Anti-VEGFR1 Antibodies and Their Uses

Abstract

Disclosed herein are antibodies or antigen-binding fragments thereof that bind to vascular endothelial growth factor receptor 1 (VEGFR1), polynucleotides, vectors, host cells, and methods of using same to treat chronic kidney disease.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 233,343, filed August 16, 2021, entitled "ANTI-VEGFR1 ANTIBODIES AND USES THEREOF," and U.S. Provisional Patent Application No. 63 / 322,273, filed March 22, 2022, each of which is incorporated by reference in its entirety herein.

[0002] (Reference to electronically submitted sequence listing) This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy was created on July 22, 2022, is named JBI6512WOPCT1_SL.xml, and is 181,681 bytes in size.

[0003] FIELD OF THEINVENTION Provided are proteins, polynucleotides, vectors, host cells comprising an antigen-binding domain that binds to vascular endothelial growth factor receptor 1 (VEGFR1) or fragments thereof, and methods of using same to treat chronic kidney disease. [Background technology]

[0004] Chronic kidney disease (CKD) is a global public health problem that continues to grow with an aging population and a diabetes / obesity epidemic. CKD increases the risk of all-cause and cardiovascular mortality, as well as end-stage renal disease requiring dialysis or kidney transplantation for survival. Patients with CKD stages 4-5 are at highest risk of renal failure, with a 5-year mortality rate of approximately 40%. Diabetes (38%) and hypertension (25%) are the leading causes of late-stage and end-stage renal disease. Diabetic kidney disease, the most common form of CKD, occurs in approximately 25% of type 2 diabetes patients within 10 years of diagnosis. Despite standard treatment of renin-angiotensin-aldosterone system inhibitors, i.e., angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, or the recent introduction of SGLT2 inhibitors, most "at risk" patients or poor responders still progress to end-stage renal disease. In the United States alone, over 100,000 patients progress to dialysis annually. 70% of patients on dialysis die within 5 years.

[0005] There remains a great medical need for therapies that prevent or treat end stage renal disease by renal protection or functional restoration in patients with advanced CKD.

[0006] In the kidney, vascular endothelial growth factor A (VEGFA), produced by glomerular podocytes and tubular epithelial cells, is essential to maintain glomerular integrity, renal microvasculature, and function. In CKD patients, mRNA microarray analysis of patient kidney biopsies shows decreased VEGFA expression, and the level of VEGFA expression in the glomerulus and tubulointerstitium correlates with eGFR, proteinuria, or vascular rarefaction (Bortoloso, Del Prete et al. 2004, Martini, Nair et al. 2014, Pan, Jiang et al. 2018). In healthy kidneys, locally produced VEGFA, signaling through VEGFR2, promotes the glomerular filtration barrier that protects endothelial health. In CKD, VEGFA deficiency leads to suppression of VEGFR2 signaling along with sequestration by its decoy receptor VEGFR1. Anti-VEGFR1 blocking antibodies have been developed to block VEGFA sequestration and increase local VEGFA availability, allowing endothelial protection, preservation of the glomerular filtration barrier and recovery of renal function. Summary of the Invention

[0007] Provided herein is an isolated antibody, or antigen-binding fragment thereof, that binds to an epitope within SEQ ID NO: 173 of VEGFR1 and prevents binding of VEGFA to VEGFR1.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof binds VEGFR1 to an epitope on VEGFR1 having the amino acid sequence FPLDTL (SEQ ID NO: 143) or EIGL (SEQ ID NO: 144). In some embodiments, the antibody or antigen-binding fragment thereof binds VEGFR1 to an epitope on VEGFR1 having the amino acid sequence FPLDTL (SEQ ID NO: 143) and EIGL (SEQ ID NO: 144).

[0009] In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to human, mouse, rat and / or cynomolgus VEGFR1.

[0010] In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to human VEGFR1. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to human VEGFR1 and VEGFR1 from at least one species selected from the group consisting of cynomolgus monkey, mouse, and rat. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to human VEGFR1 and VEGFR1 from at least two species selected from the group consisting of cynomolgus monkey, mouse, and rat. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to human, cynomolgus monkey, mouse, and rat VEGFR1.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof has a molecular weight of 6×10 -8 M or less, especially 1×10 -8 M or less, more specifically 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 It binds to human VEGFR1, mouse VEGFR1, and cynomolgus VEGFR1 with a KD of less than M.

[0012] In some embodiments, the isolated antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region (VH) having a heavy chain complementarity determining region (HCDR) of the VH comprising the amino acid sequence of SEQ ID NO: 31, 33, 34, 36, 38, or 39, and a light chain variable region (VL) having a light chain complementarity determining region (LCDR) of the VL comprising the amino acid sequence of SEQ ID NO: 32, 35, 37, or 40.

[0013] In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a. the HCDR of VH having the amino acid sequence of SEQ ID NO: 31, and the LCDR of VL having the amino acid sequence of SEQ ID NO: 32; b. the HCDR of the VH having the amino acid sequence of SEQ ID NO: 33, and the LCDR of the VL having the amino acid sequence of SEQ ID NO: 32; c. an HCDR of VH having the amino acid sequence of SEQ ID NO: 34, and an LCDR of VL having the amino acid sequence of SEQ ID NO: 35; d. an HCDR of VH having the amino acid sequence of SEQ ID NO: 36, and an LCDR of VL having the amino acid sequence of SEQ ID NO: 37; e. a VH HCDR having the amino acid sequence of SEQ ID NO: 38 and a VL LCDR having the amino acid sequence of SEQ ID NO: 37; or f. A VH HCDR having the amino acid sequence of SEQ ID NO:39, and a VL LCDR having the amino acid sequence of SEQ ID NO:40.

[0014] Provided herein is an isolated antibody or antigen-binding fragment thereof, the isolated antibody or antigen-binding fragment thereof comprising: SEQ ID NOs: 7, 175, 9, 10, 11, and 12, respectively; SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively; SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively; SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively; SEQ ID NOs: 71, 176, 73, 74, 75, and 76, respectively; SEQ ID NOs: 71, 72, 73, 74, 75, and 76, respectively; SEQ ID NOs: 77, 78, 79, 80, 81, and 82, respectively; SEQ ID NOs: 83, 84, 85, 86, 87, and 88, respectively; SEQ ID NOs: 89, 90, 91, 92, 93, and 94, respectively; SEQ ID NOs: 95, 177, 97, 98, 99, and 100, respectively; SEQ ID NOs: 95, 96, 97, 98, 99, and 100, respectively; SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively; SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively; SEQ ID NOs: 113, 114, 115, 116, 117, and 118, respectively; SEQ ID NOs: 119, 178, 121, 122, amino acid sequence LNS, and SEQ ID NO: 124, SEQ ID NOs: 119, 120, 121, 122, amino acid sequence LNS, and SEQ ID NO: 124, SEQ ID NOs: 125, 126, 127, 128, amino acid sequence FNF, and SEQ ID NO: 130, SEQ ID NO: 131, 132, 133, 134, amino acid sequence YD, and SEQ ID NO: 136, respectively; or SEQ ID NOs: 137, 138, 139, 140, amino acid sequence FNS, and SEQ ID NO: 142, and the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0015] The present disclosure further provides an isolated antibody, or antigen-binding fragment thereof, that binds to VEGFR1, VH of SEQ ID NO: 31 and VL of SEQ ID NO: 32; VH of SEQ ID NO: 33 and VL of SEQ ID NO: 32; VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35; VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37; VH of SEQ ID NO: 38 and VL of SEQ ID NO: 37; or It comprises a VH of sequence number 39 and a VL of sequence number 40.

[0016] The disclosure further includes an isolated antibody or antigen-binding fragment thereof, wherein the VH is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO:39 and the VL is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO:40.

[0017] The disclosure further includes an isolated antibody or antigen-binding fragment thereof, wherein the VH is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO:31 and the VL is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO:32.

[0018] The disclosure further includes an isolated antibody or antigen-binding fragment thereof, wherein the VH is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 33 and the VL is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO: 32.

[0019] The disclosure further includes an isolated antibody or antigen-binding fragment thereof, wherein the VH is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO:34 and the VL is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO:35.

[0020] The disclosure further includes an isolated antibody or antigen-binding fragment thereof, wherein the VH is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 36 and the VL is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO: 37.

[0021] The disclosure further includes an isolated antibody or antigen-binding fragment thereof, wherein the VH is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 38 and the VL is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO: 37.

[0022] The disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60.

[0023] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 and comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:51 or 52.

[0024] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 and comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:53 or 52.

[0025] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 and comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:54 or 55.

[0026] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 and comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:56 or 57.

[0027] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 and comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:58 or 57.

[0028] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 and comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:59 or 60.

[0029] In some embodiments, the isolated antibody of the present application comprises: a. HC comprising the amino acid sequence of SEQ ID NO:51 and LC comprising the amino acid sequence of SEQ ID NO:52; b. HC comprising the amino acid sequence of SEQ ID NO:53 and LC comprising the amino acid sequence of SEQ ID NO:52; c. HC comprising the amino acid sequence of SEQ ID NO:54 and LC comprising the amino acid sequence of SEQ ID NO:55; d. HC comprising the amino acid sequence of SEQ ID NO:56 and LC comprising the amino acid sequence of SEQ ID NO:57; e. a HC comprising the amino acid sequence of SEQ ID NO:58 and a LC comprising the amino acid sequence of SEQ ID NO:57; or f. A HC comprising the amino acid sequence of SEQ ID NO:59 and a LC comprising the amino acid sequence of SEQ ID NO:60.

[0030] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 7, 175, 9, 10, 11, and 12, respectively; VH of SEQ ID NO: 31 and VL of SEQ ID NO: 32, and / or HC of SEQ ID NO: 51 and LC of SEQ ID NO: 52, The present invention includes an antibody or an antigen-binding fragment thereof that binds to VEGFR1.

[0031] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; VH of SEQ ID NO: 33 and VL of SEQ ID NO: 32, and / or HC of SEQ ID NO: 53 and LC of SEQ ID NO: 52, An antibody or an antigen-binding fragment thereof that binds to VEGFR1.

[0032] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively; VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35, and / or HC of SEQ ID NO: 54 and LC of SEQ ID NO: 55, The present invention includes an antibody or an antigen-binding fragment thereof that binds to VEGFR1.

[0033] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively; VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37, and / or HC of SEQ ID NO: 56 and LC of SEQ ID NO: 57, The present invention includes an antibody or an antigen-binding fragment thereof that binds to VEGFR1.

[0034] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively; VH of SEQ ID NO: 38 and VL of SEQ ID NO: 37, and / or HC of SEQ ID NO:58 and LC of SEQ ID NO:57, The present invention includes an antibody or an antigen-binding fragment thereof that binds to VEGFR1.

[0035] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively; VH of SEQ ID NO: 39 and VL of SEQ ID NO: 40, and / or HC of SEQ ID NO:59 and LC of SEQ ID NO:60, The present invention includes an antibody or an antigen-binding fragment thereof that binds to VEGFR1.

[0036] In some embodiments, the isolated antibody or antigen-binding fragment thereof is conjugated to a half-life extending moiety. Optionally, the half-life extending moiety is an immunoglobulin (Ig), a fragment of Ig, an Ig constant region, a fragment of an Ig constant region, an Fc region, transferrin, albumin, an albumin binding domain, or polyethylene glycol. Optionally, the fragment of an Ig constant region comprises an Fc region. Optionally, the antibody or antigen-binding fragment thereof that binds to VEGFR1 is conjugated to the C-terminus of an Ig constant region or a fragment of an Ig constant region.

[0037] Optionally, the Ig constant region or fragment of an Ig constant region is an IgG1, IgG2, IgG3, or IgG4 isotype. Optionally, the Ig constant region or fragment of an Ig constant region is an IgG1 isotype. Optionally, the Ig constant region or fragment of an Ig constant region is an IgG4 isotype. Optionally, the Ig constant region or fragment of an Ig constant region comprises at least one mutation that results in reduced binding of the protein to an Fcγ receptor (FcγR). Optionally, the at least one mutation that results in reduced binding to an FcγR is selected from the group consisting of F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331 A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, where residue numbering is according to the EU index. Optionally, the mutation that results in reduced binding of the protein to FcγR is L234A_L235A_D265S. Optionally, the Ig constant region or fragment of an Ig constant region comprises at least one mutation that modulates the half-life of the protein. Optionally, the at least one mutation that modulates the half-life of the protein is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, where residue numbering is according to the EU index.

[0038] The disclosure further provides an immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof conjugated to a therapeutic or imaging agent.

[0039] The present invention further provides a pharmaceutical composition comprising an isolated antibody, or antigen-binding fragment thereof, that binds to VEGFR1 and a pharma- ceutically acceptable carrier.

[0040] The disclosure further provides polynucleotides encoding the isolated antibodies or antigen-binding fragments thereof that bind to VEGFR1.

[0041] Optionally, the polynucleotide encoding the isolated antibody or antigen-binding fragment thereof that binds VEGFR1 comprises the polynucleotide sequence of SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 74, 65, 66, 67, 68, 69, or 70.

[0042] Optionally, a polynucleotide encoding an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a polynucleotide that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the polynucleotide sequence of SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70.

[0043] The present disclosure also provides vectors comprising the polynucleotides of the present disclosure.

[0044] The present invention further provides a host cell comprising a polynucleotide or vector of the present disclosure.

[0045] The present disclosure further provides host cells that express the isolated antibodies or antigen-binding fragments thereof and / or contain the polynucleotides or vectors of the disclosure.

[0046] The present disclosure further provides a method for preventing the binding of VEGFA to VEGFR1 in a subject in need thereof, comprising administering to the subject an effective amount of any of the isolated antibodies or antigen-binding fragments thereof of the present disclosure, any of the immunoconjugates of the present disclosure, any of the pharmaceutical compositions of the present disclosure, any of the isolated polynucleotides or vectors of the present disclosure, or any of the host cells of the present disclosure, thereby preventing the binding of VEGFA to VEGFR1.

[0047] The present disclosure further provides a method of treating and slowing the progression of chronic kidney disease (CKD) in a subject, comprising administering to the subject a therapeutically effective amount of any of the isolated antibodies or antigen-binding fragments thereof of the present disclosure, any of the immunoconjugates of the present disclosure, any of the pharmaceutical compositions of the present disclosure, any of the isolated polynucleotides or vectors of the present disclosure, or any of the host cells of the present disclosure, for a time sufficient to treat CKD. Optionally, the subject has advanced stage 4 or stage 5 chronic kidney disease. Optionally, the subject has early stage chronic kidney disease.

[0048] The present disclosure further provides a kit comprising any of the isolated antibodies or antigen-binding fragments thereof of the present disclosure, any of the immunoconjugates of the present disclosure, any of the pharmaceutical compositions of the present disclosure, any of the isolated polynucleotides or vectors of the present disclosure, or any of the host cells of the present disclosure.

[0049] The present disclosure further provides a method of treating CKD in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the isolated antibodies or antigen-binding fragments thereof of the present disclosure, any of the immunoconjugates of the present disclosure, any of the pharmaceutical compositions of the present disclosure, any of the isolated polynucleotides or vectors of the present disclosure, or any of the host cells of the present disclosure, for a time sufficient to reduce proteinuria in the subject.

[0050] The present disclosure further provides a method of reducing proteinuria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the isolated antibodies or antigen-binding fragments thereof of the present disclosure, any of the immunoconjugates of the present disclosure, any of the pharmaceutical compositions of the present disclosure, any of the isolated polynucleotides or vectors of the present disclosure, or any of the host cells of the present disclosure, for a time sufficient to reduce proteinuria in the subject. [Brief description of the drawings]

[0051] The above Summary of the Invention and the following Detailed Description of the Invention will be better understood when read in conjunction with the appended drawings. It is to be understood that the invention is not limited to the precise embodiments shown in the drawings. [Figure 1] FIG. 1 shows an amino acid sequence alignment of the ligand-binding region of VEGFR1 (Domain 2-Domain 3) from various species. Residues identical to the human sequence are dotted. Non-identical residues are explicitly indicated. FIG. 1 discloses SEQ ID NOs: 179-184, respectively, in order of appearance. [Diagram 2] Representative FACS binding data are shown showing species cross-reactivity of VEGFR1 antibodies (VGFB54, VGFB71, VGFB78 and VGFB82) to human, cynomolgus monkey and mouse VEGFR1, but retaining specificity for VEGFR1 over VEGFR2 or VEGFR3. Human VEGR1, cynomolgus monkey VEGFR1, mouse VEGFR1, human VEGFR2 and human VEGFR3 were overexpressed on the surface of cells. Mean fluorescence intensity is plotted using cell lines overexpressing the relevant VEGFR proteins. [Diagram 3] 1 shows an assessment of the relative frequency of the SHM leucine (L) compared to glutamine (Q) at position 105 of VGFB78 VH. [Figure 4A]4A shows paratope maps of the heavy chain variable domains of four anti-VEGFR1 antibodies (VGFB54, VGFB71, VGFB78, and VGFB82) against VEGFR1 (VGFW1). FIG. 4A discloses SEQ ID NOs: 31, 34, 36, and 39, respectively, in order of appearance. [Figure 4B] 4B shows the paratope map of the light chain variable domain of four anti-VEGFR1 antibody light chains (VGFB54, VGFB71, VGFB78, and VGFB82) against VEGFR1 (VGFW1). Highlighted residues represent HCDRs and LCDRs based on IMTG delineation. Residues with free energy changes of -1.0 kcal / mol or less upon binding to VEGFR1 are shown in gradient colors from medium gray to dark gray, as indicated on the scale. Residues that were not stabilized upon binding to VEGFR1 are shown in light gray. Residues that exchange too quickly or too slowly to be perturbed upon binding are shown in gray. Residues without color indicate that HDX behavior was not monitored because no peptide was present to cover the residue or because the residue is the first two residues of the peptide. FIG. 4B discloses SEQ ID NOs: 32, 35, 37, and 40, respectively, in order of appearance. [Diagram 5] FIG. 1 shows plasma concentrations (ng / mL) of total and free VGFB54 over time following a single IV / SC dose in cynomolgus monkeys. [Figure 6] FIG. 1 shows plasma concentrations (ng / mL) of total and free VGFB78 over time following a single IV / SC dose in cynomolgus monkeys. [Figure 7] FIG. 1 shows plasma concentrations (ng / mL) of total and free VGFB82 over time following a single IV / SC dose in cynomolgus monkeys. [Figure 8] FIG. 1 shows plasma concentrations (ng / mL) of total and free VGFB80 over time following a single IV / SC dose in cynomolgus monkeys. [Figure 9] 1 shows total PlGF plasma concentrations (pg / mL) over time following a single IV / SC dose of VGFB54 in cynomolgus monkeys. [Figure 10]1 shows total PlGF plasma concentrations (pg / mL) over time following a single IV / SC dose of VGFB78 in cynomolgus monkeys. [Figure 11] 1 shows total PlGF plasma concentrations (pg / mL) over time following a single IV / SC dose of VGFB82 in cynomolgus monkeys. [Figure 12] 1 shows total PlGF plasma concentrations (pg / mL) over time following a single IV / SC dose of VGFB80 in cynomolgus monkeys. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] definition The disclosed isolated anti-VEGFR1 antibodies, antigen-binding fragments thereof, polynucleotides, vectors, cells, compositions, kits, and methods may be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this disclosure: It is to be understood that the disclosed antibodies, antigen-binding domains, antibody fragments, polynucleotides, vectors, cells, compositions, kits, and methods are not limited to the isolated antibodies, antigen-binding domains, antibody fragments, polynucleotides, vectors, cells, compositions, kits, and methods specifically described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only, and is not intended to limit the claimed antibodies, antigen-binding domains, antibody fragments, polynucleotides, vectors, cells, compositions, kits, and methods.

