FLT1 binding proteins and methods of use

Abstract

Provided herein are FLT1 binding proteins, as well as related nucleic acids, vectors, host cells, pharmaceutical compositions and kits. The present disclosure additionally provides methods of treating a subject in need thereof, including subjects in need of treatment for PAD, CLI, ANOCA, or heart failure (HF). Further provided are methods of increasing VEGF-mediated and / or PlGF-mediated signal transduction in a subject, which, in turn, increases blood flow, capillary density, capillary or coronary growth, and / or cardiac perfusion in the subject. The present disclosure further provides methods of reducing blood pressure and risk of heart failure in a subject. In exemplary embodiments, each of the methods comprise administering to the subject a pharmaceutical composition of the present disclosure. In various embodiments, the pharmaceutical composition comprises a FLT1 binding protein of the present disclosure.

Description

FLT1 BINDING PROTEINS AND METHODS OF USECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 700,358 filed on September 27, 2024; and U.S. Provisional Patent Application No. 63 / 712,745, filed on October 28, 2024, is hereby claimed.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: 1 ,700,000 bytes (XML) file named "10911 -WO01 -SEC_Seqlisting.xml"; created on September 17, 2025.BACKGROUND

[0003] Microvascular dysfunction and impaired tissue perfusion are contributing factors to a variety of cardiovascular diseases. Therapeutic improvement to microvascular function and tissue perfusion in these conditions is anticipated to alleviate symptoms and alter disease progression. One such condition is peripheral artery disease (PAD), also called peripheral vascular disease (PVD) or peripheral arterial disease, which is characterized by reduced blood flowthrough blood vessels to the limbs. The reduced blood flow is commonly caused by atherosclerotic plaques that form on the inner lining of the arteries, thereby narrowing these vessels and reducing the flow of blood to the arms and legs. The most common type of PAD involves reduced blood flow to the legs and feet. More than 8 million individuals aged 40 years and older in the United States suffer from PAD. Untreated, PAD can lead to the development of sores, infections, and the loss of a limb.

[0004] Critical limb ischemia (CLI), which occurs in a sub-segment of PAD patients, is another condition where targeting microvascular dysfunction and impaired tissue perfusion would alleviate symptoms and alter disease progression. CLI occurs when blood flow is severely reduced resulting in pain at rest, ulceration, or gangrene. CLI patients are at high risk of limb amputation, as one out of five CLI patients require amputation. CLI patients are prescribed antiplatelet drugs, anticoagulants, and vasodilators to help improve blood flow and prevent clot formation. However, when these medicines fail to sufficiently restore blood flow to the affected limb, surgical revascularization is considered. However, this is not always an effective option due to factors including 1) extensive comorbidities, 2) poor overall health, 3) lack of suitable graft material (patients may lack suitable veins or arteries to use for bypassgrafting) or 4) lack of viable arteries distal to the occlusion site to serve as a target for bypass grafting. In these instances, amputation is often required.

[0005] Accordingly, there is a need for a fast and effective treatment to improve tissue perfusion in patients with PAD, CLI, and similar conditions involving microvascular dysfunction and poor tissue perfusion.SUMMARY

[0006] The present disclosure is directed to FLT1 binding proteins, in particular anti-FLT1 antibodies, that bind human FLT1 with a high affinity to inhibit FLT1 bindingto its endogenous ligand, VEGF. Inhibition of the FLT1 -VEGF binding interaction increases VEGF mediated VEGFR2 angiogenic signaling. The present disclosure additionally provides methods of administeringthe FLT1 binding proteins described herein to subjects in need thereof, including subjects having a cardiovascular condition or a condition caused by or associated with microvascular dysfunction. The present disclosure further relates to nucleic acids encoding the FLT1 binding proteins, vectors comprising these nucleic acids, host cells, pharmaceutical compositions and kits

[0007] Accordingly, a first aspect of the present disclosure is directed to a FLT1 binding protein. This FLT1 binding protein comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and / or a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1090. Preferably, the VH of this FLT1 binding protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and / or the VL of this FLT1 binding protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1090, optionally, wherein the VH of this FLT1 binding protein comprises the Tyr at position 35, the Tyr at position 52, the Tyr at position 54, the Tyr at position 60, the Leu at position 102, the Leu at position 105, and the Leu at position 106 of SEQ ID NO: 1089 and / orthe VL of this FLT1 binding protein comprises the Tyr at position 50, the Ser at position 51 , the Asn at position 53, the His at position 54, the Trp at position 92, and the Asp at position 94 of SEQ ID NO: 1090. Optionally, the VH of this FLT1 binding protein comprises the Glu at position 103 of SEQ ID NO: 1089, and the VL of this FLT1 binding protein comprises the lie at position 31 and Asn at position 32 of SEQ ID NO: 1090. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen bindingfragment.

[0008] In another embodiment, this FLT1 binding protein, comprising a VH having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1089, comprises a complementarity determining region 1 (HCDR1 ), a HCDR2, and a HCDR3 of SEQ ID NOs: 1485, 1488, and 1491 , respectively. This FLT1 binding protein, comprising a VL having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1090, comprises a complementarity determining region 1 (LCDR1), a LCDR2, and a LCDR3 of SEQ ID NOs; 1494, 1497, 1500, respectively. Preferably, the VH of this FLT1 binding protein comprises a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1487, 1490, and 1493, respectively, and the VL of this FLT1 binding protein comprises a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 1496, 1499, 1502, respectively. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen binding protein.

[0009] In another embodiment, the VH of this FLT1 binding protein, comprising a VH having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1089, comprises an HCDR1 amino acid sequence of any one of SEQ ID NOs: 7, 151 , 181 , 331 ; an HC CDR2 amino acid sequence of any one of SEQ ID NO: 8, 152, 182, 332; an HC CDR3 amino acid sequence of any one of SEQ ID NO: 9, 153, 183, 333; or a combination thereof. This FLT1 binding protein, comprising a VL having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1090, comprises a LC CDR1 amino acid sequence of any one of SEQ ID NOs: 10, 154, 184, 334; an LC CDR2 amino acid sequence of any one of SEQ ID NO: 11 , 155, 185, 335; an LC CDR3 amino acid sequence of any one of SEQ ID NO: 12, 156, 186, 336; or a combination thereof. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen binding fragment.

[0010] In one embodiment, this FLT1 binding protein, comprising a VH having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1089, comprises the HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NO: 1089 as defined by Kabat. Alternatively, this FLT1 binding protein comprises the HC CDR2, HC CDR2, HC CDR3 of SEQ ID NO: 1089 as defined by Chothia, IMGT, or Aho as described herein. This FLT1 binding protein, comprising a VL having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1090, comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NO: 1090 as defined by Kabat. Alternatively, this FLT1 binding protein comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NO: 1090 as defined by Chothia, IMGT, or Aho as described herein. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen binding fragment.

[0011] In one embodiment, this FLT1 binding protein, comprising a VH having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1089, comprises a HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 7-9, respectively. This FLT1 binding protein comprising a VL having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1090, comprises a LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 10-12, respectively. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen binding fragment.

[0012] Another aspect of the present disclosure is directed to a FLT1 binding protein comprising: a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), a HCDR2, and a HCDR3 of SEQ ID NOs: 1485, 1488, and 1491 , respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs; 1494, 1497, 1500, respectively. In a preferred embodiment, the VH of this FLT1 binding protein comprises the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 1487, 1490, and 1493, respectively, and the VL of this FLT1 binding protein comprises the LCDR1 , the LCDR2, and the LCDR3 of SEQ ID NOs: 1496, 1499, 1502, respectively. An exemplary FLT1 binding protein comprises a VH comprising the HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 7-9, respectively, and a VL comprising the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 10-12, respectively. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen bindingfragment.

[0013] Another aspect of the present disclosure is directed to an anti-FLT1 antibody comprising a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1 ), HC CDR2, and HC CDR3 of SEQ ID NOs: 7-9, respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1), LC CDR2, and LC CDR3 of SEQ ID NOs: 10-12, respectively. In one embodiment, the VH of this anti-FLT1 antibody comprises the amino acid sequence SEQ ID NO: 1089, and the VL comprises the amino acid sequence of SEQ ID NO: 1090. In one embodiment, this anti-FLT1 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 1460 or 1572 and a light chain amino acid sequence of SEQ ID NO: 1461 .

[0014] Another aspect of the present disclosure is directed to a FLT1 binding protein comprising: a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1335, and a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1336. In one embodiment, the VH of this FLT1 binding protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQID NO: 1335, and the VL of this FLT1 binding protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1336, optionally, wherein the VH of this FLT1 binding protein comprises the His at position 35, the Trp at position 52, the Asn at position 57, the Tyr at position 59, the Asp at position 62, the Tyr at position 102, the Pro at position 104, the Tyr at position 108, and the Tyr at position 109 of SEQ ID NO: 1335 and / or the VL comprises the Gin at position 27, the His at position 31 , the Phe at position 37, the Tyr at position 99, the Arg at position 101 , and the Asp at position 1 of SEQ ID NO: 1336. Optionally, the VH of this FLT1 binding protein further comprises the Vai at position 33, the lie at position 58, the Ser at position 99, and the Thr at position 100. Optionally, the VL of this FLT1 binding protein further comprises the Asn at position 33, the Leu at position 97, the Thr at position 98, and the Pro at position 100. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen bindingfragment.

[0015] In another embodiment, this FLT1 binding protein, comprising a VH having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1335, comprises a complementarity determining region 1 (HCDR1 ), a HCDR2, and a HCDR3 of SEQ ID NOs: 1512, 1515, and 1518, respectively. This FLT1 binding protein, comprising a VL having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1336, comprises a complementarity determining region 1 (LCDR1), a LCDR2, and a LCDR3 of SEQ ID NOs: 1521 , 1524, and 1527, respectively. In a preferred embodiment, the VH of this FLT1 binding protein comprises a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1514, 1517, and 1520, respectively, and the VL of this FLT1 binding protein comprises a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs: 1523, 1526, 1529, respectively. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen binding protein.

[0016] In another embodiment, this FLT1 binding protein, comprising a VH having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1335, comprises an HCDR1 1 amino acid sequence of any one of SEQ ID NOs: 733, 1027 and 1033, an HC CDR2 amino acid sequence of any one of SEQ ID NO: 734, 1028, and 1034, an HC CDR3 amino acid sequence of any one of SEQ ID NO: 735, 1029, and 1035; or a combination thereof. This FLT1 binding protein, comprising a VL having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1336, comprises an LC CDR1 amino acid sequence of any one of SEQ ID NOs: 736, 1030, and 1036, an LC CDR2 amino acid sequence of any one of SEQ ID NO: 737, 1031 , and 1037; an LC CDR3 amino acid sequence of any one of SEQ ID NO: 738, 1032, and 1038; or a combination thereof. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen binding fragment.

[0017] In one embodiment, this FLT1 binding protein, comprising a VH having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1335, comprises the HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NO: 1336 as defined by Kabat. Alternatively, this FLT1 binding protein comprises the HC CDR2, HC CDR2, HC CDR3 of SEQ ID NO: 1335 as defined by Chothia, IMGT, or Aho as described herein. This FLT1 binding protein, comprising a VL having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1336, comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NO: 1336 as defined by Kabat. Alternatively, this FLT1 binding protein comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NO: 1336 as defined by Chothia, IMGT, or Aho as described herein. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen binding fragment.

[0018] In one embodiment, this FLT1 binding protein, comprising a VH having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1335, comprises a HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 733-735, respectively. This FLT1 binding protein, comprising a VL having at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1336, comprises a LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 736-738, respectively. Preferably, this FLT1 binding protein is an antibody. In one embodiment, this FLT1 binding protein is an antigen binding fragment.

[0019] Another aspect of the present disclosure is directed to a FLT1 binding protein comprising: a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), a HCDR2, and a HCDR3 of SEQ ID NOs: 1512, 1515, 1518, respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs: 1521 , 1524, and 1527, respectively. In a preferred embodiment, the VH of this FLT1 binding protein comprises the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 1514, 1517, and 1520, respectively, and the VL of this FLT1 binding protein comprises the LCDR1 , the LCDR2, and the LCDR3 of SEQ ID NOs: 1523, 1526, 1529, respectively. An exemplary FLT1 binding protein comprises a VH comprisingthe HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 733-735, respectively, and the VL comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 736-738, respectively.

[0020] In one embodiment, the FLT1 binding protein is an anti-FLT1 antibody comprising a VH comprisingthe HCDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 733-735, respectively, and a VL comprising a LCDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 736-738, respectively. In one embodiment, the VH of this anti-FLT1 antibody comprises the amino acid sequence SEQ ID NO: 1335, and the VL comprises the amino acid sequence of SEQ ID NO: 1336. In one embodiment,this anti-FLT1 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 1462 or 1572 and a light chain amino acid sequence of SEQ ID NO: 1463.

[0021] Preferably, the FLT1 binding proteins described herein comprise or consist of an antibody, where the antibody comprises a human IgG HC constant region and a human LC constant region. More preferably, the human IgG HC constant region is a human IgGI constant region, and the human LC constant region is a human kappa LC constant region or a human lambda LC constant region. Exemplary HC constant region sequences of the FLT1 antibodies described herein comprise any one of SEQ ID NOs: 1530-1533 or an amino acid sequence which has at least 80% sequence identity to said SEQ ID NOs: 1530-1533, and / or exemplary LC constant region sequences comprise any one of SEQ ID NOs: 1546-1547 or an amino acid sequence which has at least 80% sequence identity to said SEQ ID NOs: 1546-1547.

[0022] Another aspect of the present disclosure is directed to a nucleic acid comprising a nucleotide sequence encoding a FLT1 binding protein or an anti-FLT1 antibody as described herein. Other aspects of the present disclosure are directed to vectors comprising these nucleic acid molecules and host cells comprising the nucleic acid molecules or vectors described herein. In one embodiment, the host cell comprises (A) a first nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NOs: 1089, 1137, 1197, 1147, 1277, 1293, 1283, 1437, 1435, and 1335, or an amino acid sequence comprising at least 90% sequence identity to any one of SEQ ID NOs: 1089, 1137, 1197, 1147, 1277, 1293, 1283, 1437, 1435, and 1335; and (B) a second nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NOs: 1090, 1138, 1198, 1148, 1278, 1294, 1284, 1438, 1436, and 1336 or an amino acid sequence comprising at least 90% sequence identity to any one of SEQ ID NOs: 1090, 1138, 1198, 1148, 1278, 1294, 1284, 1438, 1436, and 1336.

[0023] Another aspect of the disclosure is directed to a kit comprising: a FLT1 binding protein described herein, an anti-FLT1 antibody described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, or a combination thereof. This kit further comprises a container.

[0024] Another aspect of the present disclosure comprises a pharmaceutical composition comprising: a FLT1 binding protein described herein, an anti-FLT1 antibody described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, or a combination thereof, and a pharmaceutically acceptable carrier, excipient or diluent. Preferably the pharmaceutical composition comprises an anti-FLT1 antibodydisclosed herein, or a nucleic acid comprising a nucleotide sequence encoding an anti-FLT1 antibody as disclosed herein and a pharmaceutically acceptable carrier, excipient or diluent.

[0025] Another aspect of the present disclosure is directed to a pharmaceutical composition comprising a means for inhibiting FLT1 binding to VEGF, and a pharmaceutically acceptable carrier, excipient or diluent.

[0026] Another aspect of the present disclosure is directed to a method of producing a FLT1 binding protein. This method comprises culturing the host cell as disclosed herein to express the FLT1 binding protein and harvesting the expressed FLT1 binding protein.

[0027] Another aspect of the present disclosure is directed to a method of treating a subject in need thereof. This method comprises administeringto the subject a FLT1 binding protein as described herein or a pharmaceutical composition comprising the FLT1 binding protein. Preferably, the subject is administered an anti-FLT1 antibody as described herein or a pharmaceutical composition comprising the anti-FLT1 antibody.

[0028] Another aspect of the present disclosure is directed to a method of increasing blood flow, capillary density, capillary or coronary growth, and / or tissue perfusion in a subject in need thereof. This method comprises administering, to the subject, a FLT1 binding protein as described herein or a pharmaceutical composition comprising the FLT1 binding protein. Preferably, the subject is administered an anti-FLT1 antibody as described herein or a pharmaceutical composition comprising the anti-FLT1 antibody. In one embodiment, the subject to be treated has reduced blood flow through peripheral blood vessels, such as blood vessels to the peripheral tissues and / or limbs. In one embodiment, the reduced blood flow is caused by atherosclerosis. In one embodiment, the reduced blood flow is not caused by atherosclerosis. In one embodiment, the subject to be treated has peripheral arterial disease (PAD) (also known as peripheral vascular disease) or critical limb ischemia (CLI) associated with PAD. In one embodiment, the subject to be treated has heart failure. In one embodiment, the subject to be treated has angina, such as angina without obstructive coronary artery disease (ANOCA). In one embodiment, the subject to be treated has cardiomyopathy, such as postpartum cardiomyopathy. In one embodiment, the subject to be treated has high blood pressure.

[0029] Another aspect of the present disclosure is directed to a method of treating a microvascular dysfunction in a subject in need thereof. This method comprises administering, to the subject, a FLT1 binding protein as described herein or a pharmaceutical composition comprisingthe FLT1 binding protein. Preferably, the subject is administered an anti-FLT1antibody as described herein or a pharmaceutical composition comprising the anti-FLT1 antibody. In one embodiment, the subject has microvascular dysfunction arising as a complication from diabetes, e.g., a subject having diabetic neuropathy, diabetic neuropathy, a diabetic wound, or a diabetic ulcer, e.g., a diabetic foot ulcer. In one embodiment, the subject has a condition associated with microvascular dysfunction, e.g., severe limb ischemia, ischemic heart disease, chronic kidney disease, bronchopulmonary dysplasia, Duchenne’s muscular dystrophy, microvascular angina.

[0030] Another aspect of the present disclosure is directed to a pharmaceutical composition as described herein for use in a method of treating a cardiovascular disorder. In one embodiment, the cardiovascular disorder is selected from PAD, CLI, heart failure, angina, and postpartum cardiomyopathy. Preferably, the pharmaceutical composition comprises an anti- FLT1 antibody as described herein.

[0031] Another aspect of the present disclosure is directed to a method of treating a cardiovascular disorder in a subject. This method comprises administering, to the subject having the cardiovascular disorder, a composition comprising a means for inhibiting FLT1 binding to VEGF, and a pharmaceutically acceptable carrier, excipient or diluent.

[0032] Presented herein are data demonstratingthe generation of anti-FLT1 antibodies that bind to human FLT1 with high affinity, block binding of FLT1 to VEGF, and restore VEGF-induced signaling through VEGFR2 (KDR). These antibodies were selected for their unique crossspecies reactivity. In particular, the anti-FLT1 antibodies described herein were selected for their high affinity bindingto human, cynomolgus monkey (cyno), canine, and porcine FLT1 , which provides the means for assessing therapeutic utility of these antibodies in relevant pre- clinical models of cardiovascular and microvascular dysfunction. As also demonstrated herein, blocking VEGF-FLT1 binding interaction using the disclosed anti-FLT1 antibodies is sufficient to increase the pro-angiogenic VEGF-VEGFR2 signaling resulting in an increase in capillary density, an increase in blood flow, and lower blood pressure in vivo. These data support the utility of these anti-FLT1 antibodies for the treatment of subjects suffering a variety of cardiovascular conditions and conditions associated with microvascular dysfunction.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figures 1 A-1C is a series of illustrations that demonstrate the role of VEGF and VEGFR2 in normal microcirculatory function (Figure 1A), sequestration of VEGF by sFLT1 leading to impaired microcirculatory function (Figure 1 B), and the restoration of microcirculatory function with anti-FLT1 antibodies (Figure 1 C).

[0034] Figure 2 is a schematic of an anti-FLT1 antibody campaign.

[0035] Figure 3 is a schematic outlining the FACS workflow of hybridoma cell selection.

[0036] Figure 4 is a graph of the % inhibition of FLT1-mediated blockade of KDR autophosphorylation exhibited by cells treated with anti-FLT1 antibodies (2 ug / ml) of the indicated harvest and / or sorted fraction, as measured by the pKDR functional assay.

[0037] Cells expressing FLT1 from different species were assessed for binding by anti-FLT1 antibodies. Each antibody had a unique heavy chain. Figure 5 is a graph of the FACS signal produced when anti-FLT1 antibody bound to FLT1 and labeled secondary antibody bound to anti-FLT1 antibody.

[0038] Figure 6 is a graph of the % inhibition of biotin-labelled VEGF binding to cells expressing FLT1 when treated with the anti-FLT1 antibodies of the indicated harvest and / or sorted fraction as measured in the receptor / ligand (R / L) inhibition assay.

[0039] Figure 7A-7C is a series of graphs, each demonstrating the % inhibition of human sFLT1 (Figure 7A), dog FLT1 (Figure 7B), or pig sFLTI (Figure 7C) by the indicated anti-FLT1 antibody clone as measured by a pKDR assay in HUVEC cells. In each assay, 1 nM VEGF and 3 nM sFLT1 were used.

[0040] Each of Figures 8A-8C is a table listing HC and LC consensus variable region sequences for the variants listed in each of Table 10 (Figure 8A), Table 11 (Figure 8B), and Table 12 (Figure 8C), wherein amino acid positions that were varied among the set of variants are denoted with X’s. The consensus sequences were created upon alignment of the sequences of the variants of Tables 10-12, against the sequences of 10B4 (Figure 8A), 10A7 (Figure 8B), or 28D8 (Figure 8C). The highest observed number of amino acid substitutions in a variant and the lowest observed % sequence identity are also provided. These values are relative to the corresponding sequences of 10B4 (Figure 8A), 10A7 (Figure 8B), or 28D8 (Figure 8C).

[0041] Figure 9A is a graph demonstrating the affinity above or below the 50 pM-cutoff for human FLT1 of antibodies derived from 10A7 (bars iFv:610794-iFv:610773), 10B4 (bars iFv:605279-iFv:605017), or 28D8 (bars iFv:609378+VH S63A-iFv:609555), or antibody 10A9 (PL53462), antibody 10D10 (PL53464), antibody 11 D4 (PL53465), and antibody 12F8 (PL53467).

[0042] Figure 9B is a graph demonstrating the affinity above or below the 200 pM-cutoff for dog sFLTI of antibodies derived from 10A7 (bars iFv:610794-iFv:610773), 10B4 (bars iFv:605279-iFv:605017), or 28D8 (bars iFv:609378+VH S63A-iFv:609555), or antibody 10A9 (PL53462), antibody 10D10 (PL53464), antibody 11 D4 (PL53465), and antibody 12F8 (PL53467).

[0043] Figure 9C is a graph demonstrating the affinity above or below the 200 pM-cutoff for pig sFLTI of antibodies derived from 10A7 (bars iFv:610794-iFv:610773), 10B4 (bars iFv:605279- iFv:605017), or 28D8 (bars iFv:609378+VHS63A-iFv:609555), or antibody 10A9 (PL53462), antibody 10D10 (PL53464), antibody 11 D4 (PL53465), and antibody 12F8 (PL53467).

[0044] Figure 10A provides the sequence of the full-length heavy chain and full-length light chain of antibody 18300. Figure 10B provides the sequence of the full-length heavy chain and full-length light chain of antibody 15331 . Signal sequences are shown in italicized, lower case letters; variable regions are shown in bold capital letters; constant regions are shown in capital letters; and CDR sequences are shown in bold, italicized, and underlined capital letters.

[0045] Each of Figures 11 A-11 C is a table listing HC and LC consensus variable region sequences for the variants listed in each of Table 10 (Figure 11 A), Table 11 (Figure 11 B), and Table 12 (Figure 11C), wherein amino acid positions that were varied among the set of variants are denoted with X’s. The consensus sequences were created upon alignment of the sequences of the variants of Tables 10-12, against the sequences of 18300 (Figure 11 A), 15314 (Figure 11 B), or 15331 (Figure 11C). The highest observed number of amino acid substitutions in a variant and the lowest observed % sequence identity are also provided. These values are relative to the corresponding sequences of 18300 (Figure 11A), 15314 (Figure 11 B), or 15331 (Figure 11 C).

[0046] Each of Figures 12A-12C is a graph showing dose-dependent % restoration of FLT1- inhibited pVEGFR2 activity in HUVEC cells by FLT1 antibodies 15331 (triangles) and 18300 (open boxes), compared to isotype control antibody (circles). pVEGFR2 activity was measured in cells treated with 3nM sFltl (R&D Systems, Lot YI12122061 ) (Figure 12A), 10nM sFltl (R&D Systems, Lot YI11823021 ) (Figure 12B), and 4.5 nM sFLT1 (Sino Biologies) (Figure 12C). The experiment was performed in triplicate. Error bars are ± S.D.

[0047] Figure 13A is a schematic overview of the FLT1 NanoBiT assay. Figure 13B is a graph showing FLT1 dimerization (measured by increasing luminescence (RLU)) plotted as a function of log concentration of anti-FLT1 antibodies, i.e., 15331 (squares), 18300 (triangles), isotype control antibody (circles), or VEGF165 (inverted triangle). This graph shows that the anti-FLT1 antibodies alone do not induce VEGFR1 dimerization. The experiment was performed in quadruplicates. Error bars are ± S.E.M.

[0048] Each of Figures 13C and 13D is a graph showing FLT1 dimerization (measured by increase in luminescence (RLU)) as a function of log concentration of VEGFAI6S in the presence of anti-FLT1 antibodies 15331 (Figure 13C) and 18300 (Figure 13D), demonstrating the extent ofVEGF165-induced FLT1 dimerization in the presence of the antibody. Titrated anti-FLT1 antibodies were treated with titrated VEGF165. Isotype control antibody was included as a control. The data shown are from 2 independent experiments performed in quadruplicates. EC50 values of VEGF165 for this experiment are shown Table 24. Error bars are ± S.E.M.

[0049] Each of Figures 13E-13F is a graph showing FLT1 dimerization (measured by change in luminescence (RLU)) as a function of log concentration of anti-FLT1 antibody. Titrated anti-FLT1 antibodies, i.e., 15331 (squares) and 18300 (triangles) or isotype control antibody (circles), were treated with a low concentration of sFLT1 (0.0005nM; Figure 13E) or high concentration of sFLT1 (40nM; Figure 13F) in the presence of VEGF165 in a fixed final concentration of 0.25nM. The data shown are from 2 independent experiments performed in quadruplicates. Error bars are ± S.E.M.

[0050] Each of Figures 14A-14E is a graph of % pVEGFR2 activity plotted as a function of anti- FLT1 antibody concentration (nM) (15331 (squares), 18300 (triangles), 13477 (diamonds), or control antibody (circles and inverted triangles)) when exposed to recombinant human FLT1 (Figure 14A), cynomolgus monkey FLT1 (Figure 14B), dog FLT1 (Figure 14C), pig FLT1 (Figure 14D), or rat FLT1 (Figure 14E).

[0051] Figure 15A is a graph of the concentration of antibody 18300 in serum of male cynomolgus monkeys plotted as a function of time (hrs) upon subcutaneous administration of 5 mg / kg antibody (circles), 10 mg / kg antibody (squares), 20 mg / kg antibody (triangle) or upon intravenous injection of 20 mg / kg antibody (inverted triangles).

[0052] Figure 15B is a graph of the concentration of free sFLT1 in serum (pg / mL) in male cynomolgus monkeys plotted as a function of time (hrs) upon subcutaneous administration of 5 mg / kg antibody (circles), 10 mg / kg antibody (squares), 20 mg / kg antibody (triangle) or upon intravenous injection of 20 mg / kg antibody (inverted triangle).

[0053] Figure 15C is a graph of the concentration of VEGF (pg / mL) in serum plotted as a function of time (hrs) upon subcutaneous administration of 5 mg / kg antibody (circles), 10 mg / kg antibody (squares), 20 mg / kg antibody (triangle) or upon intravenous injection of 20 mg / kg antibody (inverted triangle).

[0054] Figure 16A is an illustration of the overall structure of human sFLT1 in complex with the Fab of 18300, based on cryoEM data. sFLT1 is shown in white, 18300 Fab is shown in darker gray, and tool binder41883 Fab is shown in lighter gray. FLT1 domains D1 and D2, Fab constant regions and variable regions are labeled.

[0055] Figure 16B is an illustration of a close-up view of the contacts made between human FLT1 and 18300 Fab. All interacting residues are shown as sticks; the back-bone nitrogen atoms are shown as sphere. Hydrogen bonds are represented as gray dashed lines. HC CDR1- HC CDR3 and LC CDR1 -LC CDR3 of the 18300 Fab are labeled. FLT1 D2 is labeled. sFLT1 is shown in white and 18300 Fab is shown in dark gray.

[0056] Figure 16C is an illustration of the overall structure of human sFLT1 in complex with the Fab of 15331 , based on cryoEM data. sFLT1 is shown in white, 15331 Fab is shown in darker gray, and tool binder41883 Fab is shown in lighter gray. FLT1 domains D1 and D2, Fab constant regions and variable regions are labeled.

[0057] Figure 16D is an illustration of a close-up view of the contacts made between human FLT1 and 15331 Fab. All interacting residues are shown as sticks; the back-bone nitrogen atoms are shown as sphere. Hydrogen bonds are represented as gray dashed lines. HC CDR1- HC CDR3 and LC CDR1 -LC CDR3 of the 18300 Fab are labeled. FLT1 D1 and D2 are labeled. sFLT1 is shown in white and 15331 Fab is shown in dark gray.

[0058] Figure 16E is the published FLT1 -ligand complex structure (PDB5T89) superimposed over the structure of Figure 16A. Structures were aligned by FLTI D1 and D2 domains for superimposition. sFLT1 is shown in white, 18300 Fab is shown in darker gray. Ligand is labeled.

[0059] Figure 16F is the published FLT1-ligand complex structure (PDB5T89) superimposed over the structure of Figure 16B. Structures were aligned by FLTI D1 and D2 domains for superimposition. sFLT1 is shown in white, 15331 Fab is shown in darker gray. Ligand is labeled.

[0060] Figures 16G-16I are structures of the D1 and D2 domains of FLT1 wherein the epitopes bound by VEGF ligand (Figure 16G), Fab 18300 (Figure 16H), or Fab 15331 (Figure 161) are colored dark gray. Figure 16G is based on crystal structure data, while Figures 16H and 161 are based on cryoEM data.

[0061] Each of Figures 17A-17D is a graph of the concentration of anti-FLT1 antibody in plasma (nM) in mice treated with 0.3 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, orwith an isotype control antibody, as measured 15 hrs postantibody administration (Figure 17A), 39 hrs post-antibody administration (Figure 17B), 72 hrs post-antibody administration (Figure 17C), or 144 hrs post-antibody administration (Figure 17D).

[0062] Each of Figures 18A-18D is a graph of the concentration of free sFLT1 (pM) in plasma in mice treated with 0.3 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, or with an isotype control antibody, as measured 15 hrs post-antibody administration (Figure 18A), 39 hrs post-antibody administration (Figure 18B), 72 hrs postantibody administration (Figure 18C), or 144 hrs post-antibody administration (Figure 18D).

[0063] Each of Figures 19A-19D is a graph of the concentration of VEGFA in plasma (pM) in mice treated with 0.3 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, or 20 mg / kg anti- FLT1 antibody, or with an isotype control antibody, as measured 15 hrs post-antibody administration (Figure 19A), 39 hrs post-antibody administration (Figure 19B), 72 hrs postantibody administration (Figure 19C), or 144 hrs post-antibody administration (Figure 19D).

[0064] Figure 20A is graph of the concentration of anti-FLT1 antibody in plasma (nM) in mice treated with 0.3 mg / mL anti-FLT1 antibody, 3 mg / mL anti-FLT1 antibody, or 20 mg / mL anti-FLT1 antibody, orwith an isotype control antibody as measured atTmax. Figure 20B is graph of the concentration of free sFLT1 in plasma (pM) in mice treated with 0.3 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, orwith an isotype control antibody as measured atTmax. Figure 20C is graph of the concentration of VEGF in plasma (pM) in mice treated with 0.3 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, orwith an isotype control antibody as measured atTmax.

[0065] Figure 21 A is graph of the concentration of anti-FLT1 antibody in plasma (nM) in mice treated with 0.3 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, orwith an isotype control antibody, as measured at Day 7. Figure 21 B is graph of the concentration of free sFLT1 in plasma (pM) in mice treated with 0.3 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, orwith an isotype control antibody, as measured at Day 7. Figure 21 C is graph of the concentration of VEGF in plasma (pM) in mice treated with 0.3 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, or with an isotype control antibody, as measured at Day 7.

