasmaSelect (Munich, Ger antibody can bind slflt-1 in a fluid sample from a subject and many), the Diapact(R) CRRT from B. Braun (Melsungen, thereby remove sRlt-1 from the fluid sample, the antibody of Germany), the COBE SPECTRAR), a product of Cobe BCT, step (a) is identified as an anti-slfilt-1 antibody suitable for 55 Incorporated, 1201 Oak Street, Lakewood, Co. 80215, and use in ex vivo methods of treating or preventing a preg the ELUTRAR) Cell Separation System of Gambro BCT, nancy-related hypertensive disorder Such as eclampsia or Inc. pre-eclampsia. In certain embodiments of the systems disclosed herein, In certain embodiments, the Subject is a mammal. In the access device for accessing a Subject's blood system certain embodiments, the Subject is a human. 60 and/or the return device for returning blood, plasma, or In certain embodiments, the fluid sample is blood, plasma, cellular components of blood to a Subject is a single lumen amniotic fluid, or urine. catheter or a double lumen catheter Such as, e.g., the single A modified dialysis or apheresis system can be used to lumen or double lumen catheters sold by Fresenius Medical practice the methods disclosed herein, wherein the modified Care (Bad Homburg, Germany). Such catheters may be dialysis or apheresis system provides the means by which 65 made of thermosensitive polyurethane that adapts to the blood is removed, passed over a solid Support containing contour of a blood vessel as the polyurethane heats to body bound anti-sflt-1 antibodies, or sPlt-1 binding fragments temperature. US 9,592,331 B2 27 28 In certain embodiments of the methods disclosed herein, Solid phase matrix (agarose beads). The agarose beads were removing blood from the Subject includes removing an chemically treated with cyanogen bromide to create a reac amount of blood from the subject sufficient to derive at least tive chemical group on the beads. These activated beads about 650 milliliters of plasma from the blood. In certain were then mixed with antibody to covalently attach the embodiments, removing the blood from the subject includes 5 antibodies to the beads. removing at least two liters of blood from the subject. In The beads with attached antibodies were then poured into certain embodiments, removing the blood from the subject a 1 ml column containing a screen/frit at the bottom, includes continuously removing blood from the Subject until retaining the beads inside the column, but allowing fluids or substantially the entire blood volume of the subject is Solutions to pass through the column. To the resulting contacted with anti-slfilt-1 antibodies, or sPlt-1 binding frag 10 device, containing anti-SElt-1 antibodies attached to beads, ments thereof, at least once, at least twice, or at least three amniotic fluid from pre-eclampsia patients was added at the times. In certain embodiments, removing the blood from the top of the device and the solution that flowed through the Subject includes continuously removing blood from the device and out the bottom of the column was collected. The subject until about two-thirds, about half, about one-fourth, amount of sRlt-1 in the amniotic fluid before and after about one-fifth, or about one-tenth of the entire blood 15 passing through the device was measured, and the '% of volume of the subject is contacted with anti-slfilt-1 antibod depleted sRlt-1 was calculated. ies, or sPlt-1 binding fragments thereof. In certain embodi Further details were as follows: ments, removing the blood from the Subject includes con (1) 0.1 ml of agarose beads coupled to anti-slfilt-1 anti tinuously removing blood from the subject until the body were added to a 1 ml column. concentration of sRlt-1 in the subject’s blood reaches a (2) 500 lug of antibody were bound to the agarose beads. preselected concentration. In certain embodiments, the pre (3) The column was washed with 4 ml of phosphate selected concentration is less than about 50 ng/ml, less than buffered saline (PBS). about 40 ng/ml, less than about 25 ng/ml, less than about 10 (4) 1 ml of amniotic fluid from pre-eclampsia patients ng/ml, less than about 5 ng/ml, less than about 4 ng/ml, less (containing approximately 40 ng of SFlt-1 protein) was than about 3 ng/ml, less than about 2 ng/ml, less than about 25 added to the top of the column. 1 ng/ml, less than about 0.75 ng/ml, or less than about 0.5 (5) The amniotic fluid was run over the column at a flow ng/ml. In certain embodiments, the preselected concentra rate of approximately 1 ml/15 min. at room temperature tion is about 40-50 ng/ml, about 30-40 ng/ml, about 20-30 (21-24°C.). ng/ml, about 10-20 ng/ml, about 5-10 ng/ml, about 5-8 (6) The amniotic fluid was collected and re-applied to the ng/ml, about 3-7 ng/ml, about 1-5 ng/ml, about 1-3 ng/ml. 30 device two times, resulting in a total of three passages over about 0.75-2 ng/ml, or about 0.5-1 ng/ml. the column. The sRlt-1 concentration can be measured automatically (7) After the third passage over the column, the flow in blood or plasma, either continuously, or at preset inter through solution was collected and tested for sPlt-1 concen vals. For example, plasma samples from the Subject can be tration. reacted with a labeled reagent that binds to sRlt-1 or particles 35 (8) The device was washed with 4 ml of buffer to remove containing SFlt-1 and the amount of SFlt-1 measures. Alter any material that bound non-specifically to the beads/col natively, a sensor with a linked reagent that specifically umn. Then 0.5 ml of 0.5 M acetic acid (pH 3.0) was added binds to SFlt-1 (including particles containing SElt-1) can be to the device to disassociate the bound sPlt-1 from the used to continuously detect the amount of bound sPlt-1. The device. The fractions of the eluted solution were collected blood filtration procedure is terminated when the concen 40 and the SFlt-1 concentration was measured to determine tration of silt-1 detected in a subject’s blood or plasma whether there was any change. drops below a predetermined value. Results for exemplary antibodies, including affinity for sElt-1 and % sRlt-1 removed from amniotic fluid are shown EXAMPLES in Table 2 below. 