[0053] Unless specifically specified otherwise, any description of possible mechanisms or modes of action, or reasons for improvements, is intended to be illustrative only, and the disclosed antibodies, antigen-binding domains, antibody fragments, polynucleotides, vectors, cells, compositions, kits and methods are not limited by the merits or demerits of any such proposed mechanisms or modes of action, or reasons for improvements.

[0054] Throughout this specification, the description refers to antibodies, antigen-binding fragments thereof, and methods of using antigen-binding domains. Where the present disclosure describes or claims features or embodiments relating to antigen-binding domains, such features or embodiments are equally applicable to methods of using antigen-binding domains. Similarly, where the present disclosure describes or claims features or embodiments relating to methods of using antigen-binding domains, such features or embodiments are equally applicable to antigen-binding domains.

[0055] When a range of numerical values ​​is recited or established herein, the range includes its endpoints, and all individual integers and rational numbers within the range, and also includes each of the narrower ranges formed by all the various possible combinations of these endpoints and internal integers and rational numbers, forming a subgroup of the larger group of values ​​within the recited range, as if each of the narrower ranges were explicitly recited. When a range of numerical values ​​is recited herein as being greater than the recited value, the range is nevertheless finite, and its upper limit is defined by a value that is operable within the context of the invention described herein. When a range of numerical values ​​is recited herein as being less than the recited value, the range is nevertheless defined by a lower limit by a non-zero value. It is not intended that the scope of the invention be limited to the specific values ​​recited in defining the range. All ranges are inclusive and combinable.

[0056] When values ​​are expressed as approximations, by use of the antecedent "about," it is to be understood that the particular value forms another embodiment. Reference to a particular numerical value is intended to include at least the particular value unless the context clearly indicates otherwise.

[0057] It is understood that certain features of the invention that are described herein for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. That is, unless expressly incompatible or specifically excluded, each individual embodiment is considered combinable with any other embodiment, and such combination is considered to be another embodiment. Conversely, different features of the invention that are described for brevity in the context of a single embodiment may be provided separately or in any subcombination. Finally, although an embodiment may be described as part of a series of steps or as part of a more general structure, each step may be considered to be an independent embodiment that can be combined with the others.

[0058] Unless otherwise stated, any description of possible mechanisms or modes of operation or reasons for improvement is intended to be illustrative only, and the methods of the present disclosure are not limited by the merits or demerits of the proposed mechanisms or modes of operation or reasons for improvement.

[0059] Various terms relating to the embodiments of the present specification are used throughout the specification and claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0060] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0061] The transitional phrases "comprising," "consisting essentially of," and "consisting of" are intended to connote their generally accepted meanings in patent language, i.e., (i) "comprising" is synonymous with "comprising," "containing," or "characterized by," is inclusive or open-ended, and does not exclude other unrecited elements or method steps, (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim, and (iii) "consisting essentially of" limits the scope of the claim to the specified materials or steps, and those that "do not materially affect the basic and novel characteristics" of the claimed disclosure. Embodiments described with the phrase "comprising" (or its equivalents) are also provided as embodiments described independently with "consisting of" and "consisting essentially of." Any embodiment described with the phrase "consisting essentially of" (or its equivalents) is also provided as an embodiment described separately below with "consisting of."

[0062] As used herein and in the appended claims, the phrase "and fragments thereof," when appended to a list, includes fragments of one or more members of the associated list. A list may include Markush groups, such as, by way of example, the phrase "the group consisting of peptides A, B, and C, and fragments thereof" designates or recites a Markush group that includes A, B, C, fragments of A, fragments of B, and / or fragments of C.

[0063] "Isolated" refers to a homogenous population of molecules (e.g., synthetic polynucleotides or polypeptides) that have been substantially separated and / or purified away from other components of the system in which they are produced, such as in a recombinant cell, and proteins that have been subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free of other cellular material and / or chemicals, and includes molecules that have been isolated to greater degrees of purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0064] "Polynucleotide" refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent bonding chemistry. cDNA is an example of a polynucleotide.

[0065] "Polypeptide" or "protein" refers to a molecule that contains at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as "peptides."

[0066] As used herein, "Tagg" refers to the temperature at which a protein begins to aggregate, either by dimerization or oligomerization. The aggregation temperature is the temperature at which the onset of aggregation is detected and indicates the tendency of a protein to aggregate. Tagg can be measured by differential scanning calorimetry (DSC), differential scanning fluorometry (DSF), or circular dichroism (CD). These techniques can detect small changes in the conformation of a protein and therefore the onset of aggregation. The Tagg value can be lower or higher than Tm. If Tagg is lower than Tm, the protein will first dimerize and / or oligomerize and then start to unfold later at a temperature higher than Tagg. If Tagg is higher than Tm, the protein will first start to unfold and then aggregate at a temperature higher than Tm. Both events are commonly observed and depend on the amino acid composition and protein conformation.

[0067] As used herein, "Tm" or "midpoint temperature" is the temperature midpoint of a thermal denaturation curve. It refers to the temperature at which 50% of the amino acid sequence is in its native conformation and the other 50% is denatured. Thermal denaturation curves are typically plotted as a function of temperature. Tm is used to measure protein stability. Generally, a higher Tm is indicative of a more stable protein. Tm can be easily measured using methods well known to those skilled in the art, such as circular dichroism spectroscopy, differential scanning calorimetry, differential scanning fluorometry (both intrinsic and extrinsic dye-based), UV spectroscopy, FT-IR, and isothermal calorimetry (ITC).

[0068] A "complementarity determining region (CDR)" is a region of an antibody that binds to an antigen. There are three CDRs (HCDR1, HCDR2, and HCDR3) in VH, and three CDRs (LCDR1, LCDR2, and LCDR3) in VL. CDRs can be defined using various descriptions, such as Kabat (Wu et al. (1970) J Exp Med 132:211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton J Bmol Biol 263:800-15, 1996). Correspondences between various descriptions and numbering of variable regions have been described (e.g., Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun (2001), J Mol Biol 309:657-70; International ImMunoGeneTics (IMGT) database, World Wide Web: imgt.org). Available programs such as abYsis by UCL Business PLC can be used to describe CDRs. As used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include CDRs defined by any of the Kabat, Chothia, IMGT or AbM methods described above, unless otherwise expressly stated in the specification. Correspondences between numbering systems, including, for example, Kabat numbering and the IMGT specific numbering system, are well known to those of skill in the art (see, e.g., Kabat, supra; Chothia, supra; Martin, supra; Lefranc et al., supra).

[0069] [Table 1]

[0070] The term "variable region" or "variable domain" refers to a heavy or light chain domain that is involved in binding an antibody to an antigen. The heavy or light chain variable domain (VH and VL, respectively) contains four framework regions (FR) and three complementarity determining regions (CDR).

[0071] A "subject" includes any human or non-human animal. A "non-human animal" includes any vertebrate, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" may be used interchangeably herein.

[0072] The terms "kit" and "article of manufacture" are used synonymously.

[0073] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if fully set forth.

[0074] Antibody that binds to VEGFR1 The present disclosure relates to isolated anti-VEGFR1 antibodies and antigen-binding fragments thereof. The terms "isolated antibody," "antigen-binding fragment thereof," and "anti-VEGFR1 antibody" and the like are used interchangeably and refer to an antibody that contains at least one binding domain that binds to and specifically binds to VEGFR1.

[0075] The antibodies of the present disclosure have one or more functional properties, including, but not limited to, high affinity binding to VEGFR1, blocking VEGFA binding to VEGFR1, and the ability to treat chronic kidney disease (CKD). In some embodiments, the present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to VEGFR1.

[0076] As used herein, the term "antibody" has a broad meaning and includes immunoglobulin molecules including monoclonal antibodies (including murine, human, humanized, and chimeric monoclonal antibodies), antigen-binding fragments, multispecific antibodies such as bispecific, trispecific, tetraspecific, etc., dimeric, tetrameric, or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified form of immunoglobulin molecule that contains an antigen-binding site of the required specificity. The term antibody includes full length antibodies, whole antibodies, intact antibodies, antibody fragments, antigen-binding fragments, and antigen-binding domains.

[0077] In general, an antibody is a protein or peptide chain that exhibits binding specificity to a specific antigen. The structure of an antibody is well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified as isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, an antibody of the invention can be of any of the five major classes or corresponding subclasses. Preferably, an antibody of the invention is IgG1, IgG2, IgG3, or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequence of their constant domain. Thus, an antibody of the invention can contain a κ or λ light chain constant domain. According to some embodiments, an antibody of the invention comprises a heavy and / or light chain constant region derived from a rat or human antibody. In addition to the heavy and light constant domains, antibodies contain an antigen-binding region consisting of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity determining regions 1-3, CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0078] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to VEGFR1 is substantially free of antibodies that do not bind to VEGFR1). In addition, an isolated antibody is substantially free of other cellular material and / or chemicals. "Isolated antibody" encompasses highly purified isolated antibodies, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0079] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the invention can be produced by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas that contain B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse or rat, and have a genome that includes a human heavy chain transgene and a light chain transgene.

[0080] As used herein, "VEGFR1" or "VEGFR-1" refers to a known protein called vascular endothelial growth factor receptor 1. VEGFR1 is encoded by the FLT1 gene. Unless otherwise specified, as used herein, VEGFR1 refers to human VEGFR1. The amino acid sequence of human VEGFR1 can be retrieved from Uniprot (Accession No. P17948). The amino acid sequence of full-length human VEGFR1 is shown in SEQ ID NO: 170. The sequence of VEGFR1 consists of an extracellular ligand-binding domain (amino acid residues 27-758) containing seven immunoglobulin (Ig)-like motifs (domain D1-domain D7: D1 residues 32-123, D2 residues 151-214, D3 residues 230-327, D4 residues 335-421, D5 residues 428-553, D6 residues 556-654, D7 residues 661-747), a single transmembrane domain and a cytoplasmic domain containing a kinase domain separated by a kinase insert (amino acid residues 827-1158), and a carboxyl terminus. The amino acid sequence of the human VEGFR1 D2 domain is shown in SEQ ID NO: 171. The amino acid sequence of the human VEGFR1 D3 domain is shown in SEQ ID NO: 172. The amino acid sequence of the human VEGFR1 D2-D3 domain is shown in SEQ ID NO: 173. VEGFR1 is produced both as a membrane receptor and as a soluble protein. Soluble VEGFR1 acts as a negative regulator of VEGFA activity by binding to this factor and preventing its interaction with its membrane receptor.

[0081] SEQ ID NO: 170, human VEGFR1 (Uniprot accession number P17948)

[0082] SEQ ID NO: 171 (Ig-like D2 domain) GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGH

[0083] SEQ ID NO: 172 (Ig-like D3 domain) IDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVH

[0084] SEQ ID NO: 173 (Ig-like D2-D3) GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVH

[0085] "Specifically binds," "specific binding," "specifically binding," or "binds" refers to a proteinaceous molecule that binds to an antigen or an epitope within an antigen with an affinity that is higher than its affinity for other antigens. Typically, a proteinaceous molecule binds to an antigen with an affinity of about 1×10 -7 M or less, e.g., about 5×10 -8 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 The equilibrium dissociation constant (K D ) binds to an antigen or an epitope within an antigen, typically Dis the K for binding to non-specific antigens (e.g., BSA, casein) D It is at least 100 times smaller than "K D The term "dissociation constant" refers to the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). D Values ​​can be determined using methods in the art in light of the present disclosure. For example, the K D can be determined by using surface plasmon resonance (SPR), such as by using a biosensor system such as the Biacore® system, or by using biolayer interferometry techniques such as the Octet RED96 system. D The smaller the value, the higher the affinity with which the antibody binds to the target antigen.

[0086] As used herein, an antibody that "binds to VEGFR1" or "specifically binds to VEGFR1" is an antibody that binds to VEGFR1 in a concentration of 1×10 -7 M or less, preferably 1×10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 K below M D and refers to an antibody that binds to VEGFR1, preferably human VEGFR1.

[0087] In some embodiments, an antibody or antigen-binding fragment thereof that "binds to VEGFR1" or "specifically binds to VEGFR1" is -7 K below M D In certain embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof preferably binds to VEGFR1 at a concentration of 1×10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 K below M Dand specifically binds to human VEGFR1.

[0088] The anti-VEGFR1 antibodies of the present disclosure include whole antibodies, antibody fragments that specifically bind to VEGFR1, and antigen-binding fragments thereof that specifically bind to VEGFR1.

[0089] In some embodiments, the anti-VEGFR1 antibodies of the present disclosure include whole or full-length antibodies, Fv fragments, single-chain scFv fragments (scFv), Fab, F(ab)2, or single-chain antibodies. In some embodiments, the anti-VEGFR1 antibodies of the present disclosure are whole or full-length antibodies.

[0090] In some embodiments, the anti-VEGFR1 antibodies of the disclosure are antibody fragments or antigen-binding domains that specifically bind to VEGFR1.

[0091] The terms "full length antibody", "whole antibody" and "intact antibody" are used interchangeably herein and refer to antibodies with a structure similar to that of a natural antibody. An "intact antibody" is composed of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0092] As used herein, the terms "antibody fragment" and "antigen-binding fragment" refer to molecules other than intact antibodies. Antigen-binding fragments may be synthetic, enzymatically obtainable, or genetically engineered polypeptides and include portions of immunoglobulins that bind to antigens, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd and Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), single domain antibodies (sdab), scFv dimers (bivalent diabodies), multispecific antibodies formed from portions of antibodies containing one or more CDRs, laminin, alpha-amyloids ... These include camelized single domain antibodies, nanobodies, domain antibodies, domain antibodies (dAbs) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, VHH domains, minimal recognition units consisting of amino acid residues mimicking the CDRs of an antibody, e.g. a FR3-CDR3-FR4 portion, HCDR1, HCDR2 and / or HCDR3 and LCDR1, LCDR2 and / or LCDR3, alternative scaffolds that bind antigen, bivalent domain antibodies, multispecific proteins comprising antigen-binding fragments, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure.

[0093] A "dAb" or "dAb fragment" refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341:544 546 (1989)).

[0094] "Fab" or "Fab fragment" refers to an antibody fragment composed of the VH, CH1, VL, and CL domains.

[0095] "F(ab')2" or "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge in the hinge region.

[0096] "Fd" or "Fd fragment" refers to an antibody fragment composed of the VH and CH1 domains.

[0097] "Fv" or "Fv fragment" refers to an antibody fragment composed of the VH and VL domains from a single arm of an antibody. Fv fragments lack the constant regions of the Fab (CH1 and CL) regions. The VH and VL in an Fv fragment are held together by non-covalent interactions.

[0098] Antigen-binding fragments (such as VH and VL) may be linked together via synthetic linkers to form various types of single-chain antibody designs, where the VH / VL domains may pair intramolecularly or intermolecularly to form monovalent antigen-binding domains, such as single-chain Fvs (scFvs) or diabodies. In recombinant expression systems, the linker is a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker must have a length that is appropriate to link the VH and VL in a manner that allows them to form the correct conformation relative to each other so as to retain the desired activity, such as binding to VEGFR1. The linker may be about 5 to 50 amino acids in length.

[0099] A "single-chain Fv" or "scFv" is a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), where the VL and VH are linked contiguously via a polypeptide linker and can be expressed as a single-chain polypeptide. As used herein, an scFv can have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and an scFv can comprise a VL-linker-VH or a VH-linker-VL.

[0100] Bivalent or bivalent single chain variable fragments (di-scFv, bi-scFv) can be engineered by linking two scFvs. "(scFv)2" or "tandem scFv" or "bis-scFv" fragments refer to a fusion protein comprising two light chain variable regions (VL) and two heavy chain variable regions (VH), where the two VL regions and the two VH regions are linked consecutively via a polypeptide linker and can be expressed as a single polypeptide chain. The two VL and the two VH are fused by a peptide linker to form a bivalent molecule VL. A -Linker-VH A -Linker-VL B -Linker-VH B (scFv)2 can be expressed as a single polypeptide chain, forming two binding sites that can bind two different antigens or epitopes simultaneously.

[0101] Any of the VH and VL domains identified herein that bind to VEGFR1 can be engineered in scFv format in either a VH-linker-VL or VL-linker-VH orientation. Any of the VH and VL domains identified herein can be used to generate sc(Fv)2 structures such as VH-linker-VL-linker-VL-linker-VH, VH-linker-VL-linker-VH-linker-VL, VH-linker-VH-linker-VL-linker-VL, VL-linker-VH-linker-VH-linker-VL, VL-linker-VH-linker-VH-linker-VL, VL-linker-VH-linker-VL-linker-VH, or VL-linker-VL-linker-VH-linker-VH.