[0066] Figure 22A is a series of exemplary images and a summary graph showing the percent positive area for Collagen I within cardiac tissue in mice treated with an isotype control antibody or with 0.3 mg / kg anti-FLT1 antibody, 1 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, 10 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, as measured atTmax.

[0067] Figure 22B is a series of exemplary images and a summary graph showing the percent positive area for CD31 within cardiac tissue in mice treated with an isotype control antibody or with 0.3 mg / kg anti-FLT1 antibody, 1 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, 10 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, as measured atTmax.

[0068] Figure 23 is a series of exemplary images and a summary graph showing the percent positive area for CD31 within cardiac tissue in mice treated with an isotype control antibody or with 0.3 mg / kg anti-FLT1 antibody, 1 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, 10 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, as measured at Day 7.

[0069] Figure 24 is a series of graphs showing the CD31 +ve vessel density within cardiac tissue at Day 7 across the various doses of anti-FLT1 antibody for vessels sized 5 pm2-500 pm2(top graph). CD31 +ve vessel density within cardiac tissue at Day 7 for vessels sized 5-10 pm2, 10-30 pm2, 30-50 pm2, 50-100 pm2, and 100-500 pm2at the various doses of anti-FLT1 antibody are shown in the boxed graphs at the bottom of Figure 24 and are labeled accordingly.

[0070] Figure 25 is a series of exemplary images and summary graph showing the percent positive area for Collagen I within cardiac tissue in mice treated with isotype control antibody, 0.3 mg / kg anti-FLT1 antibody, 1 mg / mkg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, 10 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, as measured at Day 7.

[0071] Figure 26 is a series of exemplary images and a summary graph showing the percent positive area for CD31 within cardiac tissue in mice treated with isotype control antibody or with 0.3 mg / kg anti-FLT1 antibody, 1 mg / kg anti-FLT1 antibody, 3 mg / kg anti-FLT1 antibody, 10 mg / kg anti-FLT1 antibody, or 20 mg / kg anti-FLT1 antibody, as measured at the indicated times post-administration of antibody.

[0072] Figures 27A-27D demonstrate an anti-FLT1 antibody dose-response of blood pressure and heart rate changes. The data are presented as delta change from vehicle (hourly average). Figure 27A shows change in systolic blood pressure (sBP; mmHg), Figure 27B shows change in diastolic blood pressure (dBP; mmHg), Figure 27C shows change in arterial pressure (MAP), and Figure 27D shows change in heart rate (bpm).

[0073] Figures 28A-28D demonstrate hemodynamic changes after 30mg / kg of sFLT1 treatment in telemetrized naive mice. The data are presented as delta change from vehicle (hourly average). Circadian rhythms were labeled by empty (daytime) vs. shield grey box (night) in the graph. Figure 28A shows change in systolic blood pressure (sBP; mmHg), Figure 28B shows change in diastolic blood pressure (dBP; mmHg), Figure 28C shows change in arterial pressure (MAP), and Figure 28D shows change in heart rate (bpm).

[0074] Figures 29A-29D demonstrate hemodynamic changes after 10mg / kg of sFLT1 treatment in telemetrized naive mice. The data are presented as delta change from vehicle (hourly average). Circadian rhythms were labeled by empty (daytime) vs. shield grey box (night) in the graph. Figure 29A shows change in systolic blood pressure (sBP; mmHg), Figure 29Bshows change in diastolic blood pressure (dBP; mmHg), Figure 29C shows change in arterial pressure (MAP), and Figure 29D shows change in heart rate (bpm).

[0075] Figure 30A is a graph of the percent free Ab 18300 vs. mFLT1 concentration (Molar), as determined by KinExA analysis, of on-cell antibody binding in a U2OS cell line. The overall results show the optimized parameter estimates for affinity (Kd, in units of pM), and mFLT1 expression level (EL, in # receptors per cell) determined from ‘n-curve’ analysis performed via the KinExA Pro software using all aggregated 18300 Ab binding isotherms collected. Both raw data, and the determined best fit over an individual binding experiment performed at constant 18300 Ab concentration (33pM 18300, 125pM 18300, and 250pM 18300) are given and labeled accordingly.

[0076] Figure 30B is a graph of the percent free Ab 82540 vs. mFLT1 concentration (Molar), as determined by KinExA analysis, of on-cell Ab binding in a U2OS cell line. The overall results show the optimized parameter estimates for affinity (Kd, in units of pM), and mFLT1 expression level (EL, in # receptors per cell) determined from ‘n-curve’ analysis performed via the KinExA Pro software using all aggregated 82540 Ab binding isotherms collected. Both raw data, and the determined best fit over an individual binding experiment performed at constant 18300 Ab concentration (12.5pM 82540, 33pM 82540, 125pM 82540, and 250pM 82540) are given and labeled accordingly.

[0077] Each of Figures 31 A and 31 B is a graph of the percent CD31 + area (including lumen) of mice treated with anti-FLT1 antibody or isotype control antibody for posterior muscles (Figure 31 A) or anterior muscles (Figure 31 B).

[0078] Figure 32 is a graph of the anti-FLT1 antibody level in plasma of mice treated with anti- FLT1 antibody or isotype control antibody.

[0079] Figure 33A is a graph of the VEGF level in plasma of mice treated with anti-FLT1 antibody or isotype control antibody measured at the termination of the study. Figure 33B is a graph of the PIGF level in plasma of mice treated with anti-FLT1 antibody or isotype control antibody measured at the termination of the study.

[0080] Figure 34 is a graph of the basal level of VEGF of mice treated with anti-FLT1 antibody or isotype control antibody (measured before antibody treatment).DETAILED DESCRIPTION

[0081] FLT1

[0082] The present disclosure relates to FLT1 binding proteins, i.e., proteins that bind to a “fms related receptor tyrosine kinase 1 ” or“FLT1 ”. As used herein, “FLT1 ” is synonymous with “FLT-1 ” which is also known as “VEGFR-1 ” or “VEGFR1 ” and refers to a member of the vascular endothelial growth factor receptor (VEGFR) family of receptor tyrosine kinases (RTKs). In humans, FLT1 is encoded by a gene (named FLT1) located on chromosome 13 (location: 13q12.3) of the human genome. Multiple transcript variants encoding different isoforms of the FLT1 protein, including a longer membrane-bound protein (mFLT1) and shorter soluble versions (sFLT1), are encoded by the FLT1 gene. The National Center for Biotechnology Information (NCBI) details the FLT1 protein, transcript and the gene (FLT1) at Gene ID 2321 , and the table below provides the NCBI accession numbers of different transcripts and encoded proteins of four different isoforms.★signal peptide is amino acids 1-26 for each protein sequence.

[0083] FLT1 is expressed in vascular endothelial cells and participates in the tight regulation of angiogenesis and vasculogenesis. FLT1 binds to VEGF, a molecule that is central to blood vessel formation. However, due to its low kinase activity upon VEGF binding, FLT1 is considered a decoy receptor for VEGF, sequestering it from binding to VEGFR2 (also known as KDR). Soluble FLT1 (sFLT1) is an isoform of FLT1 comprising only the extracellular ligand binding domains of FLT1 . Therefore, while sFLT1 is capable of binding FLT1 ligands, there is noligand-mediated signaling, rendering sFLT1 an endogenous inhibitor of FLT1 ligands, such as VEGF.

[0084] Signal transduction induced by VEGF binding to VEGFR2 leads to normal microcirculatory function and a maintained microvasculature. VEGF-VEGFR2 signaling also promotes coronary growth and cardiac perfusion. When FLT1 , and, in particular sFLT1 , bind and sequester VEGF, VEGF-VEGFR2 signaling is blocked, impairing microcirculatory function, vessel growth and tissue perfusion, which further leads to capillary rarefaction, angiogenic imbalance and increased ischemic injury. See, Figures 1 A and 1 B. The anti-FLT1 antibodies described herein bind to FLT1 (both membrane and soluble forms of FLT1) with high affinity to counteract FLT1-VEGF induced imbalance thereby improving oxygen and nutrient supply to tissue. Treatment with the anti-FLT1 antibodies described herein will be particular effective for the treatment of a variety of cardiovascular indications where FLT1 levels are high and / or microvascular dysfunction and impaired perfusion are contributing factors to symptoms and disease progression. As described in more detail herein, these conditions include, without limitation, peripheral artery disease (PAD) (also known as peripheral vascular disease) and, in particular, PAD patients with critical limb ischemia, postpartum cardiomyopathy, postpartum preeclampsia, angina (e.g., angina with no obstructive coronary artery disease), and heart failure. FLT1 is further described in Tanaka et al., Reprod Biol 24(1 ): 100857 (2024); Vogtmann et al., Int J Mol Sci 25(4): 2040 (2024); Sarabipour et al., PLoS Comput Biol 20(2): e1011798 (2024); Jin et al., Arthritis Res Ther 10(4): R73 (2008); Cunningham et al., J Biol Chem 273 (35): 20254-20257 (1995); Kendall et al., PNAS 90(22): 10705-10709 (1993); de Vries et al., Science 255(5047): 989-991 (1992); Shibuya et al., Oncogene 5(4): 519-524 (1990); Satoh et al., Jpn J Cancer Res 78(8): 772-775 (1987); Matsushime et al., Jpn J Cancer Res 78(7): 655-661 (1987); Fischer et al., Nature Rev Cancer 8:942-956 (2008); and Roberts et al., Am J Pathol 164(5): 1531 -1535 (2004).

[0085] Binding Activity

[0086] The present disclosure provides FLT1 binding proteins, in particular, anti-FLT1 antibodies, that bind to human FLT1 . The FLT1 binding proteins of the present disclosure bind to both soluble FLT1 (also referred to herein as “sFLT1 ”) and full-length membrane FLT1 (also referred to herein as “mFLT1 ”). Therefore, any reference to a FLT1 binding protein or anti-FLT1 antibody of the present disclosure encompasses a binding protein or antibody that binds to both sFLT1 and mFLT1 . The amino acid sequence of human mFLT1 is provided herein as SEQ ID NO: 1453. (NCBI Ref. Seq: NP_002010; UniProt Ref. No. P17948). As noted above, sFLT1 is a signaling-incompetent isoform of FLT1 comprising only the extracellular domain of FLT. Theamino acid sequence of human sFLT1 , is provided herein as SEQ ID NO: 1455. Amino acids 151-327 of FLT1 and sFLT1 comprise the extracellular domains D2-D3, which comprise the VEGF (ligand) binding site on FLT1 .

[0087] The FLT1 binding proteins of the present disclosure bind to human FLT1 with a greater affinity than VEGF or PIGF, which are the endogenous ligands for FLT1 . The affinity of the presently disclosed FLT1 binding proteins for FLT1 may be at least about 10% greater than the affinity for FLT1 of VEGF or PIGF. Preferably, the affinity of the presently disclosed FLT1 binding proteins is at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, or at least or about 50% greater than the affinityfor FLT1 of VEGF or PIGF. More preferably, the affinity of the presently disclosed FLT1 binding proteins is at least or about 55%, at least or about 60%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, or at least or about 95% greaterthan the affinity for FLT1 of VEGF or PIGF. The FLT1 binding protein of the present disclosure exhibits an affinityfor FLT1 which is 2-, 5-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100-, 105-, 110-, 115-, 120-, 125-, 130-, 135-, 140-, 145-, 150-, 175-, 200-, 225-, 250-, 275-, 300-, 325-, 350-, 375-, 400-, 425-, 450-, 475- , 500-, 525-, 550-, 575-, 600-, 625-, 650-, 675-, 700-, 725-, 750-, 775-, 800-, 825-, 850-, 875-, 900-, 925-, 950-, 975-fold, 1000-fold, or more greaterthan the affinityfor FLT1 of VEGF or PIGF.

[0088] Exemplary FLT1 binding proteins of the present disclosure were selected based on their ability to bind to FLT1 from more than one species, or their species cross-reactivity, e.g., to human FLT1 , cyno FLT1 , porcine FLT1 , and / or canine FLT1 . This profile of binding activity to FLT1 from different species, or species cross-reactivity profile, was selected to facilitate functional testing of the antibodies in porcine and canine models of human cardiovascular disease and microvascular dysfunction and for pharmacological studies in cynomolgus monkeys. Accordingly, the FLT1 binding proteins of the present disclosure, preferably anti-FLT1 antibodies, bind to human, cyno, and canine FLT1 , orto human, cyno, canine and porcine FLT1 . The amino acid sequences of the extracellular binding domains (domains 1 -6) of cyno, canine, and porcine FLT1 are provided herein as SEQ ID NOs: 1459, 1458, and 1457, respectively. The FLT1 binding proteins of the present disclosure, e.g., anti-FLT1 antibodies, bind to human, cyno, canine, and porcine FLT1 , and this binding blocks the VEGF-FLT1 binding interaction, thereby causing an increase in VEGF bindingto VEGFR2, and a corresponding increase in VEGF mediated VEGFR2 activity as measure by VEGFR2 phosphorylation (see Figures14A-14D).Thus, in one embodiment, the anti-FLT1 antibodies of the present disclosure bind human, cyno, canine, and porcine FLT1 as measured by an increase of VEGFR2 phosphorylation. In oneembodiment, the anti-FLT1 antibodies of the present disclosure bind human, cyno, canine, and porcine FLT1 as measured by an increase of VEGFR2 phosphorylation usingthe assay of Example 8. This species cross-reactivity profile is not an inherent feature of anti-FLT1 antibodies, as demonstrated by the lack of canine and porcine FLT1 binding exhibited by the reference human anti-FLT1 antibody 13477 (Bosco et al., Mol. Ther. Meth. Clin. Dev. 21 :369 (2021)) as shown in Figures 14C-14D. Exemplary FLT1 antibodies of the present disclosure comprisingthis species cross-reactivity binding profile (i.e., bindingto human, cyno, canine, and porcine FLT1 ) include antibody 18300 and antibodies or antigen binding fragments thereof comprising at least 90% or at least 95% sequence identity to the light chain and heavy chain variable regions of antibody 18300. Exemplary antibodies sharing at least 90% sequence identity to the light chain and heavy chain variable regions of Ab 18300 include the engineered variants of the 10B4 binder, see e.g., the FLT1 binders of Table 10, Table 16 and Table 20. In one embodiment, exemplary antibodies or antigen binding fragments thereof of the present disclosure that bind human, cyno, canine, and porcine FLT1 comprise the 18300 consensus CDRs of Table E (i.e. HCDRs 1-3 having SEQ ID NOs: 1487, 1490, 1493, respectively and LCDRs 1 -3 having SEQ ID NOs: 1496, 1499, and 1502, respectively). Exemplary, antibodies or antigen binding fragments thereof of the present disclosure that bind human, cyno, canine, and porcine FLT1 comprise the CDRs of antibody 18300 (i.e. HCDR 1-3 comprising SEQ ID NOs: 7-9, respectively, and LCDRs 1 -3 comprising SEQ ID NOs: 10-12, respectively).

[0089] In another embodiment, FLT1 binding proteins, preferably, anti-FLT1 antibodies, of the present disclosure comprising this species cross-reactivity profile (i.e., bindingto human, cyno, canine, and porcine FLT1 ) include antibody 15331 and antibodies or antigen binding fragments thereof comprising at least 90% or at least 95% sequence identity to the light chain and heavy chain variable regions of antibody 15331 . Exemplary anti-FLT1 antibodies sharing at least 90% sequence identity to the light and heavy chain variable regions of Ab 15331 include the engineered variants of the 28D8 family, see e.g., the antibodies of Table 12 and Table 20. In one embodiment, exemplary antibodies or antigen binding fragments thereof that bind human, cyno, canine, and porcine FLT1 comprise the 15331 consensus CDRs of Table E (i.e. HCDR 1 -3 having SEQ ID NOs: 1514, 1517, 1520, respectively and LCDRs 1 -3 having SEQ ID NOs: 1523, 1526, and 1529, respectively). Exemplary, antibodies or antigen binding fragments thereof that bind human, cyno, canine, and porcine FLT1 comprise the CDRs of antibody 15331 (i.e. HCDR 1-3 comprising SEQ ID NOs: 733-735, respectively and LCDRs 1-3 comprising SEQ ID NOs: 736- 738, respectively).

[0090] Binding strength of the presently disclosed FLT1 binding protein for FLT1 may be described in terms of its binding affinity. “Binding affinity” generally refers to the strength of the binding interaction between a single molecule (e.g., a FLT1 binding protein as described herein) and its binding partner (e.g., human FLT1). The affinity of a molecule for its partner is generally represented by the equilibrium dissociation constant (KD) and equilibrium association constant (KA). The KDis calculated from the quotient of koff / kon, whereas KAis calculated from the quotient of kon / koft . konrefers to the association rate constant of, e.g., an antibody to an antigen, and koffrefers to the dissociation of, e.g., an antibody from an antigen.

[0091] In one embodiment, the binding affinity of FLT1 binding proteins of the present disclosure to a recombinantly expressed soluble form of human FLT1 is provided as a KD. According to this embodiment, the KD of the FLT1 binding protein, preferably an anti-FLT1 antibody, of the present disclosure to the recombinantly expressed soluble form of human FLT is in the picomolar range, e.g., 0.1 pM to 999 pM. Preferably, the KDof the FLT1 binding proteins of the present disclosure for binding a recombinant expressed soluble form of human FLT1 is about 1 pM to about 1000 pM. More preferably, the KDof the FLT1 binding proteins of the present disclosure for binding a recombinantly expressed soluble form of human FLT1 is less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, or less than 10 pM. In one embodiment, the KDof the FLT1 binding proteins of the present disclosure for binding a recombinantly expressed soluble form of human FLT1 is less than 50 pM as measured using the surface plasmon resonance (SPR) or kinetic exclusion assay (KinExA) method as described in Example 9 herein. In one embodiment, the KDof the FLT1 binding proteins of the present disclosure for binding a recombinantly expressed soluble form of human FLT1 is about 1 pM to about 50 pM, about 1 pM to about 45 pM, about 1 pM to about 40 pM, about 1 pM to about 35 pM, about 1 pM to about 30 pM, about 1 pM to about 25 pM, about 1 pM to about 20 pM, about 1 pM to about 15 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 5 pM to about 50 pM, about 10 pM to about 50 pM, about 15 pM to about 50 pM, about 20 pM to about 50 pM, about 25 pM to about 50 pM, about 30 pM to about 50 pM, about 35 pM to about 50 pM, about 40 pM to about 50 pM, or about 45 pM to about 50 pM. In one embodiment, the KDof the FLT1 binding proteins, preferably the anti-FLT1 antibodies, of the present disclosure, for binding a recombinantly expressed soluble form of human FLT1 is less than 20 pM as measured using the SPR or KinExA method as described in Example 9 herein. In one embodiment, the KDof the FLT1 binding proteins, preferably the FLT1 antibodies, of the present disclosure for binding a recombinantly expressed soluble form of human FLT1 is about 0.5 pM to about 20 pM, about 0.5 pM to about 15 pM, about 0.5 pM to about 10 pM, about 0.5pM to about 5 pM, about 1 pM to about 20 pM, about 5 pM to about 20 pM, about 10 pM to about 20 pM, or about 15 pM to about 20 pM.

[0092] The FLTI binding protein of the present disclosure, in particular, an anti-FLT1 antibody of the disclosure, binds with high affinity (KD in the picomolar range) to a recombinantly expressed soluble form human FLT1 and additionally binds to recombinantly expressed soluble forms of non-human FLT1 proteins with a similar high affinity. For example, the FLT1 binding protein of the present disclosure binds with high affinity to recombinantly expressed soluble forms of human FLT1 , cynomolgus monkey (cyno) FLT1 , canine FLT1 , and porcine FLT1. In one embodiment, the FLT1 binding protein of the present disclosure binds with high affinity to both human and cyno recombinantly expressed soluble FLT1. The amino acid sequence of cyno FLT1 (D1 -D6) is provided herein as SEQ ID NO: 1459. In one embodiment, the FLT1 binding protein of the present disclosure binds to the human recombinantly expressed soluble FLT1 with a KDthat is 200 pM or less (e.g., about 150 pM or less, about 100 pM or less, about 50 pM or less, or about 10 pM or less) and the KDof the FLT1 binding protein for cyno recombinantly expressed soluble FLT1 is within about 100-fold, about 50-fold, about 25-fold, about 10-fold, about 5-fold, or about 2-fold or less, of the KDof the FLT1 binding protein for human FLT1 . Preferably, the KDof the FLT1 binding protein for human and cyno recombinantly expressed soluble FLT1 is about the same. Preferably, the FLT1 binding protein of the present disclosure is an anti-FLT1 antibody that binds to recombinantly expressed soluble human FLT1 with a KDthat is less than 20 pM (e.g., between 0.5 pM and 10 pM), and the KDof the FLT1 binding protein for recombinantly expressed soluble cyno FLT1 is also less than 20 pM (e.g., between 0.5 pM and 10 pM) as measured using the KinExA method as described in Example 9 herein.

[0093] In one embodiment, the FLT1 binding protein of the present disclosure, in particular, an anti-FLT1 antibody of the disclosure, binds with high affinity to both human and porcine recombinantly expressed soluble forms of FLT1 . The amino acid sequence of porcine FLT1 (DI DO) is provided herein as SEQ ID NO: 1457. In one embodiment, the FLT1 binding protein of the present disclosure binds to the human recombinantly expressed soluble FLT1 with a KDthat is 200 pM or less (e.g., about 150 pM or less, about 100 pM or less, about 50 pM or less, or about 10 pM or less) and the KDof the FLT1 binding protein for porcine recombinantly expressed soluble FLT1 is within about 100-fold, about 50-fold, about 25-fold, about 10-fold, about 5-fold, or about 2-fold or less, of the KDof the FLT1 binding protein for human recombinantly expressed soluble FLT1. In one embodiment, a FLT1 antibody of the present disclosure binds to recombinantly expressed soluble human FLT1 with a KD< 50 pM and recombinantly expressed soluble porcine FLT1 with a KD< 200 pM as measured using the SPR or KinExA method asdescribed in Example 9 herein. Exemplary FLT1 antibodies or antigen binding fragments thereof of the present disclosure that bind to recombinantly expressed soluble human FLT1 with a KD< 50 pM and recombinantly expressed soluble porcine FLT1 with a KD< 200 pM include antibody 18300 and antibodies or antigen binding fragments comprising at least 90% or 95% sequence identity to the light chain and heavy chain variable regions of antibody 18300 (e.g., an antibody havingthe consensus light chain variable sequence of SEQ ID NO: 1587 and the consensus heavy chain variable sequence of SEQ ID NO: 1586). In one embodiment, exemplary antibodies or antigen binding fragments thereof of the present disclosure that bind to recombinantly expressed soluble human FLT1 with a KD< 50 pM and recombinantly expressed soluble porcine FLT1 with a KD< 200 pM comprise the 18300 consensus CDRs of Table E (i.e. HCDR 1-3 having SEQ ID NOs: 1487, 1490, 1493, respectively and LCDRs 1 -3 having SEQ ID NOs: 1496, 1499, and 1502, respectively). In one embodiment, antibodies or antigen binding fragments thereof of the present disclosure that bind to recombinantly expressed soluble human FLT1 with a KD< 50 pM and recombinantly expressed soluble porcine FLT1 with a KD< 200 pM comprise the CDRs of antibody 18300 (i.e. HCDR 1 -3 having SEQ ID NOs: 7-9, respectively and LCDRs 1 -3 having SEQ ID NOs: 10-12, respectively).

[0094] In one embodiment, the FLT1 binding protein of the present disclosure, in particular, an anti-FLT1 antibody of the disclosure, binds with high affinity to both human and canine recombinantly expressed soluble forms of FLT1 . The amino acid sequence of canine FLT1 (D1 - D6) is provided herein as SEQ ID NO: 1458. In one embodiment, the FLT1 binding protein, preferably an anti-FLT1 antibody, of the present disclosure binds to the human recombinantly expressed soluble FLT1 with a KDthat is 200 pM or less (e.g., about 150 pM or less, about 100 pM or less, about 50 pM or less, or aboutI O pM or less) and the KDof the FLT1 binding protein (preferably an anti-FLT1 antibody) for canine recombinantly expressed soluble FLT1 is within about 100-fold, about 50-fold, about 25-fold, about 10-fold, about 5-fold, or about 2-fold or less, of the KDof the FLT1 binding protein for human recombinantly expressed soluble FLT1 . In one embodiment, the KDof FLT1 antibodies as disclosed herein (e.g., anti-FLT1 antibody) for human and canine recombinantly expressed soluble FLT1 is about the same. In one embodiment, an anti-FLT1 antibody of the present disclosure binds to recombinantly expressed soluble human FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, and the KDof the FLT1 binding protein for recombinantly expressed soluble canine FLT1 is also less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM as measured using the SPR or KinExA method as described in Example 9 herein. In one embodiment, exemplary FLT1 antibodies of the present disclosure that bind to human and canine recombinantly expressed soluble FLT1with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, include antibody 18300 and antibodies or antigen binding fragments comprising at least 90% or 95% sequence identity to the light chain and heavy chain variable regions of antibody 18300 (e.g., an antibody having the consensus light chain variable sequence of SEQ ID NO: 1587 and the consensus heavy chain variable sequence of SEQ ID NO: 1586). In one embodiment, exemplary antibodies or antigen binding fragments thereof of the present disclosure that bind human and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, comprise the 18300 consensus CDRs of Table E (i.e. HCDR 1-3 having SEQ ID NOs: 1487, 1490, 1493, respectively and LCDRs 1 -3 having SEQ ID NOs: 1496, 1499, and 1502, respectively). In one embodiment, exemplary antibodies or antigen binding fragments thereof of the present disclosure that bind human and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, comprise the CDRs of antibody 18300 (i.e. HCDR 1 -3 having SEQ ID NOs: 7-9, respectively and LCDRs 1-3 having SEQ ID NOs: 10-12, respectively). In another embodiment, exemplary FLT1 antibodies of the present disclosure that bind to human and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, include antibody 15331 and antibodies comprising at least 90% or 95% sequence identity to the light chain and heavy chain variable regions of antibody 15331 (e.g., an antibody having the consensus light chain variable sequence of SEQ ID NO: 1591 and the consensus heavy chain variable sequence of SEQ ID NO: 1590). In one embodiment, exemplary antibodies of the present disclosure that bind human and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, comprise the 15331 consensus CDRs of Table E (i.e. HCDR 1-3 having SEQ ID NOs: 1514, 1517, 1520, respectively and LCDRs 1 -3 having SEQ ID NOs: 1523, 1526, and 1529, respectively). In one embodiment, exemplary antibodies of the present disclosure that bind human and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, comprise the CDRs of antibody 15331 (i.e. HCDR 1 -3 having SEQ ID NOs: 733-735, respectively and LCDRs 1 -3 having SEQ ID NOs: 736-737, respectively).

[0095] As disclosed here, the FLT1 binding protein of the present disclosure is preferably an anti-FLT antibody that [1] binds to recombinantly expressed soluble human FLT1 with a KDthat is 200 pM or less (e.g., about 150 pM or less, about 100 pM or less, about 50 pM or less, or aboutIO pM or less) [2] binds to each of recombinantly expressed soluble cyno FLT1 , canine FLT1 , and porcine FLT1 with a KDthat is within 100-fold, 50-fold, 25-fold, 10-fold, 5-fold, or 2- fold of the KDof the FLT1 binding protein for human recombinantly expressed soluble FLT1 , and[3] does not bind to recombinantly expressed soluble rodent FLT1 , e.g. does not bind to mouse FLT1 and does not bind to rat FLT1 . In one embodiment, the KDof the FLT1 binding protein for human, cyno, and canine recombinantly expressed soluble FLT1 are about the same. In one embodiment, the FLT1 binding protein of the present disclosure binds to recombinantly expressed soluble human FLT1 with a KDthat is less than 20 pM (e.g., between 0.5 pM and 10 pM), and the KDof the FLT1 binding protein for recombinantly expressed soluble cyno and canine FLT1 is also less than 20 pM (e.g., between 0.5 pM and 10 pM). In one embodiment, exemplary anti-FLT1 antibodies of the present disclosure that bind to human, cyno, and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, include antibody 18300 and antibodies or antigen binding fragments comprising at least 90% or 95% sequence identity to the light chain and heavy chain variable regions of antibody 18300 (e.g., an antibody having the consensus light chain variable sequence of SEQ ID NO: 1587 and the consensus heavy chain variable sequence of SEQ ID NO: 1586). In one embodiment, exemplary antibodies of the present disclosure that bind human, cyno, and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, comprise the 18300 consensus CDRs of Table E (i.e. HCDR 1-3 having SEQ ID NOs: 1487, 1490, 1493, respectively and LCDRs 1 -3 having SEQ ID NOs: 1496, 1499, and 1502, respectively). In one embodiment, exemplary antibodies of the present disclosure that bind human, cyno, and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, comprise the CDRs of antibody 18300 (i.e. HCDR 1 -3 having SEQ ID NOs: 7-9, respectively and LCDRs 1 -3 having SEQ ID NOs: 10-12, respectively) as disclosed herein.

[0096] In another embodiment, exemplary anti-FLT1 antibodies of the present disclosure that bind to human, cyno, and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, include antibody 15331 and antibodies and antigen binding fragments comprising at least 90% or 95% sequence identity to the light chain and heavy chain variable regions of antibody 15331 (e.g., an antibody having the consensus light chain variable sequence of SEQ ID NO: 1591 and the consensus heavy chain variable sequence of SEQ ID NO: 1590). In one embodiment, exemplary antibodies of the present disclosure that bind human, cyno, and canine recombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, comprise the 15331 consensus CDRs of Table E (i.e. HCDR 1 -3 having SEQ ID NOs: 1514, 1517, 1520, respectively and LCDRs 1 -3 having SEQ ID NOs: 1523, 1526, and 1529, respectively). In one embodiment, exemplary antibodies of the present disclosure that bind human, cyno, and caninerecombinantly expressed soluble FLT1 with a KDthat is less than 20 pM, e.g., a KDof between 0.5 pM and 10 pM, comprise the CDRs of antibody 15331 as described herein (i.e. HCDR 1 -3 having SEQ ID NOs: 733-735, respectively and LCDRs 1 -3 having SEQ ID NOs: 736-738, respectively).

[0097] In another embodiment, the binding affinity of FLT1 binding proteins of the present disclosure, in particular anti-FLT1 antibodies of the present disclosure, is provided as the binding affinity to the human membrane FLT1 (mFLT1) expressed on cells (i.e., on-cell binding affinity). This measure of FLT1 binding protein binding affinity can aid in assessing in vivo pharmacokinetics of the binding proteins. While high affinity binding of an antibody to its target antigen is often desirable for optimal potency, a binding affinity that is too high can be disadvantageous to the overall therapeutic efficacy of the antibody. See, e.g., Rabia et al., Biochem Eng J 137: 365-374 (2018). Too high a binding affinity can, for example, lead to target mediated drug disposition (via internalization and degradation vs. FcRn mediate recycling) and, consequently, faster than desired antibody clearance and ultimately lower systemic exposure. This is especially true in situations where the target antigen is highly and / or ubiquitously expressed as is the case for FLT1 . Accordingly, it was desirable to select an anti-FLT1 antibody having a binding affinity to human mFLT1 that provides sufficient target engagement to antagonize FLT1 signalingyet allows enough dissociation to enable FcRn-mediated recycling, thereby avoiding rapid target-mediated clearance and supporting extended systemic exposure. Accordingly, the KDof the FLT1 binding proteins of the present disclosure, preferably the anti- FLT1 antibodies of the present disclosure, to cell membrane expressed human FLT1 is greater than 50 pM, greater than 100 pM, greater than 150 pM, or greater than 200pM, as measured using KinExA as described in Example 16 herein (see also Rathanaswami et al., Analytical Biochemistry 373(1 ):52-60 (2008)). In one embodiment, the KDof the FLT1 binding proteins of the present disclosure (in particular, the anti-FLT1 antibodies of the disclosure) to cell membrane expressed human FLT1 is between 50 pM and 500 pM as measured using KinExA as described in Example 16 herein. In one embodiment, the KDof the FLT1 binding proteins of the present disclosure (in particular, the anti-FLT1 antibodies of the disclosure) to cell membrane expressed human FLT1 is between 100 pM and 200 pM as measured using KinExA as described in Example 16 herein. In one embodiment, the KDof the FLT1 binding proteins of the present disclosure (in particular, the anti-FLT1 antibodies of the disclosure) to cell membrane expressed human FLT1 is between 100 pM and 150 pM as measured using KinExA as described in Example 16 herein. Exemplary anti-FLT1 antibodies of the present disclosure that bind to cell membrane expressed human FLT1 with a KDof greater than 100 pM, e.g., a KDof between100 pM and 500 pM, include antibody 18300 and antibodies or antigen binding fragments comprising at least 90% or 95% sequence identity to the light chain and heavy chain variable regions of antibody 18300 (e.g., an antibody having the consensus light chain variable sequence of SEQ ID NO: 1587 and the consensus heavy chain variable sequence of SEQ ID NO: 1586). In one embodiment, exemplary antibodies of the present disclosure that bind to cell membrane expressed human FLT1 with a KDof greaterthan 100 pM, e.g., a KDof between 100 pM and 500 pM, comprise the 18300 consensus CDRs of Table E (i.e. HCDR 1-3 having SEQ ID NOs: 1487, 1490, 1493, respectively and LCDRs 1 -3 having SEQ ID NOs: 1496, 1499, and 1502, respectively). In one embodiment, exemplary antibodies of the present disclosure that bind to cell membrane expressed human FLT1 with a KDof greater than 100 pM, e.g., a KDof between 100 pM and 500 pM, comprise the CDRs of antibody 18300 (i.e. HCDR 1 -3 having SEQ ID NOs: 7-9, respectively and LCDRs 1 -3 having SEQ ID NOs: 10-12, respectively) as disclosed herein.