45 Example 1 TABLE 2 Removal of silt-1 from Human Amniotic Fluid Sample Kd (M) % sElt-1 removed Using a Column Device Containing a Solid amniotic fluid before column O Support with Bound Anti-slfilt-1. Antibodies or 50 column (no antibody) O Ligands column with Factor VIII antibody <1 column with antibody 101 144E-09 53 The experimental conditions were designed to approxi column with antibody 102 2.17 E-10 85 column with antibody 103 3.12E-10 87 mate use in a clinical setting, but on a smaller scale. column with antibody 104 n.d. 85 Amniotic fluid was obtained from human pre-eclampsia 55 column with antibody 105 7.OSE-08 <1 patients with elevated sPlt-1 levels of about 40 ng/ml. column with antibody 106 1.58E-09 59 All of the antibodies used were mouse monoclonal anti column with antibody 107 8.11E-09 <1 column with antibody 108 4.99E-09 11 bodies which bind to sRlt-1, with the exception of one column with antibody 109 7.66E-10 48 control column which used polyclonal antibodies to human column with antibody 110 3.36E-10 58 Factor VIII, The anti-slt-1 monoclonal antibodies were 60 column with antibody 111 3.18E-10 50 made by immunizing mice with human SFlt-1 protein (Ig column with antibody 112 S.35E-10 28 like domains 1-3) which includes amino acids Ser27 to column with VEGF n.d. 50 Ile328. This protein also had a poly-histidine affinity tag at the carboxy terminal. 12 antibodies that bound to slfilt-1 were A similar method was used with VEGF. VEGF was selected and tested for binding affinity to slflt-1. 65 expressed in bacteria, purified by column chromatography, A device for removing sRlt-1 protein from a biological and coupled to cyanogen bromide-activated agarose beads. Solution was made by attaching anti-SElt-1 antibodies to a Otherwise, the procedure for applying amniotic fluid to the US 9,592,331 B2 29 30 column containing the agarose bead-coupled VEGF was TABLE 3 substantially the same as for the antibodies. The results for the anti-slfilt-1 antibodies and VEGF are Flow Rate (mL/min) % sRlt1depletion shown in FIG. 2. The results show that anti-sfilt-1 antibodies and sPlt-1 O.S 98% ligand bound to a solid Support were able to specifically 1.O 94% remove sRlt-1 from amniotic fluid of pre-eclamptic patients. 1.5 87% A control column device containing the matrix/beads only 3.0 770, with no antibody or ligand attached did not remove sRlt-1 10 protein from amniotic fluid. A control column device con taining an antibody that binds to coagulation Factor VIII also did not remove slfilt-1. However, when antibodies or ligands Linear Flow Rate Forty bed volumes of horse serum that bind sPlt-1 were used in the column, slfilt-1 protein spiked with 40 ng/mL of sfilt1 (Input) was applied to a levels were reduced in the flow-through amniotic fluid by up 15 column containing 1 mL of Sepharose beads coupled to to 87%. A significant variation in how effective individual AG10B monoclonal antibody (0.8 mg), and the flow antibodies were in removing slflt-1 was observed (11-87%). through fractions (FT) were collected. The sRlt1 concentra The apparent Kd of binding between purified monoclonal tions in the Input and FT fractions were determined using the antibodies and SFlt1 was measured by Surface plasmon R&D Flt-1 DuoSet kit (DY321). The % slflt1 depletion was resonance (SPR). (FIG. 2B). Antibodies were immobilized calculated by the formula, 9% silt1 depletion-(sElt1 on the solid phase and sPlt-1 (domains 1-3) was in the liquid sFlt1)/sPlt1). The variation of SElt-1 depletion with phase. There was no direct correlation between antibody linear flow rate is shown in Table 4 and FIG. 5. affinity (as measured in a kinetic binding experiment) (FIG. 2B) and effectiveness (FIG. 2A) in the device. There was TABLE 4 also no direct correlation with on-rates or off-rates and 25 device effectiveness. These results show that devices com Linear Flow Rate (cm/hr) % silt1 depletion prising anti-SElt-1 antibodies bound to a Solid Support can be used to treat pregnancy-related hypertensive disorders, 38 98% including pre-eclampsia. 76 94% 30 113 87% Example 2 230 779,
Chimerization Residence Time Forty bed volumes of horse serum 35 spiked with 40 ng/mL of sfilt1 (Input) was applied to a Chimeric monoclonal antibodies were produced having column containing 1 mL of Sepharose beads coupled to murine variable regions and human IgG1 constant regions. AG10B monoclonal antibody (0.8 mg), and the flow Several variations of chimeric antibody were produced. through fractions (FT) were collected. The sRlt1 concentra Antibody AG10A (V: SEQID NO:35; V: SEQID NO:36) tions in the Input and FT fractions were determined using the consists of the heavy and light chain variable domains of 40 R&D Flt-1 DuoSet kit (DY321). The % slflt1 depletion was antibody 102 and a human IgG1 constant region. Antibody AG10B (V: SEQ ID NO:37; V,: SEQ ID NO:36) incor calculated by the formula, 9% silt1 depletion-(sElt1 porates a mutation (N298Q) that removes a glycosylation sFlt1)/sPlt1). The variation of SElt-1 depletion with site in the constant region. Antibody AG10C (V: SEQ ID column residence time is shown in Table 5 and FIG. 6. NO:38; V: SEQ ID NO:36) incorporates a mutation 45 (1254A) that disrupts binding to FcRn. Antibody AG10D TABLE 5 (V: SEQID NO:39; V: SEQID NO:36) incorporates both Residence Time (min) % silt1 depletion of the aforementioned mutations. O.33 779, 50 O.67 87% Example 3 1.00 94% 2.00 98% Characteristics of Immobilized Antibody AG10B Monoclonal