[0102] A "diabody" is a bivalent dimer formed from two chains, each containing a VH and a VL domain. The two domains within a chain are separated by a linker that is too short to promote intrachain dimerization, resulting in the two chains dimerizing in a head-to-tail configuration. The linker can be a pentameric glycine-rich linker (G4S).

[0103] "VHH" refers to a single domain antibody or nanobody that is composed exclusively of the antigen-binding domain of the heavy chain. VHH single domain antibodies lack the CH1 domain of the heavy chain and the light chain of the traditional Fab region. In some embodiments, the anti-VEGFR1 antibodies of the present disclosure include Fv fragments, single chain scFv fragments (scFv), (scFv)2, Fab, F(ab)2, diabodies, VHH, dAb, Fd, Fv, or other single chain antibodies.

[0104] The anti-VEGFR1 antibodies of the present disclosure include chimeric, humanized, or fully human antibodies that specifically bind to VEGFR1.

[0105] "Human antibody" refers to an antibody that is optimized to have a minimal immune response when administered to a human subject. The variable regions of a human antibody are derived from human immunoglobulin sequences. If the human antibody contains a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A human antibody contains heavy and light chain variable regions that are "derived" from sequences of human origin when the variable regions of the human antibody are derived from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Exemplary such systems are phage-displayed human immunoglobulin gene libraries and transgenic non-human animals, such as mice or rats, that carry human immunoglobulin loci. "Human antibodies" typically contain amino acid differences when compared to immunoglobulins expressed in humans, due to differences in the system used to obtain the human antibodies and human immunoglobulin loci, the intentional introduction of somatic mutations or substitutions into the framework or CDRs, or both.

[0106] Typically, a "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a "human antibody" may contain a consensus framework sequence derived from human framework sequence analysis as described, for example, in Knappik et al., (2000) J Mol Biol 296:57-86, or a synthetic HCDR3 incorporated into a library of human immunoglobulin genes displayed on phage as described, for example, in Shi et al., (2010) J Mol Biol 397:385-96 and WO 2009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody."

[0107] Transgenic animals, such as mice, rats, or chickens, carrying human immunoglobulin (Ig) loci in their genome can be used to generate antigen-binding fragments that bind to VEGFR1, as described, for example, in U.S. Patent No. 6,150,584, WO 1999 / 45962, WO 2002 / 066630, WO 2002 / 43478, WO 2002 / 043478, and WO 1990 / 04036. The endogenous immunoglobulin loci of such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the animal's genome using homologous or non-homologous recombination, using a transchromosome, or using a minigene. Companies such as Regeneron (World Wide Web: regeneron.com), Harbour Antibodies (World Wide Web: harbourantibodies.com), Open Monoclonal Technology, Inc. (OMT) (World Wide Web: omtinc.net), KyMab (World Wide Web: kymab.com), Trianni (World Wide Web: trianni.com) and Ablexis (World Wide Web: ablexis.com) can be engaged to provide human antibodies against a selected antigen.

[0108] The antibody or antigen-binding fragment thereof that binds to VEGRF1 produced by immunizing a non-human animal may be humanized. Exemplary humanization techniques that include the selection of a human acceptor framework include CDR grafting (US Pat. No. 5,225,539), SDR grafting (US Pat. No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28:489-499), specificity determining residue resurfacing (US Patent Application Publication No. 2010 / 0261620), human framework adaptation (US Pat. No. 8,748,356), or superhumanization (US Pat. No. 7,709,226). In these methods, the CDRs or a subset of CDR residues of a parent antibody are grafted onto a human framework that may be selected based on overall homology to the parent framework, based on similarity in CDR length or identity of canonical structure, or a combination thereof.

[0109] The humanized antigen-binding domain may be further optimized to improve its selectivity or affinity for the desired antigen, by incorporating altered framework support residues to retain binding affinity (backmutation), or by introducing diversity into any of the CDRs, e.g. to improve the affinity of the antigen-binding domain, by techniques such as those described in WO 1090 / 007861 and WO 1992 / 22653.

[0110] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof, wherein the isolated antibody or antigen-binding fragment thereof binds to VEGFR1; a. SEQ ID NOs: 7, 175, 9, 10, 11, and 12, respectively; b. SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; c. SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively; d. SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively; e. SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively; f. SEQ ID NOs: 71, 176, 73, 74, 75, and 76, respectively; g. SEQ ID NOs: 71, 72, 73, 74, 75, and 76, respectively; h. SEQ ID NOs: 77, 78, 79, 90, 81, and 82, respectively; i. SEQ ID NOs: 83, 84, 85, 86, 87, and 88, respectively; j. SEQ ID NOs: 89, 90, 91, 92, 93, and 94, respectively; k. SEQ ID NOs: 95, 177, 97, 98, 99, and 100, respectively; l. SEQ ID NOs: 95, 96, 97, 98, 99, and 100, respectively; m. SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively; n. SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively; SEQ ID NOs: 113, 114, 115, 116, 117, and 118, respectively; p. SEQ ID NOs: 119, 178, 121, 122, amino acid sequence LNS, and SEQ ID NO: 124, respectively; q. SEQ ID NOs: 119, 120, 121, 122, amino acid sequence LNS, and SEQ ID NO: 124, respectively; r. SEQ ID NOs: 125, 126, 127, 128, amino acid sequence FNF, and SEQ ID NO: 130, respectively; s. SEQ ID NO: 131, 132, 133, 134, amino acid sequence YD, and SEQ ID NO: 136, respectively; or t. comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 137, 138, 139, 140, the amino acid sequence FNS, and SEQ ID NO: 142, respectively.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 7, 175, 9, 10, 11, and 12, respectively.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0116] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 and comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0117] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof, comprising: heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 31, and light chain complementarity determining region (HCDR) 2 of the light chain variable region (VL) of SEQ ID NO: 32 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 35; or HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 36; and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 37; or HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 38; and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 37; or HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 39; and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 40. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 39 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0118] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: a. VH of SEQ ID NO: 31 and VL of SEQ ID NO: 32; b. VH of SEQ ID NO: 33 and VL of SEQ ID NO: 32; c. VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35; d. VH of SEQ ID NO:36 and VL of SEQ ID NO:37; e. A VH of SEQ ID NO: 38 and a VL of SEQ ID NO: 37; f. a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40; or An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0119] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH of SEQ ID NO:31 and a VL of SEQ ID NO:32.

[0120] In some embodiments, the antibody or antigen-binding fragment that binds to VEGFR1 comprises a VH of SEQ ID NO:33 and a VL of SEQ ID NO:32.

[0121] In some embodiments, the antibody or antigen-binding fragment that binds to VEGFR1 comprises a VH of SEQ ID NO:34 and a VL of SEQ ID NO:35.

[0122] In some embodiments, the antibody or antigen-binding fragment that binds to VEGFR1 comprises a VH of SEQ ID NO:36 and a VL of SEQ ID NO:37.

[0123] In some embodiments, the antibody or antigen-binding fragment that binds to VEGFR1 comprises a VH of SEQ ID NO:38 and a VL of SEQ ID NO:37.

[0124] In some embodiments, the antibody or antigen-binding fragment that binds to VEGFR1 comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0125] In some embodiments, the disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60.

[0126] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:51.

[0127] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:52.

[0128] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:53.

[0129] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:54.

[0130] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:55.

[0131] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:56.

[0132] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:57.

[0133] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:58.

[0134] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:59.

[0135] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:60.

[0136] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: a. HC comprising the amino acid sequence of SEQ ID NO:51 and LC comprising the amino acid sequence of SEQ ID NO:52; b. HC comprising the amino acid sequence of SEQ ID NO:53 and LC comprising the amino acid sequence of SEQ ID NO:52; c. HC comprising the amino acid sequence of SEQ ID NO:54 and LC comprising the amino acid sequence of SEQ ID NO:55; d. HC comprising the amino acid sequence of SEQ ID NO:56 and LC comprising the amino acid sequence of SEQ ID NO:57; e. a HC comprising the amino acid sequence of SEQ ID NO:58 and a LC comprising the amino acid sequence of SEQ ID NO:57; or f. An isolated antibody or antigen-binding fragment thereof is provided, wherein the HC comprises the amino acid sequence of SEQ ID NO:59 and the LC comprises the amino acid sequence of SEQ ID NO:60.

[0137] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 7, 175, 9, 10, 11, and 12, respectively; VH of SEQ ID NO: 31 and VL of SEQ ID NO: 32, and / or HC of SEQ ID NO: 51 and LC of SEQ ID NO: 52, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0138] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; VH of SEQ ID NO: 33 and VL of SEQ ID NO: 32, and / or HC of SEQ ID NO: 53 and LC of SEQ ID NO: 52, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0139] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively; VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35, and / or HC of SEQ ID NO: 54 and LC of SEQ ID NO: 55, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0140] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively; VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37, and / or HC of SEQ ID NO: 56 and LC of SEQ ID NO: 57, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0141] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively; VH of SEQ ID NO: 38 and VL of SEQ ID NO: 37, and / or HC of SEQ ID NO:58 and LC of SEQ ID NO:57, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0142] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively; VH of SEQ ID NO: 39 and VL of SEQ ID NO: 40, and / or HC of SEQ ID NO:59 and LC of SEQ ID NO:60, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0143] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: VH of SEQ ID NO: 31 and VL of SEQ ID NO: 32; HC of SEQ ID NO: 51 and LC of SEQ ID NO: 52, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0144] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: VH of SEQ ID NO: 33 and VL of SEQ ID NO: 32; HC of SEQ ID NO: 53 and LC of SEQ ID NO: 52, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0145] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35; HC of SEQ ID NO: 54 and LC of SEQ ID NO: 55, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0146] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37; HC of SEQ ID NO: 56 and LC of SEQ ID NO: 57, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0147] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: VH of SEQ ID NO: 38 and VL of SEQ ID NO: 37; HC of SEQ ID NO:58 and LC of SEQ ID NO:57, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0148] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: VH of SEQ ID NO: 39 and VL of SEQ ID NO: 40; HC of SEQ ID NO:59 and LC of SEQ ID NO:60, An antibody or antigen-binding fragment thereof that binds to VEGFR1 is provided.

[0149] In some embodiments, the isolated antibody or antigen-binding fragment thereof is a full-length antibody. In some embodiments, the isolated antibody or isolated antigen-binding domain thereof is an antibody fragment or antigen-binding fragment.

[0150] Homologous antibodies with conservative substitutions and antigen-binding fragments thereof Derivatives, homologous antigen-binding domains, functional equivalents, or variants of the antibodies or antigen-binding fragments thereof are also objects of the present disclosure. The antibodies of the present disclosure further include homologous antibodies, homologous antigen-binding domains, functional equivalents, or variants of the disclosed antibodies or antigen-binding fragments thereof that bind to VEGFR1, including polypeptides having substantially identical amino acid sequences to the amino acid sequences of the variable or hypervariable domains of the antibodies of the present disclosure, or polypeptides having conservative substitutions. The homologous antibodies and antigen-binding domains, functional equivalents, or variants of the present disclosure have sufficient homology with the sequence of the antibodies or antigen-binding fragments thereof that bind to VEGFR1 and are functionally similar to the unmodified anti-VEGFR1 antibodies to retain binding to VEGFR1 or retain at least one activity of the unmodified antibody.

[0151] The terms "antibody derivative", "homologous antigen binding domain", "functional equivalent" or "variant" refer to an antibody comprising one or more mutations, substitutions, deletions and / or additions of one or more amino acid residues. Such additions, substitutions or deletions may be located anywhere in the molecule. When several amino acids have been added, substituted or deleted, any combination of additions, substitutions or deletions may be considered, provided that the resulting antibody still has at least the advantageous properties of the antibodies of the invention.

[0152] The sequences of the present disclosure may include amino acid sequences having at least 80% identity or homology to the above sequences. In some embodiments, the sequence identity may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the antigen-binding domain that binds to VEGFR1 of the present disclosure. A variant of the antigen-binding domain that binds to VEGFR1 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acid substitutions in the antigen-binding domain that binds to VEGFR1 and is within the scope of the present disclosure, as long as it retains or has improved functional properties when compared to the parent antigen-binding domain. A functional equivalent or variant of the antigen-binding domain that binds to VEGFR1 includes one or more deletions and / or additions of one or more amino acid residues. Such additions, substitutions, or deletions can be located anywhere in the molecule. When several amino acids are added, substituted, or deleted, any combination of additions, substitutions, or deletions can be considered, provided that the resulting antibody still has at least the advantageous properties of the antibody of the present invention.

[0153] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 31 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 32.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 33 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 32.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 34 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 35.

[0156] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 36 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 37.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 38 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 37.

[0158] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO:39 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO:40.

[0159] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO:39 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO:40.

[0160] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0161] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH of SEQ ID NO:39 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO:40.

[0162] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 95% identical to the VH of SEQ ID NO:39 and a VL that is at least 95% identical to the VL of SEQ ID NO:40.

[0163] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 95% identical to the VH of SEQ ID NO:39 and a VL that is at least 99% identical to the VL of SEQ ID NO:40.

[0164] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 99% identical to the VH of SEQ ID NO:39 and a VL that is at least 99% identical to the VL of SEQ ID NO:40.

[0165] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 99% identical to the VH of SEQ ID NO:39 and a VL that is at least 95% identical to the VL of SEQ ID NO:40.

[0166] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 39 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0167] The disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 39 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0168] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0169] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH of SEQ ID NO: 39 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to a VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0170] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 39 and a VL that is at least 95% identical to the VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0171] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 39 and a VL that is at least 99% identical to the VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0172] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 99% identical to the VH of SEQ ID NO: 39 and a VL that is at least 99% identical to the VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0173] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 99% identical to the VH of SEQ ID NO: 39 and a VL that is at least 95% identical to the VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0174] In some embodiments, the disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 59, wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0175] In some embodiments, the disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0176] In some embodiments, an isolated antibody or antigen-binding fragment thereof of the present application comprises a heavy chain (HC) having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, or SEQ ID NO:59, and a light chain (LC) having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO:60.

[0177] The disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the HC of SEQ ID NO:59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the LC of SEQ ID NO:60.

[0178] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the HC of SEQ ID NO:59 and a LC of SEQ ID NO:60.

[0179] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC of SEQ ID NO:59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the LC of SEQ ID NO:60.

[0180] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC that is at least 95% identical to the HC of SEQ ID NO:59 and a LC that is at least 95% identical to the LC of SEQ ID NO:60.

[0181] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC that is at least 95% identical to the HC of SEQ ID NO:59 and a LC that is at least 99% identical to the LC of SEQ ID NO:60.

[0182] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC that is at least 99% identical to the HC of SEQ ID NO:59 and a LC that is at least 99% identical to the LC of SEQ ID NO:60.

[0183] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC that is at least 99% identical to the HC of SEQ ID NO:59 and a LC that is at least 95% identical to the LC of SEQ ID NO:60.

[0184] The disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the HC of SEQ ID NO: 59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the LC of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0185] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the HC of SEQ ID NO: 59 and an LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0186] In some embodiments, an antibody that binds to VEGFR1 comprises a HC of SEQ ID NO: 59 and an LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0187] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 95% identical to the HC of SEQ ID NO: 59 and a LC that is at least 95% identical to the LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0188] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 95% identical to the HC of SEQ ID NO: 59 and a LC that is at least 99% identical to the LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0189] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 99% identical to the HC of SEQ ID NO: 59 and a LC that is at least 99% identical to the LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0190] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 99% identical to the HC of SEQ ID NO: 59 and a LC that is at least 95% identical to the LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0191] The disclosure further provides an isolated antibody comprising a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the HC of SEQ ID NO:59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the LC of SEQ ID NO:60, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0192] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the HC of SEQ ID NO: 59 and a LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40.

[0193] In some embodiments, an antibody that binds to VEGFR1 comprises a HC of SEQ ID NO:59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the LC of SEQ ID NO:60, and the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0194] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 95% identical to the HC of SEQ ID NO: 59 and a LC that is at least 95% identical to the LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40.

[0195] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 95% identical to the HC of SEQ ID NO: 59 and a LC that is at least 99% identical to the LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40.

[0196] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 99% identical to the HC of SEQ ID NO: 59 and a LC that is at least 99% identical to the LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40.

[0197] In some embodiments, an antibody that binds to VEGFR1 comprises a HC that is at least 99% identical to the HC of SEQ ID NO: 59 and a LC that is at least 95% identical to the LC of SEQ ID NO: 60, and the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40.

[0198] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences (e.g., anti-VEGFR1 antibodies and the polynucleotides encoding them) refers to two or more sequences or subsequences that are the same or have a specified percentage of the same amino acid residues or nucleotides when compared and aligned for maximum correspondence, as determined by one of the following sequence comparison algorithms or by visual inspection. Percent (%) amino acid sequence identity to a reference polypeptide is defined as the percentage of amino acid residues in a given sequence that are identical to the amino acid residues in the reference polypeptide sequence. The percent (%) identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The percent identity between two amino acid sequences can be determined using various algorithms that are within the skill of the art using publicly available software such as BLAS, BLAST-2, ALIGN, etc. The GAP program available in the GCG software package or Megalin (DNASTAR).

[0199] A polypeptide is typically substantially identical to a second polypeptide, e.g., the two peptides differ only by conservative substitutions. Antibodies of the present disclosure further include those whose binding, functional or physical properties have been improved by direct mutation. In some embodiments, a variant of an antigen-binding domain that binds to VEGFR1 contains one or two conservative substitutions in any of the CDR regions while retaining the desired functional properties of the parent antigen-binding fragment that binds to VEGFR1.

[0200] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modification. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is one in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Additionally, any naturally occurring residue in the polypeptide may also be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; ​​Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions to the antibodies of the invention can be made by known methods, such as PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, libraries of variants can be generated using, for example, random codons (NNK) or non-random codons, such as the DVK codons that code for 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting variants can be tested for their characteristics using the assays described herein.

[0201] Species Cross-Reactivity In some embodiments, the anti-VEGFR1 antibodies or antigen-binding fragments thereof bind to human, mouse, rat and cynomolgus VEGFR1. In some embodiments, the anti-VEGFR1 antibodies or antigen-binding fragments thereof of the present disclosure are species cross-reactive antibodies capable of binding to human, mouse, rat and cynomolgus VEGFR1 with similar affinity providing significant value to the anti-VEGFR1 antibodies disclosed herein.