[0098] Thermostability

[0099] The FLT1 binding proteins of the present disclosure, in particularthe anti-FLT1 antibodies of the present disclosure, are thermostable. Antibody thermostability refers to the ability of an antibodyto retain native, functional structure and activity when exposed to elevated temperature. A high level of thermostability is critical because it affects manufacturability, storage, distribution, and in vivo functionality of a therapeutic antibody. The high thermostability of the FLT1 antibodies of the present disclosure prevents storage related degradation, aggregation, and loss of potency overtime (during storage), and reduces unfolding and aggregation upon in vivo administration (where antibodies are exposed to body temperature, i.e., ~98°F.

[0100] Antibody thermostability, orthermal stability, as referred to herein is measured by melting temperature (Tm). Tm is the temperature at which half of the antibody unfolds or melts, where a higher melting temperature generally indicates that the antibody is more stable. In one embodiment, the FLT1 binding proteins of the present disclosure, in particular, the anti-FLT1 antibodies of the present disclosure, comprise a Tm of greater than70°C. In one embodiment, the FLT1 binding proteins of the present disclosure, in particular, the anti-FLT1 antibodies of the present disclosure, comprise a Tm of greater than 71 °C, greater than 72°C, greater than 73°C, greaterthan 74°C, or greater than 75°C. In one embodiment, the FLT1 binding proteins of the present disclosure, in particular, the anti-FLT1 antibodies of the present disclosure, comprise a Tm of about 70°C to about 85°C. In one embodiment, the FLT1 binding proteins of the present disclosure, in particular, the anti-FLT1 antibodies of the present disclosure, comprise a Tm ofabout 70°C to about 85°C as measured by differential scanning fluorimetry (DSF) as described in Example 15 herein. Exemplary anti-FLT1 antibodies of the present disclosure having a Tm of greater than 70°C, e.g., a Tm of between 70°C and 85°C (as measured by DSF), include antibody 18300 and antibodies or antigen binding fragments comprising at least 90% or 95% sequence identity to the light chain and heavy chain variable regions of antibody 18300 (e.g., an antibody havingthe consensus light chain variable sequence of SEQ ID NO: 1587 and the consensus heavy chain variable sequence of SEQ ID NO: 1586). In one embodiment, exemplary anti-FLT1 antibodies of the present disclosure having a Tm of greater than 70°C, e.g., a Tm of between 70°C and 85°C (as measured by DSF), comprise the 18300 consensus CDRs of Table E (i.e. HCDR 1 -3 having SEQ ID NOs: 1487, 1490, 1493, respectively and LCDRs 1 -3 having SEQ ID NOs: 1496, 1499, and 1502, respectively). In one embodiment, exemplary anti-FLT1 antibodies of the present disclosure having a Tm of greater than 70°C, e.g., a Tm of between 70°C and 85°C (as measured by DSF), comprise the CDRs of antibody 18300 (i.e. HCDR 1-3 having SEQ ID NOs: 7-9, respectively and LCDRs 1 -3 having SEQ ID NOs: 10-12, respectively) as disclosed herein.

[0101] In one embodiment, the FLT1 binding proteins of the present disclosure, in particular, the anti-FLT1 antibodies of the present disclosure, comprise a Tm of about 75°C to about 85°C as measured by differential scanning fluorimetry (DSF) as described in Example 15 herein. Exemplary a nti-FLT1 antibodies of the present disclosure having a Tm of greater than 75°C, e.g., a Tm of between 75°C and 85°C (as measured by DSF), include antibody 18300 and antibodies or antigen binding fragments comprising at least 90% or 95% sequence identity to the light chain and heavy chain variable regions of antibody 18300 (e.g., an antibody having the consensus light chain variable sequence of SEQ ID NO: 1587 and the consensus heavy chain variable sequence of SEQ ID NO: 1586). In one embodiment, exemplary anti-FLT1 antibodies of the present disclosure having a Tm of greater than 75°C, e.g., a Tm of between 75°C and 85°C (as measured by DSF), comprise the 18300 consensus CDRs of Table E (i.e. HCDR 1-3 having SEQ ID NOs: 1487, 1490, 1493, respectively and LCDRs 1-3 having SEQ ID NOs: 1496, 1499, and 1502, respectively). In one embodiment, exemplary anti-FLT1 antibodies of the present disclosure having a Tm of greaterthan 75°C, e.g., a Tm of between 75°C and 85°C (as measured by DSF), comprise the CDRs of antibody 18300 (i.e. HCDR 1 -3 having SEQ ID NOs: 7-9, respectively and LCDRs 1 -3 having SEQ ID NOs: 10-12, respectively) as disclosed herein.

[0102] Viscosity

[0103] In exemplary embodiments, the FLT1 binding proteins of the present disclosure, in particular the anti-FLT1 antibodies of the present disclosure, possess a viscosity that is suitable for subcutaneous administration. Antibody viscosity is a measure of the resistance of concentrated antibody to flow, which is a property that is particularly relevant at the high concentrations typically required for subcutaneous administration. Viscosity is generally assessed using rheological methods, such as capillary viscometry, rotational viscometry, or oscillatory rheometry, under conditions that approximate the intended clinical product concentration. The measurement of antibody viscosity, expressed as centipoise (cP), is an important parameter in anti-FLT1 antibody selection to ensure the antibody can be administered in concentrated, patient-friendly subcutaneous doses without compromising manufacturability or stability.

[0104] In one embodiment, the FLT1 binding proteins of the present disclosure, in particular, the anti-FLT1 antibodies of the present disclosure, comprise a viscosity at 25 °C, at a concentration of 140-150 mg / mL, of less than than 13 cP, preferably less than 12 cP, more preferably less than 11 cP, most preferably less than 10 cP. Exemplary anti-FLT1 antibodies of the present disclosure having a viscosity at 25 °C, at a concentration of 140-150 mg / mL, of less than 13 cP, e.g., a cP of 9-10, as measured by rotational viscometry, include antibody 18300 and antibodies or antigen binding fragments comprising at least 90% or 95% sequence identity to the light chain and heavy chain variable regions of antibody 18300 (e.g., an antibody having the consensus light chain variable sequence of SEQ ID NO: 1587 and the consensus heavy chain variable sequence of SEQ ID NO: 1586). In one embodiment, exemplary anti-FLT1 antibodies of the present disclosure having a viscosity at 25 °C, at a concentration of 140-150 mg / mL, of less than 13 cP, e.g., a cP of 9-10, as measured by rotational viscometry, comprise the 18300 consensus CDRs of Table E (i.e. HCDR 1 -3 having SEQ ID NOs: 1487, 1490, 1493, respectively and LCDRs 1 -3 having SEQ ID NOs: 1496, 1499, and 1502, respectively). In one embodiment, exemplary anti-FLT1 antibodies of the present disclosure having a viscosity at 25 °C, at a concentration of 140-150 mg / mL, of less than 13 cP, e.g., a cP of 9-10, as measured by rotational viscometry, comprise the CDRs of antibody 18300 (i.e. HCDR 1 -3 having SEQ ID NOs: 7-9, respectively and LCDRs 1 -3 having SEQ ID NOs: 10-12, respectively) as disclosed herein.In one embodiment, exemplary anti-FLT1 antibodies of the present disclosure having a viscosity at 25 °C, at a concentration of 140-150 mg / mL, of less than 13 cP, e.g., a cP of 9-10, as measured by rotational viscometry also have a Tm of greater than70°C, e.g., a Tm of between 70°C and 85°C (as measured by DSF),

[0105] FL T1 Inhibition and Antagonism

[0106] In exemplary embodiments, the FLT1 binding proteins of the present disclosure, in particular the anti-FLT1 antibodies, bind FLT1 to inhibit the binding interaction between FLT1 and its endogenous ligand, VEGF. In various embodiments, the FLT1 binding protein of the present disclosure competes with VEGF for binding to FLT1 . As disclosed herein, the FLT1 binding protein of the present disclosure inhibits a binding interaction between human FLT1 and VEGF as determined by the Octet-based receptor-ligand binding assay as described herein at Example 1 . As disclosed herein, the FLT1 binding protein inhibits a binding interaction between human FLT1 and VEGF and the inhibition is expressed in terms percent inhibition. In one embodiment, greater than 80% (e.g., greater than 85%, greater than 90%) of the human FLT1 and VEGF binding interactions are inhibited in the presence of a presently disclosed FLT1 binding protein, e.g., anti-FLT1 antibody. Optionally, greaterthan 95% (e.g., greater than 96%, greaterthan 97%, greaterthan 98%, greater than 99% or nearly 100%) of human FLT1 and VEGF binding interactions are inhibited in the presence of a presently disclosed FLT1 binding protein, e.g., anti-FLT1 antibody, as determined by the Octet-based receptor-ligand binding assay, such as that described herein at Example 1 .

[0107] The reduction or inhibition provided by the FLT1 binding protein of the present disclosure may not be a 100% or complete reduction or inhibition. Rather, there are varying degrees of reduction or inhibition of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this regard, the FLT1 binding protein may inhibit the FLT1-VEGF binding interactions and the FLT1-mediated inhibition of VEGF-VEGFR2 signaling to any amount or level necessary to achieve a therapeutic end point as described herein. The inhibition provided by the FLT1 binding protein disclosed herein is at least or about 10% inhibition, at least or about 20% inhibition, at least or about 30% inhibition, at least or about 40% inhibition, at least or about 50% inhibition, at least or about 60% inhibition, at least or about 70% inhibition, at least or about 80% inhibition, at least or about 90% inhibition, at least or about 95% inhibition, at least or about 98% inhibition. In exemplary embodiments, the FLT1 binding proteins, in particular the anti-FLT1 antibodies, described herein inhibit 90%, 95% or 100% of VEGF-FLT1 binding. In one embodiment, the reduction in the VEGF-FLT1 binding interaction provided by the FLT1 binding protein is at least or about 10% reduction, at least or about 20% reduction, at least or about 30% reduction, at least or about 40% reduction, at least or about 50% reduction, at least or about 60% reduction, at least or about 70% reduction, at least or about 80% reduction, at least or about 90% reduction, at least or about 95% reduction, at least or about 98% reduction. In exemplary embodiments, the FLT1 binding proteins, inparticula r the anti-FLT1 antibodies, described herein reduce 90%, 95% or 100% of VEGF-FLT1 binding

[0108] As disclosed herein, the FLT1 binding proteins of the present disclosure inhibit binding of human VEGF to human FLT1 and this inhibition is assayed by measuring the level of free VEGF (unbound to FLT1 ) in the presence of FLT1 binding protein. In one embodiment, the level of free VEGF resulting from FLT1 binding protein inhibition of FLT1 is measured in an in vitro cell based assay. In one embodiment, the level of free VEGF is measured in the cell media of cultured endothelial cells following FLT1 binding protein treatment as described in Example 5 herein. In this assay, the inhibitor activity of a FLT1 binding protein is expressed in terms of EC50, i.e., the concentration of FLT1 binding protein effective to induce half maximal free VEGF levels in the cell culture media. In one embodiment, the EC50of the FLT1 binding protein is within about 0.1 nM to about 50 nM, about 0.1 nM to about 40 nM, about O.1 nM to about 30 nM, about 0.01 nM to about 20 nM, about 0.1 nM to about 15 nM, about O.1 nM to about 10 nM, about O.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about ? nM, about O.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, about 0.1 nM to about 0.5 nM, about 1 nM to about 5 nM, about 1 nM to about 10 nM, about 1 nM to about 15 nM. In one embodiment, the EC5o of the FLT1 binding proteins, in particular the anti-FLT1 antibodies, of the present disclosure is about 0.1 nM to about 15 nM.

[0109] As disclosed herein, the FLT1 binding protein of the present disclosure inhibits binding of VEGF to human FLT1 and the inhibition is characterized in terms of a half maximal inhibitory concentration (IC50), which is a measure of the effectiveness of the FLT1 binding protein to inhibit a specific biological or biochemical function. In exemplary embodiments, the inhibition of human VEGF-FLT1 binding is measured by an increase in the level of VEGF mediated VEGFR2 (KDR) phosphorylation (see e.g., Example 6 herein). The IC50of the FLT1 binding protein to increase VEGFR2 phosphorylation is, in exemplary embodiments, less than about 10 nM, optionally, less than 5 nM. In one embodiment, the IC5o of the FLT1 binding protein is less than 2 nM or less than 1 nM. In one embodiment, the IC5o of the FLT1 binding protein is about 0.5 nM to about 2 nM. In one embodiment, the IC5o of the FLT1 binding protein is within about O.1 nM to about 10 nM, about O.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about l nM, about O.1 nM to about 0.5 nM. In exemplary embodiments, the IC5o of the FLT1 binding proteins, in particular the anti-FLT1 antibodies of the disclosure, is about 0.1 nM toabout 2 nM as measured using the VEGFR phosphorylation assay as described in Example 6 herein.

[0110] As disclosed herein, the FLT1 binding proteins of the present disclosure, e.g., anti- FLT1 antibodies, bind FLT1 to inhibit the binding interaction between FLT1 and its endogenous ligand, VEGF. However, FLT1 also binds to placental growth factor (PIGF), a growth factor active in angiogenesis and endothelial cell growth. VEGF and PIGF both bind to the second Ig- like extracellular domain 2 (D2) of FLT1 . Accordingly, the FLT1 binding proteins of the present disclosure, e.g., anti-FLT1 antibodies, bind to FLT1 and inhibit VEGF-FLT1 binding as well as PIGF-FLT1 binding as demonstrated by the increase in both VEGF and PIGF levels in cell culture media following FLT1 antibody treatment of cultured HUVECs (see Example 5). From a therapeutic perspective, FLT1 binding proteins that exhibit a minimal level of PIGF-FLT1 inhibition are desired to minimize non-specific activity of the FLT1 antibodies. As demonstrated herein, while the FLT1 binding proteins of the present disclosure inhibit PIGF-FLT1 binding interaction, the potency of this inhibition is approximately 100 to 1000-fold less than the potency of a reference FLT1 binding protein (13477) (see Example 5; EC50 values of the anti- FLT1 antibodies disclosed herein for PIGF-FLT1 inhibition are 100 to 1000 times lower than the corresponding EC50 value of reference antibody (Ab 13477)). This lower level of non-specific PIGF-FLT1 inhibition provides a therapeutic advantage of the presently disclosed FLT1 binding proteins over previously described FLT1 antibodies, because non-specific inhibition of PIGF- FLT1 activity is minimized.

[0111] FL T1 Binding Protein Types

[0112] The “FLT1 -binding protein” as referred to herein is an antibody (i.e., immunoglobulin), an antigen binding fragment thereof, or an antibody protein product.

[0113] In exemplary embodiments, the FLT1 -binding protein of the present disclosure is an antibody. Collectively, antibodies form a family of plasma proteins known as immunoglobulins and comprise of immunoglobulin domains. (Janeway et al., Immunobiology: The Immune System in Health and Disease, 4thed., Elsevier Science Ltd. / Garland Publishing, 1999). As used herein, the term “antibody” refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions. For example, an antibody may be an IgG which is a “Y-shaped” structure of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In IgG formats, the variable region is generally about 100- 110 or more amino acids, comprises three complementarity determining regions (CDRs), isprimarily responsible for antigen recognition, and substantially varies among other antibodies that bind to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region is made of the N-terminal regions of each light chain and heavy chain, while the constant region is made of the C-terminal portions of each of the heavy and light chains. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4thed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0114] The general structure and properties of CDRs of antibodies have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition. A variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917;Chothia et al., 1989, Nature 342: 877-883), within a framework region (designated framework regions 1 -4, FR1 , FR2, FR3, and FR4, by Kabat et al., 1991 ; see also Chothia and Lesk, 1987, supra). Heavy chain and light chain CDRs of anti-FLT1 antibodies of the present disclosure can be delineated using any one of the standard methods known in the art, including and without limitation, the Kabat numbering scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 5thed., U.S. Dept, of Health and Human Services, NIH (1991)), Chothia numbering scheme (Chothia et al., J. Mol. Biol. 196: 901 -917 (1987) and Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997), which are hereby incorporated by reference in their entirety), the Abhinandan numbering scheme (Abhinandan et aL, Mol. Immunol. 45: 3832-3839 (2008), which is hereby incorporated by reference in its entirety), the immunogenetic (IMGT) database numbering scheme (Giudicelli et al., Nucl. Acids Res. 34: D781-784 (2006) and Lefranc et al., Dev. Comp. Immunol. 27: 55-77 (2003), which are hereby incorporated by reference in their entirety), the Aho numbering scheme (Honegger et al., J. Mol. Biol. 309: 657-670 (2001), which is hereby incorporated by reference in its entirety), and the Contact numbering scheme (MacCallum et al., J. Mol. Biol. 262732-745 (1996), which is hereby incorporated by reference in its entirety). The residues of the heavy chain and light chain variable regions that comprise CDRs according to each system are provided below.HC and LC Variable Region CDR Residues According to Known Numbering Schemes

[0115] Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgG 1 , lgG2, lgG3, and lgG4. IgM has subclasses, including, but not limited to, IgM 1 and lgM2. In embodiments of the present invention, the anti-FLT1 antibodies are of the IgG isotype. Preferably, the anti-FLT1 antibodies are of the human IgG isotype. Preferably, the anti-FLT1 antibodies of the present disclosure are lgG1 isotype, preferably human IgG 1 isotype. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. In exemplary embodiments, the anti-FLT1 antibodies disclosed herein comprise (i) a human IgG and human kappa light chain constant region or (ii) a human IgG and human lambda light chain constant region.

[0116] In exemplary embodiments, the FLT1 binding protein of the present disclosure is an antigen binding fragment. As used herein, the term “antigen binding fragment” refers to any polypeptide fragment, region, portion or domain of an anti-FLT1 antibody that exhibits the same or substantially similar FLT1 binding properties of the anti-FLT1 antibody from which it is derived. An antigen binding fragment can be produced by cleaving an antibody into fragments by enzymes, such as, e.g., papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab’)2fragment and a pFc’ fragment. In exemplary embodiments of the present disclosure, the FLT1 binding protein of the present disclosure comprises an antigen binding fragment, such as a Fab fragment, Fab’ fragment, a F(ab’)2fragment Fv fragment, Fd fragment, or single domain fragment.

[0117] In exemplary embodiments, the FLT1 binding protein of the present disclosure is an antibody protein product. The architecture of antibodies has been exploited to create a growingrange of alternative antibody protein products that span a molecular-weight range of at least about 12-150 kDa and have a valency (n) range from monomeric (n = 1), to dimeric (n = 2), to trimeric (n = 3), to tetrameric (n = 4), and potentially higher. Antibody protein products are proteins or polypeptides that contain at least one FLT1 binding domain of an antibody and are typically formed using recombinant techniques or via chemical modification of a parent antibody or antigen binding fragment thereof. Antibody protein products may comprise one or more antigen binding fragments of an antibody, such as the Fv fragment. The smallest antigen binding fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions. A soluble, flexible amino acid peptide linker (~15 amino acids) is used to connect the HC and LC Fv fragments to form a scFv. scFvs can be easily produced in host cells, e.g., prokaryotic host cells. In exemplary embodiments, the FLT1 binding protein is a FLT1 scFv. The scFv is a building block that is frequently used to create other suitable FLT1 antibody protein products including, e.g., minibodies, diabodies, triabodies, and tetrabodies. Other FLT1 antibody protein products include disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), single chain antibody (SCA), as well as di- and multimeric antibody formats of dia-, tria- and tetra-bodies, or minibodies, and multispecific antibodies (IgGs), such as bispecific antibodies, trispecific antibodies, and the like. Bispecific antibodies can be divided into five major classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A: 97- 106 (2015).

[0118] In exemplary embodiments, the FLT1 binding protein of the present disclosure comprises or is any one of these antibody protein products. In exemplary embodiments, the FLT1 binding protein of the present disclosure comprises or is any one of an scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., a diabody, triabody, tetrabody), miniAb, VHH / VH of camelid heavy chain antibody, sdAb, diabody; a triabody; a tetrabody; a bispecific or trispecific antibody, BsIgG, appended IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate.

[0119] In exemplary embodiments, the FLT1 binding protein of the present disclosure comprises an antibody protein product in monomeric form, or polymeric, oligomeric, or multimeric form. In certain embodiments in which the antibody protein product comprises two or more distinct antigen binding regions, the antibody protein product is considered bispecific, trispecific, or multi-specific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that are recognized and bound by the antibody protein product. In exemplary embodiments, the FLT1 binding protein of the present disclosure is a bispecific antibody (bsAb)comprising two scFv, one which binds to FLT1 and one which binds to a protein other than FLT1 . In exemplary embodiments, the FLT1 binding protein of the present disclosure is a bispecific antibody (bsAb) comprising two scFv, one which binds to a first epitope of FLT1 and one which binds to a second epitope of FLT1 which is different from the first epitope of FLT1 .

[0120] Structure of FLT1 Binding Proteins

[0121] As disclosed herein, exemplary FLT1 binding proteins of the present disclosure (in particular, an anti-FLT1 antibody or antigen binding fragment thereof) comprises an antibody heavy chain (HC) variable region (VH) comprising (a) an HC complementarity-determining region (CDR) 1 amino acid sequence set forth in Table A.1 , or a variant sequence thereof which differs by only 1 -4 amino acids (e.g., 1 , 2, 3, 4 amino acids) or which has at least or about 80%, 85%, or 90% sequence identity to the HC CDR1 amino acid sequence set forth in Table A.1 ; (b) an HC CDR2 amino acid sequence set forth in Table A.1 , or a variant sequence thereof which differs by only 1 -4 amino acids or which has at least or about 80%, 85%, or 90% sequence identity to the HC CDR2 amino acid sequence set forth in Table A.1 ; (c) an HC CDR3 amino acid sequence set forth in Table A.1 , or a variant sequence thereof which differs by only 1-4 amino acids orwhich has at least or about 80%, 85%, or 90% sequence identity to the HC CDR3 amino acid sequence set forth in Table A.1 ; or (d) or a combination thereof, e.g., (a) and (b), (a) and (c), (b) and (c), (a) to (c). In exemplary embodiments, the FLT1 binding protein (in particular, an anti- FLT1 antibody or antigen binding fragment thereof) comprises an antibody light chain (LC) variable region comprising (a) an LC CDR1 amino acid sequence set forth in Table A.1 , or a variant sequence thereof which differs by only 1-4 amino acids (e.g., 1 , 2, 3, 4 amino acids) or which has at least or about 80%, 85%, or 90% sequence identity to the LC CDR1 amino acid sequence set forth in Table A.1 ; (b) an LC CDR2 amino acid sequence set forth in Table A.1 , or a variant sequence thereof which differs by only 1-4 amino acids orwhich has at least or about 80%, 85%, or 90% sequence identity to the LC CDR2 amino acid sequence set forth in Table A.1 ; (c) an LC CDR3 amino acid sequence set forth in Table A.1 , or a variant sequence thereof which differs by only 1 -4 amino acids orwhich has at least or about 80%, 85%, or 90% sequence identity to the LC CDR3 amino acid sequence set forth in Table A.1 ; or (d) or a combination thereof, e.g., (a) and (b), (a) and (c), (b) and (c), (a) to (c).TABLE A.1Single letter amino acid codes are used.

[0122] As disclosed herein, the FLT1 binding protein of the present disclosure (in particular, an anti-FLT1 antibody or antigen binding fragment thereof) comprises a LC CDR1 amino acid sequence, a LC CDR2 amino acid sequence, and / or a LC CDR3 amino acid sequence set forth in Table A.1 , and at least 1 or 2 of the HC CDR amino acid sequences set forth in Table A.1 . As disclosed herein, the FLT1 binding protein of the present disclosure (in particular, an anti-FLT1 antibody or antigen binding fragment thereof) comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, and / or a HC CDR3 amino acid sequence set forth in Table A.1 , and at least 1 or 2 of the LC CDR amino acid sequences set forth in Table A.1 . In exemplary embodiments, the FLT1 binding protein comprises each of the HC CDR amino acid sequences of a given antibody of Table A.1 and at least 1 or 2 of the LC CDR amino acid sequences of the same antibody or another antibody of Table A.1 . In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises each of the LC CDR amino acid sequences of a given antibody of Table A.1 and at least 1 or 2 of the HC CDR amino acid sequences of the same antibody or another antibody of Table A.1 . In some embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises all three LC CDRs (LC CDR1 , LC CDR2, LC CDR3) and / or all three HC CDRs (HC CDR1 , HC CDR2, HC CDR3) for a given antibody of Table A.1 . In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises all three LC CDRs (LC CDR1 , LC CDR2, LC CDR3) and all three HC CDRs (HC CDR1 , HC CDR2, HC CDR3) of a given antibody of Table A.1 . In exemplary embodiments, the FLT1 binding protein comprises any 1 , 2, 3, 4, 5, or all 6 of the amino acid sequences of an antibody of Table A.1 .

[0123] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 7-12; (b) SEQ ID NOs: 151-156; (c) SEQ ID NOs: 331 -336; (d) SEQ ID NOs: 181 -186; (e) SEQ ID NOs: 571-576; (f) SEQ ID NOs: 619-624;(g) SEQ ID NOs: 589-594; (h) SEQ ID NOs: 1033-1038, (i) SEQ ID NOs: 1027-1032, and (j) SEQ ID NOs: 733-738.

[0124] As shown in Table A.1 , many CDRs of the FLT1 antibodies are structurally similar (i.e., they share a high level of sequence identity). For instance, the HC CDR1 for antibodies 15331 , 15327, 15328, 15314, 15311 , and 15310 comprise the amino acid sequence SYAMH (SEQ ID NO: 589) or an amino acid sequence comprising at least 80% sequence identity to SYAMH. Also, for example, the amino acid sequence of the HC CDR1 for antibody 18300 has at least 85% sequence identity with the amino acid sequence of the HC CDR1 for antibody 15320. Also, the HC CDR1 for each of antibodies 18300, 15320, 15316, and 15318 comprises the sequence SX1X2X3YWS (any one of SEQ ID NO: 1464-1466), optionally, wherein X1 is an aliphatic amino acid (e.g., A or G), X2 is an aliphatic amino acid (e.g., G, V, L) or an acidic amino acid (e.g., E), and X3 is an aromatic amino acid (e.g., Y) or a basic amino acid (e.g., H).

[0125] Further, the HC CDR2 for each of antibodies 18300, 18316, 15318, and 15320 comprises the amino acid sequence YIYYSGSTYYNPSLKS (SEQ ID NO: 152) or an amino acid sequence having at least 90% sequence identity thereto. The HC CDR2 for antibodies 15310, 15311 , and 15314 comprises the amino acid sequence IISX1X2GSTX3X4YAX5X6VKG (any one of SEQ ID NO: 1467-1469), optionally wherein X1 is an aromatic amino acid (e.g., F or Y), X2 is an aromatic amino acid (e.g., Y) or an acidic amino acid (e.g., E), X3 is an aliphatic amino acid (e.g., A) or an amino acid comprising a side chain hydroxyl (e.g., S), X4 is an aromatic amino acid (e.g., F) or an aliphatic amino acid (e.g., I), X5 is an acidic amino acid (e.g., E or D) and X5 is an amino acid comprising a side chain hydroxyl (e.g., S) or an aliphatic amino acid (e.g., A). In exemplary embodiments, the HC CDR2 comprises VIWYDGSNX1 YYX2DAVKG (any one of SEQ ID NO: 1470-1472) optionally wherein X1 is a basic amino acid (e.g., K) or an aliphatic amino acid (e.g., I) and X2 is an aliphatic amino acid (e.g., A or G).

[0126] The amino acid sequence of the HC CDR3 for antibody 15316 has at least 90% sequence identity to the amino acid sequences of the HC CDR3 for antibodies 15318 and 15320. The HC CDR3 for each of antibodies 15310, 15311 and 15314 comprise the amino acid sequence GLELQDY (SEQ ID NO: 573). Also, the HC CDR3 for each of antibodies 15331 , 15327 and 15328 comprises the amino acid sequence STYYIPSGSYYYYYGMDV (SEQ ID NO: 1035) or an amino acid sequence comprising at least 90% sequence identity.

[0127] In exemplary embodiments, the LC CDR1 comprises TGTSSDVGGYNYVS (SEQ ID NO: 574), RSSQSLLHSNGNNFLD (SEQ ID NO: 1036) or SGSSSNIGINX1 VN (any one of SEQ ID NO: 1473-1475), optionally, wherein X1 is an amino acid comprising a side chain amide or hydroxyl (e.g., N, T).

[0128] In exemplary embodiments, the LC CDR2 comprises EVSNRPS (SEQ ID NO: 575), LGSNRAS (SEQ ID NO: 1037), or SNX1X2RPX3 (any one of SEQ ID NO: 1476-1478), optionally, wherein X1 is a basic amino acid (e.g., R) or an amino acid comprising a side chain amide (e.g., N), X2 is a basic amino acid (e.g., H, R), and X3 is an aliphatic amino acid (e.g., A, V) or an amino acid comprising a side chain hydroxyl (e.g., S).

[0129] In exemplary embodiments, the LC CDR3 comprises SSYTSSSTWV (SEQ ID NO: 576); MQALX1YPRT (any one of SEQ ID NO: 1482-1484), optionally wherein X1 is T, Q, orY; or AAWDDX1 LX2GVX3 (any one of SEQ ID NO: 1479-1481 ), optionally wherein X1 is an aliphatic amino acid (e.g., I) or an amino acid comprising a side chain hydroxyl, X2 is N, G, or E, and X3 is an aliphatic amino acid (e.g., V, A).

[0130] Accordingly, in exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a HC CDR1 comprising (A) the amino acid sequence SYAMH (SEQ ID NO: 589) or an amino acid sequence comprising at least 80% sequence identity to SYAMH, (B) the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence comprising at least 85% sequence identity to this sequence, or (C) the amino acid sequence SX1X2X3YWS (SEQ ID NO: any one of SEQ ID NO: 1464-1466), optionally, wherein X1 is an aliphatic amino acid (e.g., A or G), X2 is an aliphatic amino acid (e.g., G, V, L) or an acidic amino acid (e.g., E), and X3 is an aromatic amino acid (e.g., Y) or a basic amino acid (e.g., H). In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a HC CDR2 comprising (A) the amino acid sequence YIYYSGSTYYNPSLKS (SEQ ID NO: 152) or an amino acid sequence having at least 90% sequence identity thereto, (B) the amino acid sequence IISX1X2GSTX3X4YAX5X6VKG (any one of SEQ ID NOs: 1467-1469), optionally, wherein X1 is an aromatic amino acid (e.g., F orY), X2 is an aromatic amino acid (e.g., Y) or an acidic amino acid (e.g., E), X3 is an aliphatic amino acid (e.g., A) or an amino acid comprising a side chain hydroxyl (e.g., S), X4 is an aromatic amino acid (e.g., F) or an aliphatic amino acid (e.g., I), X5 is an acidic amino acid (e.g., E or D) and X5 is an amino acid comprising a side chain hydroxyl (e.g., S) or an aliphatic amino acid (e.g., A), or (C) the amino acid sequence VIWYDGSNX1 YYX2DAVKG (any one of SEQ ID NO: 1470-1472) optionally wherein X1 is a basic amino acid (e.g., K) or an aliphatic amino acid (e.g., I) and X2 is an aliphatic amino acid (e.g., A or G). In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a HC CDR3 comprising (A) the amino acid sequence of SEQ ID NO: 735 or an amino acid sequence having at least 90% sequence identity thereto, (B) the amino acid sequence GLELQDY (SEQ ID NO: 573), or (C) the amino acid sequenceSTYYIPSGSYYYYYGMDV (SEQ ID NO: 1035) or an amino acid sequence having at least 90% sequence identity thereto. In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a LC CDR1 comprising (A) the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO: 574), (B) the amino acid sequence RSSQSLLHSNGNNFLD (SEQ ID NO: 1036) or (C) the amino acid sequence SGSSSNIGINX1 VN (any one of SEQ ID NO: 1473-1475), optionally, wherein X1 is an amino acid comprising a side chain amide or hydroxyl (e.g., N, T). In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a LC CDR2 comprising (A) EVSNRPS (SEQ ID NO: 575), (B) LGSNRAS (SEQ ID NO: 1037), or (C) SNX1X2RPX3 (any one of SEQ ID NO: 1476-1478), optionally wherein X1 is a basic amino acid (e.g., R) or an amino acid comprising a side chain amide (e.g., N), X2 is a basic amino acid (e.g., H, R), and X3 is an aliphatic amino acid (e.g., A, V) or an amino acid comprising a side chain hydroxyl (e.g., S). In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a LC CDR3 comprising (A) the amino acid sequence SSYTSSSTWV (SEQ ID NO: 576) or (B) the amino acid sequence MQALX1 YPRT (any one of SEQ ID NO: 1482-1484), optionally, wherein X1 is an amino acid comprising a side chain amide or hydroxyl or an aromatic amino acid (e.g., T, Q, or Y), or AAWDDX1 LX2GVX3 (any one of SEQ ID NO: 1479-1481 ), optionally wherein X1 is an aliphatic amino acid (e.g., I) or an amino acid comprising a side chain hydroxyl, X2 is N, G, or E, and X3 is an aliphatic amino acid (e.g., V, A).