Ab Density Horse serum spiked with 55 recombinant SElt1 was applied over a 1-mL column con Various tests of the sRlt-1 depletion characteristics of taining Sepharose beads (about 1.5x10 beads/ml), coupled Antibody AG 1 OB were performed. to various amounts of AG10B at flow rate of 0.5 mL/min Flow Rate Forty bed volumes of horse serum spiked with 40 ng/mL of slflt1 (Input) was applied to a column (residence time of 2 min). containing 1 mL of packed Sepharose beads coupled to 60 For 0.8 mg of Ab (about 3.2x10" molecules) per ml, this AG10B monoclonal antibody (0.8 mg), and the flow amounts to about 2.1x10 molecules per bead. Similarly, at through fractions (FT) were collected. The sRlt1 concentra 0.4 mg of Ab (1.6x10" molecules) there are about 1.05x10 tions in the Input and FT fractions were determined using the moleculesper bead. Given a bead surface area of 2.5x10' R&D Flt-1 DuoSet kit (DY321). The % slflt1 depletion was cm, 0.8 mg of Ab per 1 mL beads amounts to about 8.4xE' calculated by the formula, 9% split1 depletion-(sElt1 65 Ab molecules per cm bead surface area (not including sFlt17)/split1... The variation of SFlt-1 depletion with pores). The % sRlt1 depletion was determined by dividing flow rate is shown in Table 3 and FIG. 4. the depleted amount of slflt1 by the total slflt1 input. The US 9,592,331 B2 31 sElt1 concentrations were determined using the R&D Flt-1 TABLE 7 DuoSet kit (DY321). The variation of sRlt-1 depletion with sElt-1 levels Serum + MAb density is shown in Table 6 and FIG. 7. (ng/ml) Serum Heparin TABLE 6 Total SFlt-1 29.3 26.2 sElt-1 depleted by Ag10B 21.6 20.9 Ab density (mg/mL beads) % silt1 depletion % depletion 74% 80% O.O2S 74% O.OSO 82% AG10B bound to sRlt-1 does not block binding of slfilt-1 O.100 84% 10 to VEGF. As depicted in FIG. 12, ELISA plates were coated O.200 90% with either sRlt1 or VEGF. After washing, PBS was added O.400 96% to well coated with sElt-1, and either PBS or sPlt1 was added O.800 97% to wells coated with VEGF. After washing, AG10B or 18F1 (an antibody that blocks slflt1 VEGF interaction) was 15 added to wells containing immobilized VEGF bound to Depletion of slfilt-1 from plasma and serum. sElt-1, and sPlt1 pre-complexed with AG10B, or sPlt1 Normal human plasma spiked with recombinant slt1 was pre-complexed with 18F 1 was added to wells coated with applied over 0.1-mL columns containing Sepharose beads PBS. As indicated in FIG. 12, AG10B binds to sRlt1 and coupled to AG1 OB or Erbitux. The percent of depletion was sFlt1/VEGF complexes. Pre-complexed sRlt1/AG10B also determined for a wide range of Ab:ligand ratios. Diminished binds to VEGF. In contrast, 18F1, a blocking antibody, does capacity of the column occurs when the AG10B:sFlt1 ratio not bind to sRlt1/VEGF complexes. Similarly, addition of 18F1 to sRlt1 prevents VEGF and sPlt1 interaction. is below 200:1. (FIG. 8). Column runs were performed by Complement Activation—AG1 OB coupled to beads does gravity flow so residence time and flow rates were variable. not activate the complement system more than beads alone Amounts of slflt1 were determined by R&D Flt-1 DuoSet kit (FIG. 13). Human blood plasma spiked with purified sRlt1 (DY321). 25 was applied to 0.1-mL columns containing Sepharose beads Horse serum spiked with recombinant slflt1 was applied coupled to purified monoclonal antibody AG1 OB or a nega over 1-mL columns containing Sepharose beads coupled to tive control column without antibody (PBS pH 7.4, Beads). AG10B. The percent of depletion was determined for range Samples were heated to 37° C. and assayed for complement of MAb:ligand ratios. Diminished capacity of the column activation product C3a using Quidel MicroVue C3a Plus 30 EIA kit according to the manufacturer's instructions. C3a occurs when the AG10B:slflt1 ratio is below 25:1. (FIG. 9). standards provided in the kit were used to generate a Columns were run at 1 mL/min with a residence time of 1 standard curve used to determine the C3a concentrations in min. Amounts of slflt1 were determined by R&D Flt-1 the plasma fractions. DuoSet kit (DY321). AG10B binds to an epitope in the d1-d3 domain of slfilt-1. sElt-1 depletion by AG10B does not vary with column 35 Table 8 shows that AG10B binds to an epitope on native size. Horse serum spiked with 40 bed volumes of 40 ng/mL sElt1 forms that exist in amniotic fluid (AF) of PE patients as well as two recombinant forms (d1-d3 domain or full of slflt1 was applied at residence time of 2 min to either length) of silt1. The blocking antibody 18F1 that competes 1-mL or 50-mL columns containing Sepharose beads with VEGF for a binding site on sElt1 is not able to bind as coupled to AG10B monoclonal antibody (0.8 mg or 40 mg. efficiently to sRlt1 (d1-d3) in the presence of VEGF. The 508 respectively), and the flow-through fractions (FT) were 40 antibody cannot bind to slflt1 (d1-d3) but can bind native collected. The sRlt1 concentrations in the Input and FT SFlt1 isoforms in AF, indicating that its binding epitope on fractions were determined using the R&D Flt-1 DuoSet kit sElt1 may be located outside of the d1-d3 domain. The (DY321). The % slflt1 depletion was calculated by the negative control antibody EbX cannot bind to native or formula, % SElt1 depletion (sElt1-sFlt17)/silt1). recombinant forms of slt1. FIG. 10 shows that both 1 mL and 50 mL device columns 45 can deplete nearly all of the sRlt1 protein in serum. TABLE 8 Heparin does not interfere with sElt-1 depletion by Native Recombinant Recombinant Recombinant SFlt1 in SFlt1 sFlt1 (d1-d3) + selt1 full AG10B. Horse serum containing recombinant slt1 with or Amniotic Fluid (d1-d3) VEGF length without 0.45 U of heparin was applied over a 0.1-mL 50 column containing Sepharose beads coupled to AG10B. The AG1 OB ------18F1 n.d. -- +f- n.d. samples were assayed for sPlt1 levels before (load) and after SO8 -- n.d. (FT) flowing through the AG10B-containing columns. The EbX sElt1 levels were assayed using the R&D Flt-1 DuoSet kit (DY321). (Table 7, FIG. 11)