[0202] In some embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof binds to human and mouse VEGFR1. In some embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof binds to human and rat VEGFR1. In some embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof binds to human, mouse and rat VEGFR1. In some embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof binds to human, mouse and cynomolgus VEGFR1. In some embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof binds to human, rat and cynomolgus VEGFR1. In some embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof binds to human VEGFR1.

[0203] One way to quantify the degree of species cross-reactivity of an antibody is the fold difference in its affinity for an antigen of one species compared to that of another, e.g., the fold difference in affinity for human VEGFR1 versus mouse VEGFR1, versus rat VEGFR1, or versus cynomolgus VEGFR1. Affinity refers to the equilibrium dissociation constant of the antibody-antigen reaction, K, as determined by SPR, as described elsewhere herein. D Species cross-reactive anti-VEGFR1 antibodies may have a fold difference in affinity for binding to human and mouse VEGFR1, human and rat VEGFR1, or human and cynomolgus VEGFR1 that is 500-fold or less, 250-fold or less, 100-fold or less, 50-fold or less, 25-fold or less, 10-fold or less, or 5-fold or less. In other words, the K D is the K value for binding to mouse, rat, or cynomolgus VEGFR1. DThe affinity of the antibody may be within 500, 250, 100, 50, 25, 10, or 5 times of the affinity of the human VEGFR1. In some embodiments, an antibody that binds to human VEGFR1 may bind to mouse, rat, or cynomolgus monkey with an affinity that is 500 times weaker than the affinity for human (e.g., 500 times, 250 times, 100 times, 50 times, 25 times, 10 times, or 5 times weaker than the affinity for human VEGFR1). In some embodiments, an antibody that binds to human VEGFR1 may bind to mouse, rat, or cynomolgus monkey VEGFR1 with an affinity that is 500 times stronger than the affinity for human (e.g., 500 times, 250 times, 100 times, 50 times, 25 times, 10 times, or 5 times stronger than the affinity for human VEGFR1). The antibody may further have a K D meets a threshold value (e.g., K D and K for binding to mouse, rat, or cynomolgus VEGFR1 D K is 10 nM or less, 5 nM or less, 1 nM or less), it can be considered cross-reactive. D may be 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. D may be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less, 0.01 nM or less, 0.001 nM or less.

[0204] An alternative measure of cross-reactivity for binding to human VEGFR1 and mouse, rat or cynomolgus VEGFR1 is the ability of the antibody to block ligand binding to VEGR1, such as VEGFA binding or PlGF binding. A species cross-reactive anti-VEGFR1 antibody may have an IC50 for blocking VEGFA binding or PlGF binding to human VEGFR1 that is within 25-fold, 20-fold, 15-fold, 10-fold or 5-fold of the IC50 for blocking VEGFA binding or PlGF binding to mouse, rat or cynomolgus monkey. As another alternative, a species cross-reactive antibody may have the ability to modulate circulating or tissue VEGFA or circulating or tissue PlGF levels in an animal. For example, an antibody that binds to human VEGFR1 may modulate circulating or tissue VEGFA or PlGF levels in mouse, rat and / or cynomolgus monkey.

[0205] Species cross-reactive antibodies would enable translational studies across several species and would help demonstrate efficacy and pharmacodynamic properties in established preclinical disease models without the need for surrogate antibodies in the development phase. Early reported anti-VEGFR1 antibodies were either human or mouse specific and showed poor species cross-reactivity.

[0206] It is difficult to generate monoclonal antibodies that are species cross-reactive and bind to epitopes that are conserved between humans and other species. The species cross-reactive anti-VEGFR1 antibodies of the present disclosure were generated through a unique immunization approach that includes (1) the selection of both human and mouse VEGFR1 antigens containing VEGFR1 D2 and D3 domains, (2) the selection of chicken, a phylogenetically distant immunization species, (3) alternating immunization boosts between human VEGFR1 D2-D3 and mouse VEGFR1 D2-D3, and (4) careful monitoring and selection of serum titers that show reactivity against both human and mouse VEGFR1.

[0207] In some embodiments, an anti-VEGFR1 antibody of the present disclosure can bind to an epitope within the sequence of amino acid residues 130-331 of human VEGFR1 (Uniprot Accession No. P17948). In some embodiments, an anti-VEGFR1 antibody of the present disclosure recognizes and binds to an epitope within SEQ ID NO: 173. In some embodiments, an anti-VEGFR1 antibody of the present disclosure recognizes and binds to an epitope within SEQ ID NO: 4.

[0208] In some embodiments, the anti-VEGFR1 binds to an epitope on VEGFR1 set forth in SEQ ID NO: 143 (FPLDTL) and SEQ ID NO: 144 (EIGL). In some embodiments, the anti-VEGFR1 antibody binds to an epitope on VEGFR1 set forth in SEQ ID NO: 144.

[0209] In some embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds to human VEGFR1 and can also specifically bind to VEGFR1 from at least one non-human species selected from the group consisting of cynomolgus monkey, mouse, and rat. In certain embodiments, the isolated antibody or antigen-binding fragment thereof of the present application has a specific binding affinity of 6×10 to 10 ng / mL, as determined by surface plasmon resonance (SPR). -8 M or less, especially 1×10 -8 M or less, more specifically 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 K below M D and specifically binds to human VEGFR1, mouse VEGFR1, and cynomolgus monkey VEGFR1.

[0210] "Epitope" refers to a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of surface groupings of chemically active (such as polar, non-polar, or hydrophobic) moieties, such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes may be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. In discontinuous epitopes, amino acids in different parts of the linear sequence of the antigen are brought into close proximity in three-dimensional space due to folding of the protein molecule.

[0211] "Paratope" refers to the portion of an antibody that specifically binds to an antigen. The paratope may be linear or discontinuous in nature and may be formed by the spatial relationship of non-adjacent amino acids of an antibody, rather than a linear string of amino acids. "Light chain paratope" and "heavy chain paratope" or "amino acid residues of the light chain paratope" and "amino acid residues of the heavy chain paratope" refer to the light and heavy chain residues of an antibody, respectively, that contact the antigen, or generally "antibody paratope residues" refer to those antibody amino acids that contact the antigen.

[0212] The determination of the residue in hVEGFR1 to which the antibody binds can be determined by any technique known to those of ordinary skill in the art. In some embodiments, the residue in hVEGFR1 to which the antibody binds is determined by H / D exchange assay. In H / D exchange assay, recombinantly expressed hVEGFR1 is incubated in deuterated water with or without the antibody for a predetermined time, which results in the incorporation of deuterium at exchangeable hydrogen atoms not protected by the antibody, followed by protease digestion of the protein and analysis of peptide fragments using LC-MS. H / D exchange assay can be performed using known protocols. In some embodiments, the H / D exchange mixture is quenched by the addition of a quenching buffer (e.g., 8M urea, 1M TCEP, pH 3.0) and then passed through an equilibrated immobilized pepsin / FPXIII column at room temperature (e.g., 600 μL / min). The digested fragments are then loaded onto a reverse-phase trap column (e.g., 600 μL / min), desalted (e.g., 600 μL for 1 min), separated (e.g., a C18 column), and analyzed by mass spectrometry (e.g., using an LTQ™ Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) with a capillary temperature of 275° C., a resolution of 150,000, and a mass range (m / z) of 300-1,800).

[0213] Antibodies that block VEGFA binding to VEGFR1 In some embodiments, the anti-VEGFR1 antibodies of the present disclosure can recognize and bind to an epitope in the D2-D3 domain of VEGFR1 and prevent binding of vascular endothelial growth factor A (VEGFA).

[0214] In the kidney, VEGFA, produced by glomerular podocytes and tubular epithelial cells, is essential to maintain glomerular integrity, renal microvasculature, and function. The important role of VEGFA in kidney development, structure, and function is revealed in the severe phenotype observed in conditional or inducible VEGFA knockout (KO) mice. In patients with chronic kidney disease (CKD), mRNA microarray analysis of patient renal biopsies shows reduced VEGFA expression, and the level of VEGFA expression in the glomerulus and tubulointerstitium correlates with eGFR, proteinuria, or vascular rarefaction (Bortoloso, Del Prete et al. 2004, Martini, Nair et al. 2014, Pan, Jiang et al. 2018). VEGFA functions through two endothelium-restricted receptors: 1) the main signaling receptor VEGFR2, and 2) the decoy receptor VEGFR1, which sequesters VEGFA from VEGFR2 with 10-fold higher binding affinity, but has weak or undetectable tyrosine kinase activity (TK). The anti-VEGFR1 of the present disclosure aims to restore impaired VEGFA levels and VEGFA activity in the kidney by blocking the sequestration of VEGFA by its decoy receptor VEGFR1. The anti-VEGFR1 antibody of the present disclosure blocks VEGFA sequestration and increases local VEGFA availability, which in turn allows endothelium protection, preservation of glomerular filtration barrier (GFB), and recovery of kidney function.

[0215] Conjugation and Fc engineering In addition to the modifications described above, the anti-VEGFR1 antibodies of the present disclosure, or antigen-binding fragments thereof and their functional equivalents, may be conjugated to other antibodies, proteins, antigen-binding fragments or alternative scaffolds.

[0216] The anti-VEGFR1 antibodies, antigen-binding fragments thereof, and functional equivalents of the present disclosure may be conjugated to half-life extending moieties. Exemplary half-life extending moieties are albumin, albumin variants, albumin binding proteins and / or domains, transferrin and fragments and analogs thereof, immunoglobulins (Ig) or fragments thereof, such as Fc regions. The amino acid sequences of the aforementioned half-life extending moieties are known. Additional half-life extending moieties that can be conjugated to the anti-VEGFR1 antibodies, antigen-binding fragments thereof, and functional equivalents of the present disclosure include polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, such as laurate, myristic acid, stearate, arachidic acid, behenic acid, oleic acid, arachidonic acid, octanedioic acid, tetradecanediacid, octadecanedioic acid, docosanedioic acid, etc., polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides or polysaccharides) for desired properties. These moieties may be fused directly to the antibodies or antigen-binding fragments of the present disclosure, or may be produced by standard cloning and expression techniques. Alternatively, the moieties may be attached to recombinantly produced antibodies or antigen-binding fragments of the present disclosure using well-known chemical coupling methods.

[0217] For example, a pegylated moiety can be conjugated to an antibody or antigen-binding fragment thereof that binds VEGFR1 by incorporating or genetically engineering a cysteine ​​residue at the C-terminus of the antibody or antigen-binding fragment that binds VEGFR1 into a residue position that faces away from the VEGFR1 binding site and attaching a pegylated group to the cysteine ​​using well-known methods.

[0218] In some embodiments, the half-life extending moiety is albumin.

[0219] In some embodiments, the half-life extending moiety is an albumin binding domain.

[0220] In some embodiments, the half-life extending moiety is transferrin.

[0221] In some embodiments, the half-life extending moiety is polyethylene glycol.

[0222] In some embodiments, the conjugation further includes, but is not limited to, conjugation to a detectable receptor molecule.

[0223] In some embodiments, the half-life extending moiety is an Ig constant region or a fragment of an Ig constant region.

[0224] In some embodiments, the half-life extending moiety is Ig.

[0225] In some embodiments, the half-life extending moiety is a fragment of Ig.

[0226] In some embodiments, the half-life extending moiety is an Ig constant region.

[0227] In some embodiments, the half-life extending moiety is a fragment of an Ig constant region.

[0228] In some embodiments, the half-life extending moiety is an Fc region.

[0229] The Ig constant region or fragment of an Ig constant region, such as the Fc region, present in the antibodies or antigen-binding fragments thereof of the disclosure can be of any allotype or isotype (i.e., IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE).

[0230] In some embodiments, the Ig constant region or fragment of an Ig constant region is of the IgG1 isotype.

[0231] In some embodiments, the Ig constant region or fragment of an Ig constant region is an IgG2 isotype.

[0232] In some embodiments, the Ig constant region or fragment of an Ig constant region is of the IgG3 isotype.

[0233] In some embodiments, the Ig constant region or fragment of an Ig constant region is of the IgG4 isotype.

[0234] Allotypes are not expected to affect the properties of Ig constant regions, such as binding or Fc-mediated effector functions. Immunogenicity of therapeutic proteins containing fragment Ig constant regions is associated with an increased risk of infusion reactions and a shorter duration of therapeutic response (Baert et al., (2003) N Engl J Med 348:602-08). The extent to which therapeutic proteins containing fragment Ig constant regions induce an immune response in the host may be determined in part by the allotype of the Ig constant region (Stickler et al., (2011) Genes and Immunity 12:213-21). Ig constant region allotypes are associated with amino acid sequence variations at specific positions in the antibody constant region sequence.

[0235] The antibodies or antigen-binding fragments thereof of the present disclosure and their functional equivalents may be conjugated to an Ig constant region or a fragment of an Ig constant region to modulate the effector functions, such as ADCC, ADCP and / or ADCP, and / or pharmacokinetic properties of the antibody or antigen-binding fragment. This can be achieved by introducing mutations in the Fc that control binding of the mutated Fc to activating FcγRs (FcγRI, FcγRIIa, FcγRIII), FcγRIIb, and / or FcRn.

[0236] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 is conjugated to an Ig constant region or a fragment of an Ig constant region comprising at least one mutation in the Ig constant region or a fragment of an Ig constant region.

[0237] In some embodiments, at least one mutation is in the Fc region.

[0238] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 is conjugated to an Ig constant region or a fragment of an Ig constant region that comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations in the Fc region.

[0239] The neonatal Fc receptor (FcRn) plays a central role in cellular trafficking and serum half-life of IgG. In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 is conjugated to an Ig constant region or a fragment of an Ig constant region that includes at least one mutation in the Fc region that modulates binding of the antibody or antigen-binding fragment to FcRn and modulates the half-life of the antibody or antigen-binding fragment.

[0240] In some embodiments, the Ig constant region or fragment of the first Ig constant region comprises at least one mutation that modulates the half-life of the isolated antibody or antigen-binding fragment thereof.

[0241] Positions in Fc that can be mutated to modulate half-life (e.g., binding to FcRn) include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Exemplary mutations that can be made alone or in combination are the mutations T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R. Exemplary mutations, alone or in combination, that may be made to increase the half-life of an antibody are the mutations M428L / N434S, M252Y / S254T / T256E, T250Q / M428L, N434A, and T307A / E380A / N434A. In some embodiments, the at least one mutation that modulates the half-life of an antibody or antigen-binding fragment thereof and functional equivalents of the disclosure is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, where residue numbering is according to the EU index.

[0242] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 is conjugated to an Ig constant region or a fragment of an Ig constant region that comprises the M252Y / S254T / T256E mutations.

[0243] In some embodiments, the antibodies or antigen-binding fragments thereof and their functional equivalents are conjugated to an Ig constant region or a fragment of an Ig constant region that comprises at least one mutation in the Fc region that reduces binding of the protein to activating Fcγ receptors (FcγRs) and / or reduces an Fc effector function such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

[0244] Fc positions that may be mutated to reduce binding of the protein to activating FcγRs and subsequently reduce effector function include positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331, and 365. Exemplary mutations that may be made alone or in combination are the mutations K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S, and P331S in IgG1, IgG2, IgG3, or IgG4. Exemplary combinations of mutations that result in proteins with reduced ADCC are L234A / L235A in IgG1, L234A / L235A / D265S in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in all Ig isotypes, V234A / G237A in IgG2, K214T / E233P / L234V / L235A / G in IgG1 ... These mutations are 236 deletion / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S in IgG2, S267E / L328F in IgG1, L234F / L235E / D265A in IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG1, S228P / F234A / L235A / G237A / P238S in IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S in IgG4. A hybrid IgG2 / 4 Fc domain may also be used, such as an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.

[0245] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 is conjugated to an IgG1 heavy chain constant region or a fragment of an IgG1 heavy chain constant region. In some embodiments, the IgG1 heavy chain constant region comprises at least one mutation that results in reduced binding of the antibody to FcγR. In some embodiments, the at least one mutation that results in reduced antibody binding to an FcγR is selected from the group consisting of F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, where residue numbering is according to the EU index. In some embodiments, the first Ig constant region or fragment of a first Ig constant region and / or the second Ig constant region or fragment of a second Ig constant region comprises the following mutation: L234A_L235A_D265S, In some embodiments, the FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof.

[0246] In some embodiments, the antibodies or antigen-binding fragments thereof and their functional equivalents are conjugated to an Ig constant region or a fragment of an Ig constant region that comprises at least one mutation in the Fc region that enhances binding of the protein to Fcγ receptors (FcγRs) and / or enhances an Fc effector function, such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or phagocytosis (ADCP).

[0247] Fc positions that may be mutated to increase binding of the protein to activating FcγR and / or enhance Fc effector function include positions 236, 239, 243, 256, 290, 292, 298, 300, 305, 312, 326, 330, 332, 333, 334, 345, 360, 339, 378, 396, or 430 (residue numbering according to the EU index). Exemplary mutations that may be made singly or in combination are G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A, A330K, I332E, E333A, K334A, A339T, and P396L. Exemplary combinations of mutations that result in proteins with increased ADCC or ADCP are S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E.

[0248] Fc positions that may be mutated to enhance CDC include positions 267, 268, 324, 326, 333, 345, and 430. Exemplary mutations that may be made singly or in combination are S267E, F1268F, S324T, K326A, K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430F, and E430T. Exemplary combination mutations that result in proteins with increased CDC are K326A / E333A, K326W / E333A, H268F / S324T, S267E / H268F, S267E / S324T, and S267E / H268F / S324T.