[0131] In exemplary embodiments, the FLT1 binding protein of the present disclosure (in particular, an anti-FLT1 antibody or antigen binding fragment thereof) comprises a HC variable region presented in Table B or a HC variable region comprising an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to the HC variable region presented Table B or having up to 20 (e.g., up to 15, up to 12, up to 10) amino acid substitutions relative to the HC variable region presented Table B. In exemplary embodiments, the FLT1 binding protein (in particular, an anti-FLT1 antibody or antigen binding fragment thereof) comprises a LC variable region presented in Table B or a LC variable region comprising an amino acid sequence that has at least 80%, 85%, 90% or 95% sequence identity to the LC variable region presented in Table B or having up to 20 (e.g., up to 15, up to 12, up to 10) amino acid substitutions relative to the LC variable region presented Table B.

[0132] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular, an anti-FLT1 antibody, comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQID NO: 1089, and a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1090. In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular, an anti-FLT1 antibody, comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and a light chain variable region (VL) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1090. Exemplary anti-FLT1 antibodies comprising HC variable regions having at least 90% sequence identity to SEQ ID NO: 1089 and comprising LC variable regions having at least 90% sequence identity SEQ ID NO: 1090 are provided in Example 2 and Table 10. The structural similarity of the VH and VL domains, respectively, of the anti-FLT1 antibodies of Table and Ab 18300 is defined by the consensus VH and VL amino acid sequences of Figure 11A. Accordingly, in one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1574 and a VL amino acid sequence of SEQ ID NO: 1575. In one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1580 and a VL amino acid sequence of SEQ ID NO: 1581 . In one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1586 and a VL amino acid sequence of SEQ ID NO: 1587. In one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1580 or 1586, where said VH sequence has at least 90% sequence identity to SEQ ID NO: 1089; and a VL amino acid sequence of SEQ ID NO: 1581 or 1587, wherein said VL sequence has at least 90% sequence identity to SEQ ID NO: 1090. The consensus VH and VL amino acid sequences of Figure 11 A further support an anti-FLT1 antibody comprising (i) a HC variable region sequence comprising up to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions relative to the HC variable region sequence of 18300 (SEQ ID NO: 1089) and (ii) a LC variable region sequence comprising up to 10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions relative to the LC variable region sequence of 18300 (SEQ ID NO: 1090).

[0133] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular, an anti-FLT1 antibody, comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1335, and a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1336. In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular, an anti-FLT1 antibody, comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 1335, and a light chain variable region (VL) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1336. Exemplary FLT1 binding proteins comprising HC variable regions having at least 90% sequence identity to SEQ ID NO: 1335 and comprising LC variable regions having at least 90% sequence identity SEQ ID NO: 1336 are provided in Example 2 and Table 12. The structural similarity of the VH and VL domains, respectively, of the anti-FLT1 antibodies of Table 12 is defined by the consensus VH and VL amino acid sequences of Figure 11C. Accordingly, in one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1578 and a VL amino acid sequence of SEQ ID NO: 1579. In one embodiment, the anti- FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1584 and a VL amino acid sequence of SEQ ID NO: 1585. In one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1590 and a VL amino acid sequence of SEQ ID NO: 1591 . In one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1584 or 1590, where said VH sequence has at least 90% sequence identity to SEQ ID NO: 1335; and a VL amino acid sequence of SEQ ID NO: 1585 or 1591 , wherein said VL sequence has at least 90% sequence identity to SEQ ID NO: 1336. The consensus VH and VL amino acid sequences of Figure 11 C further support an antibody comprising (i) a HC variable region sequence comprising up to 8 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8) amino acid substitutions relative to the HC variable region sequence of 15331 (SEQ ID NO: 1335) and (ii) a LC variable region sequence comprising up to4 (e.g., 1 , 2, 3, 4) amino acid substitutions relative to the LC variable region sequence of 15331 (SEQ ID NO: 1336).

[0134] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular, an anti-FLT1 antibody, comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1283, and a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1284. In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular, an anti-FLT1 antibody, comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1283, and a light chain variable region (VL) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1284. Exemplary FLT1 binding proteins comprising HC variable regions having at least 90% sequence identity to SEQ ID NO: 1283 and comprising LC variable regions having at least 90% sequence identity SEQ ID NO: 1284 are provided in Example 2 and Table 11 . The structural similarity ofthe VH and VL domains, respectively, of the anti-FLT1 antibodies of Table 11 is defined by the consensus VH and VL amino acid sequences of Figure 11 B. Accordingly, in one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1576 and a VL amino acid sequence of SEQ ID NO: 1577. In one embodiment, the anti- FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1582 and a VL amino acid sequence of SEQ ID NO: 1583. In one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1588 and a VL amino acid sequence of SEQ ID NO: 1589. In one embodiment, the anti-FLT1 antibody of the present disclosure comprises a VH amino acid sequence of SEQ ID NO: 1582 or 1588, where said VH sequence has at least 90% sequence identity to SEQ ID NO: 1283; and a VL amino acid sequence of SEQ ID NO: 1583 or 1589, wherein said VL sequence has at least 90% sequence identity to SEQ ID NO: 1284. The consensus VH and VL amino acid sequences of Figure 11 B further support an anti-FLT1 antibody comprising (i) a HC variable region sequence comprising up to 9 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9) amino acid substitutions relative to the HC variable region sequence of 15314 (SEQ ID NO: 1283) and (ii) a LC variable region sequence comprising up to 5 (e.g., 1 , 2, 3, 4, 5) amino acid substitutions relative to the LC variable region sequence of 15314 (SEQ ID NO: 1284).

[0135] In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody or antigen binding fragment thereof) comprises a HC variable region comprising an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to a HC variable region presented Table B and comprises a HC CDR1 , HC CDR2, and HC CDR3 presented in Table A.1. In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody or antigen binding fragment thereof) comprises a LC variable region comprising an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to a LC variable region presented Table B and comprises a LC CDR1 , LC CDR2, and LC CDR3 presented in Table A.1 . In exemplary embodiments, the FLT1 binding protein of the present disclosure ( in particular, an anti-FLT1 antibody) comprises (i) a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1089, where the HC CDR1 , HC CDR2, and HC CDR3 comprise the amino acid sequences of SEQ ID NOs: 7-9, respectively, and (ii) a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1090, where the LC CDR1 , LC CDR2, and LC CDR3 comprises the amino acid sequences of SEQ ID NOs: 10-12, respectively. In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody) comprises a (i) heavy chain variable region (VH) comprising an amino acid sequencehaving at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1335, where the HC CDR1 , HC CDR2, and HC CDR3 comprise the amino acid sequences of SEQ ID NOs: 733-735, respectively; and (ii) a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1336, where the LC CDR1 , LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 736-738, respectively. In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody) comprises (i) a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1283, where the HC CDR1 , HC CDR2, and HC CDR3 comprise the amino acid sequences of SEQ ID NOs: 589-591 , respectively; and (ii) a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1284, where the LC CDR1 , LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 592-594, respectively.

[0136] In exemplary embodiments, the FLT1 binding protein (in particular, an anti-FLT1 antibody) comprises a HC variable region comprising an amino acid sequence of any one of SEQ ID NOs 1574, 1576, and 1578 and / or a LC variable region comprising an amino acid sequence of any one of SEQ ID NOs 1575, 1577, and 1579. In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a HC variable region comprising an amino acid sequence of SEQ ID NO: 1574 and a LC variable region comprising an amino acid sequence of SEQ ID NO: 1575. Preferably, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a HC variable region comprising an amino acid sequence of SEQ ID NO: 1580 and a LC variable region comprising an amino acid sequence of SEQ ID NO: 1581. More preferably, the FLT1 binding protein, in particular an anti-FLT1 antibody of the disclosure, comprises a HC variable region comprising an amino acid sequence of SEQ ID NO: 1586 and a LC variable region comprising an amino acid sequence of SEQ ID NO: 1587. In exemplary embodiments, the FLT1 binding protein, in particular an anti-FLT1 antibody of the disclosure, comprises the HC variable region and LC variable region of any one of the anti-FLT1 binders of Table 10 herein. In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a HC variable region and LC variable region of (a) SEQ ID NOs: 1089 and 1090, respectively; (b) SEQ ID NOs: 1137 and 1138, respectively; (c) SEQ ID NOs: 1197 and 1198, respectively; or (d) SEQ ID NOs: 1147 and 1148, respectively.

[0137] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a HC variable region comprisingan amino acid sequence of SEQ ID NO: 1576 and a LC variable region comprising an amino acid sequence of SEQ ID NO: 1577. Preferably, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a HC variable region comprising an amino acid sequence of SEQ ID NO: 1582 and a LC variable region comprising an amino acid sequence of SEQ ID NO: 1583. More preferably, the FLT1 binding protein, in particular an anti- FLT1 antibody of the disclosure, comprises a HC variable region comprising an amino acid sequence of SEQ ID NO: 1588 and a LC variable region comprising an amino acid sequence of SEQ ID NO: 1589. In exemplary embodiments, the FLT1 binding protein in particular, an anti- FLT1 antibody comprises the HC variable region and LC variable region of any one of the FLT1 binders of Table 11 herein. In exemplary embodiments, the FLT1 binding protein (in particular, an anti-FLT1 antibody) comprises a HC variable region and LC variable region of (a) SEQ ID NOs: 1277 and 1278, respectively; (b) SEQ ID NOs: 1293 and 1294, respectively; or (c) SEQ ID NOs: 1283 and 1284, respectively.

[0138] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a HC variable region comprising an amino acid sequence of SEQ ID NO: 1578 and a LC variable region comprising an amino acid sequence of SEQ ID NO: 1579. Preferably, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a HC variable region comprising an amino acid sequence of SEQ ID NO: 1584 and a LC variable region comprising an amino acid sequence of SEQ ID NO: 1585. More preferably, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a HC variable region comprising an amino acid sequence of SEQ ID NO: 1590 and a LC variable region comprising an amino acid sequence of SEQ ID NO: 1591 . In exemplary embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises the HC variable region and LC variable region of any one of the FLT1 binders of Table 12 herein. In exemplary embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a HC variable region and LC variable region of (a) SEQ ID NOs: 1437 and 1438, respectively, (b) SEQ ID NOs: 1435 and 1436, respectively, or (c) SEQ ID NOs: 1335 and 1336, respectively.

[0139] In exemplary embodiments, the FLT1 binding protein (in particular, an antibody or antigen binding fragment thereof) comprises a HC CDR1 , HC CDR2, and HC CDR3 of a HC variable region presented in Table B and / or a LC CDR1 , LC CDR2, and LC CDR3 of a LC variable region presented in Table B. Any one of the known CDR numbering schemes may be used to determine the HC CDR1 , HC CDR2, and HC CDR3 of the HC variable region presented in Table B and / or LC CDR1 , LC CDR2, and LC CDR3 of a LC variable region presented in Table B. TheCDRs of the anti-FLT1 antibodies disclosed herein can be determined by any of the methods known in the art, including, but not limited to Kabat, Chothia, IMGT, AbM, Martin, Aho, and Gelfand as described and provided supra. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept, of Health and Human Services, NIH (1991 ); Chothia et al., J Mol Biol 196: 901-917 (1987); Al-Lazikani et al., J Mol Biol 273: 927-948 (1997); Abhinandan et al., Mol Immunol 45: 3832-3839 (2008); Lefranc et al., The Immunologist 7: 132-136 (1999); Lefranc et aL, Dev Comp Immunol 27: 55-77 (2003); Honegger et al., J Mol Biol 309: 657-670 (2001); Dondelinger at al., Front Immunol. 9:2278 (2018); Sela-Chulang et al., Front Immunol 4:302 (2013); North et al., J Mol Biol 4:901 -917 (1987); Kelow et al., Mabs (2020); 10.1080 / 19420862.2020.1840005; Brennan et al., Bioinformatics 38(7): 1877-1880 (2022); Wong et al., Bioinformatics 35(10): 1774-1776 (2019); Jumper et al., Nature 596: 583-589 (2021); Dunbar et aL, Bioinformatics 32(2): 298-300 (2016).

[0140] In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 7-9, respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1089 in accordance with Kabat numbering, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 10-11 , respectively) of the LC variable region comprisingthe amino acid sequence of SEQ ID NO: 1090 in accordance with Kabat numbering. In exemplary embodiments, the FLT1 binding protein (in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 1606-1608, respectively) of the HC variable region comprisingthe amino acid sequence of SEQ ID NO: 1089 in accordance with Chothia numbering, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 1609-1611 , respectively) of the LC variable region comprisingthe amino acid sequence of SEQ ID NO: 1090 in accordance with Chothia numbering. In exemplary embodiments, the FLT1 binding protein (in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 1612-1614, respectively) of the HC variable region comprisingthe amino acid sequence of SEQ ID NO: 1089 in accordance with IMGT numbering, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 1615-1617, respectively) of the LC variable region comprising the amino acid sequence of SEQ ID NO: 1090 in accordance with IMGT numbering. In exemplary embodiments, the FLT1 binding protein (in particular, an anti- FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 1618-1620, respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1089 in accordance with AHonumbering, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 1621 -1623, respectively) of the LC variable region comprising the amino acid sequence of SEQ ID NO: 1090 in accordance with AHo numbering

[0141] In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 733-735, respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1335 in accordance with the Kabat numbering scheme, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 736-738, respectively) of the LC variable region comprising the amino acid sequence of SEQ ID NO: 1336 in accordance with Kabat numbering scheme. In exemplary embodiments, the FLT1 binding protein (in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 1624-1626, respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1335 in accordance with the Chothia numbering scheme, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 1627-1629, respectively) of the LC variable region comprising the amino acid sequence of SEQ ID NO: 1336 in accordance with Chothia numbering scheme. In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 1630-1632, respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1335 in accordance with the IMGT numbering scheme, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 1633-1635, respectively) of the LC variable region comprising the amino acid sequence of SEQ ID NO: 1336 in accordance with IMGT numbering scheme. In exemplary embodiments, the FLT1 binding protein (in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 1636-1638, respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1335 in accordance with the Aho numbering scheme, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 1639-1641 , respectively) of the LC variable region comprisingthe amino acid sequence of SEQ ID NO: 1336 in accordance with Aho numbering scheme.

[0142] In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 589-591 , respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1283 in accordance with Kabat numbering scheme, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 592-594, respectively) of the LCvariable region comprising the amino acid sequence of SEQ ID NO: 1284 in accordance with Kabat numbering scheme. In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 1642-1644, respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1283 in accordance with Chothia numbering scheme, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 1645-1647, respectively) of the LC variable region comprising the amino acid sequence of SEQ ID NO: 1284 in accordance with Chothia numbering scheme. In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibodyor antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 1648-1650, respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1283 in accordance with IMGT numbering scheme, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 1651-1653, respectively) of the LC variable region comprising the amino acid sequence of SEQ ID NO: 1284 in accordance with IMGT numbering scheme. In exemplary embodiments, the FLT1 binding protein ( in particular, an anti-FLT1 antibody or antigen binding fragment thereof as disclosed herein) comprises the HC CDR1 , HC CDR2, and HC CDR3 (SEQ ID NOs: 1654-1656, respectively) of the HC variable region comprising the amino acid sequence of SEQ ID NO: 1283 in accordance with Aho numbering scheme, and the LC CDR1 , LC CDR2, and LC CDR3 (SEQ ID NOs: 1657-1659, respectively) of the LC variable region comprising the amino acid sequence of SEQ ID NO: 1284 in accordance with Aho numbering scheme.Table A.2TABLE BSingle letter amino acid codes are used. SEQ ID NOs are provided in () following the amino acid sequence.

[0143] In exemplary embodiments, the FLT1 binding protein comprises an antibody heavy chain variable region and / or light chain variable region as described supra and further comprises one or more heavy chain constant regions coupled thereto. For instance, in exemplary embodiments, the FLT1 binding protein comprises a LC variable region of Table B and a light chain constant region of a human kappa light chain. In exemplary embodiments, the FLT1 binding protein comprises a LC variable region of Table B and a light chain constant region of a human lambda light chain. Also, in exemplary embodiments, the FLT1 binding protein comprises a HC variable region of Table B and a constant region of a human IgGI heavy chain. In exemplary embodiments, the FLT1 binding protein comprises a HC variable region of Table B and a constant region of a human lgG2 heavy chain. In exemplary embodiments, the FLT1 binding protein comprises a HC variable region of Table B and a constant region of a human lgG3 heavy chain. In exemplary embodiments, the FLT1 binding protein comprises a HC variable region of Table B and a constant region of a human lgG4 heavy chain.

[0144] In exemplary embodiments, the FLT1 binding protein comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 1530, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1530. In exemplary embodiments, the C-terminal lysine undergoes cleavage by carboxypeptidase during expression. A heavy chain constant region lackingthe C-terminal Lys advantageously prevents carboxypeptidase from reactingwith the heavy chain of the FLT1 binding protein. Accordingly, in exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 1530, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1530, with the C-terminal Lysclipped or removed. In this regard, in some embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a heavy chain constant region lacking the C-terminal Lys and comprises the amino acid sequence of SEQ ID NO: 1531 , or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1531 .

[0145] In exemplary embodiments, the FLT1 binding protein of the present disclosure comprises one or more amino acid modifications, relative to the naturally-occurring counterpart, in order to improve half-life / stability or to render the binding protein ( in particular, the anti-FLT1 antibody of the present disclosure) more suitable for expression / manufacturability. In exemplary embodiments, the FLT1 binding protein is an antibody comprising one or more constant regions designed to prevent or reduce interaction between itself and Fc receptors. In exemplary embodiments, the FLT1 binding protein is a Stable Effector Functionless (SEFL) antibody comprising a constant region that lacks the ability to interact with Fey receptors. SEFL antibodies are known in the art. See, e.g., Liu et aL, J Biol Chem 292: 1876-1883 (2016); and Jacobsen et al., J. Biol. Chem. 292: 1865-1875 (2017). In exemplary embodiments, the SEFL antibody comprises one or more of the following heavy chain mutations, numbered according to the EU system: L242C, A287C, R292C, N297G, V302C, L306C, and / or K334C. In exemplary embodiments, the SEFL antibody comprises N297G. In exemplary embodiments, the SEFL antibody comprises A287C, N297G, and L306C. In other exemplary embodiments, the SEFL antibody comprises R292C, N297G, and V302C (i.e., SEFL2-2).

[0146] As disclosed herein, an exemplary FLT1 binding protein, in particular, an exemplary anti-FLT1 antibody of the present disclosure, comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 1530, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1530, with one or more SEFL mutations which prevent or reduce interaction between the FLT1 binding protein and Fc receptors, including but not limited to L242C, A287C, R292C, N297G, V302C, L306C, and / or K334C. In exemplary embodiments, the SEFL mutations are SEFL2-2 mutations: R292C, N297G, and V302C, such that the FLT1 binding protein, in particularthe anti-FLT1 antibody, comprises a heavy chain constant region aminoacid sequence of SEQ ID NO: 1532, or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1532. In exemplary embodiments, the FLT1 binding protein comprises a heavy chain constant region with SEFL2-2 mutations and with the C-terminal Lys clipped or removed. Such a heavy chain constant region may comprise the sequence of SEQ ID NO: 1533.

[0147] The FLT1 binding protein may comprise other half-life extension (HLE) modifications. In exemplary embodiments, the HLE modification occurs in the heavy chain constant region and comprises one or more of the following mutations, numbered according to the EU system: M252Y, S254T, and T256E. In exemplary embodiments, the FLT1 binding protein comprises one or two of M252Y, S254T, and T256E. In exemplary instances, the FLT1 protein comprises all three of M252Y, S254T, and T256E. In exemplary embodiments, the heavy chain constant region of an anti-FLT1 antibody of the present disclosure comprises an amino acid sequence of SEQ ID NO: 1534 or SEQ ID NO: 1535 or SEQ ID NO: 1536 or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1534 or SEQ ID NO: 1535 or SEQ ID NO: 1536. In exemplary embodiments, the HLE modification occurs in the heavy chain constant region and comprises one or more of the following mutations, numbered according to the EU system: L309D, Q311 H, and N434S. In exemplary embodiments, the FLT1 binding protein comprises one, two or all three of L309D, Q311 H, and N434S. In exemplary embodiments, the FLT1 binding protein comprises all three of L309D, Q311 H, and N434S. In exemplary embodiments, the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 1537 or SEQ ID NO: 1538 or SEQ ID NO: 1539 or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1537 or SEQ ID NO: 1538 or SEQ ID NO: 1539. In exemplary embodiments, the FLT1 binding protein comprises SEFL2-2 modifications and HLE modifications. In some embodiments, the HLE modifications comprise one or two or all three of M252Y, S254T, and T256E. In exemplary embodiments, the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 1540 or SEQ ID NO: 1541 or SEQ ID NO: 1542 or an amino acid sequence which has at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1540 or SEQ ID NO: 1541 or SEQ ID NO: 1542. In some embodiments, the HLE modifications comprise one or two or all three of L309D, Q311 H, and N434S. In exemplary embodiments, the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 1543 or SEQ ID NO: 1544 or SEQ ID NO: 1545 or an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 1543 or SEQ ID NO: 1544 or SEQ ID NO: 1545.

[0148] Exemplary FLT1 binding protein of the present disclosure, in particular an exemplary anti-FLT1 antibody of the disclosure, comprises a HC constant region and a LC constant region listed in Table C.TABLE C

[0149] In exemplary embodiments, the FLT1 binding protein is an antibody comprising a heavy chain comprising HC CDR1 , HC CDR2, and HC CDR3 as presented in Table A.1 and a light LC CDR1 , LC CDR2, and LC CDR3 as presented in Table A.1 . In exemplary embodiments, the FLT1 binding protein is an antibody comprising a HC variable region and a LC variable region as presented in a single row of Table B. In exemplary embodiments, the FLT1 binding protein is an antibody comprising the heavy chain sequence and light chain sequence as detailed in Table D. In exemplary embodiments, the FLT1 binding protein is an antibody comprising the heavy chain sequence and light chain sequence as detailed in Table D but with the C-terminal Lys of the heavy chain clipped or removed. In exemplary embodiments, the FLT1 binding protein is an antibody comprising the heavy chain sequence of SEQ ID NO: 1572 or 1573 and a light chain sequence as detailed in Table D. In exemplary embodiments, the FLT1 binding protein is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 1460 and a light chain amino acid sequence of SEQ ID NO: 1461 . In exemplary embodiments, the FLT1 binding protein is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 1572 and a light chain amino acid sequence of SEQ ID NO: 1461 . In exemplary embodiments, the FLT1 binding protein is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 1462 and a light chain amino acid sequence of SEQ ID NO: 1463. In exemplary embodiments, the FLT1 binding protein is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 1573 and a light chain amino acid sequence of SEQ ID NO: 1463.TABLE DSEQ ID NO: 1460 without C-terminal Lys is SEQ ID NO: 1572. SEQ ID NO: 1462 without C- terminal Lys is SEQ ID NO: 1573.

[0150] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence which is structurally similar to any one or more of the above amino acid sequences, while retaining the same biological functions (e.g., binding to human, cyno, canine, and porcine FLT1 , inhibiting binding interactions between FLT1 and VEGF, increasing free VEGF levels, and increasing signal transduction mediated by VEGF binding to VEGFR2) of its most closely related counterpart (e.g., parent). In exemplary embodiments, the FLT1 binding protein comprises an amino acid sequence which has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to any one or more of the above detailed amino acid sequences, e.g., those presented in Table A.1 , Table B, Table C, orTable D. In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence which differs by only 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, relative to a parent amino acid sequence having an amino acid sequence referenced in Table A.1 orTable B. In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a variant sequence of the parent sequence, which variant sequence differs by only one or two amino acids, relative to the parent sequence. In exemplary embodiments, the FLT1 binding protein comprises one or more amino acid substitutions that occur outside of the CDRs, e.g., the one or more amino acid substitutions occurwithin the framework region(s) of the heavy chain or light chain. In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises one or more amino acid substitutions, yet the FLT1 binding protein retains the amino acid sequences of the six CDRs. Inexemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence having only 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more conservative amino acid substitutions, relative to the parent sequence(s). As used herein, the term "conservative amino acid substitution" refers to the substitution of one amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes exchanges within one of the following five groups:I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly;II. Polar, negatively charged residues and their amides and esters: Asp, Asn, Glu, Gin, cysteic acid and homocysteic acid;III. Polar, positively charged residues: His, Arg, Lys; Ornithine (Orn)IV. Large, aliphatic, nonpolar residues: Met, Leu, lie, Vai, Cys, Norleucine (Nle), homocysteineV. Large, aromatic residues: Phe, Tyr, Trp, acetyl phenylalanine.

[0151] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence comprising at least one amino acid substitution relative to the parent sequence, and the amino acid substitution(s) is / are non-conservative amino acid substitution(s). As used herein, the term "non-conservative amino acid substitution" is defined herein as the substitution of one amino acid with another amino acid having different properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes exchanges outside the above five groups.

[0152] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence comprising at least one amino acid substitution relative to the parent sequence, and the substitute amino acid is a naturally-occurring amino acid. By “naturally-occurring amino acid” or “standard amino acid” or “canonical amino acid” is meant one of the 20 alpha amino acids found in eukaryotes encoded directly by the codons of the universal genetic code (Ala, Vai, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Arg, His, Lys, Asp, Glu). In exemplary embodiments , the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence comprising at least one amino acid substitution relative to the parent sequence, and the substitute amino acid is a nonstandard amino acid, or an amino acid which is not incorporated into proteins during translation. Non-standard amino acids include, but are not limited to: selenocysteine, pyrrolysine, ornithine, norleucine, p-amino acids (e.g., p-alanine, p-aminoisobutyric acid, p-phenlyalanine, 0-homophenylalanine, 0-glutamic acid, 0-glutamine, 0-homotryptophan, 0- leucine, 0-lysine), homo-amino acids (e.g., homophenylalanine, homoserine, homoarginine, monocysteine, homocystine), A / -methyl amino acids (e.g., L-abrine, A / -methyl-alanine, N- methyl-isoleucine, A / -methyl-leucine), 2-aminocaprylic acid, 7-aminocephalosporanic acid, 4- aminocinnamic acid, alpha-aminocyclohexanepropionic acid, amino-(4-hydroxyphenyl)acetic acid, 4-amino-nicotinic acid, 3-aminophenylacetic acid, and the like.

[0153] In exemplary embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence which has greater than or about 70% sequence identity, greater than or about 75% sequence identity, greater than or about 80% sequence identity, greater than or about 85% sequence identity, greater than or about 90% sequence identity, or greater than or about 95% sequence identity, to the parent amino acid sequence(s), e.g., those presented in Table A.1 , Table A.2, Table B, Table C, orTable D. In exemplary embodiments, the FLT1 binding protein, in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence which has at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity to the parent amino acid sequence. In exemplary embodiments, the FLT1 binding protein, in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity along the full-length of the parent amino acid sequence. In exemplary embodiments, the FLT1 binding protein in particular an anti-FLT1 antibody of the disclosure, comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity along the full-length of the parent amino acid sequence.

[0154] In various embodiments, the FLT1 binding protein of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises a variant sequence of a HC variable region amino acid sequence or a variant sequence of a LC variable region amino acid sequence listed in Table B which variant sequence differs from the sequence of Table B by only 1 to 12 amino acids (e.g., 1 to 11 , 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) or which has at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity). In various embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a variant sequence of a HC constant region amino acid sequence or a variant sequence of a LC constant region amino acid sequence listed in Table C which variant sequence differs from the sequence of Table C by only 1 to 46 amino acids or which has at leastor about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity).

[0155] As disclosed herein, the FLT1 binding protein comprises (a) a heavy chain variable region comprising a 18300 consensus HC CDR1 , 18300 consensus HC CDR2, and 18300 consensus HC CDR3 in accordance with Table E and / or a light chain variable region comprising a 18300 consensus LC CDR1 , 18300 consensus LC CDR2, and 18300 consensus LC CDR3 in accordance with Table E, (b) a heavy chain variable region comprising a 15314 consensus HC CDR1 , 15314 consensus HC CDR2, and 15314 consensus HC CDR3 in accordance with Table E and / or a light chain variable region comprising a 15314 consensus LC CDR1 , 15314 consensus LC CDR2, and 15314 consensus LC CDR3 in accordance with Table E, or (c) a heavy chain variable region comprising a 15331 consensus HC CDR1 , 15331 consensus HC CDR2, and 15331 consensus HC CDR3 in accordance with Table E and / or a light chain variable region comprising a 15331 consensus LC CDR1 , 15331 consensus LC CDR2, and 15331 consensus LC CDR3 in accordance with Table E. The consensus CDR sequences of Table E were developed from the CDR engineering of parent antibodies 10B4, 28D8 and 10A7 as described in Example 2 herein and alignment of engineered antibodies to their respective parent antibodies and, in some instances, antibodies in the same VDJ family as the parent antibody. For example, the 18300 Consensus CDRs represent the CDR structure shared by 18300 (11 D4; VDJ sibling of 10B4), 10B4 (VDJ sibling of 18300) and the 10B4 engineered variants of Table 10. As described herein this shared CDR structure is associated with high affinity human FLT1 binding as well as canine and porcine FLT1 binding. The 15331 Consensus CDRs represent the CDR structure shared by 15331 and the 28D8 engineered variants of Table 12 that is associated with high affinity human FLT1 binding as well as canine and porcine FLT1 binding. The 15314 Consensus CDs represent the CDR structure shared by 15314 and the 10A7 engineered variants of Table 11 that is associated with high affinity human FLT1 binding. Exemplary antibodies comprisingthe consensus CDR sequences of Table E are provided in Tables 10-12.TABLE E*sequence of SEQ ID NOs: are same, but definitions of X’s differ.