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 39
<21 Oc SEO ID NO 1 <211 LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Mus musculus <22 Os FEATURE; NAME/KEY: CDS
US 9,592,331 B2 35 36 - Continued
<4 OO > SEQUENCE: 7 aag gcc agt cag agt gtg agt att gat gta gct 33 Lys Ala Ser Glin Ser Val Ser Ile Asp Wall Ala 1. 5 1O
<210s, SEQ ID NO 8 &211s LENGTH: 11 212. TYPE: PRT &213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 8 Lys Ala Ser Glin Ser Val Ser Ile Asp Wall Ala 1. 5 1O
<210s, SEQ ID NO 9 &211s LENGTH: 21 &212s. TYPE: DNA &213s ORGANISM: Mus musculus 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (21)
<4 OOs, SEQUENCE: 9 cat goa to c aat cqg tac act 21 His Ala Ser Asn Arg Tyr Thr 1. 5
<210s, SEQ ID NO 10 &211s LENGTH: 7 212. TYPE PRT &213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 10 His Ala Ser Asn Arg Tyr Thr 1. 5
<210s, SEQ ID NO 11 &211s LENGTH: 27 &212s. TYPE: DNA &213s ORGANISM: Mus musculus 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (27)
<4 OOs, SEQUENCE: 11
Cag cag aat tat gac t ct c catt c acg 27 Glin Glin Asn Tyr Asp Ser Pro Phe Thr 1. 5
<210s, SEQ ID NO 12 &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 12
Glin Glin Asn Tyr Asp Ser Pro Phe Thr 1. 5
<210s, SEQ ID NO 13 &211s LENGTH: 366 &212s. TYPE: DNA &213s ORGANISM: Mus musculus 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (366) US 9,592,331 B2 37 38 - Continued
<4 OOs, SEQUENCE: 13 Cag gtg cag ctgaag cag tica gga cct ggc cta gtg cag ccc. tca cag 48 Glin Val Glin Leu Lys Glin Ser Gly Pro Gly Lieu Val Glin Pro Ser Glin 1. 5 1O 15 agc ctd to c ct c acc togc aca gtc. tct ggit titc. tca tta act aat tat 96 Ser Leu Ser Lieu. Thr Cys Thr Val Ser Gly Phe Ser Lieu. Thir Asn Tyr 2O 25 3O ggt gta cat tig att CC cag tot coa gga aag ggit Ctg gag tog Ctg 144 Gly Val His Trp Ile Arg Glin Ser Pro Gly Lys Gly Lieu. Glu Trp Lieu. 35 4 O 45 gga gtg at a tigg agt ggt gga agc at C gat tat aat gca gtt tt C aca 192 Gly Val Ile Trp Ser Gly Gly Ser Ile Asp Tyr Asn Ala Val Phe Thr SO 55 6 O tcc aga titg acc atc acc aag gac cat to c aag agc caa gtt titc titt 24 O Ser Arg Lieu. Thir Ile Thr Lys Asp His Ser Lys Ser Glin Val Phe Phe 65 70 7s 8O aaa gtgaac agt Ctg gaa agt aat gac aca gcc at a tat tac tit gcc 288 Llys Val Asn. Ser Lieu. Glu Ser Asn Asp Thir Ala Ile Tyr Tyr Cys Ala 85 90 95 aga aat gtg ggc tat agg tac gaC gac ggc tat gtt atg gac tac tig 336 Arg Asn Val Gly Tyr Arg Tyr Asp Asp Gly Tyr Val Met Asp Tyr Trip 1OO 105 11 O ggit caa gga acc tica gtc atc gtc. tcc to a 366 Gly Glin Gly Thr Ser Val Ile Val Ser Ser 115 12 O
<210s, SEQ ID NO 14 <211 LENGTH: 122 212. TYPE: PRT <213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 14 Glin Val Glin Leu Lys Glin Ser Gly Pro Gly Lieu Val Glin Pro Ser Glin 1. 5 1O 15 Ser Leu Ser Lieu. Thr Cys Thr Val Ser Gly Phe Ser Lieu. Thir Asn Tyr 2O 25 3O Gly Val His Trp Ile Arg Glin Ser Pro Gly Lys Gly Lieu. Glu Trp Lieu. 35 4 O 45 Gly Val Ile Trp Ser Gly Gly Ser Ile Asp Tyr Asn Ala Val Phe Thr SO 55 6 O Ser Arg Lieu. Thir Ile Thr Lys Asp His Ser Lys Ser Glin Val Phe Phe 65 70 7s 8O Llys Val Asn. Ser Lieu. Glu Ser Asn Asp Thir Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Asn Val Gly Tyr Arg Tyr Asp Asp Gly Tyr Val Met Asp Tyr Trip 1OO 105 11 O Gly Glin Gly Thr Ser Val Ile Val Ser Ser 115 12 O
<210s, SEQ ID NO 15 &211s LENGTH: 327 &212s. TYPE: DNA <213s ORGANISM: Mus musculus 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (327)
<4 OOs, SEQUENCE: 15 agt att gtg atg acc cag act coc aaa titc ctd citt gta to a gca gga 48 Ser Ile Val Met Thr Glin Thr Pro Llys Phe Leu Lleu Val Ser Ala Gly US 9,592,331 B2 39 40 - Continued
1. 5 1O 15 gac agg gtt acc ata acc tic aag gcc agt cag agt gtg agt att gat 96 Asp Arg Val Thir Ile Thr Cys Lys Ala Ser Glin Ser Val Ser Ile Asp 2O 25 3O gta gct tog tac Caa Cag aag cca ggg cag tdt CCt aaa citt Ctg at a 144 Val Ala Trp Tyr Glin Gln Llys Pro Gly Glin Ser Pro Llys Lieu. Lieu. Ile 35 4 O 45 tat cat gca to c aat cqg tac act gga gtc. cct gat cqc ttic att gga 192 Tyr His Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ile Gly SO 55 6 O agt aga tat ggg acg gat titc act tt C acc atc agc act gtg cag gct 24 O Ser Arg Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Glin Ala 65 70 7s 8O gaa gac ct g goa gtt tat titc tdt cag cag aat tat gac tot coa titc 288 Glu Asp Leu Ala Val Tyr Phe Cys Glin Glin Asn Tyr Asp Ser Pro Phe 85 90 95 acg tt C ggc ticg ggg aca aag ttg gala tta aaa C9g gct 327 Thir Phe Gly Ser Gly Thr Llys Lieu. Glu Lieu Lys Arg Ala 1OO 105
<210s, SEQ ID NO 16 &211s LENGTH: 109 212. TYPE: PRT <213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 16 Ser Ile Val Met Thr Glin Thr Pro Llys Phe Leu Lleu Val Ser Ala Gly 1. 5 1O 15 Asp Arg Val Thir Ile Thr Cys Lys Ala Ser Glin Ser Val Ser Ile Asp 2O 25 3O Val Ala Trp Tyr Glin Gln Llys Pro Gly Glin Ser Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr His Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ile Gly SO 55 6 O Ser Arg Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Glin Ala 65 70 7s 8O Glu Asp Leu Ala Val Tyr Phe Cys Glin Glin Asn Tyr Asp Ser Pro Phe 85 90 95 Thir Phe Gly Ser Gly Thr Llys Lieu. Glu Lieu Lys Arg Ala 1OO 105
<210s, SEQ ID NO 17 &211s LENGTH: 30 &212s. TYPE: DNA <213s ORGANISM: Mus musculus 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (3 O
<4 OOs, SEQUENCE: 17 gga tac acatt c act gac tat gtt ata agt 3 O Gly Tyr Thr Phe Thr Asp Tyr Val Ile Ser 1. 5 1O
<210s, SEQ ID NO 18 &211s LENGTH: 10 212. TYPE: PRT <213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 18 Gly Tyr Thr Phe Thr Asp Tyr Val Ile Ser
US 9,592,331 B2 43 44 - Continued