[0249] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., an IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0250] In some embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0251] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 39 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment binds to HCDR1, HCDR2, HCDR3, L and L of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively. and wherein the antibody or antigen-binding fragment comprises CDR1, LCDR2, and LCDR3, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, for example, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0252] The disclosure further relates to an isolated antibody or antigen-binding fragment thereof comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO: 39 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, HCDR2, HCDR3, LCDR4, HCDR5, HCDR6, HCDR7, HCDR8, HCDR9, HCDR10, HCDR11, HCDR12, HCDR13, HCDR14, HCDR15, HCDR16, HCDR17, HCDR18, HCDR19, HCDR110, HCDR120, HCDR130, HCDR140, HCDR150, HCDR160, HCDR171, HCDR182, HCDR193, HCDR194, HCDR195, HCDR196, HCDR197, HCDR198, HCDR199, HCDR199, HCDR199, HCDR199, HCDR191, HCDR199, HCDR199, HCDR191, HCDR192, HCDR193, HCDR194, HCDR195, HCDR196, HCDR197, HCDR198, HCDR199, HCDR199, HCDR199, HCDR199, HCDR199, HCDR199, An isolated antibody or antigen-binding fragment thereof is provided, comprising an LCDR2 and an LCDR3, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., an IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g. the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0253] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively. and optionally, the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0254] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH of SEQ ID NO: 39 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to a VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively. The fragment is an IgG1 (e.g., IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0255] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 39 and a VL that is at least 95% identical to the VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively, and wherein the antibody or antigen-binding fragment binds to an IgG1 (e.g., IgG1λ), and optionally wherein the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0256] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 39 and a VL that is at least 99% identical to the VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively, and wherein the antibody or antigen-binding fragment binds to an IgG1 (e.g., IgG1λ), and optionally wherein the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0257] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 99% identical to the VH of SEQ ID NO: 39 and a VL that is at least 99% identical to the VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively, and wherein the antibody or antigen-binding fragment binds to an IgG1 (e.g., IgG1λ), and optionally wherein the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0258] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a VH that is at least 99% identical to the VH of SEQ ID NO: 39 and a VL that is at least 95% identical to the VL of SEQ ID NO: 40, wherein the antibody or antigen-binding fragment comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively, and wherein the antibody or antigen-binding fragment binds to an IgG1 (e.g., IgG1λ), and optionally wherein the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0259] In some embodiments, the disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 59, wherein the antibody or antigen-binding fragment thereof binds to VEGFR1, and the antibody or antigen-binding fragment thereof is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0260] In some embodiments, the disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment thereof binds to VEGFR1, and the antibody or antigen-binding fragment thereof is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0261] The disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the HC of SEQ ID NO: 59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the LC of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0262] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises an HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the HC of SEQ ID NO: 59 and an LC of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0263] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC of SEQ ID NO: 59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0264] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises an HC that is at least 95% identical to the HC of SEQ ID NO: 59 and an LC that is at least 95% identical to the LC of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0265] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC that is at least 95% identical to the HC of SEQ ID NO: 59 and a LC that is at least 99% identical to the LC of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0266] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises an HC that is at least 99% identical to the HC of SEQ ID NO: 59 and an LC that is at least 99% identical to the LC of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0267] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises an HC that is at least 99% identical to the HC of SEQ ID NO: 59 and an LC that is at least 95% identical to the LC of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0268] The disclosure further relates to an isolated antibody or antigen-binding fragment thereof comprising a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the HC of SEQ ID NO:59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the LC of SEQ ID NO:60, wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, HCDR2, HCDR3, LCDR4, HCDR5, HCDR6, HCDR7, HCDR8, HCDR9, HCDR10, HCDR11, HCDR12, HCDR13, HCDR14, HCDR15, HCDR16, HCDR17, HCDR18, HCDR19, HCDR110, HCDR111, HCDR120, HCDR130, HCDR14, HCDR150, HCDR160, HCDR171, HCDR182, HCDR193, HCDR194, HCDR195, HCDR196, HCDR197, HCDR198, HCDR199, HCDR199, HCDR199, HCDR199, HCDR191, HCDR199, HCDR199, HCDR191, HCDR192, HCDR193, HCDR194, HCDR195, HCDR196, HCDR197, HCDR198, HCDR199, HCDR199, HCDR199, HCDR199, HCDR199, HCDR An isolated antibody or antigen-binding fragment thereof is provided, comprising an LCDR2 and an LCDR3, wherein the antibody or antigen-binding fragment is an IgG1 (e.g., an IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g. the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0269] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the HC of SEQ ID NO: 59 and a LC of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively. and optionally, the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0270] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC of SEQ ID NO:59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the LC of SEQ ID NO:60, wherein the antibody or antigen-binding fragment comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs:25, 26, 27, 28, 29, and 30, respectively. The fragment is an IgG1 (e.g., IgG1λ) and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0271] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC that is at least 95% identical to the HC of SEQ ID NO: 59 and a LC that is at least 95% identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0272] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC that is at least 95% identical to the HC of SEQ ID NO: 59 and a LC that is at least 99% identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0273] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC that is at least 99% identical to the HC of SEQ ID NO: 59 and a LC that is at least 99% identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0274] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC that is at least 99% identical to the HC of SEQ ID NO: 59 and a LC that is at least 95% identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0275] The disclosure further provides an isolated antibody or antigen-binding fragment thereof comprising a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the HC of SEQ ID NO:59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the LC of SEQ ID NO:60, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40, The binding fragment is an IgG1 (e.g., IgG1λ) and optionally wherein the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0276] In some embodiments, an antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the HC of SEQ ID NO: 59 and a LC of SEQ ID NO: 60, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0277] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC of SEQ ID NO: 59 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0278] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC that is at least 95% identical to the HC of SEQ ID NO: 59 and a LC that is at least 95% identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0279] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC that is at least 95% identical to the HC of SEQ ID NO: 59 and a LC that is at least 99% identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0280] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC that is at least 99% identical to the HC of SEQ ID NO: 59 and a LC that is at least 99% identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0281] In some embodiments, an antibody or antigen-binding fragment thereof comprises a HC that is at least 99% identical to the HC of SEQ ID NO: 59 and a LC that is at least 95% identical to the LC of SEQ ID NO: 60, and binds to VEGFR1, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40, and the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), and optionally the first Ig constant region or fragment of the first Ig constant region and / or the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S, e.g., the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region comprise the following mutation: L234A_L235A_D265S.

[0282] Polynucleotides Polynucleotides encoding the anti-VEGFR1 antibodies or antigen-binding fragments of the present disclosure and their functional equivalents are also provided. The present disclosure provides isolated polynucleotides encoding any of the anti-VEGFR1 antibodies or antigen-binding fragments thereof of the present disclosure.

[0283] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:31.

[0284] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:33.

[0285] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:34.

[0286] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:36.

[0287] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:38.

[0288] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:39.

[0289] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VL of SEQ ID NO:32.

[0290] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VL of SEQ ID NO:35.

[0291] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VL of SEQ ID NO:37.

[0292] In some embodiments, the disclosure provides an isolated polynucleotide encoding the VL of SEQ ID NO:40.

[0293] In some embodiments, the disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:51.

[0294] In some embodiments, the disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:53.

[0295] In some embodiments, the disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:54.

[0296] In some embodiments, the disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:56.

[0297] In some embodiments, the disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:58.

[0298] In some embodiments, the disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:59.

[0299] In some embodiments, the disclosure provides an isolated polynucleotide encoding the light chain of SEQ ID NO:52.

[0300] In some embodiments, the disclosure provides an isolated polynucleotide encoding the light chain of SEQ ID NO:55.

[0301] In some embodiments, the disclosure provides an isolated polynucleotide encoding the light chain of SEQ ID NO:57.

[0302] In some embodiments, the disclosure provides an isolated polynucleotide encoding the light chain of SEQ ID NO:60.

[0303] In some embodiments, the disclosure provides isolated polynucleotide sequences that encode the polynucleotide sequences of SEQ ID NOs:59 and 60.

[0304] In some embodiments, the disclosure provides an isolated polynucleotide of SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70.

[0305] In some embodiments, the disclosure provides an isolated polynucleotide comprising a polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the polynucleotide sequence of SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70.

[0306] Polynucleotides encoding the anti-VEGFR1 antibodies or antigen-binding fragments of the present disclosure include polynucleotides having substantially the same nucleic acid sequence as the nucleic acid sequence of the polynucleotides of the present disclosure. A "substantially the same" nucleic acid sequence is defined herein as a sequence that has at least 80% identity to another nucleic acid sequence when the two sequences are aligned. Two nucleic acid sequences are substantially identical if the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0307] Modified nucleotides can be used to generate the polynucleotides of the present disclosure. Exemplary modified nucleotides include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, N 6-Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5"-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyladenine, uracil-5-oxyacetate (v), wybutosine, pseudouracil, queuosine, beta-D-galactosylqueuosine, inosine, N 6 -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0308] Vectors Containing Polynucleotides Encoding Anti-VEGFR1 Antibodies Also provided is a vector comprising DNA encoding the anti-VEGFR1 antibody or antigen-binding fragment of the present disclosure. The disclosed vector can be used, for example, to produce any of the anti-VEGFR1 antibodies or antigen-binding fragments thereof disclosed above. The polynucleotide encoding any of the anti-VEGFR1 antibodies or antigen-binding fragments thereof of the present disclosure can be incorporated into a vector using standard molecular biology methods.

[0309] In some embodiments, the present disclosure provides an expression vector comprising the polynucleotide of the present invention. Such vectors can be plasmid vectors, viral vectors, baculovirus expression vectors, transposon-based vectors, or any other vector suitable for introducing the synthetic polynucleotide of the present invention into a given organism or genetic background by any means. The vector of the present disclosure can be an expression vector for efficient synthesis of VEGFR1 antibody polypeptide, and for expression of the VEGFR1 antibody polypeptide of the present disclosure in prokaryotic and eukaryotic systems, including but not limited to yeast and mammalian cell culture.

[0310] Exemplary vectors that can be used are: bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia), pEE6.4 (Lonza), and pEE12.4 (Lonza). Additional vectors include the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can be used. Exemplary plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Exemplary animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The expression vector can be a viral vector, e.g., a retroviral vector, e.g., a gamma retroviral vector.

[0311] The vector of the present disclosure may contain promoter and enhancer sequences. The polynucleotide encoding the VEGFR1-binding protein of the present disclosure may be operably linked to a control sequence in an expression vector that ensures the expression of the VEGFR1-binding protein. Such regulatory elements may include a transcription promoter, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls the termination of transcription and translation. The expression vector may further include one or more non-transcriptional elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' adjacent non-transcriptional sequences, 5' or 3' non-translation sequences (e.g., essential ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or a transcription termination sequence. An origin of replication that confers the ability to replicate in a host may also be incorporated.

[0312] The vectors of the present disclosure may also contain one or more Internal Ribosome Entry Sites (IRES). The inclusion of an IRES sequence in a fusion vector may be beneficial for enhancing expression of some proteins. In some embodiments, the vector system includes one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. The components of the vector may be closely linked, or may be positioned to provide optimal spacing for expression of the gene product (i.e., by introducing "spacer" nucleotides between ORFs), or may be otherwise positioned. Regulatory elements, such as IRES motifs, may also be positioned to provide optimal spacing for expression.

[0313] The vectors of the present disclosure may be circular or linear. They may be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. Replication systems may be derived, for example, from ColE1, SV40, 2μ plasmid, lambda, bovine papilloma virus, and the like.

[0314] Recombinant expression vectors can be designed for either transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0315] Vectors may also contain selection markers well known in the art. Selection markers include positive and negative selection markers. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation to provide prototrophy in auxotrophic hosts, etc. Exemplary marker genes include antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene, histidinol resistance gene, histidinol x resistance gene), glutamine synthetase gene, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleic acid sequence or cloning site encoding the selection marker may be upstream or downstream of the nucleic acid sequence or cloning site encoding the polypeptide of interest.

[0316] host cell The present disclosure further provides a host cell comprising any of the vectors of the present disclosure. "Host cell" refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell, as well as to stable cell lines generated from the particular subject cell. Since certain modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic, prokaryotic, plant, or archaeal cells. Examples of prokaryotic host cells are bacilli such as Escherichia coli, Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. Other microbes, such as yeast, are also useful for expression. Examples of suitable yeast host cells are Saccharomyces (e.g., S. cerevisiae) and Pichia. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines such as hybridoma or myeloma cell lines, such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61), or DG44.

[0317] The present disclosure further provides a recombinant host cell comprising any of the expression vectors of the present disclosure. The nucleic acid encoding any of the VEGFR1 binding proteins or fragments thereof can be used to transform a suitable mammalian host cell. Transformation of host cells, culture, antibody expression, and purification are carried out using well-known methods.

[0318] The cell line can be selected based on high level expression of the VEGFR1 antibody of interest and minimal contamination from host cell proteins. Mammalian cell lines available as host cells for expression are well known in the art and include, but are not limited to, Chinese hamster ovary (CHO) cells (e.g., CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61), or CHO DG44) and baby hamster kidney (BHK) cell derived. These cell lines can be used to produce any of the anti-VEGFR1 antibodies or antibody fragments of the present disclosure by culturing the cells under conditions suitable for antibody expression and purifying the antibody from the host cells or the medium surrounding the host cells.

[0319] The disclosure further provides a method of producing an anti-VEGFR1 binding protein of the disclosure, comprising culturing a host cell of the disclosure under conditions in which the anti-VEGFR1 binding protein is expressed, and recovering the anti-VEGFR1 binding protein produced by the host cell using methods known in the art. The protein of interest can be substantially pure, e.g., at least about 80%-85% pure, at least about 85%-90% pure, at least about 90%-95% pure, or at least about 98%-99% pure or more, and can be free of contaminants, such as cellular debris, macromolecules other than the protein of interest, etc.

[0320] In some embodiments, the disclosure provides a host cell expressing any of the isolated antibodies or antigen-binding fragments thereof of the disclosure.

[0321] In some embodiments, the disclosure provides a host cell comprising a vector of the disclosure.

[0322] Pharmaceutical Compositions Also provided is the use of any of the disclosed antibodies for the preparation of a medicament for treating chronic kidney disease.

[0323] Also provided is the use of any of the disclosed antibodies for the preparation of a pharmaceutical composition for treating chronic kidney disease.

[0324] Additionally, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment thereof of the present disclosure and a pharma- ceutically acceptable carrier.

[0325] For therapeutic applications, the anti-VEGFR1 antibodies and antigen-binding fragments thereof of the present disclosure can be prepared as pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharma- ceutically acceptable carrier.

[0326] "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the antibody of the invention is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The composition may contain pharma- ceutical acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickening agents, lubricants, colorants, and the like. The concentration of the antibody of the invention in such pharmaceutical formulations may vary from less than about 0.5% by weight, usually at least about 1% by weight, up to as much as 15 or 20% by weight, and may be selected primarily based on the required dose, fluid volumes, viscosities, and the like, according to the method of administration selected. Suitable vehicles and formulations (including other human proteins such as human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DBed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see in particular pp. 958-989.

[0327] Pharmaceutically acceptable carriers can include buffers, excipients, stabilizers, or preservatives. Examples of pharma-ceutically acceptable carriers include physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like, such as salts, buffer solutions, antioxidants, sugars, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants, or emulsifiers, or combinations thereof. The amount of pharma-ceutically acceptable carrier in a pharmaceutical composition can be empirically determined based on the activity of the carrier and the desired properties of the formulation, such as stability and / or minimal oxidation.

[0328] Pharmaceutical compositions may include buffers such as acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffer, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), antibacterial and antifungal agents; and preservatives.

[0329] The pharmaceutical compositions of the present disclosure can be formulated for various means of parenteral or oral administration. In one embodiment, the compositions can be formulated for injection or intravenous administration. The pharmaceutical compositions disclosed herein can be provided, for example, as sterile liquid preparations, such as isotonic aqueous solutions, emulsions, suspensions, dispersions, or viscous compositions, which can be buffered to a desired pH. Preparations suitable for oral administration can include liquid solutions, capsules, sachets, tablets, drops, and lozenges, liquid suspensions of powders in appropriate liquids and emulsions.

[0330] As used herein with respect to pharmaceutical compositions, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals and / or humans.

[0331] Treatment and Methods of Use Use of any of the disclosed antibodies, antigen-binding fragments thereof, or pharmaceutical compositions for treating chronic kidney disease is also provided.

[0332] "Treating," "treating," or "treatment" of a disease or disorder, such as chronic kidney disease, hereinafter refers to achieving one or more of: reducing the severity and / or duration of the disorder, delaying the progression of the disorder, slowing the progression of the disorder, inhibiting the worsening of symptoms characteristic of the disorder being treated, limiting or preventing the recurrence of the disorder in a subject who previously had the disorder, or limiting or preventing the recurrence of symptoms in a subject who was previously symptomatic for the disorder. As used herein, the term "delaying its progression" or "slowing its progression" is intended to include (a) delaying or slowing the onset of one or more symptoms or complications of the disease, condition, or disorder, (b) delaying or slowing the onset of one or more new / further symptoms or complications of the disease, condition, or disorder, and / or (c) delaying or slowing the progression of the disease, condition, or disorder to a later or more severe form of the disease, condition, or disorder.

[0333] A "subject" includes any human or non-human animal. A "non-human animal" includes all vertebrates, e.g., mammals and non-mammals. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, non-human primates, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like. The terms "subject" and "patient" can be used interchangeably herein. In some embodiments, the subject or patient is a human.

[0334] The isolated antibody or antigen fragment thereof of the present application can prevent VEGFA from binding to VEGFR1 in a subject in need thereof. Accordingly, another general aspect of the present application relates to a method of preventing VEGFA from binding to VEGFR1 in a subject in need thereof, comprising administering to the subject an effective amount of the isolated antibody or antigen fragment thereof, immunoconjugate, pharmaceutical composition, isolated polynucleotide, vector, or host cell of the present application, thereby preventing VEGFA from binding to VEGFR1. The subject may require treatment for a disease, disorder, or medical condition associated with VEGFA binding to VEGFR1.

[0335] In some embodiments, the subject is in need of treatment for chronic kidney disease (CKD), in need of reducing proteinuria, has advanced stage 4 or stage 5 chronic kidney disease, or has CKD associated with proteinuria, albuminuria, or diabetes.

[0336] In some embodiments, a method for preventing binding of VEGFA to VEGFR1 in a subject in need thereof comprises administering to the subject an effective amount of any of the isolated antibodies or antigenic fragments thereof disclosed herein, any of the immunoconjugates disclosed herein, any of the pharmaceutical compositions disclosed herein, any of the isolated polynucleotides disclosed herein, any of the vectors disclosed herein, or any of the host cells disclosed herein, thereby preventing binding of VEGFA to VEGFR1.