[0156] In exemplary embodiments, the FLT1 binding protein, of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises an antibody heavy chain variable region and antibody light chain variable region, wherein the HC variable region comprises a HC CDR1 , HC CDR2, and HC CDR3 in accordance with Table E and the LC variable region comprises a LC CDR1 , LC CDR2, and LC CDR3 in accordance with Table E. In exemplary embodiments, any of the amino acids designated with an “X” followed by a number is anyamino acid, e.g., any naturally occurring amino acid. In various embodiments, (1 ) the HC variable region of the FLT1 binding protein (in particular, an anti-FLT1 antibody or antigen binding fragment) comprises (A) a HC CDR1 comprising SX1X2X3YWS (any one of SEQ ID NO : 1485-1487), optionally wherein X1 is an aliphatic amino acid (e.g., A, G), X2 is an acidic, basic, or aliphatic amino acid or an amino acid comprising a side chain amide (e.g., E, G, L, Q, V, R), and X3 is an aromatic or basic amino acid (e.g., Y, H), (B) a HC CDR2 comprising YIYYSGSX1YYNPSLKS (any one of SEQ ID NOs: 1488-1490), optionally wherein X1 is an aliphatic amino acid or an amino acid comprising a side chain hydroxyl (e.g., T, A), and (C) a HC CDR3 comprising GX1 LX2X3LLGX4X5DX6 (any one of SEQ ID NOs: 1491-1493), optionally wherein X1 is an aliphatic, acidic or basic amino acid or an amino acid comprising a side chain hydroxyl (e.g., D, E, G, I, K, N, R, S, V), X2 is an aliphatic, acidic or basic amino acid or an amino acid comprising a side chain hydroxyl (e.g., A, E, G, K, R, T), X3 is an aromatic amino acid (e.g., W, Y), X4 is an aliphatic amino acid (e.g., A, G), X5 is an aliphatic amino acid or an amino acid comprising a side chain sulfur (e.g., M, I, L), and X6 is an aliphatic or an imino acid (e.g., A, L P, V), and / or (2) the LC variable region of the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises (A) a LC CDR1 comprising SGSSSNIGIX1X2VX3 (any one of SEQ ID NOs: 1494-1496), optionally, wherein each of X1 , X2, and X3, is independently an amino acid comprising a side chain amide or a side chain hydroxyl (e.g., N, T, S), (B) a LC CDR2 comprising SNX1X2RPX3 (any one of SEQ ID NO: 1497-1499) , optionally wherein X1 is a basic amino acid or an amino acid comprising a side chain amide or hydroxyl (e.g., H, N, R, S), X2 is a basic amino acid (e.g., R, H), and X3 is an aliphatic or acidic amino acid or an amino acid comprising a side chain hydroxyl (e.g., A, D, V, L, S), (C) a LC CDR3 comprising AX1WDX2X3LX4X5VX6 (any one of SEQ ID NOs: 1500-1502) optionally wherein X1 is an aliphatic amino acid or an amino acid comprising a side chain hydroxyl (e.g., A, S), X2 is an acidic or aliphatic amino acid (e.g., D, V), X3 is an aliphatic amino acid or an amino acid comprising a side chain hydroxyl (e.g., I, S), X4 is an acidic, basic, or aliphatic amino acid or an amino acid comprising a side chain amide (e.g., E, G, K, R, N), X5 is an aliphatic amino acid or amino acid comprising a side chain hydroxyl (e.g., G, S), and X6 is an aliphatic amino acid (e.g., A, V). In exemplary embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1485, 1488, and 1491 , respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 1494, 1497, 1500, respectively. In exemplary embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and aHCDR3 of SEQ ID NOs: 1487, 1490, and 1493, respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 1496, 1499, 1502, respectively, as defined above. Exemplary FLT1 binding proteins, in particular anti-FLT1 antibodies of the present disclosure, comprising the shared CDR sequence structure set forth in these consensus heavy chain and light chain CDRs include antibody 18300 and the binding proteins of Table 10 (derived from the 10B4 antibody), which share at least 90% sequence identity across the entirety of their VH and VL domains.

[0157] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment thereof comprising a heavy chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and (ii) comprising a HC CDR1 of any one of SEQ ID NOs: 1485-1487, a HC CDR2 of any one of SEQ ID NOs: 1488-1490 and a HC CDR3 of any one of SEQ ID NOs: 1491-1493. This FLT1 antibody or antigen binding fragment further comprises a light chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1090, and (ii) comprising a LC CDR1 of any one of SEQ ID NOs: 1494-1496, a LC CDR2 of any of SEQ ID NOs: 1497-1499, and a LC CDR3 of SEQ ID NO: 1500- 1502.

[0158] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment thereof comprising a heavy chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and (ii) comprising the HC CDR1 of SEQ ID NO: 1487, the HC CDR2 of SEQ ID NO: 1490 and the HC CDR3 of SEQ ID NO: 1493. This FLT1 antibody or antigen binding fragment thereof further comprises a light chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1090, and (ii) comprising the LC CDR1 of SEQ ID NO: 1496, the LC CDR2 of SEQ ID NO: 1499, and the LC CDR3 of SEQ ID NO: 1502.

[0159] In exemplary embodiments, (1) the HC variable region of the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises (A) a HC CDR1 comprising SYVX1 H (any one of SEQ ID NO: 1512-1514) optionally wherein X1 is an aliphatic or basic amino acid (e.g., M, L), (B) a HC CDR2 comprising VX1WX2X3GSNX4YYX5DX6VKG (any one of SEQ ID NOs: 1515-1517), optionally wherein X1 is an aliphatic, acidic or basic amino acid, an amino acid comprising a side chain amide or hydroxyl, or an imino acid (e.g., I, A, Q, V, E, K, L, P, T), X2 is an acidic, aromatic, or basic amino acid, or an amino acid comprising side chain amide (e.g., E, N, Y, D, K), X3 is an acidic amino acid (e.g., D, E), X4 is a basic or aliphatic amino acid, animino acid, or an amino acid comprising a side chain hydroxyl or amide (e.g., I, K, P, T, L, Q), X5 is an aliphatic, acidic, or basic amino acid, an amino acid comprising a side chain amide or hydroxyl, or an imino acid (e.g., A, G, H, S, D, N, P, R, T), X6 is an aliphatic or basic amino acid, or amino acid comprising a side chain hydroxyl (e.g., A, R, K, S), and (C) a HC CDR3 comprising STYYX1X2SGSYYYYYX3MDV (any one of SEQ ID NOs: 1518-1520), optionally wherein X1 is an aromatic, aliphatic, acidic, or basic amino acid or an amino acid comprising a side chain hydroxyl (e.g., Y, A, E, H, I, L, R, S, T, V), X2 is an aliphatic or basic amino acid, an imino acid, or an amino acid comprising a side chain amide (e.g., G, H, L, P, Q), and X3 is an aliphatic amino acid (e.g., G, V), and / or (2) the LC variable region comprises (A) a LC CDR1 comprising RSSX1X2X3X4HSX5GX6X7FLD (any one of SEQ ID NOs: 1521-1523), optionally wherein X1 is an aliphatic, basic, or acidic amino acid or an amino acid comprising a side chain amide (e.g., G, Q, R, E), X2 is an amino acid comprising a side chain hydroxyl (e.g., S, T), X3 is an aliphatic amino acid or imino acid (e.g., L, P), X4 is an aliphatic, acidic or basic amino acid (e.g., L, E, R, G, V), X5 is a basic amino acid or an amino acid comprising a side chain amide (e.g., H, N), X6 is an amino acid comprising a side chain amide or a basic amino acid (e.g., N, K), and X7 is a basic, acidic or aliphatic amino acid, imino acid, or an amino acid comprising a side chain amide (e.g., H, N, P, V, D, I, L), (B) a LC CDR2 comprising LGX1 NRAX2 (any one of SEQ ID NOs: 1524-1526) optionally wherein X1 is an aliphatic amino acid or an amino acid comprising a side chain hydroxyl (e.g., A, V, S, L), X2 is an aliphatic amino acid or amino acid comprising a side chain hydroxyl (e.g., T, A, S), and (C) a LC CDR3 comprising MQALX1X2X3RT (any one of SEQ ID NOs: 1527-1529), optionally wherein X1 is an aliphatic or aromatic amino acid or an amino acid comprising a side chain hydroxyl or amide (e.g., G, S, T, Q, Y, A), X2 is an aromatic amino acid or an amino acid comprising a side chain amide or hydroxyl (e.g., T, Y, N, S), and X3 is an aliphatic or basic amino acid, imino acid, or an amino acid comprising a side chain hydroxyl (e.g., I, P, R, V, A, G, L, T). In exemplary embodiments, the FLT1 binding protein comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1512, 1515, and 1518, respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 1521 , 1524, and 1527, respectively. In exemplary embodiments, the FLT1 binding protein comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1514, 1517, and 1520, respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs: 1523, 1526, 1529, respectively. Exemplary FLT1 binding proteins, in particular anti-FLT1 antibodies of the present disclosure, comprising these heavy chain and light chain CDRs are disclosed herein in Table 12.

[0160] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment comprising a heavy chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1335, and (ii) comprises a HC CDR1 of any one of SEQ ID NOs: 1512-1514, a HC CDR2 of any one of SEQ ID NOs: 1515-1517, and a HC CDR3 of any one of SEQ ID NOs: 1518-1520. This FLT1 antibody or antigen binding fragment further comprises a light chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1336, and (ii) comprises a LC CDR1 of any one of SEQ ID NOs: 1521- 1523, a LC CDR2 of any one of SEQ ID NOs: 1524-1526, and a LC CDR3 of any one of SEQ ID NOs: 1527-1529.

[0161] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment comprising a heavy chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1335, and (ii) comprising the HC CDR1 of SEQ ID NO: 1514, the HC CDR2 of SEQ ID NO: 1517, and the HC CDR3 of SEQ ID NO: 1520. This FLT1 antibody or antigen binding protein further comprises a light chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1336, and (ii) comprisingthe LC CDR1 of SEQ ID NO: 1523, the LC CDR2 of SEQ ID NO: 1526, and the LC CDR3 of SEQ ID NO: 1529.

[0162] In exemplary embodiments, (1) the HC variable region of the FLT1 binding protein, of the present disclosure, in particular an anti-FLT1 antibody of the disclosure, comprises (A) a HC CDR1 comprising X1YAX2H (any one of SEQ ID NO: 1503-1505) optionally, wherein X1 is an acidic, aliphatic, or basic amino acid or an amino acid comprising a side chain hydroxyl or amide (e.g., D, E, G, K, N, R, S) and X2 is an aliphatic amino acid or an amino acid comprising a side chain sulfur (e.g., I, M), (B) HC CDR2 is IISX1X2GSTX3X4YAX5X6VKG (any one of SEQ ID NOs: 1506-1508) optionally wherein X1 is an aromatic amino acid (e.g., F, Y); X2 is an aromatic, basic, or acidic amino acid (e.g., F, H, L, Y, E); X3 is an aliphatic, acidic, or basic amino acid or an amino acid comprising a side chain amide or hydroxyl (e.g., A, D, E, G, K, R, N, R, S, T); X4 is an aromatic or aliphatic amino acid or an amino acid comprising a side chain amide (e.g., F, I, N, Y); X5 is an acidic amino acid (e.g., D, E); and X6 is an aliphatic amino acid or an amino acid comprising a side chain hydroxyl (e.g., A, S); (C) HC CDR3 comprising GLELQDY (SEQ ID NO: 573), and / or (2) a LC variable region comprising (A) a LC CDR1 comprising TGTSSDVGGYNYVS (SEQ ID NO: 592), (B) a LC CDR2 comprising EVSNRPS (SEQ ID NO: 593), and (C) a LC CDR3 comprising SX1YTX2X3X4X5X6X7 (any one of SEQ ID NOs: 1509-1511) optionally, wherein X1 isan aliphatic amino acid or an amino acid comprising a side chain hydroxyl (e.g., G, A, S, T); X2 is an aliphatic, acidic, or basic amino acid, or an amino acid comprising a side chain amide or hydroxyl (e.g., A, D, E, G, K, N, R, S, T); X3 is a basic amino acid or an amino acid comprising a side chain amide or hydroxyl (e.g., H, K, N, Q, R, S); X4 is an aliphatic amino acid or an amino acid comprising a side chain hydroxyl (e.g., A, S, T); X5 is an aliphatic amino acid or an amino acid comprising a side chain hydroxyl (e.g., T, A, G, I, S, V); X6 is an aromatic amino acid (e.g., W, F, Y); X7 is an aliphatic aa or acidic amino acid or an amino acid comprising a side chain amide or hydroxyl (e.g., V, A, I, D, N, T). In exemplary embodiments, the FLT1 binding protein, in particular an anti-FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1503, 1506, and 573, respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 592, 593, and 1509, respectively. In exemplary embodiments, the FLT1 binding protein, in particular an anti-FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1505, 1508, and 573, respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 592, 593, and 1511 , respectively. Exemplary FLT1 binding proteins, in particular anti- FLT1 antibodies of the present disclosure, comprising these heavy chain and light chain CDRs are disclosed herein in Table 11 .

[0163] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment comprising a heavy chain variable region (i) having 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1283, and (ii) comprising a HC CDR1 of any one of SEQ ID NOs: 1503-1505, a HC CDR2 of any one of SEQ ID NOs: 1506-1508, and a HC CDR3 of SEQ ID NO: 573. This FLT1 antibody or antigen binding fragment further comprises a light chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1284, and (ii) comprising a LC CDR1 of SEQ ID NO: 592, a LC CDR2 of SEQ ID NO: 593, and a LC CDR3 of SEQ ID NO: 1509-1511.

[0164] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment comprising a heavy chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1283, and (ii) comprising the HC CDR1 of SEQ ID NO: 1505, the HC CDR2 of SEQ ID NO: 1508, and the HC CDR3 of SEQ ID NO: 573. This FLT1 antibody or antigen binding fragment further comprises an antibody light chain variable region (ii) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1284, and (ii)comprising the LC CDR1 of SEQ ID NO: 592, the LC CDR2 of SEQ ID NO:593, and the LC CDR3 of SEQ ID NO: 1511.

[0165] In various embodiments, (1) the HC variable region of the FLT1 binding protein (in particular, an anti-FLT1 antibody or antigen binding fragment) comprises (A) a HC CDR1 comprisingX1X2GYYWS (any one of SEQ ID NO : 1882, 1886, and 1890), optionally wherein each of X1 and X2 is an aliphatic amino acid or an amino acid with a side chain hydroxyl (e.g., G, S), (B) a HC CDR2 comprising YIYYSGSAYYNPSLKS (SEQ ID NO: 8) and (C) a HC CDR3 comprisingX1X2LEX3LLGGMDV (any one of SEQ ID NOs: 1883, 1887, and 1891 ), optionally wherein X1 is an aliphatic or an amino acid comprising a side chain hydroxyl (e.g., G, S), X2 is an aliphatic amino acid (e.g., V, A), X3 is an aromatic amino acid (e.g., W, Y) and / or (2) the LC variable region of the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises (A) a LC CDR1 comprising SGSSX1 NIGX2NTVN (any one of SEQ ID NOs: 1884, 1888, 1892), optionally, wherein each of X1 and X2 is independently an aliphatic amino acid or an amino acid with a side chain hydroxyl (e.g., S,G, I), (B) a LC CDR2 comprising SNNHRPS (SEQ ID NO: 11 ); and (C) a LC CDR3 comprising AAWDDSLXGW (any one of SEQ ID NOs: 1885, 1889, 1893) optionally wherein X is an aliphatic amino acid or an amino acid comprising a side chain amide (e.g., N,G). In exemplary embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1182, 8, and 1883, respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs: 1884, 11 , and 1885, respectively. In exemplary embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1886, 8, and 1887, respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 1888, 11 , and 1889, respectively, as defined above. In exemplary embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1890, 8, and 1891 , respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 1892, 11 , and 1893, respectively, as defined above. Exemplary FLT1 binding proteins, in particular anti-FLT1 antibodies of the present disclosure, comprising the shared CDR sequence structure set forth in these consensus heavy chain and light chain CDRs include the binding proteins of Tables 43A and 43B (derived from the 18300 antibody), which share at least 90% sequence identity across the entirety of their VH and VL domains.

[0166] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment thereof comprising a heavy chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and (ii) comprising a HC CDR1 of any one of SEQ ID NOs: 1882, 1886, and 1890, a HC CDR2 of SEQ ID NO: 8 and a HC CDR3 of any one of SEQ ID NOs: 1883, 1887, and 1891 . This FLT1 antibody or antigen binding fragment further comprises a light chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1090, and (ii) comprising a LC CDR1 of any one of SEQ ID NOs: 1884, 1888, and 1892, a LC CDR2 of SEQ ID NO: 11 , and a LC CDR3 of SEQ ID NO: 1885, 1889, and 1893.

[0167] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment thereof comprising a heavy chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and (ii) comprising the HC CDR1 of SEQ ID NO: 1890, the HC CDR2 of SEQ ID NO: 8 and the HC CDR3 of SEQ ID NO: 1891. This FLT1 antibody or antigen binding fragment thereof further comprises a light chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1090, and (ii) comprising the LC CDR1 of SEQ ID NO: 1892, the LC CDR2 of SEQ ID NO: 11 , and the LC CDR3 of SEQ ID NO: 1893.

[0168] In various embodiments, (1) the HC variable region of the FLT1 binding protein (in particular, an anti-FLT1 antibody or antigen binding fragment) comprises (A) a HC CDR1 comprising SYVMH (SEQ ID NO : 733), (B) a HC CDR2 comprising VIWYDGSNX1 YYX2DAVKG (any one of SEQ ID NO: 1894, 1897, 1900) optionally wherein X1 is an aliphatic amino acid or a basic amino acid (e.g., K, I), X2 is an aliphatic amino acid (e.g., G, A), and (C) a HC CDR3 comprising STYYX1X2SGSYYYYYGMDV (any one of SEQ ID NOs: 1895, 1898, 1901), optionally wherein X1 is a basic amino acid or aromatic amino acid (e.g., R, Y), X2 is an aliphatic amino acid or an amino acid with a side chain imine (e.g., p, G), and / or (2) the LC variable region of the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises (A) a LC CDR1 comprising RSSQSLLHSNGNNFLD (SEQ ID NOs: 736), (B) a LC CDR2 comprising LGSNRAS (SEQ ID NO: 737); and (C) a LC CDR3 comprising MQALX1X2PRT (any one of SEQ ID NOs: 1896, 1899, 1902) optionally wherein X1 is an amino acid with a side chain hydroxyl or a side chain amide (e.g., Q, T), X2 is an aromatic amino acid or amino acid with a side chain hydroxyl (e.g., T, Y). In exemplary embodiments, the FLT1 binding protein, in particular, the anti- FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 ,a HCDR2, and a HCDR3 of SEQ ID NOs: 733, 1894, and 1895, respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs: 736, 737, and 1896, respectively. In exemplary embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 733, 1897, and 1898, respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 736, 737, and 1899, respectively, as defined above. In exemplary embodiments, the FLT1 binding protein, in particular, the anti-FLT1 antibody of the disclosure, comprises a heavy chain variable region comprising a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 733, 1900, and 1901 , respectively, and light chain variable region comprising a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 736, 737, and 1902, respectively, as defined above. Exemplary FLT1 binding proteins, in particular anti-FLT1 antibodies of the present disclosure, comprisingthe shared CDR sequence structure set forth in these consensus heavy chain and light chain CDRs include the binding proteins of Tables 43A and 43B (derived from the 15331 antibody), which share at least 90% sequence identity across the entirety of their VH and VL domains.

[0169] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment thereof comprising a heavy chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1335, and (ii) comprising a HC CDR1 of SEQ ID NO: 733, a HC CDR2 of any one of SEQ ID NOs: 1894, 1897, and 1900 and a HC CDR3 of any one of SEQ ID NOs: 1895, 1898, and 1901 . This FLT1 antibody or antigen binding fragment further comprises a light chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1336, and (ii) comprising a LC CDR1 of SEQ ID NO: 736, a LC CDR2 of SEQ ID NO: 737, and a LC CDR3 of any one of SEQ ID NOs: 1896, 1899, and 1902.

[0170] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment thereof comprising a heavy chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1335, and (ii) comprisingthe HC CDR1 of SEQ ID NO: 733, the HC CDR2 of SEQ ID NO: 1900 and the HC CDR3 of SEQ ID NO: 1901. This FLT1 antibody or antigen binding fragment thereof further comprises a light chain variable region (i) having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1336, and (ii) comprisingthe LC CDR1 of SEQ ID NO: 736, the LC CDR2 of SEQ ID NO: 737, and the LC CDR3 of SEQ ID NO: 1902.

[0171] Epitopes and Paratopes

[0172] In exemplary embodiments, the binding interaction of the presently disclosed FLT1 binding protein and FLT1 is mediated through intermolecular interactions between amino acids of FLT1 and amino acids of the FLT1 binding protein. Generally, the amino acids of FLT1 that have intermolecular interactions with the amino acids of the FLT1 binding protein form the "epitope" and the amino acids of the FLT1 binding protein that have intermolecular interactions with the amino acids of FLT1 form the “paratope”. In various instances, the intermolecular interactions between the amino acids of the epitope and the amino acids of the paratope are one or more of hydrogen bonds, van der Waals interactions, electrostatic bonds, ionic bonds, hydrophobic interactions, or hydrophilic interactions. The epitope may be a “linear epitope” in which the epitope is composed of amino acids that are contiguous within the FLT1 amino acid sequence, orthe epitope may be a “conformational epitope” in which the epitope is composed of amino acids that are non-contiguous within the FLT1 amino acid sequence. Unless expressly stated otherwise herein, epitopes will be understood to referto epitopes on native FLT1 .

[0173] In various embodiments, the epitope of FLT1 to which an exemplary FLT1 binding protein of the present disclosure, in particular, an exemplary anti-FLT1 antibody of the disclosure binds comprises amino acids within the extracellular domain D2 of human FLT1 . In various embodiments, the epitope comprises a combination of at least 5 of the amino acids E141 , 1142, P143, K171, F172, P173, L174, L204, L215, K217, and L221 of human FLT1 of SEQ ID NO: 1453. In one embodiment, the epitope comprises or consists of amino acid residues correspondingto residues El 41 , P143, F172, P173, and L204 of SEQ ID NO: 1453, which have been identified as the first tier amino acid residues involved to the binding interaction using cryoEM as described in Example 11 herein. In one embodiment, the epitope comprises or consists of amino acid residues correspondingto residues E141 , P143, F172, P173, L204, 1142, and K217 of SEQ ID NO: 1453, which include the first tier amino acid residues of the binding interaction as well as second tier amino acid residues that contribute to the binding interaction (as identified using cryoEM as described in Example 11 ). In one embodiment, the epitope comprises or consists of the amino acids correspondingto the amino acid residues of F172, P173, L174, L215, K217, and L221 of human FLT1 of SEQ ID NO: 1453, which have been identified as unique epitope residues bound by the FLT1 binding proteins disclosed herein. An exemplary FLT1 binding protein of the present disclosure that binds the aforementioned epitope residues is the anti-FLT1 antibody 18300.

[0174] In various embodiments, the paratope resides of an exemplary FLT1 binding protein of the present disclosure that binds to the FLT1 epitope described in the preceding paragraphcomprises amino acids residues that form hydrogen bonds, van der Waals interactions, and / or hydrophobic interactions with the epitope residues. In various embodiments, the FLT1 binding protein is an antibody, or an antigen binding fragment thereof, wherein the paratope comprises amino acid residues of the VH and amino acid residues of the VL. In various embodiments, and as shown in Example 11 herein, the paratope comprises particular amino acid residues of the HC CDR1 , HC CDR2, HC CDR3 and particular amino acids of at least two of the LC CDR1 , LC CDR2, and LC CDR3 of the FLT1 binding protein. In exemplary embodiments, the FLT1 antibody or antigen binding fragment thereof comprises (A) a VH having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 1089, and comprising the following amino acids: a Tyr, Phe, or Met at position 35 (HCDR1 ), a Tyr, Phe, orTrp at position 52 (HCDR2), a Tyr at position 54 (HCDR2), a Tyr, Gin, Arg, Lys, Met, Phe, orTrp at position 60 (HCDR2), a Leu or lie at position 102 (HCDR3), an Ala, Asp, Glu, Phe, Gly, His, Lys, Asn, Gin, Arg, Ser, Thr, Vai, Trp, Tyr at position 103 (HCDR3), a Leu, Phe, Trp, or Try at position 105 (HCDR3), and a Leu or Met at position 106 (HCDR3), and (B) a VL having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 1090, and comprising the following amino acids: an lie, Ala, Lys, Leu, Met, Asn, Gin, Arg, Thr, or Vai at position 31 (LCDR1 ), a Ser, Ala, Gly, lie, Asn, orThr at position 51 (LCDR2), an Asn, Ala, Asp, Lys, Gin, Arg, Ser, Thr at position 53 (LCDR2), a His, Met, Gin, Arg, orTrp at position 54 (LCDR2), a Trp, Phe, Arg, or Tyr at position 92 (LCDR3), an Asp, Glu, lie, or Gin at position 94 (LCDR3), and a Tyr, Phe, His, lie, Lys, Arg, Trp at position 50 (FR2).

[0175] In exemplary embodiments, the FLT1 binding protein is an anti-FLT1 antibody or antigen binding fragment thereof that comprises (A) a VH having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 1089 and comprising the following amino acids: the Tyr at position 35, the Tyr at position 52, the Tyr at position 54, the Tyr at position 60, the Leu at position 102, the Leu at position 105, and the Leu at position 106 of SEQ ID NO: 1089 and (B) a VL having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 1090 and comprising the following amino acids: the Tyr at position 50, the Ser at position 51 , the Asn at position 53, the His at position 54, the lie at position 31 , and the Trp at position 92, and the Asp at position 94 of SEQ ID NO: 1090.

[0176] Table F below provides details regarding the FLT1 -anti-FLT1 antibody (18300) residues involved in the binding interface and Example 11 ranks the contributions of these interactions to the binding interaction.Table F: Summary of sFltl epitope residues interacting with Fab 18300 paratope residues

[0177] In another embodiment, the epitope of FLT1 to which an exemplary FLT1 binding protein of the present disclosure binds comprises amino acids within the extracellular domain D2 of human FLT1 . In various embodiments, the epitope comprises a combination of at least 5 of the amino acids M138, Y139, S140, E141 , 1142, P143, F172, K217, N100 (via linked glycan), L204, Y216, N219, K69, R133, R134, and E144 of human FLT1 of SEQ ID NO: 1453. In various embodiments, the epitope comprises amino acid residues correspondin to residues M138, Y139, S140, E141 , 1142, P143, F172, K217 of SEQ ID NO: 1453, which have been identified as first tier amino acid residues contributing to the binding interaction using cryoEM as described in Example 11 herein. In various embodiments, the epitope comprises amino acid residues corresponding to residues M138, Y139, S140, E141 , 1142, P143, F172, K217, N100 (via linked glycans), L204, Y216, and N219 of SEQ ID NO: 1453, which include the first tier amino acid residues of the binding interaction as well as second tier amino acid residues that contribute tothe binding interaction (as also identified using cryoEM as described in Example 11 herein). In various embodiments, the epitope comprises the amino acids corresponding to the amino acid residues of M138, Y139, S140, F172, Y216, and K217 of human FLT1 of SEQ ID NO: 1453, which have been identified as unique epitope residues bound by the FLT1 binding proteins disclosed herein. An exemplary FLT1 binding protein of the present disclosure that binds the aforementioned epitope residues is the anti-FLT1 antibody 15331 .

[0178] In various embodiments, the paratope resides of an exemplary FLT1 binding protein of the present disclosure that binds to the FLT1 epitope described in the preceding paragraph comprises amino acids residues that form hydrogen bonds, van der Waals interactions, and / or hydrophobic interactions with the epitope residues. In various embodiments, the FLT1 binding protein is an antibody, or an antigen binding fragment thereof, wherein the paratope comprises amino acid residues of the VH and amino acid residues of the VL. In various embodiments, and as shown in Example 11 herein, the paratope comprises particularamino acid residues of the HC CDR1 , HC CDR2, HC CDR3 and particular amino acids of the LC CDR1 and LC CDR3 of the FLT1 binding protein. In exemplary embodiments, the FLT1 binding protein is an anti-FLT1 antibody or antigen binding fragment there of that comprises (A) a VH having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 1335 and comprising the following amino acids: a His or Asn at position 35 (HCDR1 ), a Trp, Phe, orTrp at position 52 (HCDR2), an Asn or Thr at position 57 (HCDR2), a Tyr or Phe at position 59 (HCDR2), an Asp, Glu, or Asn at position 62 (HCDR2), a Tyr, Phe, His, Met, Leu, or Trp at position 102 (HCDR3), a Pro, Gly, Asp, Glu, Asn, Gin, or Vai at position 104 (HCDR3), a Tyr, Phe, or Trp at position 108 (HCDR3), and a Tyr at position 109 (HCDR3); and (B) a VL having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 1336 and comprising the following amino acids: a Gin, Asn, Asp, Glu, Arg, or His at position 27 (LCDR1 ), a His, Arg, Lys, Phe, Trp, orTyr at position 31 (LCDR1 ), a Phe, Trp, Vai, or Leu at position 37 (LCDR1 ), a Tyr, Gin, His, Met, or Trp at position 99 (LCDR3), an Arg, Lys, or Met at position 101 (LCDR3), and an Asp, Asn, Glu, Phe, or Trp at position 1 . Optionally, the VH of this FLT1 binding protein further comprises a Vai, Ala, lie, or Ser at position 33 (HCDR1 ), an lie, Ala, Cys, Gly, Lys, Met, Asn, Gin, Arg, Ser, Thr, or Vai at position 58 (HCDR2), a Ser, Asp, Asn, or Thr at position 99, and a Thr, Ala, or Ser at position 100 (HCDR3). Optionally, the VL of this FLT1 binding protein further comprises a Asn, Ala, Cys, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Gin, Arg, Ser, Thr, Vai, Trp, Tyr at position 33 (LCDR1), a Leu, Ala, Glu, Phe, His, lie, Lys, Met, Thr, Vai, Trp, or Tyr at position 97 (LCDR3), a Thr, Ala, Cys, Gly, Vai, Ser, Asn, Asp, lie, or Leu at position 98 (LCDR3), and a Pro, Ala, Arg, Gin, His, Lys, Met, Trp, or Tyr at position 100 (LCDR3).

[0179] In exemplary embodiments, the FLT1 binding protein is an antibody or antigen binding fragment thereof that comprises (A) a VH having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 1335 and comprising the following amino acids: the His at position 35, the Trp at position 52, the Asn at position 57, the Tyr at position 59, the Asp at position 62, the Tyr at position 102, the Pro at position 104, the Tyr at position 108, and the Tyr at position 109 of SEQ ID NO: 1335; and (B) a VL having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 1336 and comprising the following amino acids: the Gin at position 27, the His at position 31 , the Phe at position 37, the Tyr at position 99, the Arg at position 101 , and the Asp at position 1 of SEQ ID NO: 1336. Optionally, the VH of this FLT1 binding protein further comprises the Vai at position 33 (HCDR1), the lie at position 58 (HCDR2), the Ser at position 99, and the Th rat position 100 (HCDR3). Optionally, the VL of this FLT1 binding protein further comprises the Asn at position 33 (LCDR1), the Leu at position 97 (LCDR3), the Thr at position 98 (LCDR3), and the Pro at position 100 (LCDR3).

[0180] Table G below provides details regarding the FLT1-anti-FLT1 antibody (15331) residues involved in the binding interface and Example 11 ranks the contributions of these interactions to the binding interaction.Table G. Summary of sFltl epitope residues interacting with Fab 15331 paratope residues

[0181] Nucleic Acids

[0182] The present disclosure further provides nucleic acids comprising a nucleotide sequence encoding a FLT1 binding protein of the present disclosure, in particular anti-FLT1 antibodies of the present disclosure,. By "nucleic acid" as used herein includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, or modified forms thereof, which can be single-stranded or doublestranded, synthesized or obtained (e.g., isolated and / or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, nonnatural or altered inter-nucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. The nucleic acid can comprise any nucleotide sequence which encodes any of the antigen-binding proteins or polypeptides of the present disclosure. In some embodiments, the nucleic acid does not comprise any insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid comprises one or more insertions, deletions, inversions, and / or substitutions.

[0183] In some embodiments, the nucleic acids of the present disclosure are recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those describedin (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.