212. TYPE: PRT &213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 24 Lys Ala Ser Glin Asp Wall. Thir Ile Thir Wall Ala Trp Tyr 1. 5 1O
SEQ ID NO 25 LENGTH: 33 TYPE: DNA ORGANISM: Mus musculus FEATURE: NAME/KEY: CDS LOCATION: . (33)
<4 OOs, SEQUENCE: 25 citt ctd att tac tcg gca toc tac cqg tac act 33 Lieu. Lieu. Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr 1. 1O
<210s, SEQ ID NO 26 &211s LENGTH: 11 212. TYPE: PRT &213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 26
Lieu. Lieu. Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr 1. 5 1O
SEQ ID NO 27 LENGTH: 27 TYPE: DNA ORGANISM: Mus musculus FEATURE: NAME/KEY: CDS LOCATION: (1) . . (27)
<4 OOs, SEQUENCE: 27 cag caa cat tat act act ccd tig acg 27 Gln Gln His Tyr Thir Thr Pro Trp Thr 1. 5
<210s, SEQ ID NO 28 &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 28 Gln Gln His Tyr Thir Thr Pro Trp Thr 1.
SEQ ID NO 29 LENGTH: 35 4. TYPE: DNA ORGANISM: Mus musculus FEATURE: NAME/KEY: CDS LOCATION: (1) ... (354)
<4 OOs, SEQUENCE: 29
Cag gtt cag Ctg Cag Cag tot gga cott gag Ctg gtg aag cott ggg gct 48 Glin Wall Gln Lieu. Glin Glin Ser Gly Pro Glu Luell Wall Pro Gly Ala 1. 1O 15 toa gtg aag atg t cc tgc aag gct tot gga tac a Ca tto act gac tat 96 Ser Wall Lys Met Ser Cys Ala Ser Gly Tyr Thir Phe Thir Asp Tyr 2O 25 3O US 9,592,331 B2 45 46 - Continued gtt at a agt tog gtg aaa cag aga act gga cag ggc Ctt gag tog att 144 Val Ile Ser Trp Val Lys Glin Arg Thr Gly Glin Gly Lieu. Glu Trp Ile 35 4 O 45 gga gag att tat cct gga agt ggit agt att tac tac aat gag aag titc 192 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ile Tyr Tyr Asn Glu Lys Phe SO 55 6 O aag ggc aag goc aca citg act gca gac aca toc toc aac aca gcc tac 24 O Lys Gly Lys Ala Thr Lieu. Thir Ala Asp Thir Ser Ser Asn. Thir Ala Tyr 65 70 7s 8O atg cag ct c agc agc ctg aca ttt gag gac tot gcg gtc att tt C tit 288 Met Glin Leu Ser Ser Lieu. Thir Phe Glu Asp Ser Ala Val Ile Phe Cys 85 90 95 gca aga ggg cat tat tac ggit tac titt gac tac tig ggc caa ggc acc 336 Ala Arg Gly His Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Glin Gly Thr 1OO 105 11 O act ct c aca gtc. tcc tica 3.54 Thir Lieu. Thir Wal Ser Ser 115
<210s, SEQ ID NO 3 O &211s LENGTH: 118 212. TYPE: PRT <213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 30 Glin Val Glin Lieu. Glin Glin Ser Gly Pro Glu Lieu Val Llys Pro Gly Ala 1. 5 1O 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 2O 25 30 Val Ile Ser Trp Val Lys Glin Arg Thr Gly Glin Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ile Tyr Tyr Asn Glu Lys Phe SO 55 6 O Lys Gly Lys Ala Thr Lieu. Thir Ala Asp Thir Ser Ser Asn. Thir Ala Tyr 65 70 7s 8O Met Glin Leu Ser Ser Lieu. Thir Phe Glu Asp Ser Ala Val Ile Phe Cys 85 90 95 Ala Arg Gly His Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Glin Gly Thr 1OO 105 11 O
Thir Lieu. Thir Wal Ser Ser 115
<210s, SEQ ID NO 31 &211s LENGTH: 327 &212s. TYPE: DNA <213s ORGANISM: Mus musculus 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (327)
<4 OOs, SEQUENCE: 31 gac att gtg atg acc cag tot cac aaa titc atgtcc aca to a gta gga 48 Asp Ile Val Met Thr Glin Ser His Llys Phe Met Ser Thr Ser Val Gly 1. 5 1O 15 gac agg gt C agc atc acc tic aag gcc agt cag gat gtg act att act 96 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Glin Asp Val Thir Ile Thr 2O 25 3O gta gcc tigg tat caa cag aaa cca gga caa tot cot aaa citt ctd att 144 Val Ala Trp Tyr Glin Gln Llys Pro Gly Glin Ser Pro Llys Lieu. Lieu. Ile 35 4 O 45
US 9,592,331 B2 55 56 - Continued tgt gat titt ggc citt gcc cgg gat att tat aag aac ccc gat tat 31.59 Cys Asp Phe Gly Luell Ala Arg Asp Ile Lys Asn Pro Asp Tyr O4 O O45 OSO gtg a.a.a. gga gat act citt cott Ctg a.a.a. tgg atg gct cott 3204 Wall Gly Asp Thir Luell Pro Luell Trp Met Ala Pro O65 gaa atc. titt gac a.a.a. tac agc acc aag agc gac gtg tgg 3249 Glu Ile Phe Asp Tyr Ser Thir Lys Ser Asp Wall Trp O8O tot tac gga gta ttg Ctg gala at C titc t cc tta get gig tot 3.294 Ser yr Gly Wall Luell Luell Glu Ile Phe Ser Lieu. Gly Gly Ser O85 O95
C Ca tac C Ca gga gta Cala gat gag gac titt tgc agt cqc Ctg 3339 Pro Pro Gly Wall Glin Asp Glu Asp Phe Cys Ser Arg Lell OO 10 agg ggc atg agg atg gct cott gag tac tot act cott gaa 3384 Arg Gly Met Arg Met Ala Pro Glu Ser Thr Pro Glu 25 atc. Cag atc. atg Ctg tgc tgg CaC aga gac cca aaa gaa 3.429 Ile Glin Ile Met Luell Cys Trp His Arg Asp Pro Llys Glu 4 O agg aga titt gca gala gtg gala a.a.a. Cta ggt gat ttg citt 3474 Arg Arg Phe Ala Glu Wall Glu Lell Gly Asp Lieu. Lell 55
Cala aat gta Cala cag ggt a.a.a. gac atc Coa atc. aat 3519 Glin Asn Wall Glin Glin Gly Asp e Pro Ile Asn 70 gCC Ctg ca 99a aat 999 titt Ca teca act cot gCC 3564 Ala Lell Thir Gly Asn Gly Phe Thir Ser Thr Pro Ala 85 tto gag gac ttic ttic gala agt att toa gct cog aag titt 3. 609 Phe Glu Asp Phe Phe Glu Ser Ile Ser Ala Pro Llys Phe 2OO aat gga agc tot gat gt C aga tat gta aat gct titc aag 3.654 Asn Gly Ser Ser Asp Wall Arg Wall Asn Ala Phe Lys 215 tto agc Ctg gala aga a.a.a. acc titt gaa gaa citt tta cc.g 3699 Phe Ser Lell Glu Arg Thir Phe Glu Glu Lieu. Lieu. Pro 23 O aat a CC t cc atg titt gac tac cag ggc gac agc agc act 3744 Asn Thir Ser Met Phe Asp Glin Gly Asp Ser Ser Thir 245
Ctg gcc tot cc c atg aag cgc titc a CC tgg act gac agc 3789 Lell Ala Ser Pro Met Lys Arg Phe Thir Trp. Thir Asp Ser 26 O
aag gcc tog citc. att gac ttg aga gta acc agt a.a.a. 383 4 Ala Ser Luell Ile Asp Luell Arg Wall. Thir Ser Lys 27s
gag tcg 999 Ctg gat gt C agc agg c cc agt titc tgc 3879 Glu Ser Gly Luell Asp Wall Ser Arg Pro Ser Phe Cys 29 O
Cat agc 999 CaC gt C agc gala ggc aag cgc agg ttc a CC 392.4 His Ser Gly His Wall Ser Glu Gly Arg Arg Phe Thir 3OO 305 tac CaC gct gag Ctg gala agg a.a.a. atc. gcg tgc tigc ticc cc.g 3969 His Ala Glu Luell Arg Ile Ala Cys Cys Ser Pro 315 32O c cc gac tac aac tog gtg gt C Ctg tac t cc a CC Coa C. CC atc. 4 O14 Pro Asp Asn Ser Wall Wall Luell Ser Thir Pro Pro Ile 33 O 335 US 9,592,331 B2 57 - Continued tag 4 O17