[0337] Also provided is a method of treating a medical condition by administering a therapeutically effective amount of an anti-VEGFR 1 antibody or an antigen-binding fragment thereof to a subject in need thereof. In some embodiments, the medical condition is chronic kidney disease. A "therapeutically effective amount" refers to an amount effective to obtain a desired therapeutic result at the dosage and duration required. The therapeutically effective amount may vary depending on factors such as the individual disease state, age, sex, and weight.

[0338] In some embodiments, the medical condition is chronic kidney disease. In some embodiments, the present disclosure provides a method of treating chronic kidney disease in a subject, comprising administering to the subject a therapeutically effective amount of a disclosed pharmaceutical composition.

[0339] "Chronic kidney disease" is meant to include all types of chronic kidney disease (CKD) caused by any type of condition related to chronic kidney disease, of any etiology, or resulting in all types of symptoms related to chronic kidney disease. CKD includes primary glomerular disease (including, but not limited to, nephropathy and focal segmental glomerulosclerosis), secondary glomerular disease (including, but not limited to, lupus nephritis), thrombotic microangiopathy, tubulointerstitial disease, ischemic nephropathy, diabetic nephropathy, polycystic kidney disease, hypertensive nephropathy, focal segmental glomerulosclerosis, nephrotic syndrome, and obstructive uropathy (including, but not limited to, reflux nephropathy).

[0340] In some embodiments, the present disclosure provides a method for treating diabetic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of any of the anti-VEGFR1 antibodies or antigen-binding fragments thereof of the present disclosure, or any of the disclosed pharmaceutical compositions of the present disclosure.

[0341] In some embodiments, the disclosure provides a method for treating chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the chronic kidney disease is diabetic nephropathy.

[0342] In some embodiments, the disclosure provides a method for delaying or slowing the progression of chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the chronic kidney disease is diabetic nephropathy.

[0343] In some embodiments, the disclosure provides a method of treating chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody of the disclosure, wherein the chronic kidney disease is focal segmental glomerulosclerosis.

[0344] In some embodiments, the disclosure provides a method for delaying or slowing the progression of chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody of the disclosure, wherein the chronic kidney disease is focal segmental glomerulosclerosis.

[0345] In some embodiments, the disclosure provides a method for treating chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the chronic kidney disease is hypertensive nephropathy.

[0346] In some embodiments, the disclosure provides a method for delaying or slowing the progression of chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the chronic kidney disease is hypertensive nephropathy.

[0347] In some embodiments, the subject has chronic kidney disease (CKD). Chronic kidney disease has been classified into five stages of kidney disease. The stages of kidney disease are classified based on the rate at which the kidneys can filter waste and excess fluid from the blood. Estimated glomerular filtration rate (eGFR) numbers are used to classify five stages of kidney damage, from very mild damage in stage 1 to renal failure in stage 5, based on the levels of creatine and waste products in the blood.

[0348] In some embodiments, a method of treating chronic kidney disease (CKD) in a subject in need thereof comprises administering to the subject a therapeutically effective amount of any of the isolated antibodies or antigenic fragments thereof of this disclosure, any of the immunoconjugates of this disclosure, any of the pharmaceutical compositions of this disclosure, any of the isolated polynucleotides of this disclosure, any of the vectors of this disclosure, or any of the host cells of this disclosure for a time sufficient to treat CKD.

[0349] In some embodiments, the disclosure provides a VEGFR1 antibody for use in reducing glomerular filtration rate (GFR) loss.

[0350] In some embodiments, the disclosure provides a method for reducing loss of glomerular filtration rate (GFR), comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment of the disclosure.

[0351] In some embodiments, the subject receives a blood glucose level of 60 mL / min / 1.73 m 2 In some embodiments, the subject has advanced stage 3, 4, or 5 chronic kidney disease with an eGFR (estimated glomerular filtration rate) of less than 30 mL / min / 1.73 m 2 The subject has advanced stage 4 or 5 chronic kidney disease with a measured eGFR of 30-60 mL / min / 1.73 m 2 In some embodiments, the subject has stage 3 chronic kidney disease with a measured eGFR of 30-45 mL / min / 1.73 m 2 In some embodiments, the subject has stage 3b chronic kidney disease with a measured eGFR of 0.01 to 0.01. In some embodiments, the subject has advanced stage 4 or stage 5 chronic kidney disease and is not on dialysis. In some embodiments, the subject has advanced stage 4 or stage 5 chronic kidney disease and is on dialysis.

[0352] In some embodiments, the disclosure provides a method of treating chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the subject has an average blood flow rate of 30 mL / min / 1.73 m 2 The method provides for the treatment of chronic kidney disease in a subject having stage 4 or stage 5 chronic kidney disease with a measured eGFR of

[0353] In some embodiments, the disclosure provides a method of delaying or slowing the progression of chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the subject receives blood flow at a rate of 30 mL / min / 1.73 m 2 The method provides for the treatment of chronic kidney disease in a subject having stage 4 or stage 5 chronic kidney disease with a measured eGFR of

[0354] In some embodiments, the disclosure provides a method of treating chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the subject receives an IV flow rate of 30-60 mL / min / 1.73 m 2 The method provides for the treatment of chronic kidney disease in a subject having stage 3 chronic kidney disease with a measured eGFR of 0.1 to 0.2 mg / kg.

[0355] In some embodiments, the present disclosure provides a method of delaying or slowing the progression of chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the present disclosure, wherein the subject receives blood at a rate of 30-60 mL / min / 1.73 m 2 The method provides for the treatment of chronic kidney disease in a subject having stage 3 chronic kidney disease with a measured eGFR of 0.1 to 0.2 mg / kg.

[0356] In some embodiments, the subject has chronic kidney disease accompanied by proteinuria. In some embodiments, the subject has chronic kidney disease accompanied by albuminuria. In some embodiments, the subject has chronic kidney disease accompanied by diabetes.

[0357] In some embodiments, the disclosure provides a method of treating chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the subject has chronic kidney disease accompanied by proteinuria.

[0358] In some embodiments, the disclosure provides a method for delaying or slowing the progression of chronic kidney disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the subject has chronic kidney disease accompanied by proteinuria.

[0359] In some embodiments, the present disclosure provides a method of reducing proteinuria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the isolated antibodies or antigenic fragments thereof of this disclosure, any of the immunoconjugates of this disclosure, any of the pharmaceutical compositions of this disclosure, any of the isolated polynucleotides of this disclosure, any of the vectors of this disclosure, or any of the host cells of this disclosure, for a time sufficient to reduce proteinuria in the subject.

[0360] In some embodiments, the disclosure provides a method of treating chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the subject has chronic kidney disease accompanied by albuminuria.

[0361] In some embodiments, the disclosure provides a method for delaying or slowing the progression of chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the subject has chronic kidney disease accompanied by albuminuria.

[0362] In some embodiments, the disclosure provides a method for treating chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the subject has chronic kidney disease associated with diabetes.

[0363] In some embodiments, the disclosure provides a method for delaying or slowing the progression of chronic kidney disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure, wherein the subject has chronic kidney disease associated with diabetes.

[0364] "Proteinuria" refers to the presence of increased protein in the urine. Proteinuria may reflect abnormal loss of plasma proteins due to a) increased glomerular permeability to high molecular weight proteins (albuminuria or glomerular proteinuria), b) incomplete tubular reabsorption of normally filtered low molecular weight proteins (tubular proteinuria), or c) increased plasma concentrations of low molecular weight proteins (overproduction proteinuria, such as immunoglobulin light chains). Proteinuria may also reflect abnormal loss of proteins originating from the kidney (renal tubular cellular components due to tubular damage) and lower urinary tract.

[0365] "Albuminuria" is the condition in which albumin is present in the urine. In healthy individuals, albumin is filtered by the kidneys. When the kidneys do not properly filter larger molecules (such as albumin) from the urine, albumin is excreted in the urine and is typically a sign of kidney damage. Albuminuria is a common but heterogeneous condition in CKD patients. Albuminuria is the earliest marker of glomerular disease, including diabetic nephrosclerosis, and generally appears before a decline in eGFR. Albuminuria is a marker of hypertensive nephrosclerosis, but may not appear until after a decline in eGFR.

[0366] Diabetic nephropathy is one of the microvascular complications of diabetes and is characterized by persistent albuminuria and a progressive decline in renal function.

[0367] In some embodiments, the disclosure provides a method for reducing proteinuria in a patient with chronic kidney disease, comprising administering to a patient in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure.

[0368] In some embodiments, the disclosure provides a method of reducing proteinuria, comprising administering to a patient in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment of the disclosure to the patient. In some embodiments, the proteinuria is caused by chronic kidney disease. In other embodiments, the proteinuria is caused by diabetic nephropathy.

[0369] In some embodiments, the disclosure provides a method for reducing albuminuria in a patient with chronic kidney disease, comprising administering to a patient in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the disclosure.

[0370] Further provided is a method for detecting VEGFR1 in a sample, comprising obtaining a sample, contacting the sample with an anti-VEGFR1 antibody or antigen-binding fragment thereof of the present disclosure, and detecting bound VEGFR1 in the sample.

[0371] In some embodiments, samples may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, synovial fluid, circulating cells, cells not associated with tissue (i.e., free cells), tissue (e.g., surgically removed tissue, biopsy material including fine needle aspirate), tissue preparations, etc. VEGFR1 can be detected using known methods. Exemplary methods include directly labeling the antibody using a fluorescent or chemiluminescent label, or a radiolabel, or conjugating the antibody of the invention with a readily detectable moiety such as biotin, an enzyme, or an epitope tag. Exemplary labels and moieties include ruthenium, 111 In-DOTA, 111In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, and Alexafluor® dyes.

[0372] The antibodies or antigen-binding fragments thereof of the present disclosure may be used in a variety of assays to detect VEGFR1 in a sample. Exemplary assays are Western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS), or ELISA assays.

[0373] kit Kits comprising the antibodies or antigen-binding fragments of the disclosure, or functional equivalents thereof, are described herein.

[0374] The present invention further provides kits comprising an antibody or antigen-binding fragment thereof that binds to VEGFR1.

[0375] The kits can be used for therapeutic applications and as diagnostic kits.

[0376] The kit can be used to detect the presence of VEGFR1 in a sample.

[0377] In some embodiments, the kit comprises an anti-VEGFR1 antibody or antigen-binding fragment of the present disclosure and a reagent for detecting a VEGFR1 binding protein. The kit may include one or more other components, including instructions for use, other reagents, such as a label, a therapeutic agent, or an agent useful for chelating or otherwise coupling, an antibody to a label or therapeutic agent, or a radioprotective composition, a device or other material for preparing the antibody for administration, a pharma- ceutically acceptable carrier, and a device or other material for administration to a subject.

[0378] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to VEGFR1 in a container, and instructions for using the kit.

[0379] In some embodiments, the present disclosure comprises a kit comprising any of the isolated antibodies or antigen-binding fragments of this disclosure, any of the immunoconjugates of this disclosure, any of the pharmaceutical compositions of this disclosure, any of the isolated polynucleotides of this disclosure, any of the vectors of this disclosure, or any of the host cells of this disclosure.

[0380] In some embodiments, the antibody or antigen-binding fragment thereof that binds VEGFR1 in the kit is labeled.

[0381] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to VEGFR1; VH of SEQ ID NO: 31 and VL of SEQ ID NO: 32; VH of SEQ ID NO: 33 and VL of SEQ ID NO: 32; VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35; VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37; VH of SEQ ID NO: 38 and VL of SEQ ID NO: 37; or It comprises a VH of sequence number 39 and a VL of sequence number 40.

[0382] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to VEGFR1, comprising a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0383] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to VEGFR1, comprising the amino acid sequence of SEQ ID NO:51, 52, 53, 54, 55, 56, 57, 59, or 60.

[0384] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to VEGFR1 and comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:51 and SEQ ID NO:52; (b) SEQ ID NO:53 and SEQ ID NO:52, respectively; (c) SEQ ID NO:54 and SEQ ID NO:55, respectively; (d) SEQ ID NO:56 and SEQ ID NO:57, respectively; (e) SEQ ID NO:58 and SEQ ID NO:57, and SEQ ID NO:59 and SEQ ID NO:60.

[0385] Embodiment The following list of embodiments is intended to complement, not replace or supplant, the foregoing description.

[0386] Embodiment 1. An isolated antibody or antigen-binding fragment thereof that binds to an epitope within the amino acid sequence of SEQ ID NO:173 or SEQ ID NO:4, wherein the antibody or antigen-binding fragment thereof prevents binding of VEGFA to VEGFR1.

[0387] Embodiment 2. An isolated antibody or antigen-binding fragment thereof that binds to an epitope within the amino acid sequence of SEQ ID NO: 173, wherein the antibody or antigen-binding fragment thereof prevents binding of VEGFA to VEGFR1.

[0388] Embodiment 3. An isolated antibody or antigen-binding fragment thereof that binds to an epitope within the amino acid sequence of SEQ ID NO:4, wherein the antibody or antigen-binding fragment thereof prevents binding of VEGFA to VEGFR1.

[0389] Embodiment 4. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, which binds to an epitope on VEGFR1 having the amino acid sequence FPLDTL (SEQ ID NO: 143) or EIGL (SEQ ID NO: 144), or binds to an epitope having the amino acid sequence FPLDTL (SEQ ID NO: 143) and EIGL (SEQ ID NO: 144).

[0390] Embodiment 5. The isolated antibody or antigen-binding fragment thereof of embodiment 4, wherein the antibody or antigen-binding fragment thereof binds to an epitope on VEGFR1 having the amino acid sequence FPLDTL (SEQ ID NO: 143).

[0391] Embodiment 6. The isolated antibody or antigen-binding fragment thereof of embodiment 4, wherein the antibody or antigen-binding fragment thereof binds to an epitope on VEGFR1 having the amino acid sequence EIGL (SEQ ID NO: 144).

[0392] Embodiment 7. The isolated antibody or antigen-binding fragment thereof of embodiment 4, wherein the antibody or antigen-binding fragment thereof binds to human, mouse, rat and / or cynomolgus VEGFR1.

[0393] Embodiment 8. The isolated antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment thereof binds to human VEGFR1.

[0394] Embodiment 9. The isolated antibody or antigen-binding fragment thereof described in embodiment 8, wherein the antibody or antigen-binding fragment thereof binds to human VEGFR1 and VEGFR1 derived from at least one species selected from the group consisting of cynomolgus monkey, mouse and rat.

[0395] Embodiment 10. The isolated antibody or antigen-binding fragment thereof of embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human and mouse VEGFR1.

[0396] Embodiment 11. The isolated antibody or antigen-binding fragment thereof of embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human and rat VEGFR1.

[0397] Embodiment 12. The isolated antibody or antigen-binding fragment thereof of embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human, mouse and rat VEGFR1.

[0398] Embodiment 13. The isolated antibody or antigen-binding fragment thereof of embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human and cynomolgus VEGFR1.

[0399] Embodiment 14. The isolated antibody or antigen-binding fragment thereof of embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human, mouse, rat and cynomolgus VEGFR1.

[0400]

[0043] Embodiment 15. The antibody or antigen-binding fragment thereof is determined using surface plasmon resonance (SPR), 6×10 -8 M or less, especially 1×10 -8 M or less, more specifically 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 K below M D 10. The isolated antibody or antigen-binding fragment thereof of embodiment 9, which binds to human VEGFR1, mouse VEGFR1, rat VEGFR1, and cynomolgus VEGFR1.

[0401]

[0046] Embodiment 16. The antibody or antigen-binding fragment thereof is determined using surface plasmon resonance (SPR), 6×10 -8 M or less, especially 1×10 -8 M or less, more specifically 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 K below M D 10. The isolated antibody or antigen-binding fragment thereof of embodiment 9, which binds to human VEGFR1, mouse VEGFR1, and cynomolgus VEGFR1.

[0402]

[0043] Embodiment 17. The antibody or antigen-binding fragment thereof is determined using surface plasmon resonance (SPR), and the antibody or antigen-binding fragment thereof is 6×10 -8 M or less, especially 1×10 -8 M or less, more specifically 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 K below M D 10. The isolated antibody or antigen-binding fragment thereof of embodiment 9, which binds to human VEGFR1 and mouse VEGFR1 at

[0403]

[0043] Embodiment 18. The antibody or antigen-binding fragment thereof is determined using surface plasmon resonance (SPR), and the antibody or antigen-binding fragment thereof is 6×10 -8 M or less, especially 1×10 -8 M or less, more specifically 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 K below M D 10. The isolated antibody or antigen-binding fragment thereof of embodiment 9, which binds to human VEGFR1 and rat VEGFR1 at

[0404]

[0036] Embodiment 19. The antibody or antigen-binding fragment thereof is determined using surface plasmon resonance (SPR), and the antibody or antigen-binding fragment thereof is 6×10 -8 M or less, especially 1×10 -8 M or less, more specifically 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 K below M D 10. The isolated antibody or antigen-binding fragment thereof of embodiment 9, which binds to human VEGFR1 and cynomolgus VEGFR1 at

[0405] Embodiment 20. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 19, comprising a heavy chain complementarity determining region (HCDR) of VH of SEQ ID NO: 31, 33, 34, 36, 38 or 39, and a light chain complementarity determining region (LCDR) of VL of SEQ ID NO: 32, 35, 37 or 40.

[0406] EMBODIMENT 21. a. the HCDR of VH having the amino acid sequence of SEQ ID NO: 31, and the LCDR of VL having the amino acid sequence of SEQ ID NO: 32; b. the HCDR of the VH having the amino acid sequence of SEQ ID NO: 33, and the LCDR of the VL having the amino acid sequence of SEQ ID NO: 32; c. an HCDR of VH having the amino acid sequence of SEQ ID NO: 34, and an LCDR of VL having the amino acid sequence of SEQ ID NO: 35; d. an HCDR of VH having the amino acid sequence of SEQ ID NO: 36, and an LCDR of VL having the amino acid sequence of SEQ ID NO: 37; e. a VH HCDR having the amino acid sequence of SEQ ID NO: 38 and a VL LCDR having the amino acid sequence of SEQ ID NO: 37; or f. The isolated antibody or antigen-binding fragment thereof of embodiment 20, comprising an HCDR of VH having the amino acid sequence of SEQ ID NO: 39, and an LCDR of VL having the amino acid sequence of SEQ ID NO: 40.