[0184] The nucleic acids in some embodiments are constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra; and Ausubel et al., supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridme, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N -substituted adenine, 7-methylguanine, 5-methylammomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5- oxyacetic acid (v), wybutoxosine, pseudouratil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3- (3- amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present disclosure can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0185] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an FLT1 binding protein of the present disclosure. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence(s) having a SEQ ID NO: listed in Tables A-E. In various embodiments, the nucleotide sequence encodes an amino acid sequence comprising the six CDRs of an antibody as defined in Table A.1 with intervening amino acids. In various embodiments, the nucleotide sequence encodes only one of the HC variable or LC variable amino acid sequences presented in a single row of Table B. In various embodiments, the nucleotide sequence encodes both the HC variable and LC variable amino acid sequences presented in a single row of Table B fused as one long nucleotide sequence, optionally, wherein the HC variable and LC variable amino acid sequences are linked together by a linker sequence. In various embodiments, the nucleotide sequence encodes a polypeptide described herein. In various embodiments, the nucleotide sequence comprises a nucleotide sequence encodingthe HC and / or LC constant regions of Table C. Invarious embodiments, the nucleotide sequence encodes an amino acid sequence of Table D or E. In various embodiments, the nucleotide sequence encodes an amino acid sequence which differs by only 1 , 2, 3, 4, 5, 6, or more amino acids, relative to an amino acid sequence presented in any one of Tables A-E. In exemplary embodiments, the nucleotide sequence encodes a variant sequence which differs by only one or two amino acids, relative to an amino acid sequence presented in any one of Tables A-E. In exemplary embodiments, the nucleotide sequence encodes a variant sequence having only 1 , 2, 3, 4, 5, 6, or more conservative amino acid substitutions, relative to an amino acid sequence presented in any one of Tables A-E. In exemplary embodiments, the nucleic acid comprises a nucleotide sequence of any one of SEQ ID NOs: 1548-1567, as shown in Table H. In various embodiments, the nucleic acid of the present disclosure comprises a nucleotide sequence (e.g., any one of SEQ ID NOs: 1548-1571) comprising a nucleotide sequence encoding a signal sequence (e.g., the signal sequence of SEQ ID NO: 1596 or 1597). In various embodiments, the nucleic acid of the present disclosure comprises a nucleotide sequence (e.g., SEQ ID NOs: 1548-1571 ) without a nucleotide sequence encoding the signal sequence. In various embodiments, the nucleic acid comprises a nucleotide sequence of any one of SEQ ID NOs: 1568-1571 without the first 60, 63, 66, or 69 nucleic acids of any one of SEQ ID NOs: 1568-1571 or without the signal sequence (e.g., the signal sequence of SEQ ID NO: 1596 or 1597).TABLE H

[0186] Vectors

[0187] The nucleic acids of the present disclosure in some embodiments are incorporated into a vector. In this regard, the present disclosure provides vectors comprising any of the presently disclosed nucleic acids. In exemplary embodiments, the vector is a recombinant expression vector. For purposes herein, the term "recombinant expression vector" means agenetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the present disclosure are not naturally- occurring as a whole. However, parts of the vectors can be naturally-occurring. The presently disclosed vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single- stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The vectors can comprise naturally-occurring or non-naturally-occurring internucleotide linkages, or both types of linkages. In some embodiments, the altered nucleotides or non-natu rally occurring internucleotide linkages do not hinder the transcription or replication of the vector.

[0188] The vector of the present disclosure can be any suitable vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, Wl), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as AGTIO, AGTl 1 , AZapll (Stratagene), AEMBL4, and ANMl 149, also can be used. Examples of plant expression vectors include pBIOl, pB1101 .2, pB1101 .3, pBII 21 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). In some embodiments, the vector is a viral vector, e.g., a retroviral vector.

[0189] The vectors of the present disclosure can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 p plasmid, A, SV40, bovine papilloma virus, and the like.

[0190] In some embodiments, the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.

[0191] The vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the presently disclosed expression vectors include, for instance, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0192] The vector can comprise a native or normative promoter operably linked to the nucleotide sequence encoding the polypeptide (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the antigen binding protein. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental- specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.

[0193] In some embodiments, the vector encodes an antibody light chain, an antibody heavy chain, or an antibody light chain and heavy chain. Avector encoding an antibody light chain may be useful for producing an antibody of the present invention when expressed in a cell that further contains a vector encoding an antibody heavy chain. Likewise, a vector encoding an antibody heavy chain may be useful for producing an antibody of the present invention when expressed in a cell that further contains a vector encoding an antibody light chain. Thus, whereas both the light chain and the heavy chain may be encoded on a single vector, in certain embodiments the vector encodes the light chain but does not encode the heavy chain. In other embodiment, the vector encodes the heavy chain but does not encode the light chain.

[0194] In various embodiments, the vector of the present comprises a nucleotide sequence encoding HC variable region or a full-length HC and a nucleotide sequence encoding LC variable region or a full-length LC. In alternative embodiments, the vector of the present disclosure comprises a nucleotide sequence encoding HC variable region or a full-length HC.

[0195] Host cells

[0196] Provided herein are host cells comprising one or more nucleic acids or vectors of the present disclosure. As used herein, the term "host cell" refers to any type of cell that can contain the presently disclosed vector or vectors and is capable of producing an expression product encoded by the nucleic acid(s) (e.g., mRNA, protein). The host cell in some embodimentsis an adherent cell or a suspended cell, i.e. , a cell that grows in suspension. The host cell in exemplary embodiments is a cultured cell or a primary cell, i.e., isolated directlyfrom an organism. The host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage.

[0197] In exemplary embodiments, the cell is a eukaryotic cell, including, but not limited to, a yeast cell, filamentous fungi cell, protozoa cell, algae cell, insect cell, or mammalian cell. Such host cells are described in the art. See, e.g., Frenzel, et al., Front Immunol 4: 217 (2013). In exemplary embodiments, the eukaryotic cells are mammalian cells. In exemplary embodiments, the mammalian cells are non-human mammalian cells. In some embodiments, the cells are Chinese Hamster Ovary (CHO) cells and derivatives thereof (e.g., CHO-K1 , CHO pro-3, CS9), mouse myeloma cells (e.g., NSO, GS-NSO, Sp2 / 0), cells engineered to be deficient in di hydro folate reductase (DHFR) activity (e.g., DUKX-X11 , DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinomic human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryo fibroblast cells NIH 3T3, mouse fibroblast cells L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2): 257-263 (2013)). In a particular embodiment, the host cell is CS9 (a CHO cell line).

[0198] For purposes of amplifying or replicating the vector, the host cell is in some embodiments a prokaryotic cell, e.g., a bacterial cell.

[0199] Also provided by the present disclosure is a population of cells comprising at least one host cell described herein. The population of cells in some embodiments is a heterogeneous population comprising the host cell comprising vectors described, in addition to at least one other cell, which does not comprise any of the vectors. Alternatively, in some embodiments, the population of cells is a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the vector. The population in some embodiments is a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a vector, such that all cells of the population comprise the vector. In exemplary embodiments of the present disclosure, the population of cells is a clonal population comprising host cells comprising a vector as described herein.

[0200] In various embodiments of the present disclosure, the host cells comprise a first vector comprising a nucleotide sequence encoding HC variable region or a full-length HC asdescribed in Table B and a second vector comprising a nucleotide sequence encoding LC variable region or a full-length LC as described in Table B.

[0201] Pharmaceutical Compositions

[0202] Pharmaceutical compositions comprising a FLT1 binding protein, an anti-FLT1 antibody, a nucleic acid, a vector, a host cell, of the present disclosure, or a combination thereof, are provided herein. The pharmaceutical compositions in some embodiments comprise the FLT1 binding protein, anti-FLT1 antibody, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof, in isolated and / or purified form. In some embodiments, the composition comprises a single type (e.g., structure) of FLT1 binding protein, anti-FLT1 antibody, nucleic acid, vector, or host cell of the present disclosure, or comprises a combination of two or more different types (e.g., different structures) of FLT1 binding proteins, anti-FLT1 antibodies, nucleic acids, vectors or host cells of the present disclosure.

[0203] In some embodiments, the pharmaceutical composition of the present disclosure comprises a means for inhibiting FLT1 binding to VEGF, and a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition of the present disclosure comprises a means for increasing plasma VEGF levels in a subject and a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition of the present disclosure comprises a means for increasing VEGFR2 phosphorylation in a subject, and a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment, the means for inhibiting FLT1 binding to VEGF, the means for increasing plasma VEGF levels in a subject, and the means for increasing VEGFR2 phosphorylation in a subject comprise an anti-FLT1 antibody having the structure of the anti- FLT1 antibody 18300 or an equivalent structure thereof as depicted in Figures 16A and 16B and described in Example 11 herein. As depicted in Figure 16E, this antibody binds to the same domain of FLT1 (i.e., D2) as VEGF, the natural ligand of FLT1 , with a 71 % overlap in the residues of FLT1 bound by the antibody and bound by VEGF. This data confirms that an anti-FLT1 antibody having the 18300 antibody structure, or an equivalent structure thereof, blocks VEGF bindingto FLT1. Blocking VEGF bindingto FLT1 using an antibody having the structure of antibody 18300 increases the amount of plasma VEGF in a subject available to bind to VEGFR2 resulting in an increase in VEGFR2 phosphorylation. In one embodiment, the means for blocking VEGF bindingto FLT1 comprises an antibody having the paratope structure of antibody 18300, or an equivalent structure thereof, as provided in Figure 16B. As shown in Figure 16B, heavy chain residues Y35, Y52, Y54, L102, L105, L106, and Y60 (SEQ ID NO: 1089), and light chain residues S51 , N53, H54, D94, Y50 and W92 (SEQ ID NO: 1090) are the paratope interfaceresidues directly involved in contacting D2 of FLT1 to block VEGF binding to FLT1 . Additional heavy chain residues contributing to the binding interaction with FLT1 include E103 (SEQ ID NO: 1089) and light chain residues contributing to the binding interaction with FLT1 include 131 and N32 (SEQ ID NO: 1090). Table 36 provides a listing of acceptable amino acid substitutions at the aforementioned paratope interface residues that are expected to maintain an equivalent antibody paratope structure capable of binding D2 of FLT1 to block VEGF binding to FLT1 .

[0204] In another embodiment, the means for inhibiting FLT1 bindingto VEGF, the means for increasing plasma VEGF levels in a subject, and the means for increasing VEGFR2 phosphorylation in a subject comprises an anti-FLT1 antibody having the structure of the anti- FLT1 antibody 15331 or an equivalent structure thereof as depicted in Figures 16C and 16D. As depicted in Figure 16F, the 15331 antibody binds to the same domain of FLT1 (D2) as VEGF, the natural ligand of FLT1 , with an 86% overlap in the residues of FLT1 bound by the antibody and bound by VEGF. This data confirms that an anti-FLT1 antibody having the 15331 antibody structure, or an equivalent structure thereof, blocks VEGF binding to FLT1. Blocking VEGF binding to FLT1 using an antibody having the structure of antibody 1533, or an equivalent structure thereof, increases the amount of plasma VEGF in a subject available to bind to VEGFR2 resulting in an increase in VEGFR2 phosphorylation. In one embodiment, the means for blocking VEGF bindingto FLT1 comprises an antibody having the paratope structure of antibody 15331 , or an equivalent structure thereof, as provided in Figure 16D. As shown in Figure 16D, heavy chain residues N57, Y59, Y108, Y109, H35, W52, D62, Y102, and P104 (SEQ ID NO: 1335), and light chain residues H31 , Y99, R101 , D1 , Q27, and F37 (SEQ ID NO: 1336) are the paratope interface residues directly involved in contacting D2 of FLT1 to block VEGF binding to FLT1 . Additional heavy chain residues contributing to the binding interaction with FLT1 include V33, 158, S99, and T100 (SEQ ID NO: 1335) and light chain residues contributing to the binding interaction with FLT1 include N33, L97, and T98 (SEQ ID NO: 1336). Table 40 provides a listing of acceptable amino acid substitutions at the aforementioned paratope interface residues that maintain an equivalent antibody paratope structure capable of binding D2 of FLT1 to block VEGF bindingto FLT1 .

[0205] In exemplary embodiments of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the FLT1 binding protein, in particular anti-FLT1 antibody or nucleic acid encoding the same, as presently disclosed (hereinafter referred to as “active agents”) are formulated into a pharmaceutical composition comprising the active agent, alongwith a pharmaceutically acceptable carrier, diluent, or excipient. In this regard, the presentdisclosure further provides pharmaceutical compositions comprising an active agent which pharmaceutical composition is intended for administration to a subject, e.g., a mammal. As used herein, the term “pharmaceutically acceptable carrier” includes any pharmaceutical carrier used in the field, such as a phosphate buffered saline solution, water, emulsions such as an oil / water orwater / oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.

[0206] In some embodiments, the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%. In various embodiments, the pharmaceutical composition is sterile and manufactured following good manufacturing practices (GMP).

[0207] Routes of Administration

[0208] With regard to the present disclosure, the FLT1 binding protein, in particular anti- FLT1 antibody, or pharmaceutical composition comprising the same, can be administered to the subject via any suitable route of administration. For example, the active agent can be administered to a subject via parenteral administration. In exemplary embodiments, the pharmaceutical compositions provided herein are suitable for subcutaneous administration or intravenous administration. In a preferred embodiment, the pharmaceutical compositions provided herein are administered subcutaneously. In a preferred embodiment, the pharmaceutical compositions provided herein are administered intravenously. Injectable formulations are in accordance with the present disclosure. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0209] Kits

[0210] The present disclosure additionally provides kits comprising an FLT1 binding protein, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof. The kit in exemplary embodiments comprises at least one FLT1 binding protein, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof, in a container. In exemplary embodiments, the at least one FLT1 binding protein, nucleic acid, vector, or host cell of thepresent disclosure, is provided in the kit as a unit dose. For purposes herein “unit dose" refers to a discrete amount dispersed in a suitable carrier. In exemplary embodiments, the unit dose is the amount sufficient to provide a subject with a desired effect, e.g., treatment of peripheral arterial disease. In exemplary embodiments, the kit comprises several unit doses, e.g., a week or month supply of unit doses, optionally, each of which is individually packaged or otherwise separated from other unit doses. In some embodiments, the components of the kit / unit dose are packaged with instructions for administration to a patient. In some embodiments, the kit comprises one or more devices for administration to a patient, e.g., a needle and syringe, and the like. In some embodiments, the at least one FLT1 binding protein, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof, is / are pre-packaged in a ready to use form, e.g., a syringe, an intravenous bag, etc. In exemplary embodiments, the ready to use form is for a single use. In exemplary embodiments, the kit comprises multiple single use, ready to use forms of the at least one FLT1 binding protein, nucleic acid, vector, or host cell of the present disclosure. In some embodiments, the kit further comprises other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein.

[0211] Methods of Treatment

[0212] Methods of treatment are additionally provided by the present disclosure. The method, in exemplary embodiments, is a method of treating a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition of the present disclosure in an amount effective to treat the subject. In exemplary embodiments, the FLT1 binding proteins, in particular, the anti-FLT1 antibodies, and pharmaceutical compositions comprising the same as described herein are effective for treating a subject suffering from a cardiovascular disorder or dysfunction or a condition associated with microvascular dysfunction. In exemplary embodiments, the cardiovascular disorder or dysfunction or a condition associated with microvascular dysfunction is associated with increased plasma levels of soluble FLT1 . Treatment with the FLT1 binding proteins, in particular, the anti-FLT1 antibodies, as described herein will increase blood flow, capillary density, capillary or coronary growth, tissue perfusion (e.g., muscle perfusion, skin perfusion, etc.) in the treated subject.

[0213] In exemplary embodiments, the FLT1 binding proteins, in particular, the anti-FLT1 antibodies, and pharmaceutical compositions comprising the same as described herein are effective for treating a condition that involves or is associated with reduced blood flowthrough blood vessels to the limbs or other organs. In exemplary embodiments, the reduced blood flowis caused by atherosclerotic plaques that form on the inner lining of the arteries. In exemplary embodiments, the subject suffers from peripheral artery disease (PAD), also known as peripheral vascular disease (PVD). In exemplary embodiments, the subject suffers from severe arterial blockage in lower extremities. In various embodiments, the subject suffers from critical limb ischemia (CLI), also known as chronic limb threatening ischemia (CLTI). CLI occurs in subjects having severe stages of PAD and involves severe blockage of blood flow to the subject’s arms, legs, or feet. In various embodiments, the subject has chronic ischemic ulcers, and treatment with an anti-FLT1 antibody of the disclosure improves blood flow and perfusion to the affected area. In various embodiments, the subject needs a limb amputation or surgical revascularization and treatment with an anti-FLT1 antibody is administered as an alternative to amputation or surgical revascularization to provide fast onset of tissue perfusion to the affected limb or other tissue. In various embodiments, the subject has an increased sFLT1 plasma level as compared to the average sFLT1 plasma level of corresponding healthy subjects.

[0214] In exemplary embodiments, the FLT1 binding proteins, in particular, the anti-FLT1 antibodies, and pharmaceutical compositions comprising the same as described herein are effective for treating a subject suffering from heart failure (HF), a medical condition in which the heart is unable to pump the amount of blood required by the body. In some embodiments, the HF is congestive heart failure, systolic heart failure, diastolic heart failure, or right ventricle heart failure. In various embodiments, the HF is Stage A HF, Stage B HF, Stage C HF, or Stage D HF. In various embodiments, the Stage C or Stage D HF is further classified by the New York Heart Association (NYHA) Functional Classification system and is one of Class I, Class II, Class III, or Class IV HF. The signs and symptoms of heart failure include dyspnea (e.g., orthopnea, paroxysmal nocturnal dyspnea), coughing, cardiac asthma, wheezing, dizziness, confusion, cool extremities at rest, chronic venous congestion, ankle swelling, peripheral edema or anasarca, nocturia, ascites, heptomegaly, jaundice, coagulopathy, fatigue, exercise intolerance, jugular venous distension, pulmonary rales, peripheral edema, pulmonary vascular redistribution, interstitial edema, pleural effusions, or a combination thereof. In exemplary embodiments, the heart failure is a systolic heart failure, which is heart failure caused or characterized by a systolic dysfunction. Systolic dysfunction is a condition in which the pump function or contraction of the heart (i.e., systole), fails. Systolic dysfunction may be characterized by a decreased or reduced ejection fraction, e.g., an ejection fraction which is less than 45%, and an increased ventricular end- diastolic pressure and volume. In some embodiments, the strength of ventricular contraction is weakened and insufficient for creating an appropriate stroke volume, resulting in less cardiac output. In some embodiments, thesystolic heart failure is an ischemic heart failure. In alternative embodiments, the systolic heart failure is a nonischemic heart failure. In various embodiments, the subject has an increased sFLT1 plasma level as compared to the average sFLT1 plasma level of corresponding healthy subjects.

[0215] In exemplary embodiments, the FLT1 binding proteins, in particular, the anti-FLT1 antibodies, and pharmaceutical compositions comprising the same as described herein are effective for treating cardiomyopathy, a condition that affects the heart muscle, making it harder for the heart to pump blood. In exemplary embodiments, the anti-FLT1 antibody is useful for treating subject with peripartum cardiomyopathy or postpartum cardiomyopathy (PPCM). In various embodiments, the subject has an increased sFLT1 plasma level as compared to the average sFLT1 plasma level of corresponding healthy subjects.

[0216] In exemplary embodiments, the FLT1 binding proteins, in particular, the anti-FLT1 antibodies, and pharmaceutical compositions comprising the same as described herein are effective for treating a subject suffering microvascular dysfunction. Microvascular dysfunction encompasses a varied set of conditions which cause small-vessel obstruction that results in tissue damage and organ disfunction. In exemplary embodiments, the microvascular dysfunction involves the cardiovascular system, including, e.g., ischemic heart injury or disease and infarction. In exemplary embodiments, the subject to be treated suffers from microvascular infarction without obstructive CAD (MINOCA). In exemplary embodiments, the subject to be treated with the FLT1 binding proteins described herein, in particular, the anti- FLT1 antibodies of the disclosure, has angina. Angina is chest pain or discomfort caused by a lack of oxygen-rich blood reaching the heart. In exemplary embodiments, the subject to be treated suffers from microvascular angina or Angina without Obstructive CAD (ANOCA). In various embodiments, the subject has an increased sFLT1 plasma level as compared to the average sFLT1 plasma level of corresponding healthy subjects.

[0217] In exemplary embodiments, the FLT1 binding proteins, in particular the anti-FLT1 antibodies, and pharmaceutical compositions comprising the same as described herein are effective for treating microvascular dysfunction associated with diabetes. In various embodiments, the subject has an increased sFLT1 plasma level as compared to the average sFLT1 plasma level of corresponding healthy subjects. For example, the anti-FLT1 antibody is useful for treating diabetic neuropathy and diabetic nephropathy. The anti-FLT1 antibody of the disclosure is also useful for treating diabetic ulcers (e.g., diabetic foot ulcers) and other diabetic wounds, including severe limb ischemia. In some embodiments, the anti-FLT1 antibody treatment is administered to a subject in combination with other revascularizationtechniques, such as stenting or bypass grafting to improve major adverse limb events (MALE) outcomes. In certain embodiments, the subject is not a good candidate for surgical revascularization, due to one or more of: 1 ) extensive comorbidities, 2) poor overall health, 3) lack of suitable graft material (patients may lack suitable veins or arteries to use for bypass grafting) or 4) lack of viable arteries distal to the occlusion site to serve as a target for bypass grafting. Treatment with a FLT1 binding protein, in particular an anti-FLT1 antibody, as described herein provides a preferred alternative to surgical revascularization or amputation to provide fast onset of tissue perfusion to the affected limb or other tissue.

[0218] In exemplary embodiment, the FLT1 binding protein, in particular, the anti-FLT1 antibodies, and pharmaceutical compositions comprising the same as described herein are effective for treating chronic kidney disease (CKD), which is a microvascular disease of kidney involving impaired vasoreactivity, fibrotic remodeling and microvascular rarefaction. In various embodiments, the subject has an increased sFLT1 plasma level as compared to the average sFLT1 plasma level of corresponding healthy subjects.

[0219] In exemplary embodiments, the FLT1 binding proteins, e.g., the anti-FLT1 antibodies, and pharmaceutical compositions comprising the same as described herein are effective for treating bronchopulmonary dysplasia, a clinical syndrome of lung injury that disrupts alveolarization and microvascular development. In exemplary embodiments, the FLT1 binding proteins, e.g., the anti-FLT1 antibodies, and pharmaceutical compositions comprising the same as described herein are effective for treating Duchenne’s muscular dystrophy (DMD), a genetic disorder characterized by a progressive muscle wasting. Tissue ischemia is a primary manifestation of DMD, and increasing tissue perfusion using an anti-FLT1 antibody of the present disclosure will aid in the treatment and reversal of this tissue ischemia in subjects with DMD. In various embodiments, the subject has an increased sFLT1 plasma level as compared to the average sFLT1 plasma level of corresponding healthy subjects.

[0220] Accordingly, the present disclosure provides methods of treating a medical condition associated with reduced blood flow and / or tissue ischemia in a subject. In various embodiments, the medical condition is a cardiovascular disorder such as PAD, CLI, ANOCA, PPMC, and HF as described above. In various embodiments, the medical condition arises from or is associated with microvascular dysfunction, such as, diabetic neuropathy, diabetic nephropathy, diabetic wound healing, DMD, bronchopulmonary dysplasia, and chronic kidney disease. In one embodiment, the medical condition that arises from or is associated with microvascular dysfunction and is treatable with an anti-FLT1 antibody as disclosed herein is diabetic ulcers. In various embodiments, the subject has an increased sFLT1 plasma level ascompared to the average sFLT1 plasma level of corresponding healthy subjects. Treatment of these conditions with a FLT1 binding protein of the present disclosure provides a means for increasing VEGF-mediated signal transduction in a subject, which, in turn, increases blood flow, capillary density, capillary or coronary growth, and / or tissue perfusion in the subject. The present disclosure further provides methods of reducing blood pressure and risk of heart failure in a subject. In exemplary embodiments, each of the methods comprise administering to the subject a pharmaceutical composition of the present disclosure. In various embodiments, the pharmaceutical composition comprises a FLT1 binding protein of the present disclosure. In various embodiments, the FLT1 binding protein comprises or is an antibody, or an antigenbindingfragment thereof, comprising an antibody HC variable region and an antibody LC variable region as described herein. In various embodiments, the FLT1 binding protein comprises or is an antibody, or an antigen-binding fragment thereof, comprising an antibody HC variable region and an antibody LC variable region, wherein the HC variable region comprises a HC CDR1 , HC CDR2, HC CDR3 as described in Table A.1 , Table A.2, or E, wherein the LC variable region comprises a LC CDR1 , LC CDR2, LC CDR3 as described in Table A.1 , Table A.2, orTable E. In one embodiment, the FLT1 binding protein comprises an antibody, or an antigen-bindingfragment thereof, comprising an antibody HC variable region and an antibody LC variable region, wherein the HC variable region comprises a HC CDR1 , HC CDR2, HC CDR3 of SEQ ID NOs: 7-9, respectively, and LC variable region comprises a LC CDR1 , LC CDR2, LC CDR3 of SEQ ID NOs: 10-12, respectively. In another embodiment, the FLT1 binding protein comprises an antibody or antigen-binding fragment thereof, comprising an antibody HC variable region and an antibody LC variable region, wherein the HC variable region comprises a HC CDR1 , HC CDR2, HC CDR3 of SEQ ID NOs: 733-735, respectively, and LC variable region comprises a LC CDR1 , LC CDR2, LC CDR3 of SEQ ID NOs: 736-738, respectively. In various embodiments, the FLT1 binding protein comprises or is an antibody, or an antigen-binding fragment thereof, comprising an antibody HC variable region comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO :1089, and an antibody LC variable region comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 1090. In various embodiments, the FLT1 binding protein comprises or is an antibody, or an antigen-binding fragment thereof, comprising an antibody HC variable region of SEQ ID NO :1089 and an antibody LC variable region of SEQ ID NO: 1090. In various embodiments, the FLT1 binding protein comprises an antibody having a HC amino acid sequence of SEQ ID NO: 1460 and a LC amino acid sequence of SEQ ID NO: 1461 . In various embodiments, the FLT1 binding protein comprises or is an antibody, or an antigen-binding fragment thereof, comprising an antibody HC variable region comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO :1335 and an antibody LC variable region comprising an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 1336. In various embodiments, the FLT1 binding protein comprises or is an antibody, or an antigen-binding fragment thereof, comprising an antibody HC variable region of SEQ ID NO :1335 and an antibody LC variable region of SEQ ID NO: 1336. In various embodiments, the FLT1 binding protein comprises an antibody having a HC amino acid sequence of SEQ ID NO: 1462 and a LC amino acid sequence of SEQ ID NO: 1463.

[0221] In some embodiments, the methods of treating a subject as described supra, e.g., methods of treating a subject having a cardiovascular condition or microvascular dysfunction associated with reduced blood flow such as, but not limited, PAD, CLI, angina, cardiomyopathy, HF, diabetic neuropathy, diabetic nephropathy, diabetic wound healing, diabetic ulcers, DMD, bronchopulmonary dysplasia, or chronic kidney disease involve administering a pharmaceutical composition that comprises (i) a means for inhibiting FLT1 binding to VEGF and (ii) a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition of the present disclosure comprises a means for increasing plasma VEGF levels in a subject and a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition of the present disclosure comprises a means for increasing VEGFR phosphorylation in a subject, and a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment, the means for inhibiting FLT1 binding to VEGF, the means for increasing plasma VEGF levels in a subject, and the means for increasing VEGFR2 phosphorylation in a subject comprises an anti-FLT1 antibody having the structure of the anti-FLT1 antibody 18300 or an equivalent structure thereof as depicted in Figures 16A and 16B. As depicted in Figure 16E, antibody 18300 binds to the same domain of FLT1 (D2) as VEGF, the natural ligand of FLT1 , with a 71 % overlap in the residues of FLT1 bound by antibody 18300 and VEGF. This data confirms that an anti-FLT1 antibody having the 18300 antibody structure, or an equivalent structure thereof, blocks VEGF binding to FLT1. Blocking VEGF binding to FLT1 using an antibody having the structure of antibody 18300 (or an equivalent structure thereof) increases the amount of plasma VEGF in a subject available to bind to VEGFR2 resulting in an increase in VEGFR2 phosphorylation. In one embodiment, the means for blocking VEGF binding to FLT1 comprises an antibody having the paratope structure of antibody 18300, or an equivalent structure thereof, as provided in Figure 16B. As shown in Figure 16B and described herein in Example 11 , heavy chain residues Y35, Y52, Y54, L102, L105, L106, and Y60 (SEQ ID NO: 1089, and light chain residues S51 , N53, H54, D94, Y50 andW92 (SEQ ID NO: 1090) are directly involved in contacting D2 of FLT1 to block VEGF binding to FLT1 . Table 36 provides a listing of acceptable amino acid substitutions at the aforementioned contact points that maintain an equivalent antibody paratope structure capable of binding D2 of FLTI to block VEGF binding to FLT1.

[0222] In another embodiment, the means for inhibiting FLT1 bindingto VEGF, the means for increasing plasma VEGF levels in a subject, and the means for increasing VEGFR2 phosphorylation in a subject comprises an anti-FLT1 antibody having the structure of the anti- FLT1 antibody 15331 or an equivalent structure thereof as depicted in Figures 16C and 16D. As depicted in Figure 16F, this antibody binds to the same domain of FLT1 (D2) as VEGF, the natural ligand of FLT1 , with a 86% overlap in the residues of FLT1 bound by the antibody and VEGF. This data confirms that an anti-FLT1 antibody having the 15331 antibody structure, or an equivalent structure thereof, blocks VEGF binding to FLT1 . Blocking VEGF bindingto FLT1 using an antibody having the structure of antibody 15331 (or an equivalent structure thereof) increases the amount of plasma VEGF in a subject available to bind to VEGFR2 resulting in an increase in VEGFR2 phosphorylation. In one embodiment, the means for blocking VEGF bindingto FLT1 comprises an antibody having the paratope structure of antibody 15331 , or an equivalent structure thereof, as provided in Figure 16D. As shown in Figure 16D, heavy chain residues N57, Y59, Y108, Y109, H35, W52, D62, Y102, and P104 (SEQ ID NO: 1335), and light chain residues H31 , Y99, R101 , D1 , Q27, and F37 (SEQ ID NO: 1336) are the paratope interface residues directly involved in contacting D2 of FLT1 to block VEGF binding to FLT1 . Additional heavy chain residues contributing to the binding interaction with FLT1 include V33, 158, S99, and T100 (SEQ ID NO: 1335) and light chain residues contributing to the binding interaction with FLT1 include N33, L97, and T98 (SEQ ID NO: 1336). Table 40 provides a listing of acceptable amino acid substitutions at the aforementioned contact points that maintain an equivalent antibody paratope structure capable of binding D2 of FLT1 to block VEGF bindingto FLT1 .

[0223] As used herein, the terms “treat” and words stemming therefrom may not be a 100% or complete treatment. Rather, there are varying degrees of treatment which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the pharmaceutical compositions of the present disclosure may treat PAD, CLI, ANOCA, HF and other medical conditions arising from or is associated with microvascular dysfunction, such as, diabetic neuropathy, diabetic nephropathy, diabetic wound healing, diabetic ulcers, DMD, bronchopulmonary dysplasia, and chronic kidney disease, to any amount or level.Furthermore, the treatment provided by the method of the present disclosure can include treatment or a reduction of one or more signs or symptoms of the condition being treated. Forinstance, the treatment provided herein in various embodiments treats or reduces the signs and symptoms of HF (e.g., dyspnea, (orthopnea, paroxysmal nocturnal dyspnea), coughing, cardiac asthma, wheezing, dizziness, confusion, cool extremities at rest, chronic venous congestion, ankle swelling, peripheral edema or anasarca, nocturia, ascites, heptomegaly, jaundice, coagulopathy, fatigue, exercise intolerance, and the like. Also, the treatment provided by the methods of the present disclosure can encompass slowing the progression of the condition. For instance, the treatment provided by the methods of the present disclosure may slow the progression of CLI by delaying or effectively preventing amputation of a limb or the development of gangrene. Also, in this respect, the pharmaceutical compositions of the present disclosure may increase, e.g., blood flow, capillary density, capillary or coronary growth, and / or cardiac perfusion, to any amount or level. In exemplary embodiments, the increase provided by the methods of the present disclosure is at least or about a 10% increase (e.g., at least or about a 20% increase, at least or about a 30% increase, at least or about a 40% increase, at least or about a 50% increase, at least or about a 60% increase, at least or about a 70% increase, at least or about an 80% increase, at least or about a 90% increase, at least or about a 95% increase, at least or about a 98% increase).

[0224] Subjects

[0225] In some embodiments of the present disclosure, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some embodiments, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). Preferably, the subject is a human.

[0226] Methods of Manufacture

[0227] The FLT1 binding proteins of the present disclosure may be obtained by methods known in the art. Suitable methods of de novo synthesizing polypeptides are described in, for example, Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U.S. Patent No. 5,449,752. Additional exemplary methods of making the peptides of the invention are set forth herein.

[0228] In some embodiments, the FLT1 binding proteins described herein are commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp.(Gaithersburg, MD), Multiple Peptide Systems (San Diego, CA), Peptide 2.0 Inc. (Chantilly, VA), and American Peptide Co. (Sunnyvale, CA). In this respect, the FLT1 binding proteins can be synthetic, recombinant, isolated, and / or purified.