<210s, SEQ ID NO 34 &211s LENGTH: 1338 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 34 Met Val Ser Tyr Trp Asp Thr Gly Val Lieu. Lieu. Cys Ala Lieu. Lieu. Ser 1. 5 1O 15 Cys Lieu. Lieu. Lieu. Thr Gly Ser Ser Ser Gly Ser Llys Lieu Lys Asp Pro 2O 25 3O Glu Lieu Ser Leu Lys Gly Thr Gln His Ile Met Glin Ala Gly Glin Thr 35 4 O 45 Lieu. His Lieu. Glin Cys Arg Gly Glu Ala Ala His Llys Trp Ser Lieu Pro SO 55 6 O Glu Met Val Ser Lys Glu Ser Glu Arg Lieu. Ser Ile Thr Lys Ser Ala 65 70 7s 8O Cys Gly Arg Asn Gly Lys Glin Phe Cys Ser Thr Lieu. Thir Lieu. Asn Thr 85 90 95 Ala Glin Ala Asn His Thr Gly Phe Tyr Ser Cys Llys Tyr Lieu Ala Val 1OO 105 11 O Pro Thir Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 12 O 125 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 13 O 135 14 O Ile Ile His Met Thr Glu Gly Arg Glu Lieu Val Ile Pro Cys Arg Val 145 150 155 160 Thir Ser Pro Asn Ile Thr Val Thr Lieu Lys Llys Phe Pro Leu. Asp Thr 1.65 17O 17s Lieu. Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 18O 185 19 O Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Lieu. Lieu. Thir Cys Glu 195 2OO 2O5 Ala Thr Val Asn Gly His Lieu. Tyr Lys Thr Asn Tyr Lieu. Thr His Arg 21 O 215 22O Gln Thr Asn. Thir Ile Ile Asp Val Glin Ile Ser Thr Pro Arg Pro Val 225 23 O 235 24 O Llys Lieu. Lieu. Arg Gly His Thr Lieu Val Lieu. Asn. Cys Thr Ala Thir Thr 245 250 255 Pro Leu. Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 26 O 265 27 O Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Glin Ser Asn. Ser His 27s 28O 285
Ala Asn. Ile Phe Tyr Ser Val Lieu. Thir Ile Asp Llys Met Glin Asn Lys 29 O 295 3 OO
Asp Llys Gly Lieu. Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 3. OS 310 315 32O
Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 3.25 330 335 Llys His Arg Lys Glin Glin Val Lieu. Glu Thr Val Ala Gly Lys Arg Ser 34 O 345 35. O
Tyr Arg Lieu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365 US 9,592,331 B2 59 60 - Continued
Trp Luell Asp Gly Lell Pro Ala Thir Glu Lys Ser Ala Arg Tyr Luell 37 O 375 38O
Thir Arg Ser Lell Ile Ile Asp Wall Thir Glu Glu Asp Ala 385 390 395 4 OO
Gly Asn Thir Ile Lell Lell Ser Ile Lys Glin Ser Asn Wall Phe 4 OS 415
Asn Luell Thir Ala Thir Lell Ile Wall Asn Wall Pro Glin Ile Glu 425 43 O
Ala Wall Ser Ser Phe Pro Asp Pro Ala Luell Pro Luell Gly Ser 435 44 O 445
Arg Glin Ile Luell Thir Thir Ala Gly Ile Pro Glin Pro Thir Ile 450 45.5 460
Lys Trp Phe Trp His Pro Asn His Asn His Ser Glu Ala Arg Cys 465 470
Asp Phe Ser Asn Asn Glu Glu Ser Ser Ile Lell Asp Ala Asp Ser 485 490 495
Asn Met Gly Asn Arg Ile Glu Ser Ile Thir Glin Arg Met Ala Ile Ile SOO 505
Glu Gly Lys Asn Met Ala Ser Thir Luell Wall Wall Ala Asp Ser Arg 515 525
Ile Ser Gly Ile Ile Cys Ile Ala Ser ASn Lys Wall Gly Thir Wall 53 O 535 54 O
Gly Arg Asn Ile Ser Phe Ile Thir Asp Wall Pro Asn Gly Phe His 5.45 550 555 560
Wall Asn Luell Glu Lys Met Pro Thir Glu Gly Glu Asp Lell Luell Ser 565 st O sts
Thir Wall Asn Phe Lell Arg Asp Wall Thir Trp Ile Luell Luell 585 59 O
Arg Thir Wall Asn Asn Arg Thir Met His Ser Ile Ser Glin 595 605
Met Ala Ile Thir Glu His Ser Ile Thir Luell Asn Lell Thir Ile Met 610 615
Asn Wall Ser Luell Glin Asp Ser Gly Thir Ala Arg Ala Arg Asn 625 630 635 64 O
Wall Thir Gly Glu Glu Ile Luell Glin Lys Glu Ile Thir Ile Arg 645 650 655
Asp Glin Glu Ala Pro Lell Luell Arg Asn Luell Ser Asp His Thir Wall 660 665 67 O
Ala Ile Ser Ser Ser Thir Thir Luell Asp His Ala Asn Gly Wall Pro 675 685
Glu Pro Glin Ile Thir Trp Phe Asn Asn His Lys Ile Glin Glin Glu 69 O. 695 7 OO
Pro Gly Ile Ile Lell Gly Pro Gly Ser Ser Thir Lell Phe Ile Glu Arg 7 Os
Wall Thir Glu Glu Asp Glu Gly Wall His Lys Ala Thir Asn Glin 72 73 O 73
Gly Ser Wall Glu Ser Ser Ala Tyr Luell Thir Wall Glin Gly Thir Ser 740 74. 7 O
Asp Ser Asn Lell Glu Lell Ile Thir Luell Thir Cys Thir Wall Ala 760 765
Ala Thir Luell Phe Trp Lell Lell Luell Thir Luell Phe Ile Arg Met 770 775 78O US 9,592,331 B2 61 - Continued Arg Ser Ser Ser Glu Ile Llys Thr Asp Tyr Lieu. Ser Ile Ile Met Asp 78s 79 O 79. 8OO Pro Asp Glu Val Pro Lieu. Asp Glu Glin Cys Glu Arg Lieu Pro Tyr Asp 805 810 815 Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg Lieu Lys Lieu. Gly Lys Ser 82O 825 83 O Lieu. Gly Arg Gly Ala Phe Gly Llys Val Val Glin Ala Ser Ala Phe Gly 835 84 O 845 Ile Llys Llys Ser Pro Thr Cys Arg Thr Val Ala Wall Lys Met Lieu Lys 850 855 860 Glu Gly Ala Thr Ala Ser Glu Tyr Lys Ala Lieu Met Thr Glu Lieu Lys 865 87O 87s 88O Ile Lieu. Thir His Ile Gly His His Lieu. Asn Val Val Asn Lieu. Lieu. Gly 885 890 895 Ala Cys Thr Lys Glin Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys 9 OO 905 91 O Llys Tyr Gly Asn Lieu. Ser Asn Tyr Lieu Lys Ser Lys Arg Asp Lieu. Phe 915 92 O 925 Phe Lieu. Asn Lys Asp Ala Ala Lieu. His Met Glu Pro Llys Lys Glu Lys 93 O 935 94 O Met Glu Pro Gly Lieu. Glu Glin Gly Llys Llys Pro Arg Lieu. Asp Ser Val 945 950 955 96.O Thir Ser Ser Glu Ser Phe Ala Ser Ser Gly Phe Glin Glu Asp Llys Ser 965 97O 97. Lieu Ser Asp Val Glu Glu Glu Glu Asp Ser Asp Gly Phe Tyr Lys Glu 98O 985 99 O Pro Ile Thr Met Glu Asp Lieu. Ile Ser Tyr Ser Phe Glin Val Ala Arg 995 1OOO 1005 Gly Met Glu Phe Lieu. Ser Ser Arg Lys Cys Ile His Arg Asp Lieu. O1O O15 O2O Ala Ala Arg Asn. Ile Lieu. Lieu. Ser Glu Asn. Asn Val Val Lys Ile O25 O3 O O35 Cys Asp Phe Gly Lieu Ala Arg Asp Ile Tyr Lys Asn. Pro Asp Tyr O4 O O45 OSO Val Arg Lys Gly Asp Thr Arg Lieu Pro Lieu Lys Trp Met Ala Pro O55 O6 O O65 Glu Ser Ile Phe Asp Llys Ile Tyr Ser Thr Lys Ser Asp Val Trp Of O O7 O8O Ser Tyr Gly Val Lieu. Leu Trp Glu Ile Phe Ser Lieu. Gly Gly Ser O85 O9 O O95 Pro Tyr Pro Gly Val Glin Met Asp Glu Asp Phe Cys Ser Arg Lieu. OO O5 10 Arg Glu Gly Met Arg Met Arg Ala Pro Glu Tyr Ser Thr Pro Glu
Ile Tyr Glin Ile Met Lieu. Asp Cys Trp His Arg Asp Pro Llys Glu
Arg Pro Arg Phe Ala Glu Lieu Val Glu Lys Lieu. Gly Asp Lieu. Lieu.