[0407] Embodiment 22. A VH having HCDR1, HCDR2 and HCDR3, and a VL having LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are a. SEQ ID NOs: 7, 175, 9, 10, 11, and 12, respectively; b. SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; c. SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively; d. SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively; e. SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively; f. SEQ ID NOs: 71, 176, 73, 74, 75, and 76, respectively; g. SEQ ID NOs: 71, 72, 73, 74, 75, and 76, respectively; h. SEQ ID NOs: 77, 78, 79, 80, 81, and 82, respectively; i. SEQ ID NOs: 83, 84, 85, 86, 87, and 88, respectively; j. SEQ ID NOs: 89, 90, 91, 92, 93, and 94, respectively; k. SEQ ID NOs: 95, 177, 97, 98, 99, and 100, respectively; l. SEQ ID NOs: 95, 96, 97, 98, 99, and 100, respectively; m. SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively; n. SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively; SEQ ID NOs: 113, 114, 115, 116, 117, and 118, respectively; p. SEQ ID NOs: 119, 178, 121, 122, amino acid sequence LNS, and SEQ ID NO: 124, respectively; q. SEQ ID NOs: 119, 120, 121, 122, amino acid sequence LNS, and SEQ ID NO: 124; r. SEQ ID NOs: 125, 126, 127, 128, amino acid sequence FNF, and SEQ ID NO: 130, respectively; s. SEQ ID NO: 131, 132, 133, 134, amino acid sequence YD, and SEQ ID NO: 136, respectively; or t. The isolated antibody or antigen-binding fragment thereof of embodiment 21, comprising SEQ ID NO: 137, 138, 139, 140, amino acid sequence FNS, and SEQ ID NO: 142, respectively.

[0408] Embodiment 23. An isolated antibody or antigen-binding fragment thereof according to embodiment 22, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 175, 9, 10, 11 and 12, respectively.

[0409] Embodiment 24. The isolated antibody or antigen-binding fragment thereof of embodiment 22, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 14, 15, 16, 17 and 18, respectively.

[0410] Embodiment 25. The isolated antibody or antigen-binding fragment thereof of embodiment 22, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively.

[0411] Embodiment 26. The isolated antibody or antigen-binding fragment thereof of embodiment 22, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 8, 9, 10, 11 and 12, respectively.

[0412] Embodiment 27. The isolated antibody or antigen-binding fragment thereof of embodiment 22, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 20, 21, 22, 23 and 24, respectively.

[0413] Embodiment 28. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 27, comprising a heavy chain variable domain (VH) having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 31, 33, 34, 36, 38, or SEQ ID NO: 39, and a light chain variable domain (VL) having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 32, 35, 37, or 40.

[0414] 29. a. SEQ ID NO:31 and SEQ ID NO:32, respectively; b. SEQ ID NO:33 and SEQ ID NO:32, respectively; c. SEQ ID NO:34 and SEQ ID NO:35, respectively; d. SEQ ID NO:36 and SEQ ID NO:37, respectively; e. SEQ ID NO:38 and SEQ ID NO:37, respectively; or f. The isolated antibody or antigen-binding fragment thereof of embodiment 28, comprising a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 39 and 40, respectively.

[0415] Embodiment 30. An isolated antibody or antigen-binding fragment thereof described in embodiment 29, comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 39, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 40.

[0416] Embodiment 31. An isolated antibody or antigen-binding fragment thereof according to embodiment 30, comprising a VH having the amino acid sequence of SEQ ID NO: 39 and a VL having the amino acid sequence of SEQ ID NO: 40.

[0417] Embodiment 32. An isolated antibody or antigen-binding fragment thereof according to embodiment 29, comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 33, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 32.

[0418] Embodiment 33. An isolated antibody or antigen-binding fragment thereof according to embodiment 32, comprising a VH comprising the amino acid sequence of SEQ ID NO: 33 and a VL comprising the amino acid sequence of SEQ ID NO: 32.

[0419] Embodiment 34. An isolated antibody or antigen-binding fragment thereof according to embodiment 29, comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 38, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 37.

[0420] Embodiment 35. An isolated antibody or antigen-binding fragment thereof according to embodiment 34, comprising a VH comprising the amino acid sequence of SEQ ID NO: 38 and a VL comprising the amino acid sequence of SEQ ID NO: 37.

[0421] Embodiment 36. An isolated antibody or antigen-binding fragment thereof according to embodiment 29, comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 31, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 32.

[0422] Embodiment 37. An isolated antibody or antigen-binding fragment thereof according to embodiment 36, comprising a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 32.

[0423] Embodiment 38. An isolated antibody or antigen-binding fragment thereof according to embodiment 29, comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 36, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 37.

[0424] Embodiment 39. An isolated antibody or antigen-binding fragment thereof according to embodiment 38, comprising a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 37.

[0425] Embodiment 40. An isolated antibody or antigen-binding fragment thereof according to embodiment 29, comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 34, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 35.

[0426] Embodiment 41. An isolated antibody or antigen-binding fragment thereof according to embodiment 40, comprising a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35.

[0427] Embodiment 42. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 41, comprising a heavy chain (HC) having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, or SEQ ID NO:59, and a light chain (LC) having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO:60.

[0428] Embodiment 43. The isolated antibody or antigen-binding fragment thereof of embodiment 42, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 59, and 60.

[0429] Embodiment 44. An isolated antibody, comprising: a. a HC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the amino acid sequence of SEQ ID NO:51, and a LC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the amino acid sequence of SEQ ID NO:52; b. a HC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:53, and a LC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:52; c. a HC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:54, and a LC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:55; d. a HC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:56, and a LC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:57; e. a HC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 58, and a LC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 57, or f. The isolated antibody of embodiment 43, comprising an HC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:59, and an LC comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:60.

[0430] Embodiment 45 The isolated antibody of embodiment 44, comprising a HC comprising the amino acid sequence of SEQ ID NO:51 and a LC comprising the amino acid sequence of SEQ ID NO:52.

[0431] Embodiment 46 The isolated antibody of embodiment 44, comprising an HC comprising the amino acid sequence of SEQ ID NO:53 and an LC comprising the amino acid sequence of SEQ ID NO:52.

[0432] Embodiment 47 The isolated antibody of embodiment 44, comprising a HC comprising the amino acid sequence of SEQ ID NO:54 and a LC comprising the amino acid sequence of SEQ ID NO:55.

[0433] Embodiment 48 The isolated antibody of embodiment 44, comprising a HC comprising the amino acid sequence of SEQ ID NO:56 and a LC comprising the amino acid sequence of SEQ ID NO:57.

[0434] Embodiment 49 The isolated antibody of embodiment 44, comprising a HC comprising the amino acid sequence of SEQ ID NO:58 and a LC comprising the amino acid sequence of SEQ ID NO:57.

[0435] Embodiment 50 The isolated antibody of embodiment 44, comprising a HC comprising the amino acid sequence of SEQ ID NO:59 and a LC comprising the amino acid sequence of SEQ ID NO:60.

[0436] Embodiment 51. An isolated antibody or antigen-binding fragment thereof, comprising: a. the HCDR of VH comprising the amino acid sequence of SEQ ID NO: 33, and the LCDR of VL having the amino acid sequence of SEQ ID NO: 32; b. a VH comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and a VL comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; c. a VH having the amino acid sequence of SEQ ID NO: 33 and a VL having the amino acid sequence of SEQ ID NO: 32, and / or d. An isolated antibody or antigen-binding fragment thereof, comprising: a HC having the amino acid sequence of SEQ ID NO:53; and a LC having the amino acid sequence of SEQ ID NO:52.

[0437] Embodiment 52. An isolated antibody or antigen-binding fragment thereof, comprising: a. the HCDR of VH comprising the amino acid sequence of SEQ ID NO: 34, and the LCDR of VL having the amino acid sequence of SEQ ID NO: 35; b. VH comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and VL comprising LCDR1, LCDR2, and LCDR3 of the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively; An isolated antibody or antigen-binding fragment thereof, comprising: c. a VH having the amino acid sequence of SEQ ID NO: 34 and a VL having the amino acid sequence of SEQ ID NO: 35; and / or d. a HC having the amino acid sequence of SEQ ID NO: 54 and a LC having the amino acid sequence of SEQ ID NO: 55.

[0438] Embodiment 53. An isolated antibody or antigen-binding fragment thereof, comprising: a. a heavy chain variable region (VH) having an HCDR of the VH comprising the amino acid sequence of SEQ ID NO: 38, and a light chain variable region (VL) having an LCDR of the VL comprising the amino acid sequence of SEQ ID NO: 37; b. a VH comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively, and a VL comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively; c. a VH having the amino acid sequence of SEQ ID NO: 38 and a VL having the amino acid sequence of SEQ ID NO: 37, and / or d. An isolated antibody or antigen-binding fragment thereof, comprising: a HC having the amino acid sequence of SEQ ID NO:58; and a LC having the amino acid sequence of SEQ ID NO:57.

[0439] Embodiment 54. An isolated antibody or antigen-binding fragment thereof, comprising: a. a heavy chain variable region (VH) having an HCDR of the VH comprising the amino acid sequence of SEQ ID NO: 39, and a light chain variable region (VL) having an LCDR of the VL comprising the amino acid sequence of SEQ ID NO: 40; b. a VH comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively, and a VL comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively; c. a VH having the amino acid sequence of SEQ ID NO: 39 and a VL having the amino acid sequence of SEQ ID NO: 40, and / or d. An isolated antibody or antigen-binding fragment thereof, comprising: a HC having the amino acid sequence of SEQ ID NO:59; and a LC having the amino acid sequence of SEQ ID NO:60.

[0440] Embodiment 55. An isolated antibody or antigen-binding fragment thereof, comprising: a. a heavy chain variable region (VH) having an HCDR of the VH comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region (VL) having an LCDR of the VL comprising the amino acid sequence of SEQ ID NO: 32; b. a VH comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 7, 175, and 9, respectively, and a VL comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; c. a VH having the amino acid sequence of SEQ ID NO: 31 and a VL having the amino acid sequence of SEQ ID NO: 32, and / or d. An isolated antibody or antigen-binding fragment thereof, comprising: a HC having the amino acid sequence of SEQ ID NO:51; and a LC having the amino acid sequence of SEQ ID NO:52.

[0441] Embodiment 56. An isolated antibody or antigen-binding fragment thereof, comprising: a. a heavy chain variable region (VH) having an HCDR of the VH comprising the amino acid sequence of SEQ ID NO: 36, and a light chain variable region (VL) having an LCDR of the VL comprising the amino acid sequence of SEQ ID NO: 37; b. a VH comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively, and a VL comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively; c. a VH having the amino acid sequence of SEQ ID NO: 36 and a VL having the amino acid sequence of SEQ ID NO: 37, and / or d. An isolated antibody or antigen-binding fragment thereof, comprising: a HC having the amino acid sequence of SEQ ID NO:56; and a LC having the amino acid sequence of SEQ ID NO:57.

[0442] Embodiment 57. An isolated antigen-binding fragment according to any one of embodiments 1 to 56, wherein the antigen-binding fragment is an scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, VHH or a single chain antibody.

[0443] Embodiment 58. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 57, wherein the antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0444] Embodiment 59. The isolated antibody or antigen-binding fragment thereof of embodiment 58, wherein the antibody or antigen-binding fragment thereof is of the IgG1 isotype.

[0445] Embodiment 60. An isolated antibody or antigen-binding fragment thereof according to embodiment 59, comprising an Ig constant region or a fragment of an Ig constant region.

[0446] Embodiment 61. The isolated antibody or antigen-binding fragment thereof of embodiment 60, wherein the Ig constant region or fragment of the Ig constant region comprises at least one mutation that results in reduced binding of the isolated antibody or antigen-binding fragment thereof to an Fcγ receptor (FcγR).

[0447]

[0046] Embodiment 62. The isolated antibody or antigen-binding fragment thereof has at least one mutation that results in reduced binding to FcγR, and is selected from the group consisting of F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D3 62. The isolated antibody or antigen-binding fragment thereof of embodiment 61, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of 65E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236deletion / G237A / P238S, where residue numbering is according to the EU index.

[0448] Embodiment 63. An isolated antibody or antigen-binding fragment thereof according to embodiment 62, wherein the Ig constant region or fragment of the constant region comprises the following mutations: L234A_L235A_D265S.

[0449] Embodiment 64. An isolated antibody or antigen-binding fragment thereof according to embodiment 62, wherein the FcγR is FcγRI, FcγRIIA, FcγRIIB or FcγRIII, or any combination thereof.

[0450] Embodiment 65. The isolated antibody or antigen-binding fragment thereof of embodiment 64, wherein the Ig constant region or fragment of the Ig constant region further comprises at least one mutation that modulates the half-life of the antibody or antigen-binding fragment thereof.

[0451] Embodiment 66. An isolated antibody or antigen-binding fragment thereof according to embodiment 65, wherein at least one mutation that modulates the half-life of the antibody or antigen-binding fragment thereof is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, and wherein residue numbering is according to the EU index.

[0452] Embodiment 67. An immunoconjugate comprising an isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 66, conjugated to a therapeutic or imaging agent.

[0453] Embodiment 68. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment according to any one of embodiments 1 to 66, or an immunoconjugate according to embodiment 67, and a pharma- ceutical acceptable carrier.

[0454] Embodiment 69. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 66.

[0455] Embodiment 70. The isolated polynucleotide of embodiment 69, comprising a polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the polynucleotide sequence of SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70.

[0456] Embodiment 71. A vector comprising the polynucleotide of embodiment 69 or 70.

[0457] Embodiment 72. A host cell expressing the isolated antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 70, such as a host cell comprising the vector described in embodiment 71.

[0458] Embodiment 73. A method for preventing the binding of VEGFA to VEGFR1 in a subject in need thereof, comprising administering to the subject an effective amount of an isolated antibody or antigen fragment thereof described in any one of embodiments 1 to 66, an immunoconjugate described in embodiment 67, a pharmaceutical composition described in embodiment 68, an isolated polynucleotide described in embodiment 69 or 70, a vector described in embodiment 71, or a host cell described in embodiment 72, thereby preventing the binding of VEGFA to VEGFR1.

[0459] Embodiment 74. The method of embodiment 73, wherein the subject is in need of treatment for chronic kidney disease (CKD), in need of reducing proteinuria, has advanced stage 4 or stage 5 chronic kidney disease, or has CKD associated with proteinuria, albuminuria, or diabetes.

[0460] Embodiment 75. A method for treating chronic kidney disease (CKD) in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an isolated antibody or antigenic fragment thereof described in any one of embodiments 1 to 66, an immunoconjugate described in embodiment 67, a pharmaceutical composition described in embodiment 68, an isolated polynucleotide described in embodiment 69 or 70, a vector described in embodiment 71, or a host cell described in embodiment 72 for a time sufficient to treat CKD.

[0461] Embodiment 76. The method of embodiment 75, wherein the subject has advanced stage 4 or 5 chronic kidney disease.

[0462] Embodiment 77. The method of embodiment 75, wherein the subject has stage 3 chronic kidney disease.

[0463] Embodiment 78. The method of embodiment 75, wherein the subject has CKD with proteinuria, albuminuria, or diabetes.

[0464] Embodiment 79. A method for reducing proteinuria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated antibody or antigenic fragment thereof described in any one of embodiments 1 to 66, an immunoconjugate described in embodiment 67, a pharmaceutical composition described in embodiment 68, an isolated polynucleotide described in embodiment 69 or 70, a vector described in embodiment 71, or a host cell described in embodiment 72 for a time sufficient to reduce proteinuria in the subject.

[0465] Embodiment 80. A method for reducing albuminuria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated antibody or antigenic fragment thereof described in any one of embodiments 1 to 66, an immunoconjugate described in embodiment 67, a pharmaceutical composition described in embodiment 68, an isolated polynucleotide described in embodiment 69 or 70, a vector described in embodiment 71, or a host cell described in embodiment 72 for a time sufficient to reduce proteinuria in the subject.

[0466] Embodiment 81. A kit comprising an isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 66, an immunoconjugate according to embodiment 67, a pharmaceutical composition according to embodiment 68, an isolated polynucleotide according to embodiment 69 or 70, a vector according to embodiment 71, or a host cell according to embodiment 72. EXAMPLES

[0467] Example 1. Antigen production Vascular endothelial growth factor receptor 1 (VEGFR1) is a member of the V-type subfamily of receptor tyrosine kinases (RTKs) consisting of seven extracellular immunoglobulin (Ig)-like domains, a single transmembrane domain, and a cytoplasmic kinase domain. Structural studies have demonstrated that ligand binding to VEGFR1 is mediated by Ig domains 2 and 3 (D2 and D3), with the two domains spanning approximately 800 and 700 Å, respectively, at the binding interface. 2It has been previously demonstrated that hVEGFR1 contributes to the binding of VEGF165 (R&D Systems) to the VEGF165 domain, which has been shown to be significantly lower than hVEGFR1 (D1-D3) and hVEGFR1 (D2-D3) (R&D Systems) in the absence of a VEGF165 domain. This was confirmed by binding analysis in which the affinity of recombinant human VEGFR1 (D1-D3) and human VEGFR1 (D2-D3) for human VEGF165 (R&D Systems) was determined by SPR to be <45 pM and <30 pM, respectively. Domain 2 of hVEGFR1 alone showed a significantly lower affinity of 22 nM (approximately a 1,000-fold decrease in affinity), consistent with previously published biochemical data demonstrating that D2 of VEGFR1 alone (Flt-1(2)-IgG) shows substantially reduced affinity for VEGF165 (Davis-Smyth, T. et al, EMBO Journal, 15(18), 4919-4927, 1996).