[0229] Also, in some embodiments, the FLT1 binding proteins are recombinantly produced using a nucleic acid encoding the amino acid sequence of the peptide using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001 ; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

[0230] In exemplary embodiments, the method of producing the presently disclosed FLT1 binding proteins comprises culturing a presently disclosed host cell so as to express the FLT1 binding protein and harvesting the expressed FLT1 binding protein. The host cell can be any of the host cells described herein. In exemplary embodiments, the host cell is selected from the group consisting of: CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In exemplary embodiments, the step of culturing a host cell comprises culturing the host cell in a growth medium to support the growth and expansion of the host cell. In exemplary embodiments, the growth medium increases cell density, culture viability and productivity in a timely manner. In exemplary embodiments, the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. In exemplary embodiments, the growth medium is a fully chemically defined media consisting of amino acids, vitamins, trace elements, inorganic salts, lipids and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also helps maintain pH and osmolality.Several growth medias are commercially available and are described in the art. See, e.g., Arora, “Cell Culture Media: A Review” MATER METHODS 3:175 (2013).

[0231] In exemplary embodiments, the method of making a FLT1 binding protein of the present disclosure comprises culturingthe host cell in a feed medium. In exemplary embodiments, the method comprises culturing in a feed medium in a fed-batch mode. Methods of recombinant protein production are known in the art. See, e.g., Li et al., “Cell culture processes for monoclonal antibody production” MAbs 2(5): 466-477 (2010).

[0232] The method making a FLT1 binding protein can comprise one or more steps for purifying the protein from a cell culture or the supernatant thereof and preferably recovering thepurified protein. In exemplary embodiments, the method comprises one or more chromatography steps, e.g., affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. In exemplary embodiments, the method comprises purifying the protein using a Protein A affinity chromatography resin.

[0233] In exemplary embodiments, the method further comprises steps for formulating the purified protein, etc., thereby obtaining a formulation comprising the purified protein. Such steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0234] Exemplary Embodiments

[0235] The following is a listing of exemplary embodiments of the present disclosure:1. A FLT1 binding protein comprising: a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1090.2. The FLT1 binding protein of embodiment 1 , wherein, the VH comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and the VL comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1090.3. The FLT1 binding protein of embodiment 1 or embodiment 2, wherein the VH comprises the Tyr at position 35, the Tyr at position 52, the Tyr at position 54, the Tyr at position 60, the Leu at position 102, the Leu at position 105, and the Leu at position 106 of SEQ ID NO: 1089; and the VL comprises the Gly at position 30, the Tyr at position 50, the Ser at position 51 , the Asn at position 53, the His at position 54, the T rp at position 92, and the Asp at position 94 of SEQ ID NO: 1090.4. The FLT1 binding embodiment of claim 3, wherein the FLT1 binding protein binds to an epitope comprising residues F172, P173, K217, L174, L215, and L221 of human FLT1 (SEQ ID NO: 1453).5. The FLT1 binding protein of any one of embodiments 1-4, wherein the VH comprises a complementarity determining region 1 (HCDR1 ), a HCDR2, and a HCDR3 of SEQ ID NOs:1485, 1488, and 1491 , respectively, and the VL comprises a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs; 1494, 1497, 1500, respectively.6. The FLT1 binding protein of any one of embodiments 1-5, wherein the VH comprises a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1487, 1490, and 1493, respectively, and the VL comprises a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 1496, 1499, 1502, respectively.7. The FLT1 binding protein of embodiment 1 or embodiment 2, wherein the VH comprises a. an HC complementarity-determining region (CDR) 1 amino acid sequence of any one of SEQ ID NOs: 7, 151 , 181 , 331 ; b. an HC CDR2 amino acid sequence of any one of SEQ ID NO: 8, 152, 182, 332; c. an HC CDR3 amino acid sequence of any one of SEQ ID NO: 9, 153, 183, 333; or d. a combination thereof, and the VL comprises a. an LC CDR1 amino acid sequence of any one of SEQ ID NOs: 10, 154, 184, 334; b. an LC CDR2 amino acid sequence of any one of SEQ ID NO: 11 , 155, 185, 335; c. an LC CDR3 amino acid sequence of any one of SEQ ID NO: 12, 156, 186, 336; or d. a combination thereof.8. The FLT1 binding protein of any one of embodiments 1-7, wherein the VH comprises HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NO: 1089; and the VL comprises LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 1090.9. The FLT1 binding protein of any one of embodiments, 1-8, wherein the VH comprises a HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 7-9, respectively, and the VL comprises a LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 10-12, respectively.10. A FLT1 binding protein comprising:a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), a HCDR2, and a HCDR3 of SEQ ID NOs: 1485, 1488, and 1491 , respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs; 1494, 1497, 1500, respectively.11 . The FLT1 binding protein of embodiment 10, wherein the VH comprises the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 1487, 1490, and 1493, respectively, and the VL comprises the LCDR1 , the LCDR2, and the LCDR3 of SEQ ID NOs; 1496, 1499, 1502, respectively.12. The FLT1 binding protein of embodiment 10 or 11 , wherein the VH comprises the HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 7-9, respectively, and the VL comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 10-12, respectively.13. A FLT1 antibody comprising: a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), HC CDR2, and HC CDR3 of SEQ ID NOs: 7-9, respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), LC CDR2, and LC CDR3 of SEQ ID NOs: 10-12, respectively.14. The FLT1 antibody of embodiment 13, wherein the VH comprises the amino acid sequence SEQ ID NO: 1089, and the VL comprises the amino acid sequence of SEQ ID NO: 1090.15. The FLT1 antibody of embodiment 13, wherein the antibody comprises: a heavy chain amino acid sequence of SEQ ID NO: 1460 or 1572 and a light chain amino acid sequence of SEQ ID NO: 1461 .16. A FLT1 binding protein comprising: a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1335, and a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1336.17. The FLT1 binding protein of embodiment 16, wherein, the VH comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1335, and the VL comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1336.18. The FLT1 binding protein of embodiment 16 or embodiment 17, wherein the VH comprises the His at position 35, the Trp at position 52, the Asn at position 57, the Tyr at position 59, the Asp at position 62, the Tyr at position 102, the Pro at position 104, the Tyr at position 108, and the Tyr at position 109 of SEQ ID NO: 1335; and the VL comprises the Gin at position 27, the His at position 31 , the Phe at position 37, the Tyr at position 99, the Arg at position 101 , and the Asp at position 1 of SEQ ID NO: 1336.19. The FLT1 binding protein of embodiment 18, wherein said binding protein binds to an epitope comprising M138, Y139, S140, F172, L215, Y216, K217, and glycans linked to N100 of human FLT1 (SEQ ID NO: 1453).20. The FLT1 binding protein of any one of embodiments 16-19, wherein the VH comprises a complementarity determining region 1 (HCDR1 ), a HCDR2, and a HCDR3 of SEQ ID NOs: 1512, 1515, and 1518, respectively, and the VL comprises a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs; 1521 , 1524, and 1527, respectively.21 . The FLT1 binding protein of any one of embodiments 16-20, wherein the VH comprises a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1514, 1517, and 1520, respectively, and the VL comprises a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 1523, 1526, 1529, respectively.22. The FLT1 binding protein of embodiment 16 or embodiment 17, wherein the VH comprises a. an HC complementarity-determining region (CDR) 1 amino acid sequence of any one of SEQ ID NOs: 733, 1027 and 1033, b. an HC CDR2 amino acid sequence of any one of SEQ ID NO: 734, 1028, and 1034, c. an HC CDR3 amino acid sequence of any one of SEQ ID NO: 735, 1029, and 1035; or d. a combination thereof, and the VL comprisese. an LC CDR1 amino acid sequence of any one of SEQ ID NOs: 736, 1030, and 1036, f. an LC CDR2 amino acid sequence of any one of SEQ ID NO: 737, 1031 , and 1037; g. an LC CDR3 amino acid sequence of any one of SEQ ID NO: 738, 1032, and 1038; or h. or a combination thereof.23. The FLT1 binding protein of any one of embodiments of 16-22, wherein the VH comprises HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NO: 1335; and the VL comprises LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 1336.24. The FLT1 binding protein of any one of embodiments of 16-23, wherein the VH comprises the HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 733-735, respectively, and the VL comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 736-738, respectively.25. A FLT1 binding protein comprising: a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), a HCDR2, and a HCDR3 of SEQ ID NOs: 1512, 1515, 1518, respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs; 1521 , 1524, and 1527, respectively.26. The FLT1 binding protein of embodiment 25, wherein the VH comprises the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 1514, 1517, and 1520, respectively, and the VL comprises the LCDR1 , the LCDR2, and the LCDR3 of SEQ ID NOs: 1523, 1526, and 1529, respectively.27. The FLT1 binding protein of embodiment 26, wherein the VH comprises the HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 733-735, respectively, and the VL comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 736-738, respectively28. A FLT1 binding antibody comprising: a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), HC CDR2, and HC CDR3 of SEQ ID NOs: 733-735, respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), LC CDR2, and LC CDR3 of SEQ ID NOs: 736-738, respectively.29. The FLT1 antibody of embodiment 28, wherein the VH comprises the amino acid sequence SEQ ID NO: 1335, and the VL comprises the amino acid sequence of SEQ ID NO: 1336.30. A FLT1 antibody of embodiment 28 or embodiment 29, wherein the antibody comprises: a heavy chain amino acid sequence of SEQ ID NO: 1462 or 1573 and a light chain amino acid sequence of SEQ ID NO: 1463.31 . A FLT1 binding protein comprising:I. an antibody heavy chain (HC) variable region comprising a) an HC complementarity-determining region (CDR) 1 amino acid sequence of any one of SEQ ID NOs: 7, 151 , 181 , 331 , 571 , 589, 619, 733, 1027, and 1033, or a variant sequence thereof which differs by 1 , 2, 3, or 4 amino acids or which has at least or about 80% sequence identity to the HC CDR1 amino acid sequence of any one of SEQ ID NOs: 7, 151 , 181 , 331 , 571 , 589, 619, 733, 1027, and 1033; b) an HC CDR2 amino acid sequence of any one of SEQ ID NO: 8, 152,182, 332, 572, 590, 620, 734, 1028, and 1034, or a variant sequence thereof which differs by 1 , 2, 3, or4 amino acids orwhich has at least or about 80% sequence identity to the HC CDR2 amino acid sequence of any one of SEQ ID NO: 8, 152, 182, 332, 572, 590, 620, 734, 1028, and 1034; c) an HC CDR3 amino acid sequence of any one of SEQ ID NO: 9, 153,183, 333, 573, 591 , 621 , 735, 1029, and 1035, or a variant sequence thereof which differs by 1 , 2, 3, or4 amino acids orwhich has at least or about 80% sequence identity to the HC CDR3 amino acid sequence of any one of SEQ ID NO: 9, 153, 183, 333, 573, 591 , 621 , 735, 1029, and 1035; or d) a combination thereof, andII. an antibody light chain (LC) variable region comprising a) an LC CDR1 amino acid sequence of any one of SEQ ID NOs: 10, 154,184, 334, 574, 592, 622, 736, 1030, and 1036, or a variant sequence thereof which differs by 1 , 2, 3, or4 amino acids orwhich has at least or about 80% sequence identity to the LC CDR1 amino acid sequenceof any one of SEQ ID NOs: 10, 154, 184, 334, 574, 592, 622, 736, 1030, and 1036; b) an LC CDR2 amino acid sequence of any one of SEQ ID NO: 11 , 155,185, 335, 575, 593, 623, 737, 1031 , and 1037, or a variant sequence thereof which differs by 1 , 2, 3, or 4 amino acids or which has at least or about 80% sequence identity to the LC CDR2 amino acid sequence of any one of SEQ ID NO: 11 , 155, 185, 335, 575, 593, 623, 737, 1031 , and 1037; c) an LC CDR3 amino acid sequence of any one of SEQ ID NO: 12, 156,186, 336, 576, 594, 624, 738, 1032, and 1038, or a variant sequence thereof which differs by 1 , 2, 3, or 4 amino acids or which has at least or about 80% sequence identity to the LC CDR3 amino acid sequence of any one of SEQ ID NO: 12, 156, 186, 336, 576, 594, 624, 738, 1032, and 1038; or d) or a combination thereof. he FLT1 binding protein of embodiment 31 comprising:III. an antibody heavy chain (HC) variable region comprising a) a HC CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 7, 151 , 181 , 331 , 571 , 589, 619, 733, 1027, and 1033; b) a HC CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 8, 152, 182, 332, 572, 590, 620, 734, 1028, and 1034; c) a HC CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 9, 153, 183, 333, 573, 591 , 621 , 735, 1029, and 1035; or d) a combination thereof; andIV. an antibody light chain (LC) variable region comprising a) a LC CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 10, 154, 184, 334, 574, 592, 622, 736, 1030, and 1036; b) a LC CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 11 , 155, 185, 335, 575, 593, 623, 737, 1031 , and 1037; c) a LC CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 12, 156, 186, 336, 576, 594, 624, 738, 1032, and 1038; ord) a combination thereof.33. The FLT1 binding protein of embodiment 31 or embodiment 32 comprisingV. an antibody heavy chain (HC) variable region comprising a HC CDR1 , HC CDR2, and HC CDR3 of any one of SEQ ID NOs: 1089, 1137, 1197, 1147, 1277, 1293,1283. 1437. 1435, and 1335; andVI. an antibody heavy chain (LC) variable region comprising a LC CDR1 , LC CDR2, and LC CDR3 of any one of SEQ ID NOs: 1090, 1138, 1198, 1148, 1278, 1294,1284. 1438. 1436, and 1336.34. The FLT1 binding protein of any one of embodiments 31-33 comprising six CDRs of (a) SEQ ID NOs: 7-12; (b) SEQ ID NOs: 151-156; (c) SEQ ID NOs: 331 -336; (d) SEQ ID NOs: 181- 186; (e) SEQ ID NOs: 571 -576; (f) SEQ ID NOs: 619-624; (g) SEQ ID NOs: 589-594; (h) SEQ ID NOs: 1033-1038; (i) SEQ ID NOs: 1027-1032; or (j) SEQ ID NOs: 733-738.35. The FLT1 binding protein of any one of embodiments 1 -12, 16-27, or 31 -34, wherein the binding protein comprises or is an antigen-binding antibody fragment.36. The FLT1 binding protein of any one of embodiments 1 -12, 16-27, or 31-34, wherein the binding protein is an antibody.37. The FLT1 binding protein of embodiment 36, wherein the antibody comprises a human IgG HC constant region and a human LC constant region.38. The FLT1 binding protein of embodiment 37, wherein the human IgG HC constant region is a human IgG 1 constant region and the human LC constant region is a human kappa LC constant region or a human lambda LC constant region.39. The FLT1 binding protein of embodiment claim 37 wherein the HC constant region comprises any one of SEQ ID NOs: 1530-1533 or an amino acid sequence which has at least 80% sequence identity to said SEQ ID NOs: 1530-1533, and / or the LC constant region comprises any one of SEQ ID NOs: 1546-1547 or an amino acid sequence which has at least 80% sequence identity to said SEQ ID NOs: 1546-1547.40. A nucleic acid comprising a nucleotide sequence encoding a FLT1 binding protein of any one of claims 1 -12, 16-27, or 31-35 or a FLT1 antibody of any one of claims 13-15 and 28-30, or 36-39.41 . A vector comprising the nucleic acid of embodiment 40.42. A host cell comprising the nucleic acid of embodiment 40 or the vector of embodiment41.The host cell of embodiment 42, comprising (A) a first nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NOs: 1089, 1137, 1197, 1147, 1277, 1293, 1283, 1437, 1435, and 1335, or an amino acid sequence comprising at least 90% sequence identity to any one of SEQ ID NOs: 1089, 1137, 1197, 1147, 1277, 1293, 1283, 1437, 1435, and 1335; and (B) a second nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NOs: 1090, 1138, 1198, 1148, 1278, 1294, 1284, 1438, 1436, and 1336 or an amino acid sequence comprising at least 90% sequence identity to any one of SEQ ID NOs: 1090, 1138, 1198, 1148, 1278, 1294, 1284, 1438, 1436, and 1336. A kit comprising: a FLT1 binding protein of any one of embodiments 1 -12, 16-27, or 31-35, a FLT1 antibody of any one of claims 13-15, 28-30, or 36-39, the nucleic acid of embodiment 40, the vector of embodiment 41 , the host cell of embodiment 42 or43, or a combination thereof, and a container. A pharmaceutical composition comprising: the FLT1 binding protein of any one of claims 1-12, 16-27, or 31 -35, the FLT1 antibody of any one of claims 13-15, 28-30, or 36-39, the nucleic acid of embodiment 40, the vector of embodiment 41 , the host cell of embodiment 42 or 43, or a combination thereof, and a pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutical composition comprising: a means for inhibiting FLT1 bindingto VEGF, and a pharmaceutically acceptable carrier, excipient or diluent. A method of producing a FLT1 binding protein, said method comprising: culturing the host cell of embodiment 42 or 43 to express the FLT1 binding protein and harvesting the expressed FLT1 binding protein. A method of treating a subject in need thereof, comprising administering to the subject the FLT1 binding protein of any one of embodiments 1 -12, 16-27, or 31 -35, the FLT1 antibody of any one of claims 13-15, 28-30, or 36-39, orthe pharmaceutical composition of embodiment 45 or embodiment 46.A method of increasing blood flow, capillary density, capillary or coronary growth, and / ortissue perfusion in a subject in need thereof, said method comprising administeringto the subject the FLT1 binding protein of any one of embodiments 1 -12, 16-27, or 31 -35, the FLT1 antibody of any one of claims 13-15, 28-30, or 36-39, or the pharmaceutical composition of embodiment 45 or embodiment 46. The method of embodiment 48 or embodiment 49, wherein the subject has reduced blood flowthrough blood vessels to the limbs. The method of embodiment 50, wherein the reduced blood flow is caused by atherosclerosis. The method of embodiment 50, wherein the reduced blood flow is not caused by atherosclerosis. The method of any one of embodiments 48-50, wherein the subject has peripheral arterial disease (PAD) or critical limb ischemia (CLI). The method of any one of embodiments 48-49, wherein the subject has heart failure. The method of any one of embodiments 48-49, wherein the subject has angina. The method of any one of embodiments 48-49, wherein the subject has postpartum cardiomyopathy. The method of any one of embodiments 48-50, wherein the subject has high blood pressure. A method of treating microvascular dysfunction in a subject in need thereof, said method comprising administeringto the subject the FLT1 binding protein of any one of embodiments 1-12, 16-27, or 31 -35, the FLT1 antibody of any one of claims 13-15, 28- 30, or 36-39, orthe pharmaceutical composition of embodiment 45 or embodiment 46. The method of embodiment 58, wherein the subject has chronic kidney disease, diabetic neuropathy, diabetic neuropathy, a diabetic wound, or bronchopulmonary dysplasia. The pharmaceutical composition of claim 45 for use in a method of treating a cardiovascular disorder. he pharmaceutical composition of embodiment 60, wherein the cardiovascular disorder is selected from PAD, CLI, heart failure, angina, and postpartum cardiomyopathy.62. The pharmaceutical composition of embodiment 45 for use in a method of treating a microvascular dysfunction.63. The pharmaceutical composition of embodiment 62, wherein the subject has chronic kidney disease, diabetic neuropathy, diabetic neuropathy, a diabetic wound, or bronchopulmonary dysplasia.64. A method of treating a cardiovascular disorder in a subject, said method comprising: administeringto the subject havingthe cardiovascular disorder a composition comprising: a means for inhibiting FLT1 bindingto VEGF, and a pharmaceutically acceptable carrier, excipient or diluent.65. The method of embodiment 64, wherein the cardiovascular disorder is selected from PAD, CLI, heart failure, angina, and postpartum cardiomyopathy.66. A method of treating a microvascular dysfunction in a subject, said method comprising: administeringto the subject having a microvascular dysfunction a composition comprising: a means for inhibiting FLT1 bindingto VEGF, and a pharmaceutically acceptable carrier, excipient or diluent.67. The FLT1 binding protein of any one of embodiments 1-15, wherein said binding protein is an antibody, and said antibody comprises (i) a Tm of greater than 70°C, (ii) a viscosity at 150 mg / mL of <10 cP, (iii) an on-cell binding affinity (KD) to human FLT1 of greater than 100 pM, or (iv) any combination of (i)-(iii).68. The FLT1 binding protein of any one of embodiments 16-30, wherein said binding protein is an antibody, and said antibody comprises (i) a Tm of greater than 70°C, (ii) a viscosity at 150 mg / mL of <10 cP, or (iii) the combination of (i) and (ii).69. The FLT1 binding protein of any one of embodiments 16-30, wherein said binding protein is an antibody and said antibody comprises a binding affinity KDfor cell membrane expressed human FLT1 of between 50 pM and 500 pM as measured by KinExA.

[0236] The following examples are given merely to illustrate the present invention and not in any way to limit its scope.EXAMPLESEXAMPLE 1

[0237] This example describes the generation and screening of anti-FLT1 antibodies.

[0238] VEGF-induced signaling through VEGFR2 (also known as KDR) is critical for angiogenesis and the proliferation of card io myocytes and pericytes, and also is necessary for coronary growth and cardiac perfusion (Figure 1A). FLT1 (also known as VEGFR1) is a member of the VEGFR family of receptor tyrosine kinases (RTKs) that is, in some instances, expressed as a membrane receptor to which VEGF can bind. Due to its low kinase activity, FLT1 is considered as a decoy receptor for VEGF, sequestering VEGF and impeding VEGF-mediated signaling through VEGFR2 (De Vries et al., Science 255: 989-91 (1992); Kendall et al., PNAS 90: 10705-9 (1993); Hiratsuka et al., PNAS 95: 9349-54 (1998); Fong et al., Nature 376: 66-70 (1995); Shalaby et al., Nature 376: 62-6 (1995); Terman et al., Biochem Biophys Res Commun 187: 1579-86 (1992); and Gille et al., EMBO J 19: 4064-73 (2000). FLT1 alternatively is expressed as a secreted, soluble isoform and is noted herein as “sFLT1 ”. sFLT1 comprises the ligand binding domain of the membrane form of FLT1 and acts as an inhibitor of VEGF signaling, due to its ability to sequester VEGF and prevent it from binding VEGFR2. Reduced signaling through VEGFR2 results in impaired coronary growth and cardiac perfusion, capillary rarefaction and an angiogenic imbalance (Yu et al., Hypertension 68: 749-59 (2016); Grunewald et al., Science 373 (2021); and Zhao, et al., Aging Dis 8: 287-300 (2017)) which can lead to heart failure (Figure 1 B).

[0239] It was hypothesized that antibodies that could bind FLT1 (anti-FLT1 antibodies that bind to mFLT1 and sFLT1 ) and prevent FLT1 from binding to VEGF would fully restore VEGF- VEGFR2 signaling (Figure 1C). Antibodies that bind FLT1 and antagonize the FLT1 binding interactions with VEGF to promote VEGF / VEGFR2 signaling were hypothesized to be effective treatments for conditions involving impaired microcirculatory function.

[0240] Thus, a campaign to generate anti-FLT1 antibodies suitable as a human therapeutic was designed and implemented. More particularly, the campaign was designed to generate anti-FLT1 antibodies that would (a) block FLT1 from binding VEGF and / or liberate bound VEGF from FLT1-VEGF complexes, (b) bind to neither VEGFR2 (KDR) or VEGFR3, (c) cross-react with pig (porcine) FLT1 , dog (canine) FLT1 , and cynomolgus monkey FLT1 , and (d) exhibit high affinity for human sFLT1 (e.g., KD < about 20 pM) and pig sFLTI (e.g., KD < about 200 pM). The crossspecies reactivities would facilitate functional testing of the antibodies in pig and / or dog models of microcirculatory disorders and pharmacological studies of the antibodies in cynomolgus monkeys. Figure 2 illustrates several steps of the anti-FLT1 antibody campaign.

[0241] Immunization and Hybridoma Generation

[0242] Antibodies to human FLT1 were generated by immunizingXENOMOUSE® transgenic mice (U.S. Pat. Nos. 6,114,598; 6,162,963;6,833,268; 7,049,426; 7,064,244, which are incorporated herein by reference in their entirety; Green et al., 1994, Nature Genetics 7:13-21 ; Mendez et al., 1997, Nature Genetics 15:146-156; Green and Jakobovits, 1998, J. Ex. Med, 188:483-495; Kellerman and Green, Current Opinion in Biotechnology 13, 593-597, 2002) with immunogen comprising full length human FLT1 (amino acids 1-1338), or a portion thereof (comprising D1 -D3, the extracellular domains comprising the VEGF binding site (VEGF binds between D2 and D3)). A subset of mice were subsequently given a boost with r pig FLT1 . Mice of the XENOMOUSE® strains XMG2-K and XMG2-KL, which produce fully human lgG2 antibodies with kappa light chains (XMG2-K) or both kappa and lambda light chains (XMG2-KL), mice of the XENOMOUSE® strains XMG4-K and XMG4-KL, which produce fully human lgG4 antibodies with kappa light chains (XMG4-K) or both kappa and lambda light chains (XMG4-KL), among other mice, were immunized. Animals were bled, and plasma was collected at various time points during the immunization studies to identify animals producing FLT1 -specific titers.

[0243] FLT1 -specific serum titers were monitored by FACS. Briefly, cells were mock- transfected or transiently transfected to express FLT1 . Serum from immunized animals was diluted and incubated on the transfected cells. The cells were then washed to remove unbound antibodies and a secondary anti-human IgG Fc-specific antibody labeled with Cy5 was incubated on the cells. The cells were washed to remove unbound secondary antibody and the fluorescent signal on the cells was quantitated by FACS.

[0244] Animals with the highest antigen-specific serum native titers directed against FLT1 were identified through the FACS analysis described above and were selected for traditional hybridoma generation. Briefly, the spleen and / or draining lymph nodes were harvested from the selected animals and lymphocytes were dissociated from these lymphoid tissues by grinding in a suitable medium (for example, Dulbecco’s Modified Eagle Medium (DMEM); Invitrogen, Carlsbad, CA). The lymphocytes from each harvest were pooled together. B cells were selected and / or expanded using standard methods and then fused with a suitable fusion partner using techniques that were known in the art to generate hybridomas. The hybridomas were subsequently plated using FACS-based antigen specific sorting or by standard polyclonal plating techniques.

[0245] FACS Sorting of Hybridoma Cells

[0246] Hybridoma cells were removed from the flask and washed in sterile FACS buffer (2% FBS PBS). The cells were then incubated with a high concentration (200 ng / mL) or low concentration (4 ng / mL) of biotin-labeled antigen (D1 -D3 of sFLT1), washed again with FACS buffer and then stained with a detection cocktail containing Alexa Fluor 488 conjugated F(ab’)2 fragment goat anti-human IgG Fc (Jackson, Cat: 109-546-098) and Alexa Fluor 647 conjugated streptavidin (Jackson, Cat: 016-600-084). Cells were washed again in FACS buffer, resuspended in media and then put through a 40-micron cell strainer to remove aggregated cells. Antigen-specific cells were sorted using BD FACSAria 3 by gating on population exhibiting both Alexa Fluor 488 and Alexa Fluor 647 fluorescence (lgG+ and antigen binding cells). The sorted cells were cultured for a few days in hybridoma media. The hybridomas were then single cell sorted into multi-well microtiter plates using BD FACSAria 3. In some embodiments, hybridomas that expressed antibodies that bound to the high concentration of antigen (200 ng / mL) were also sorted based on their ability to function as competitive blockers of VEGF binding to sFLT1 . In other instances, hybridoma that expressed antibodies that bound to a low concentration of antigen (4 ng / ml) were sorted based on their ability to function as high affinity binders able to bind with limited amount of antigen available. A summary of the FACS workflow is shown in Figure 3 and the number of hits (by harvest and sort) is shown in Table 1 .TABLE 1

[0247] Quantification / Normalization

[0248] As shown in Table 1 , over 9600 binders across five harvests were identified by the FACS-based screen. Due to the expansion of the B cells prior to fusion, only about one-third of the hybridoma was selected for further analysis, as this was considered representative of the unique repertoire found in the hybridoma pool. The concentration of antibody in the collected hybridoma media supernatant for the selected binders was quantified using an Octet®-based method . Of 2714 binders selected to proceed to functional screening, more than 95% were at a concentration in the collected media greater than 10 pg / ml with the remainder greater than 7 pg / ml. The binders were normalized to 10 pg / ml with those binders less than 10 pg / ml being undiluted for use in subsequent assays.

[0249] pKDR Functional Assay in HUVEC Cells

[0250] Quantified / normalized binders (n=2714) were screened in a phosphorylated-VEGFR2 (pKDR) functional assay in HUVEC cells at a single point concentration (2 pg / mL). Briefly, HUVEC cells were cultured in endothelial basal medium (EBM) supplemented with EGM endothelial cell growth medium SingleQuots supplements. For cell treatment, HUVEC cells were cultured in 96-well plates and starved in EBM2 medium without supplements overnight. The next day, hybridoma supernatant comprising anti-FLT1 antibodies were prepared to a 2ug / ml final concentration in EBM2 medium and equilibrated with a final concentration of 3 nM of sFltl and 1 nM VEGF165 (active splice variant of VEGF-A that binds FLT1 and VEGFR2) for 30 minutes at room temperature. 1 nM VEGF165 alone or 1 nM VEGF165 and 3 nM sFltl without antibody were included as controls. The HUVEC cell culture plates were equilibrated to room temperature, the medium was withdrawn, and the treatment mixture was added. After incubation for 10 minutes at room temperature, the treatment mixture was removed, and cells were lysed. The cell lysate was used for quantification of phosphorylated VEGFR2 using a sandwich ELISA. Briefly, a 96-well immune plate was coated with the capture antibody for VEGFR2, and nonspecific binding sites were blocked. Cell lysate from treatment was added to the plates and incubated overnight at 4 °C, washed, incubated with the phosphor-tyrosine detection antibody, washed again, then incubated with a peroxidase-conjugated secondary antibody, and subsequently with a substrate solution. The absorbance was read in a spectrophotometer at 450 nm. The data were presented as a percentage of the restoration ofpVEGFR2 activity by a nti-sFlt1 antibody. Figure 4 shows exemplary results of the pKDR functional assay by sorted fraction and / or harvest.

[0251] Potency, Sequence, and Species Cross-Reactivity Analyses

[0252] Of the hits identified by the pKDR single point functional assay, 145 were further analyzed for potency, sequence, and species cross-reactivity. Potency was assayed using the same conditions as the single point pKDR functional assay but with the antibody concentration titrated from 13 nM to 0.00017 nM. Most hits lost activity after the first dilution step. Forty-five (45) were selected based on activity seen at the start concentration (greater than 40% at 13 nM) and retaining activity at the next dilution (greater than 20% at 2.6 nM).

[0253] A subset of hits identified by the pKDR functional assay were subjected to sequence analysis wherein the heavy chain and light chain of each were sequenced. Analysis of the heavy chain sequence identified 36 unique heavy chains having 16 different VDJ recombinations, 22 different CDR3 sequences and 29 different groups of three HC CDRs.

[0254] Thirty-six antibodies each having a unique heavy chain were tested for species crossreactivity. Cells expressing either cynomolgus monkey FLT1 , dog FLT1 , pig FLT1 , or rat FLT1 were used to assay antibody binding by a FACS-based screen. Briefly, cells expressing the species specific FLT1 were incubated with individual antibodies at 5 ug / ml for 1 hr at 4C. Following 2x wash with FACS buffer (1xPBS + 2% FBS), Alexa Fluor 647-conjugated affiniPure Goat anti-human IgG (Jackson 1009-605-098) was added as secondary and incubated for 15 minutes at 4C. Following a wash with FACS buffer, the cells were read on an Accuri FACS machine with I ntellicyte autosampler. Exemplary results are shown in Figure 5.

[0255] The same 36 antibodies were tested in a FLT1 receptor / ligand (R / L) inhibition assay. Briefly, 293T cells expressing human FLT1 were incubated with human anti-FLT1 antibodies and biotinylated human VEGF was added. Following a wash step, streptavidin Alexa Fluor-647 (SA- 647) and 7-aminoactinomycin D (7AAD) were added. Cells were washed and samples were analyzed using a BD Accuri™ Flow Cytometer with an I ntellicyt HyperCyt autoSampler. Of the 36 antibodies tested, 21 induced at least a 40% inhibition of receptor / ligand activity. Exemplary results are shown in Figure 6.

[0256] Octet Assays, Cross-Species Functional Assay and KinExA Affinity Measurements

[0257] The above assays led to the selection of 13 binders for further analysis.