Glin Ala Asn Val Glin Glin Asp Gly Lys Asp Tyr e Pro Ile Asn
Ala e Lieu. Thr Gly Asn Ser Gly Phe Thr Tyr Ser Thr Pro Ala
Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile Ser Ala Pro Llys Phe US 9,592,331 B2
- Continued
190 195 2OO Asn Ser Gly Ser Ser Asp Asp Val Arg Tyr Val Asn Ala Phe Llys 2O5 21 O 215 Phe Met Ser Lieu. Glu Arg Ile Llys Thr Phe Glu Glu Lieu Lleu Pro 22O 225 23 O Asn Ala Thir Ser Met Phe Asp Asp Tyr Glin Gly Asp Ser Ser Thr 235 24 O 245 Lieu. Leu Ala Ser Pro Met Leu Lys Arg Phe Thr Trp Thr Asp Ser 250 255 26 O Llys Pro Lys Ala Ser Lieu Lys Ile Asp Lieu. Arg Val Thir Ser Lys 265 27 O 27s Ser Lys Glu Ser Gly Leu Ser Asp Val Ser Arg Pro Ser Phe Cys 28O 285 29 O His Ser Ser Cys Gly His Val Ser Glu Gly Lys Arg Arg Phe Thr 295 3OO 305 Tyr Asp His Ala Glu Lieu. Glu Arg Lys Ile Ala Cys Cys Ser Pro 310 315 32O Pro Pro Asp Tyr Asn Ser Val Val Lieu. Tyr Ser Thr Pro Pro Ile 3.25 33 O 335
<210s, SEQ ID NO 35 &211s LENGTH: 447 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chimeric antibody heavy chain
<4 OOs, SEQUENCE: 35 Glin Val Glin Lieu. Glin Glin Ser Gly Pro Glu Lieu Val Llys Pro Gly Ala 1. 5 1O 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 2O 25 3O Val Ile Ser Trp Val Lys Glin Arg Thr Gly Glin Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ile Tyr Tyr Asn Glu Lys Phe SO 55 6 O Lys Gly Lys Ala Thr Lieu. Thir Ala Asp Thir Ser Ser Asn. Thir Ala Tyr 65 70 7s 8O Met Glin Leu Ser Ser Lieu. Thir Phe Glu Asp Ser Ala Val Ile Phe Cys 85 90 95 Ala Arg Gly His Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Glin Gly Thr 1OO 105 11 O Thr Lieu. Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 12 O 125 Lieu Ala Pro Ser Ser Lys Ser Thir Ser Gly Gly Thr Ala Ala Lieu. Gly 13 O 135 14 O
Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160
Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val Lieu. Glin 1.65 17O 17s
Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 18O 185 19 O
Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Llys Pro Ser 195 2OO 2O5
Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys Asp Llys Thr US 9,592,331 B2 65 - Continued
21 O 215 22O His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro Ser 225 23 O 235 24 O Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 26 O 265 27 O Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 27s 28O 285 Lys Thr Llys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 29 O 295 3 OO Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr 3. OS 310 315 32O Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys Thr 3.25 330 335 Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu. 34 O 345 35. O Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thir Cys 355 360 365 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 37 O 375 38O Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 385 390 395 4 OO Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys Ser 405 410 415 Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 42O 425 43 O Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 44 O 445
<210s, SEQ ID NO 36 &211s LENGTH: 214 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chimeric antibody light chain <4 OOs, SEQUENCE: 36 Asp Ile Val Met Thr Glin Ser His Llys Phe Met Ser Thr Ser Val Gly 1. 5 1O 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Glin Asp Val Thir Ile Thr 2O 25 3O Val Ala Trp Tyr Glin Gln Llys Pro Gly Glin Ser Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly SO 55 6 O
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Glin Ala 65 70 7s 8O Glu Asp Leu Ala Val Tyr Tyr Cys Glin Glin His Tyr Thr Thr Pro Trp 85 90 95
Thir Phe Gly Gly Gly Thr Llys Lieu. Glu Ile Lys Arg Thr Val Ala Ala 1OO 105 11 O
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 12 O 125
Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu Ala US 9,592,331 B2 67 - Continued
13 O 135 14 O Llys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser Glin 145 150 155 160 Glu Ser Val Thr Glu Glin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 1.65 17O 17s Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Llys Val Tyr 18O 185 19 O Ala Cys Glu Val Thr His Glin Gly Lieu Ser Ser Pro Val Thr Lys Ser 195 2OO 2O5 Phe Asn Arg Gly Glu. Cys 21 O
<210s, SEQ ID NO 37 &211s LENGTH: 447 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chimeric antibody heavy chain 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (298) ... (298) <223> OTHER INFORMATION: N298Q mutant not glycosylated <4 OO > SEQUENCE: 37 Glin Val Glin Lieu. Glin Glin Ser Gly Pro Glu Lieu Val Llys Pro Gly Ala 1. 5 1O 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 2O 25 3O Val Ile Ser Trp Val Lys Glin Arg Thr Gly Glin Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ile Tyr Tyr Asn Glu Lys Phe SO 55 6 O Lys Gly Lys Ala Thr Lieu. Thir Ala Asp Thir Ser Ser Asn. Thir Ala Tyr 65 70 7s 8O Met Glin Leu Ser Ser Lieu. Thir Phe Glu Asp Ser Ala Val Ile Phe Cys 85 90 95 Ala Arg Gly His Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Glin Gly Thr 1OO 105 11 O Thr Lieu. Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 12 O 125 Lieu Ala Pro Ser Ser Lys Ser Thir Ser Gly Gly Thr Ala Ala Lieu. Gly 13 O 135 14 O Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val Lieu. Glin 1.65 17O 17s Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 18O 185 19 O Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Llys Pro Ser 195 2OO 2O5
Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys Asp Llys Thr 21 O 215 22O His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro Ser 225 23 O 235 24 O
Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser Arg 245 250 255 US 9,592,331 B2 69 - Continued Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 26 O 265 27 O Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 27s 28O 285 Lys Thr Llys Pro Arg Glu Glu Gln Tyr Glin Ser Thr Tyr Arg Val Val 29 O 295 3 OO Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr 3. OS 310 315 32O Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys Thr 3.25 330 335 Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu. 34 O 345 35. O Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thir Cys 355 360 365 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 37 O 375 38O Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 385 390 395 4 OO Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys Ser 4 OS 41O 415 Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 42O 425 43 O Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 44 O 445
<210s, SEQ ID NO 38 &211s LENGTH: 447 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chimeric antibody heavy chain 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (254) ... (254) <223> OTHER INFORMATION: I254A mutant does not bind FocRn
<4 OOs, SEQUENCE: 38 Glin Val Glin Lieu. Glin Glin Ser Gly Pro Glu Lieu Val Llys Pro Gly Ala 1. 5 1O 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 2O 25 3O Val Ile Ser Trp Val Lys Glin Arg Thr Gly Glin Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ile Tyr Tyr Asn Glu Lys Phe SO 55 6 O Lys Gly Lys Ala Thr Lieu. Thir Ala Asp Thir Ser Ser Asn. Thir Ala Tyr 65 70 7s 8O
Met Glin Leu Ser Ser Lieu. Thir Phe Glu Asp Ser Ala Val Ile Phe Cys 85 90 95 Ala Arg Gly His Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Glin Gly Thr 1OO 105 11 O
Thr Lieu. Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 12 O 125
Lieu Ala Pro Ser Ser Lys Ser Thir Ser Gly Gly Thr Ala Ala Lieu. Gly 13 O 135 14 O
Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 US 9,592,331 B2 71 - Continued
Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val Lieu. Glin 1.65 17O 17s Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 18O 185 19 O Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Llys Pro Ser 195 2OO 2O5 Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys Asp Llys Thr 21 O 215 22O His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro Ser 225 23 O 235 24 O Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ala Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 26 O 265 27 O Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 27s 28O 285 Lys Thr Llys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 29 O 295 3 OO Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr 3. OS 310 315 32O Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys Thr 3.25 330 335 Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu. 34 O 345 35. O Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thir Cys 355 360 365 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 37 O 375 38O Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 385 390 395 4 OO Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys Ser 4 OS 41O 415 Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 42O 425 43 O Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 44 O 445
<210s, SEQ ID NO 39 &211s LENGTH: 447 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chimeric antibody heavy chain 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (254) ... (254) <223> OTHER INFORMATION: I254A mutant does not bind FocRn 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (298) ... (298) <223> OTHER INFORMATION: N298Q mutant not glycosylated
<4 OOs, SEQUENCE: 39 Glin Val Glin Lieu. Glin Glin Ser Gly Pro Glu Lieu Val Llys Pro Gly Ala 1. 5 1O 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 2O 25 3O US 9,592,331 B2 73 74 - Continued
Wall Ile Ser Trp Wall Lys Glin Arg Thir Gly Glin Gly Lell Glu Trp Ile 35 4 O 45
Gly Glu Ile Pro Gly Ser Gly Ser Ile Tyr Asn Glu Lys Phe SO 55 6 O
Lys Gly Ala Thir Lell Thir Ala Asp Thir Ser Ser Asn Thir Ala Tyr 65 70 7s
Met Glin Luell Ser Ser Lell Thir Phe Glu Asp Ser Ala Wall Ile Phe 85 90 95
Ala Arg Gly His Tyr Gly Phe Asp Trp Gly Glin Gly Thir 105 11 O
Thir Luell Thir Wall Ser Ser Ala Ser Thir Gly Pro Ser Wall Phe Pro 115 12 O 125
Lell Ala Pro Ser Ser Lys Ser Thir Ser Gly Gly Thir Ala Ala Luell Gly 13 O 135 14 O
Cys Luell Wall Asp Tyr Phe Pro Glu Pro Wall Thir Wall Ser Trp Asn 145 150 155 160
Ser Gly Ala Luell Thir Ser Gly Wall His Thir Phe Pro Ala Wall Luell Glin 1.65 17s
Ser Ser Gly Luell Ser Lell Ser Ser Wall Wall Thir Wall Pro Ser Ser 18O 185 19 O
Ser Luell Gly Thir Glin Thir Ile Asn Wall Asn His Pro Ser 195
Asn Thir Wall Asp Arg Wall Glu Pro Ser Asp Thir 21 O 215 22O
His Thr Pro Pro Cys Pro Ala Pro Glu Lieu. Lieu Gly Gly Pro Ser 225 23 O 235 24 O
Wall Phe Luell Phe Pro Pro Pro Asp Thir Lell Met Ala Ser Arg 245 250 255
Thir Pro Glu Wall Thir Wall Wall Wall Asp Wall Ser His Glu Asp Pro 26 O 265 27 O
Glu Wall Lys Phe Asn Trp Wall Asp Gly Wall Glu Wall His Asn Ala 27s 285
Thir Pro Arg Glu Glu Glin Glin Ser Thir Arg Wall Wall 29 O 295 3 OO
Ser Wall Luell Thir Wall Lell His Glin Asp Trp Luell Asn Gly Glu Tyr 3. OS 310 315 32O
Wall Ser Asn Ala Luell Pro Ala Pro Ile Glu Lys Thir 3.25 330 335
Ile Ser Ala Gly Glin Pro Arg Glu Pro Glin Wall Tyr Thir Luell 34 O 345 35. O
Pro Pro Ser Arg Glu Glu Met Thir Asn Glin Wall Ser Luell Thir 355 360 365
Lell Wall Gly Phe Pro Ser Ile Ala Wall Glu Trp Glu Ser 37 O 375
Asn Gly Glin Pro Glu Asn Asn Thir Thir Pro Pro Wall Luell Asp 385 390 395 4 OO
Ser Asp Gly Ser Phe Phe Lell Ser Lys Luell Thir Wall Asp Lys Ser 4 OS 415
Arg Trp Glin Glin Gly Asn Wall Phe Ser Ser Wall Met His Glu Ala 425 43 O
Lell His Asn His Tyr Thir Glin Lys Ser Luell Ser Lell Ser Pro Gly 435 44 O 445 US 9,592,331 B2 75 76 What is claimed is: prises removing a volume of the subject’s blood and sepa 1. An anti-sfilt-1 antibody which comprises heavy chain rating the blood into plasma and cellular components and CDRs having the amino acid sequence SEQID NO:18, SEQ passing the plasma over the solid support. ID NO:20, and SEQID NO:22 and light chain CDRs having 14. A system comprising: the amino acid sequence SEQ ID NO:24, SEQID NO:26, 5 (a) anti-slfilt-1 antibodies, wherein the anti-slfilt-1 antibod and SEQ ID NO:28. ies comprise the anti-sflt-1 antibodies of claim 1, or 2. The anti-sfilt-1 antibody of claim 1, wherein the heavy sElt-1 binding fragments thereof; chain comprises the amino acid sequence SEQID NO:30 or (b) first means for conveying blood or a component a sequence at least 85% identical thereto and the light chain thereof from a subject to the anti-slfilt-1 antibodies, or comprises the amino acid sequence SEQ ID NO:32 or a 10 sElt-1 binding fragments thereof, bound to the solid sequence at least 85% identical thereto. Support so as to contact the blood or a component 3. The anti-slfilt-1 antibody of claim 1, wherein the anti thereof with the anti-sPlt-1 antibodies, or sPlt-1 binding sFlt-1 antibody binds to one or more of domains 1-3 of fragments thereof, and thereby remove sRlt-1 from the sFlt-1. blood or a component thereof; and 4. The anti-slfilt-1 antibody of claim 1, wherein the anti 15 (c) second means for conveying the blood or a component sFlt-1 antibody does not block ligand binding to sRlt-1. thereof to the subject following contact of the blood or 5. A method of treating eclampsia or pre-eclampsia in a a component thereof with the anti-slfilt-1 antibodies, or Subject comprising providing ex vivo to the subject an sElt-1 binding fragments thereof. anti-sfit-1 antibody, or sPlt-1 binding fragment thereof, 15. An anti-sfilt-1 antibody or sPlt-1 binding fragment which comprises the anti-slfilt-1 antibody of claim 1. thereof, which competes for binding with an antibody which 6. The method of claim 5, wherein the heavy chain comprises heavy chain CDRs having the amino acid comprises the amino acid sequence SEQ ID NO:30 or a sequence SEQ ID NO:18, SEQ ID NO:20, and SEQ ID sequence at least 85% identical thereto and the light chain NO:22 and light chain CDRs having the amino acid comprises the amino acid sequence SEQ ID NO:32 or a sequence SEQ ID NO:24, SEQ ID NO:26, and SEQ ID sequence at least 85% identical thereto. 25 NO:28. 7. The method of claim 5, wherein the anti-sPlt-1 antibody 16. The anti-slfilt-1 antibody or sPlt-1 binding fragment of binds to one or more of domains 1-3 of sRlt-1. claim 15, wherein the anti-slfilt-1 antibody, or sPlt-1 binding 8. The method of claim 5, wherein the anti-sPlt-1 antibody fragment thereof, competes for binding to sfilt-1 with an does not block ligand binding to sRlt-1. antibody which comprises a heavy chain. 9. The method of claim 5, wherein the pregnancy-related 30 17. A method of treating eclampsia or pre-eclampsia in a hypertensive disorder is pre-eclampsia. Subject comprising providing ex vivo to the subject an 10. The method of claim 9, wherein the subject is a anti-sfit-1 antibody, or sPlt-1 binding fragment thereof pregnant human or a post-partum human. which competes for binding with the anti-sfit-1 antibody or 11. The method of claim 10, wherein, the subject is a sElt-1 binding fragment thereof of claim 15. pregnant human. 35 18. The method of claim 17, wherein the anti-slfilt-1 12. The method of claim 5, which comprises: antibody, or sPlt-1 binding fragment thereof, competes for (a) removing blood from the subject, binding to split-1 with an antibody which comprises the (b) passing the blood or a component thereof over a solid amino acid sequence SEQ ID NO:30. Support to which are attached anti-slt-1 antibodies, or 19. The method of claim 17, wherein the anti-sflt-1 sElt-1 binding fragments thereof, to decrease the level 40 antibody, or sPlt-1 binding fragment thereof, competes for of silt-1 in the blood or component thereof, and binding to sRlt-1 with an antibody which comprising a heavy (c) returning the blood or component thereof to the chain having the amino acid sequence SEQ ID NO:30 and subject's body. a light chain having the amino acid sequence SEQ ID 13. The method of claim 12, wherein the blood or a NO:32. component thereof comprises plasma and the method com