[0258] The 13 antibodies were assessed for their ability to block VEGF and Placental Growth Factor (PIGF) binding to FLT1 in an Octet assay. Briefly, the individual antibodies werecaptured on anti-HuFc sensors (AHQ) cat#18-5005. sFLT1 (D1-D3 Promokine C64430) was bound to the captured antibodies with the subsequent binding of either VEGF or PIGF to the sFLT1 assessed. The percent of VEGF binding inhibited by the antibody and the percent of PIGF binding inhibited by the antibody are shown in Table 2 (2ndcolumn from left). These data support that, the antibodies bind to sFLT1 , which renders it unable to bind to VEGF or PIGF. Thes results confirm the ability of the antibodies to block the ligand binding activity of FLT1 .

[0259] The 13 antibodies were additionally assayed for cross-reactivity with KDR or FLT4 using an Octet assay. Briefly, the individual antibodies were captured on anti-HuFc sensors (AHQ) cat#18-5005 and assessed for binding to 50 nM_KDR or 50 nM FLT4. Results are provided in Table 2, 3rd column from left. As indicated here, none of the 13 antibodies demonstrated binding to KDR or FLT4.

[0260] To assess function across species, FLT1 protein from human, dog or pig were incubated with one of the 13 anti-FLT1 antibodies and the extent of KDR phosphorylation in the presence of 1 nM VEGF was measured. Exemplary results are shown in Figures 7A-7C. All 13 antibodies demonstrated activity against human FLT1 (with IC50s ranging from 0.6 nM to 2.1 nM), and also exhibited activity against dog FLT1 (with IC50s ranging from IC500.5 nM to 512 nM). The antibodies demonstrated lower activity against pig FLT1 (with IC50s ranging from 10.4 nM to 522 nM).

[0261] The Fabs of seven of the 13 antibodies were constructed and tested for affinity for human FLT1 (D1 -D3) using the KinExA® method, which is a solution-based determination of formal affinity measurements at equilibrium. The method was performed using an automated flow immunoassay system and poly(methyl methacrylate) or PMMA beads coated with biotinylated human FLT1 , which served as the solid phase. Briefly, Fabs were incubated with titrating concentrations of human FLT1 in sample buffer comprising BSA. FLT1 / Fab complexes were incubated at RT for several hours to allow equilibrium to be reached. The mixture was drawn through the FLT1 beads to accumulate unbound Fab. The captured Fab was detected using solutions containing a labeled secondary antibody in sample buffer. The bound signals were converted into relative values as a proportion of control in the absence of human FLT1 . The equilibrium dissociation constant (Kd) was obtained from nonlinear regression analysis of the data using a one-site homogeneous binding model contained within the KinExA n-curve analysis software. The software calculates the Kd and determines the 95% confidence interval by fitting the data points to a theoretical Kd curve. The 95% confidence interval is given as Kd low and Kd high. The method was repeated using pig FLT1 (D1-D3) in place of the human FLT1 for a subset of Fabs. The Kd values as measured by KinExA are reported in Table 2.

[0262] A summary of results from the cross-species functional assay, octet assays, and kinexa affinity assays is provided in Table 2.TABLE 2*The affinity of the Fabs of selected antibody leads for human FLT1 and / or pig FLT1 was ranked using a KinExA method. **Group 1 HC germline (VH3 / D7 / JH4); Group 2 HC germline (VH3 / D5 / JH3);Group 5 HC germline (VH3 / D3 / JH6); Group 8 HC germline (VH4 / D1 / JH6)

[0263] As shown in Table 2, only one antibody (11 D4) met the initial high affinity goal for human FLT1 (e.g., KD less than about 20 pM), though none of the antibodies demonstrated the initial design goal affinity for pig FLT1 (KD less than about 200 pM). Antibodies 10D10 and 10B4 were among 11 D4 as exhibiting high affinity for human FLT1 wherein the KD was less than about50 pM. Interestingly, each of these antibodies belonged to VDJ Group 8 and, thus, the HC and LC CDR sequences of these three antibodies (11 D4, 10D10 and 10B4) were very similar. The HC CDR1 and HC CDR3 were identical among the three antibodies, while the HC CDR2 varied at 3 positions, the LC CDR1 and LC CDR2 varied at 2 positions, and the LC CDR3 varied at one position. Table 3A lists consensus CDR sequences for 11 D4, 10D10 and 10B4, Table 3B lists the actual CDR sequences for each of these antibodies, and Table 3C provides the heavy chain (HC) and light chain (LC) variable region sequences.TABLE 3ATABLE 3BTABLE 3C

[0264] Taken together, this example demonstrated the generation of anti-FLT1 antibodies, and the selection of lead antibodies based on structural and functional characteristics. The data further support the structural similarity among the antibodies that exhibit the highest affinity for human FLT1 .EXAMPLE 2

[0265] This example demonstrates hotspot remediation and affinity maturation of FLT1 antibodies identified during screening.

[0266] The FLT1 antibody campaign described in Example 1 yielded an initial panel of lead antibodies that blocked the VEGF-FLT1 interaction, neutralized sFLT1 inhibition of VEGFR2 signaling in a functional assay and demonstrated potential to cross-react to some extent with FLT1 from dog and pig, the model species for cardiovascular and circulatory studies. However, none of the antibody leads met both the initial affinity design goals for human FLT1 (KD= 20 pM) and the initial affinity design goal for pig FLT1 (KD= 200 pM), when the Fab-FLT1 D1 -D3 interactions were assessed by KinExA (Table 2). Therefore, a single representative antibody from each of three major families of the leads (VDJ Groups 1 , 5, 8) was chosen for affinity maturation via CDR engineering as described below. Predicted chemical hotspot remediation was also addressed during the CDR engineering process. “Hotspots” are residues or sequence motifs that may isomerize, deamidate, and oxidize, and may negatively impact the molecule’s expression, purification, thermal stability, colloidal stability, long-term storage stability, in vivo pharmacokinetics, and / or immunogenicity. Among several siblings, 10B4 was chosen to represent VDJ group 8, because it had the fewest predicted hotspots and the highest sequenceidentity to germline. 28D8 was chosen to represent VDJ group 5 instead of 12F8, because 28D8 exhibited detectable binding to pig FLT1 . 10B4 had the best potential for meeting human affinity goals, but its binding affinity for pig FLT1 required improvement. Therefore, the focus for engineering 10B4 was improvement of binding affinity for pig FLT1 . The focus for engineering 10A7 and 28D8 was to improve their affinities for human FLT1 .

[0267] Affinity maturation involved constructingyeast-displayed Fab libraries and enriching for the mutants with enhanced binding as assessed by FACS using the following methods.

[0268] Yeast display binding experiments . The propagation and induction of yeast cells displaying antibodies for binding experiments have been described by Luna V. et al. (Luna V. et al., European Polymer Journal 109 (2018) 483-488). Flow cytometry experiments measuring binding of Fab-yeast cells to His-tagged or biotinylated antigens were conducted as described by Chao et al. (Chao et al., Nature Protocols 1 (2006) 755-768) in a phosphate-buffered salinebased binding buffer supplemented with blocking agent and detergent to minimize non-specific sticking to yeast cells. The employed Flt1 antigens foryeast display are composed of the first three domains (D1 -D3) or the second and third domains (D2-D3) of the total Flt1 extracellular domain. After incubation with the Fltl antigens, the yeast cells were washed thoroughly with binding buffer before stainingwith a Dylight650-conjugated anti-Fab antibody to detect surface display and a phycoerythrin (PE)-conjugated streptavidin or anti-His antibody to detect Fltl binding. The cells were washed additionally with binding buffer before flow cytometry analysis using a FACSCanto, LSRII, or Fortessa (BD Biosciences). Allophycocyanin (APC) and PE median fluorescence intensities (MFI) were recorded to measure surface display and binding, respectively.

[0269] FACS library sorting. At least 107cells were incubated with antigen as described above. Following fluorescent antibody staining, the cells were sorted using a FACS Aria (BD Biosciences) using the appropriate settings for allophycocyanin and phycoerythrin detection.

[0270] Design and construction of Fab libraries foryeast display affinity maturation. Deep mutational scanning of each anti-hu Fltl antibody was carried out to inform the design of affinity maturation libraries. Deep mutational scanning entails generating comprehensive libraries of point mutations, performing binding selections under different conditions, and evaluating changes in mutation frequencies after selections with high-throughput sequencing. (See e.g., Araya, C. L. and Fowler, D. M., Trends Biotechnol. 2011 Sep; 29(9): 435- 442). Identification of the subset of mutations associated with improved hu FLT1 bindingenables focused combinatorial library designs for affinity maturation without the benefit of a high-resolution antibody-antigen co-crystal structure.

[0271] For each parent antibody, CDR positions within the heavy chain (HC) and light chain (LC) were chosen for single-site saturation mutagenesis using oligos containing NNK codons. Yeast-displayed Fab libraries were generated wherein the comprehensive set of point mutations in the heavy and light chain CDRs were split into three libraries (two for heavy and one for light chain). Each library was sorted using FACS for surface expression and separately for binding to hu Fltl D1 -D3 at two different concentrations. After FACS, the plasmids from the growth-amplified pools were harvested using a Zymoprep II kit (Zymo Research) and amplicons were generated by minimal PCR amplification of the recovered plasmids.

[0272] Illumina MiSeq was used to assess the binding fitness of each generated point mutation by comprehensively assessing changes in the frequencies of mutations within the binding-sorted gates vs their starting frequencies (freq) within an expression-sorted reference pool. Binding enrichment (EF) factors for each mutation were calculated based on NGS analysis using the expression-sorted pools as reference samples (EF = freqbnd / freqexPreSSion ). CDR mutations that resulted in larger, comparable, and smaller binding enrichment factors to hu Fltl (D1 -D3) compared to the parent antibody are referred to as beneficial, neutral, and deleterious mutations, respectively.

[0273] The sequence-binding fitness maps generated for each CDR position were used to identify the most promising CDR positions to mutate for enhancing binding and to suggest beneficial and neutral mutations for incorporation into combinatorial library designs. The prioritized mutations are expected to enhance binding or remediate predicted chemical hotspots (asparagine deamidation, aspartate isomerization, tryptophan and methionine oxidation) without significant detriment to binding.

[0274] For each parent antibody, at least two combinatorial Fab-yeast libraries were designed for affinity maturation based on sequence-binding fitness data. Combinations of the desired mutations were introduced using PCR amplification with oligos containing multiple degenerate codons. Each degenerate codon representing a specific position was chosen to maximize the representation of the desired mutations within the encoded codon diversity. Mutagenized PCR products containing full-length Fab chain coding sequences were transformed into yeast along with linearized expression vectors for to construct the yeast display libraries by in vivo homologous recombination (Chao et al., Nature Protocols 1 (2006) 755-768). To keep the combinatorial theoretical diversities manageable for coverage by aroutine yeast library transformation, each library explored mutations within HC CDR loops paired with the parental LC or LC CDR loop mutations paired with the parental HC.

[0275] Tables 4 and 5 depict the positions for diversification and the diversification strategy at each chosen position within the light chain (Table 4) and heavy chain (Table 5) of 10B4, within the light chain (Table 6) and heavy chain (Table 7) of 10A7, and within the light chain (Table 8) and heavy chain (Table 9) of 28D8. Theoretical combinatorial diversity is provided in each table.

[0276] Sorting and screening libraries for improved binders. The constructed Fab-yeast libraries were enriched for cells exhibiting equivalent or improved binding relative to the parental Fab following established FACS methods (Chao et al., Nature Protocols 1 (2006) 755- 768). The surviving HC or LC mutations were recovered from the yeast pools and shuffled together during yeast transformation to construct a new set of libraries. For the 28D8 campaign, additional mutational diversity (italicized in Tables 8 and 9) was introduced at themutation-shuffling stage to further improve the binding. Higher-stringency selections for improved binders with slower binding off-rates were implemented on the shuffled libraries (Boder, E.T. and Wittrup, K.D. Methods Enzymol 328 (2000) 430-44).

[0277] Individual yeast clones from binding-enriched pools were plated for screening, induced for Fab display, and tested for binding to FLT1 antigens as described above. For each binding condition, the median PE fluorescence intensity of binding is divided by the median fluorescence intensity of Fab display to calculate a normalized binding value enabling comparison of yeast clones. Additionally, clones were optionally tested for slower apparent binding off-rate following an established competition method (Boder, E.T. and Wittrup, K.D. Methods Enzymol 328 (2000) 430-44). The yeast cells were first incubated with saturating concentrations (5-15 nM) of His-tagged or biotinylated FLT1 antigen, washed thoroughly, and resuspended in 125 nM of untagged or unlabeled FLT1 protein for at least two hours of binding competition. Yeast cells were stained and prepared for flow cytometry analysis as described above. The fraction of remaining bindingwas calculated by dividing the remaining normalized binding after competition by the initial normalized binding value. Multiple parameters governed top clone selection from the yeast display campaigns, including higher normalized FLT1 binding vs the parental Fab, slower binding off-rate (higher remaining binding after competition), preservation of lack of non-specific binding to huVEGFR2, and reduction of predicted chemical hotspots within CDR loops.

[0278] Through implementing the process above, in the first round of screening and selection, 70 variants of 10B4, 44 variants of 10A7, and 56 variants of 28D8 were identified as having as good or better binding to human FLT1 (relative to the corresponding parent clone, 10B4, 10A7, or 28D8). The SEQ ID NOs of the CDRs and variable regions for each variant, as well as the normalized bindingto human FLT1 (D2D3), are shown in Tables 10-12. The mutations found in these variants exhibiting improved human FLT1 binding are described in Tables 13-15. Amino acids shown in bold, underlined were found in greater than 85% of the variants, while italicized positions were mutated (relative to the parent) in greater than 85% of these variants.TABLE 10: 10B4 Variant CDR SEQ ID NOs and Normalized Binding Activityformalized to 10B4 on yeast binding to human FLT1TABLE 11 : 10A7 Variant CDR SEQ ID NOs and Normalized Binding Activityformalized to 10A7 on yeast bindingto human FLT1TABLE 12: 28D8 Variant CDR SEQ ID NOs and Normalized Binding Activityformalized to 28D8 on yeast binding to human FLT1TABLE 13: 10B4 MUTATIONS FOUND IN IMPROVED HU BINDERSPositions are in reference to SEQ ID NOs: 1088 and 1087 for 10B4 LC variable and 10B4 HC variable, respectively.TABLE 14: 10A7 MUTATIONS FOUND IN IMPROVED HU BINDERS*Positions are in reference to SEQ ID NOs: 1232 and 1231 for 10A7 LC variable and 10A7 HC variable, respectively.TABLE 15: 28D8 MUTATIONS FOUND IN IMPROVED HU BINDERS*Positions are in reference to SEQ ID NOs: 1322 and 1321 for 28D8 LC variable and 28D8 HC variable, respectively.

[0279] Analyses of the HC and LC variable region sequences of the variants listed in each of Tables 10-12 support the high structural similarity among the set of variants within each table (Tables 10-12). The sequence analyses involved aligning the HC variable region sequences of the variants of Table 10 to the HC variable region sequence of the parent (10B4) and separately aligning the LC variable region sequences of the variants of Table 10 to the LC variable region sequence of the parent (10B4). A consensus sequence for each of the HC variable region and the LC variable region were created, wherein the positions modified in at least one variant of Table 10, relative to the parent (10B4) are denoted with an X (Figure 8A). As shown in Figure 8A, there were 14 X’s in the consensus HC variable region, denoting 14 positions that were variedamong the variants in Table 10, and 13 X’s in the consensus LC variable region, denoting 13 positions that were varied among the variants in Table 10. As all the variants in Table 10 demonstrated binding to human FLT1 that was the same or better than the parent 10B4, amino acid substitutions at one or more of the positions denoted with an X may be made without losing activity and may improve binding activity. As shown in Figure 8A, the most amino acid substitutions within a HC variable region observed for any one variant of Table 10 was 9, and the most amino acid substitutions within a LC variable region observed for any one variant of Table10 was 8, supportingthat the variants of Table 10 had at least 90% sequence identity to the sequence of 10B4- each of the variants of Table 10 had a HC variable region sequence having at least 92.6% sequence identity to the 10B4 HC variable region sequence and a LC variable region sequence having at least 92.8% sequence identity to the 10B4 LC variable region sequence.

[0280] Analyses involving HC variable region and LC variable region sequence alignments were carried out for (A) the variants of Table 11 , wherein the reference sequences were the HC variable region and LC variable region sequences of the parent 10A7, and (B) the variants of Table 12, wherein the reference sequences were the HC variable region and LC variable region sequences of the parent 28D8. The results are shown in Figures 8B and 8C for the 10A7 variants of Table 11 and the 28D8 variants of Table 12, respectively.

[0281] Figure 8B provides a consensus sequence for each of the HC variable region and the LC variable region created upon alignment. The positions modified in at least one variant of Table 11 , relative to the parent 10A7 are denoted with an X (Figure 8B). As shown in Figure 8B, there were 11 X’s in the consensus HC variable region, denoting 11 positions that were varied among the variants in Table 11 , and 7 X’s in the consensus LC variable region, denoting 7 positions that were varied among the variants in Table 11 . As all the variants in Table 11 demonstrated binding to human FLT1 that was the same or better than the parent 10A7, amino acid substitutions at one or more of the positions denoted with an X may be made without losing activity and may improve binding activity. As shown in Figure 8B, the most amino acid substitutions within a HC variable region for any one variant of Table 11 was observed as 10, and the most amino acid substitutions within a LC variable region for any one variant of Table11 was observed as 5, supporting that the variants of Table 11 had at least 90% sequence identity to the sequence of 10A7 - each of the variants of Table 11 had a HC variable region sequence having at least 91 .4% sequence identity to the 10A7 HC variable region sequence and a LC variable region sequence having at least 95.5% sequence identity to the 10A7 LC variable region sequence.

[0282] Figure 8C provides a consensus sequence for each of the HC variable region and the LC variable region created upon alignment. The positions modified in at least one variant of Table 12, relative to the parent 28D8 are denoted with an X (Figure 8C). As shown in Figure 8C, there were 10 X’s in the consensus HC variable region, denoting 10 positions that were varied among the variants in Table 12, and 7 X’s in the consensus LC variable region, denoting 7 positions that were varied among the variants in Table 12. As all the variants in Table 12 demonstrated binding to human FLT1 that was the same or better than the parent 28D8, amino acid substitutions at one or more of the positions denoted with an X may be made without losing activity and may improve binding activity. As shown in Figure 8C, the most amino acid substitutions within a HC variable region for any one variant of Table 12 was observed as 8, and the most amino acid substitutions within a LC variable region for any one variant of Table 11 was observed as 5, supportingthat the variants of Table 12 had at least 90% sequence identity to the sequence of 28D8 - each of the variants of Table 12 had a HC variable region sequence having at least 93.7% sequence identity to the 28D8 HC variable region sequence and a LC variable region sequence having at least 95.6% sequence identity to the 28D8 LC variable region sequence.

[0283] Interestingly, many of the X’s of the consensus sequences in Figures 8A-8C fall within a CDR of the HC variable or LC variable regions, supporting that amino acid modifications may be made in the CDRs without loss of FLT1 binding activity.

[0284] This example de...

Claims

1. WHAT IS CLAIMED IS:

1. A FLT1 binding protein comprising: a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1090.

2. The FLT1 binding protein of claim 1 , wherein: the VH comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1089, and the VL comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1090.

3. The FLT1 binding protein of claim 1 or claim 2, wherein the VH comprises the Tyr at position 35, the Tyr at position 52, the Tyr at position 54, the Tyr at position 60, the Leu at position 102, the Leu at position 105, and the Leu at position 106 of SEQ ID NO: 1089; and the VL comprises the Tyr at position 50, the Ser at position 51 , the Asn at position 53, the His at position 54, the Trp at position 92, and the Asp at position 94 of SEQ ID NO: 1090.

4. The FLT1 binding protein of claim 3, wherein the FLT1 binding protein binds to an epitope comprising residues F172, P173, K217, L174, L215, and L221 of human FLT1 (SEQ ID NO: 1453).

5. The FLT1 binding protein of claim 1 or claim 2, wherein the VH comprises a complementarity determining region 1 (HCDR1 ), a HCDR2, and a HCDR3 of SEQ ID NOs: 1485, 1488, and 1491 , respectively, and the VL comprises a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs; 1494, 1497, 1500, respectively.

6. The FLT1 binding protein of claim 1 or claim 2, wherein the VH comprises a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1487,1490, and 1493, respectively, andthe VL comprises a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs; 1496, 1499, 1502, respectively.

7. The FLT1 binding protein of claim 1 or claim 2, whereinI. the VH comprises a. an HC complementarity-determining region (CDR) 1 amino acid sequence of any one of SEQ ID NOs: 7, 151 , 181 , 331 ; b. an HC CDR2 amino acid sequence of any one of SEQ ID NO: 8, 152, 182, 332; c. an HC CDR3 amino acid sequence of any one of SEQ ID NO: 9, 153, 183, 333; or d. a combination thereof, andII. the VL comprises a. an LC CDR1 amino acid sequence of any one of SEQ ID NOs: 10, 154, 184, 334; b. an LC CDR2 amino acid sequence of any one of SEQ ID NO: 11 , 155, 185, 335; c. an LC CDR3 amino acid sequence of any one of SEQ ID NO: 12, 156, 186, 336; or d. a combination thereof.

8. The FLT1 binding protein of claim 1 or claim 2, wherein the VH comprises HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NO: 1089; and the VL comprises LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 1090.

9. The FLT1 binding protein of claim 1 or claim 2, wherein the VH comprises a HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 7-9, respectively, and the VL comprises a LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 10-12, respectively.

10. A FLT1 binding protein comprising:a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), a HCDR2, and a HCDR3 of SEQ ID NOs: 1485, 1488, and 1491 , respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs; 1494, 1497, 1500, respectively.11 . The FLT1 binding protein of claim 10, wherein the VH comprises the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 1487, 1490, and 1493, respectively, and the VL comprises the LCDR1 , the LCDR2, and the LCDR3 of SEQ ID NOs; 1496, 1499, 1502, respectively.

12. The FLT1 binding protein of claim 11 , wherein the VH comprises the HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 7-9, respectively, and the VL comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 10-12, respectively.

13. A FLT1 antibody comprising: a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), HC CDR2, and HC CDR3 of SEQ ID NOs: 7-9, respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), LC CDR2, and LC CDR3 of SEQ ID NOs: 10-12, respectively.

14. The FLT1 antibody of claim 13, wherein the VH comprises the amino acid sequence SEQ ID NO: 1089, and the VL comprises the amino acid sequence of SEQ ID NO: 1090.

15. The FLT1 antibody of claim 13, wherein the antibody comprises: a heavy chain amino acid sequence of SEQ ID NO: 1460 or 1572 and a light chain amino acid sequence of SEQ ID NO: 1461 .

16. A FLT1 binding protein comprising: a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1335, anda light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1336.

17. The FLT1 binding protein of claim 16, wherein: the VH comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1335, and the VL comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1336.

18. The FLT1 binding protein of claim 16 or claim 17, wherein the VH comprises the His at position 35, the Trp at position 52, the Asn at position 57, the Tyr at position 59, the Asp at position 62, the Tyr at position 102, the Pro at position 104, the Tyr at position 108, and the Tyr at position 109 of SEQ ID NO: 1335; and the VL comprises the Gin at position 27, the His at position 31 , the Phe at position 37, the Tyr at position 99, the Arg at position 101 , and the Asp at position 1 of SEQ ID NO: 1336.

19. The FLT1 binding protein of claim 18, wherein said binding protein binds to an epitope comprisingY139, S140, F172, L204, Y216, K217, N219, and N100 of human FLT1 (SEQ ID NO: 1453).

20. The FLT1 binding protein of claim 16 or claim 17, wherein the VH comprises a complementarity determining region 1 (HCDR1 ), a HCDR2, and a HCDR3 of SEQ ID NOs: 1512, 1515, and 1518, respectively, and the VL comprises a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs; 1521 , 1524, and 1527, respectively.

21. The FLT1 binding protein of claim 16 or claim 17, wherein the VH comprises a HCDR1 , a HCDR2, and a HCDR3 of SEQ ID NOs: 1514, 1517, and 1520, respectively, and the VL comprises a LCDR1 , a LCDR2, and a LCDR3 of SEQ ID NOs: 1523, 1526, 1529, respectively.

22. The FLT1 binding protein of claim 16 or claim 17, whereinI. the VH comprisesa. an HC complementarity-determining region (CDR) 1 amino acid sequence of any one of SEQ ID NOs: 733, 1027 and 1033, b. an HC CDR2 amino acid sequence of any one of SEQ ID NO: 734, 1028, and 1034, c. an HC CDR3 amino acid sequence of any one of SEQ ID NO: 735, 1029, and 1035; or e. a combination thereof, andII. the VL comprises a. an LC CDR1 amino acid sequence of any one of SEQ ID NOs: 736, 1030, and 1036, b. an LC CDR2 amino acid sequence of any one of SEQ ID NO: 737, 1031 , and 1037; c. an LC CDR3 amino acid sequence of any one of SEQ ID NO: 738, 1032, and 1038; or d. or a combination thereof.

23. The FLT1 binding protein of claim 16 or claim 17, wherein the VH comprises HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NO: 1335; and the VL comprises LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 1336.

24. The FLT1 binding protein of claim 16 or claim 17, wherein the VH comprises the HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 733- 735, respectively, and the VL comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 736- 738, respectively.

25. A FLT1 binding protein comprising: a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), a HCDR2, and a HCDR3 of SEQ ID NOs: 1512, 1515, 1518, respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), a LCDR2, and a LCDR3 of SEQ ID NOs; 1521 , 1524, and 1527, respectively.

26. The FLT1 binding protein of claim 25, whereinthe VH comprises the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 1514, 1517, and 1520, respectively, and the VL comprises the LCDR1 , the LCDR2, and the LCDR3 of SEQ ID NOs: 1523, 1526, and 1529, respectively.

27. The FLT1 binding protein of claim 26, wherein the VH comprises the HC CDR1 , HC CDR2, and HC CDR3 of SEQ ID NOs: 733-735, respectively, and the VL comprises the LC CDR1 , LC CDR2, and LC CDR3 of SEQ ID NOs: 736-738, respectively28. A FLT1 antibody comprising: a heavy chain variable region (VH) comprising a complementarity determining region 1 (HCDR1), HC CDR2, and HC CDR3 of SEQ ID NOs: 733-735, respectively, and a light chain variable region (VL) comprising a complementarity determining region 1 (LCDR1 ), LC CDR2, and LC CDR3 of SEQ ID NOs: 736-738, respectively.

29. The FLT1 antibody of claim 28, wherein the VH comprises the amino acid sequence SEQ ID NO: 1335, and the VL comprises the amino acid sequence of SEQ ID NO: 1336.

30. A FLT1 antibody of claim 28, wherein the antibody comprises: a heavy chain amino acid sequence of SEQ ID NO: 1462 or 1573 and a light chain amino acid sequence of SEQ ID NO: 1463.31 . The FLT1 binding protein of any one of claims 1 -12 and 16-27, which comprises or is an antigen-binding antibody fragment.

32. The FLT1 binding protein of any one of claims 1 -12 and 16-27, which comprises or is an antibody.

33. The FLT1 binding protein of claim 32, wherein the antibody comprises a human IgG HC constant region and a human LC constant region.

34. The FLT1 binding protein of claim 33, wherein the human IgG HC constant region is a human IgGI constant region and the human LC constant region is a human kappa LC constant region or a human lambda LC constant region.

35. The FLT1 binding protein of claim 32 wherein the HC constant region comprises any one of SEQ ID NOs: 1530-1533 or an amino acid sequence which has at least 80% sequence identity to said SEQ ID NOs: 1530-1533, and / or the LC constant region comprises any one of SEQ ID NOs: 1546-1547 or an amino acid sequence which has at least 80% sequence identity to said SEQ ID NOs: 1546-1547.

36. A nucleic acid comprising a nucleotide sequence encoding a FLT1 binding protein of any one of claims 1 -12, 16-27, and 31-35 or a FLT1 antibody of any one of claims 13-15 and 28-30.

37. Avector comprising the nucleic acid of claim 36.

38. A host cell comprising the nucleic acid of claim 36 orthe vector of claim 37.

39. The host cell of claim 38, comprising (A) a first nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NOs: 1089, 1137, 1197, 1147, 1277, 1293, 1283, 1437, 1435, and 1335, or an amino acid sequence comprising at least 90% sequence identity to any one of SEQ ID NOs: 1089, 1137, 1197, 1147, 1277, 1293, 1283, 1437, 1435, and 1335; and (B) a second nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NOs: 1090, 1138, 1198, 1148, 1278, 1294, 1284, 1438, 1436, and 1336 or an amino acid sequence comprising at least 90% sequence identity to any one of SEQ ID NOs: 1090, 1138, 1198, 1148, 1278, 1294, 1284, 1438, 1436, and 1336.

40. A kit comprising: a FLT1 binding protein of any one of claims 1-12, 16-27, and 31 -35, a FLT1 antibody of any one of claims 13-15 and 28-30, a nucleic acid of claim 36, a vector of claim 37, a host cell of claim 38 or 39, or a combination thereof, and a container.41 . A pharmaceutical composition comprising: the FLT1 binding protein of any one of claims 1 -12, 16-27, and 31 -35, the FLT1 antibody of any one of claims 13-15 and 28-30, the nucleic acid of claim 36, a vector of claim 37, the host cell of claim 38 or 39, or a combination thereof, and a pharmaceutically acceptable carrier, excipient or diluent.

42. A pharmaceutical composition comprising: a means for inhibiting FLT1 binding to VEGF, anda pharmaceutically acceptable carrier, excipient or diluent.

43. A method of producing a FLT1 binding protein, said method comprising: culturing the host cell of claim 38 or 39 to express the FLT1 binding protein and harvesting the expressed FLT1 binding protein.

44. A method of treating a subject in need thereof, comprising administering to the subject the FLT1 binding protein of any one of claims 1 -12, 16-27, and 31 -35, the FLT1 antibody of any one of claims 13-15 and 28-30, orthe pharmaceutical composition of claim 41 or claim 42.

45. A method of increasing blood flow, capillary density, capillary or coronary growth, and / or tissue perfusion in a subject in need thereof, said method comprising administering to the subject the FLT1 binding protein of any one of claims 1-12, 16-27, and 31-35, the FLT1 antibody of any one of claims 13-15 and 28-30, or the pharmaceutical composition of claim 41 or claim 42.

46. The method of claim 44 or 45, wherein the subject has reduced blood flow through blood vessels to the limbs.

47. The method of claim 46, wherein the reduced blood flow is caused by atherosclerosis.

48. The method of claim 46, wherein the reduced blood flow is not caused by atherosclerosis.

49. The method of any one of claims 44-45, wherein the subject has peripheral arterial disease (PAD) or critical limb ischemia (CLI).

50. The method of any one of claims 44-45, wherein the subject has heart failure.51 . The method of any one of claims 44-45, wherein the subject has angina.

52. The method of any one of claims 44-45, wherein the subject has postpartum cardiomyopathy.

53. The method of any one of claims 44-50, wherein the subject has high blood pressure.

54. A method of treating microvascular dysfunction in a subject in need thereof, said method comprising administering to the subject the FLT1 binding protein of any one of claims 1-12, 16-27, and 31 -35, the FLT1 antibody of any one of claims 13-15 and 28-30, or the pharmaceutical composition of claim 41 or claim 42.

55. The method of claim 54, wherein the subject has chronic kidney disease, diabetic neuropathy, diabetic neuropathy, a diabetic wound, or bronchopulmonary dysplasia.

56. The pharmaceutical composition of claim 42 for use in a method of treating a cardiovascular disorder.

57. The pharmaceutical composition of claim 54, wherein the cardiovascular disorder is selected from PAD, CLI, heart failure, angina, and postpartum cardiomyopathy.

58. The pharmaceutical composition of claim 42 for use in a method of treating a microvascular dysfunction.

59. The pharmaceutical composition of claim 57, wherein the subject has chronic kidney disease, diabetic neuropathy, diabetic neuropathy, a diabetic wound, or bronchopulmonary dysplasia.

60. A method of treating a cardiovascular disorder in a subject, said method comprising: administeringto the subject havingthe cardiovascular disorder a composition comprising: a means for inhibiting FLT1 bindingto VEGF, and a pharmaceutically acceptable carrier, excipient or diluent.61 . The method of claim 54, wherein the cardiovascular disorder is selected from PAD, CLI, heart failure, angina, and postpartum cardiomyopathy.

62. A method of treating a microvascular dysfunction in a subject, said method comprising: administeringto the subject having a microvascular dysfunction a composition comprising: a means for inhibiting FLT1 bindingto VEGF, and a pharmaceutically acceptable carrier, excipient or diluent.

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