[0468] Therefore, to identify antibodies capable of blocking ligand binding to VEGFR1, truncated versions of the VEGFR1 extracellular domain (ECD) containing only D2-D3 or D1-D3 were used as primary immunogens during antibody discovery. Fc chimeras of the second and third immunoglobulin (Ig)-like domains of human VEGFR1 (hVEGFR1 D2-D3-Fc-6xHis) were generated by fusing amino acid residues 130-331 (UniProt accession number P17948) to residues 100-330 of human immunoglobulin heavy chain constant gamma 1 (UniProt accession number P01857) via the HRV3C protease recognition sequence "LEVLFQGP" (SEQ ID NO: 174). For flexible purification options, a 6XHis tag was added to the C-terminus of the construct (construct VGFW8, SEQ ID NO: 1). Additionally, an Fc chimera of the first to third immunoglobulin (Ig)-like domains of mouse VEGFR1 (mVEGFR1 D1-D3-Fc-6xHis) was generated by linking residues 23-332 (UniProt accession number P35969) to residues 100-330 of human immunoglobulin heavy chain constant gamma 1 (UniProt accession number P01857) via the HRV3C protease recognition sequence "LEVLFQGP" (sequence number 174).

[0469] A similar construct (mFlt(1-3-IgG) was previously described as a potent inhibitor of VEGF and / or PlGF activity (Ferrara, N. et al, Nature Medicine, 4(3), 336-340 1998). In addition, a 6XHis tag was added to the C-terminus of the construct for flexible purification methods (construct VGFW9, SEQ ID NO:2). Additional constructs were designed to add part or the entire VEGFR1 extracellular domain (ECD) from human, mouse, or cynomolgus monkey (cynomolgus monkey) to the C-terminus with Avi-tag and 6xHis-tag sequences. These were the Ig-like domain 2 of human VEGFR1 (residues 130-225 of Uniprot accession number P17948, construct VGFW2, SEQ ID NO:3), the Ig-like domain 2 of human VEGFR1 (residues 130-225 of Uniprot accession number P17948, construct VGFW2, SEQ ID NO:3), and domain 3 (residues 130-331 of Uniprot accession number P17948, construct VGFW3, SEQ ID NO: 4), Ig-like domains 1-3 of mouse VEGFR1 (residues 23-332 of Uniprot accession number P35969, construct VGFW4, SEQ ID NO: 5), and the entire extracellular domain of monkey VEGFR1 (residues 27-758 of accession number XP_005585612.1, construct VGFW5, SEQ ID NO: 6).

[0470] VEGFR1 ECD expression constructs were transiently transfected into HEK 293-6E cells using polyethyleneimine. When the viability of the cultures dropped to <80%, the cells were incubated at 37°C with 5% CO2 in a batch-fed Wave bag (Culti bag) 20L [Sartorius, Flexsafe® RM 20L optical Cat#DFO020L] for 6 days before harvesting. The cells were removed by centrifugation, and the His-tagged soluble VEGFR1 protein was purified from the medium using a Superdex 200 column (GE Healthcare) equilibrated with immobilized metal affinity chromatography (IMAC) using Ni Sepharose 6 Fast Flow resin (GE Healthcare), followed by preparative size-exclusion chromatography (SEC) in Dulbecco's phosphate saline buffer pH 7.2 (1xDPBS). Alternatively, for VGFW8 and VGFW9, recombinant proteins were captured from spent supernatants by application to and elution from MabSelect SuRe Protein A chromatography resin (GE Healthcare). The amino acid sequences of the generated antigens are shown in Table 2.

[0471] [Table 2-1]

[0472] [Table 2-2]

[0473] Example 2. Generation and preliminary characterization of VEGFR1 antibodies in scFv-Fc format Immunization method It was highly preferred to identify a ligand-blocking anti-VEGFR1 antibody that exhibited broad cross-reactivity across various species (in addition to humans), including cynomolgus monkeys as well as rodents, to enable both non-clinical safety and pharmacology / efficacy studies in well-established rodent models of diabetic kidney disease (DKD). The ability to generate species-cross-reactive antibodies at the initiation of a discovery campaign eliminated the need to develop surrogate antibodies that may not mimic the behavior of the antibody of interest, given the complexity of biology.

[0474] Obtaining antibodies with broad species cross-reactivity (including rodents) may represent a stringent design requirement. Indeed, previously reported anti-VEGFR1 antibodies have been either strictly mouse-specific or strictly human-specific, and have shown poor or no cross-species reactivity. VGFB80, used herein as a reference, is a potent human VEGFR1 binder, but does not show binding to rodent-derived VEGFR1. It is well established that the generation of broadly cross-reactive antibodies is often hindered by immunological tolerance to epitopes that are conserved across mammalian species typically used for antibody discovery (e.g., mouse, rat, and rabbit). Conservation of the ligand-binding domain of VEGFR1 across typical antibody discovery species (i.e., mouse and rat) is evidenced by the amino acid sequence identity shown in Figure 1 and Table 3. Approximately 80% and 67% sequence identity is observed between human and mouse / rat for VEGFR1 domain 2 and domain 3, respectively.

[0475] [Table 3]

[0476] Alternatively, immunization of more phylogenetically distant species such as chickens may be able to overcome immune tolerance to "pan-mammalian" epitopes (Ching, KHet al, MAbs, 10(1), 71-80, 2018). Transgenic Omnichicken, which are phylogenetically distant from humans compared to mice and rats, have demonstrated robust immune responses to conserved human proteins. However, the use of Omnichicken alone does not guarantee high affinity species cross-reactive antibodies.

[0477] The high affinity and species cross-reactive anti-VEGFR1 antibodies described herein were generated using a unique antibody production methodology that includes (1) selection and design of a unique antigen (ligand-binding subdomain of VEGFR1), (2) selection of a phylogenetically distant species (chicken) for immunization, (3) a unique immunization procedure, and (4) a rigorous serum titer screening process to identify anti-VEGFR1 antibodies with broad species reactivity. Transgenic chickens (n=12) carrying humanized immunoglobulin genes (OmniChicken; Ligand Pharmaceuticals; Emeryville, CA and Schusser, B. et al, Proc. Natl. Acad. Sci. USA 110, 20170-20175, 2013) were immunized with either hVEGFR1 D2-D3-Fc-6xHis (VGFW8) or alternating boosts of VGFW8 and mVEGFR1 D1-D3-Fc-6xHis (VGFW9). Bird serum titers were monitored by ELISA using VGFW2 (hVEGFR1_D2-Avi-His), VGFW3 (hVEGFR1_D2-3 Avi His), VGFW4 (mVEGFR1_D1-3_Avi-6xHis), and VGFW5 (cynoVEGFR1_ECD_Avi-6xHis) purified recombinant proteins. Spleens were harvested from birds with the best titers to both human and mouse VEGFR1 recombinant proteins. Lymphocytes were isolated from the spleen and examined for positive binding to both VGFW4 and VGFW3 and / or VGFW2 coated beads. Single cell reverse transcriptase PCR was performed on lymphocytes to recover v-gene sequences. VH and VL sequences were assembled as scFv-Fc (human IgG1) molecules in mammalian expression vectors. Mammalian expression constructs were used to produce antibodies on a small scale (96-well format) by transient transfection. Supernatants from small scale expression were screened for the presence of recombinant antibodies with specificity for VEGFR1 by ELISA using purified antigen.Reconfirmed positive clones were expanded into 6-well plates (2 mL) and ELISA and FACS analysis was repeated using serial dilutions of the supernatants to confirm binding to human, cynomolgus, and mouse VEGFR1 both as soluble purified recombinant protein of the extracellular domain and as full-length cell surface bound receptor on HEK cells engineered to overexpress either human, cynomolgus, or mouse VEGFR1. Additionally, expanded clones were counterscreened on HEK and K562 cells engineered to overexpress hVEGFR2 and hVEGFR3, respectively (Figure 2).

[0478] A subset of clones that displayed specific binding to human, cynomolgus monkey, and mouse VEGFR1 by ELISA and FACS were sequenced and reformatted as human monoclonal IgG1 (L234A / L235A / D265S) molecules (2H:2L) to abolish any Fc receptor effector function while preserving FcRn affinity. Of approximately 800 lymphocytes initially examined, 43 unique molecules resulted (5.6%) that met the above criteria. The remaining clones generally lacked cross-reactivity to mouse VEGFR1 or did not show specific binding to VEGFR1 (i.e., they also bound to human VEGFR2 or human VEGFR3 on cells). In contrast, a more conventional rodent immunization campaign was performed in parallel (n=42 animals immunized) in which approximately 2,200 MSD primary hits were screened against cells, yielding only four clones that demonstrated broad species cross-reactivity with Hu / Cy / Mo VEGFR1 (0.18% hit rate); these molecules ultimately proved less effective in blocking ligand binding or possessed undesirable sequence disorders.

[0479] Forty-three unique Omnichicken-derived clones reformatted as human monoclonal IgG1 (L234A / L235A / D265S) molecules were expressed and purified at small scale (2 mL) and subjected to extensive additional characterization including (1) affinity determination for human, cynomolgus monkey, and mouse VEGFR1 by SPR, (2) ligand blocking potency (both biochemical and cell-based), (3) temperature stability, (4) surface hydrophobicity, (5) nonspecific binding, and (6) lack of modification after stress / forced degradation studies. Twenty-eight of the 43 molecules (~65%) were identified as potent ligand blockers representing 10-11 sequence families. Eight anti-VEGFR1 mAbs were evaluated in single-dose PK-TE-PD studies in db / db mice. VGFB54, VGFB71, VGFB78 and VGFB82 were evaluated in a single-dose PK-TE study in cynomolgus monkeys and subjected to an extensive panel of intrinsic properties and structural characterization.

[0480] Design of VGFB883 and VGFB400 VGFB54 was observed to contain a "NG" deamidation sequence lesion in the heavy chain CDR2, and was observed by peptide mapping to undergo significant deamidation (14.7%) when maintained at pH 8.5 (40C) for 1 week. To address this risk, a small library of variants was generated in which either N54 or G55 was replaced with every possible alternative amino acid (except Cys or Trp). The library of variants was then evaluated for retained binding to the target in both biochemical ligand blocking assays and cell binding assays. Nearly all variants showed retained or slightly improved binding to VEGFR1. The N54Q variant of VGFB54 (VGFB883) was selected for further development as it is one of the most chemically conservative amino acid substitutions, resolved the post-translational modification risk, and showed retained binding to the target (Table 4).

[0481] [Table 4]

[0482] The antibody sequences obtained from the transgenic animals may contain somatic hypermutations in the framework and CDR regions. Somatic hypermutations result in unusual or low frequency residues in human framework regions, which may affect the stability and immunogenicity of biotherapeutics. The parent anti-VEGFR1 antibody VGFB78 heavy chain variable region contained a somatic hypermutation at position 110 (105Chothia delineation) within the framework region. The leucine residue at position 110 (105Chothia) is typically a low frequency residue (frequency <1%) compared to the most frequently found glutamine (Q) residue at that position (frequency 76%) (Figure 3). Thus, an L11Q mutation was engineered in the heavy chain of VGFB78 to result in VGFB400, representing a reversion to germline identity at this position. This substitution was chosen to reflect the most common identity at this position.

[0483] ExpiCHO small scale transfection and purification Antibodies identified from the immunization campaign were cloned, expressed at a 2 ml scale as IgG1-AAS (L234A / L235A / D265S), and purified. ExpiCHO™ cells (ThermoFisher Scientific) were cultured in ExpiCHO™ Expression Medium at 37°C and 7% CO2. Cells were subcultured, in this case at a density of 4-6 × 10 with 98-99% viability. 6 Logarithmic growth was reached in viable cells / mL. On the day of transfection, viable cell density and viability were determined. Cells were transfected at 6 × 10 6Transfections were performed at a density of 10000 viable cells / mL. Culture supernatants were collected by centrifugation at 850 × g for 15 min on day 7 post-transfection and then purified. Antibodies were purified from the clarified supernatants using MabSelect Sure resin (GE Healthcare) and dialyzed into PBS. Protein concentrations were determined by measuring the absorbance of the filtrates at 280 nm using a DropSense Instrument (Trinean NV / SA).

[0484] Anti-VEGFR1 antibody sequence Table 5 shows Kabat HCDR1, HCDR2, and HCDR3 of selected anti-VEGFR1 antibodies. Table 6 shows Kabat LCDR1, LCDR2, and LCDR3 of selected anti-VEGFR1 antibodies. Table 7 shows Chothia HCDR1, HCDR2, and HCDR3 of selected anti-VEGFR1 antibodies. Table 8 shows Chothia LCDR1, LCDR2, and LCDR3 of selected anti-VEGFR1 antibodies. Table 9 shows ABM HCDR1, HCDR2, and HCDR3 of selected anti-VEGFR1 antibodies. Table 10 shows ABM LCDR1, LCDR2, and LCDR3 of selected anti-VEGFR1 antibodies. Table 11 shows IMTG HCDR1, HCDR2, and HCDR3 of selected anti-VEGFR1 antibodies. Table 12 shows IMTG LCDR1, LCDR2, and LCDR3 of selected anti-VEGFR1 antibodies. Table 13 shows the VH and VL amino acid sequences of selected anti-VEGFR1 antibodies. Table 14 shows the VH nucleic acid sequences of selected anti-VEGFR1 antibodies. Table 15 shows the VL nucleic acid sequences of selected anti-VEGFR1 antibodies.

[0485] Table 16 shows the HC amino acid sequences of selected anti-VEGFR1 antibodies. Table 17 shows the LC amino acid sequences of selected anti-VEGFR1 antibodies. Table 18 shows the HC nucleotide sequences of selected anti-VEGFR1 antibodies. Table 19 shows the LC nucleotide sequences of selected anti-VEGFR1 antibodies. Table 20 summarizes the SEQ ID NOs assigned to selected anti-VEGFR1 antibodies.

[0486] [Table 5]

[0487] [Table 6]

[0488] [Table 7]

[0489] [Table 8]

[0490] [Table 9]

[0491] [Table 10]

[0492] [Table 11]

[0493] [Table 12] NA=Not Applicable

[0494] [Table 13]

[0495] [Table 14-1]

[0496] [Table 14-2]

[0497]

Table 15-1

[0498]

Table 15-2

[0499]

Table 16-1

[0500]

Table 16-2

[0501]

Table 17

[0502]

Table 18-1

[0503]

Table 18-2

[0504]

Table 18-3

[0505]

Table 19-1

[0506]

Table 19-2

[0507] [Table 20]

[0508] Example 3. Biophysical characterization and in vitro activity profiling of anti-VEGFR1 antibodies Confirmation of binding to cells overexpressing VEGFR1 Parental CHO-K1 cells, CHO-K1 cells stably expressing human VEGFR1, Ba / F3 parental cells, and Ba / F3 cells stably expressing mouse VEGFR1 were stained with either no stain, CSFE, CTV, or CTV+CSFE, respectively, for 10 min at 4°C. Cells were then washed with medium containing heat-inactivated FBS, collected by centrifugation, combined, resuspended in staining buffer, and plated (95 μL / well, 200,000 of each cell type per well). Antibody samples were added to a final concentration of 10 nM (5 μL / well) and incubated for 60 min at 4°C protected from light. Cells were washed twice with 150 μL / well of staining buffer. AlexaFluor 647-conjugated anti-human IgG1 secondary antibody was added (100 μL / well) and incubated for 30 min at 4°C protected from light. The cells were washed twice with 150 μL staining buffer, and finally resuspended in 30 μL running buffer and read on an iQue flow cytometer. This single concentration assay was used as a quick confirmation of binding to human and mouse VEGFR1 on cells. A similar assay was performed to confirm the binding of candidate antibodies to HEK293 cells stably overexpressing cynomolgus VEGFR1 at a single concentration of 10 nM. Furthermore, to demonstrate specificity for VEGFR1, a similar counter-screening assay was also performed to confirm the lack of binding of anti-VEGFR1 antibodies to either HEK293 cells stably expressing human VEGFR2 or K562 cells stably expressing human VEGFR3 at a single high concentration (10 nM) (Table 21).

[0509] [Table 21] The threshold for positivity for CyVEGFR1 was S / B>3. ND = Not Determined Goat anti-human VEGFR2 = R&D Systems Catalog No. AF357 Goat anti-human VEGFR2 = R&D Systems Catalog No. AF349

[0510] A full dose-response curve for binding of anti-VEGFR1 antibodies to VEGFR1 on cells was also generated. Parental CHO-K1 cells and CHO-K1 cells stably expressing human VEGFR1 (CHO-K1\hVEGFR1) were cultured in aMEM+10% FBS and aMEM+10% FBS containing 10 μg / mL puromycin, respectively. CHOK1 / hVEGFR1 were stained with CSFE as above, while parental CHO-K1 cells were left unstained. Cells were washed and combined (approximately 0.5×10 6 Cells were plated at approximately 40,000 cells per well (75 μL / well) at 1:1000 cells / mL. Serially diluted antibody samples were then added to the cells and incubated at 37° C. for 1 hour. Cells were washed twice with staining buffer and then stained with AF647-conjugated secondary antibody for 30 minutes at 4° C. Plated cells were washed twice, followed by the addition of read buffer containing Sytox Blue (1:1000) and read on an iQue flow cytometer. The data was fitted to determine EC 50 The values ​​were obtained (Table 22).

[0511] [Table 22]

[0512] Ligand blockade: biochemical and cellular basis The ability of anti-VEGFR1 antibodies to prevent binding of VEGFA or placental growth factor (PlGF) ligands to VEGFR1 was determined in both a biochemical electrochemiluminescence (ECL) assay and in a cell-based assay format.

[0513] Briefly, MSD MA6000 384 plates were coated with 0.5ug / ml Protein A / G (Thermo Fisher Scientific) or anti-His mAb (R&D) in TBS for 16 hours in the cold room. Plates were washed twice with 1x TBS and blocked with 35μl / well Blocker A for 1 hour at room temperature. Recombinant human or mouse VEGFR1-Fc protein (R&D Systems) or His-tagged cynomolgus VEGFR1 was added to the plates at 0.5ug / ml and incubated for 1 hour at room temperature. Unbound VEGFR1 protein was washed away using 70ul / well 1x MSD Tris Wash buffer. Half-log serial dilutions of unlabeled VEGFR1 ligand or anti-VEGFR1 antibody were pre-incubated with bound VEGFR1 for 30 min at room temperature, then diluted biotin-labeled human VEGFA165 (0.2 nM), mouse VEGFA164 (0.2 nM) or PLGF (2 nM) were added for binding. Of note, the amino acid sequences of cynomolgus and human VEGFA and PlGF are identical, and biotin-labeled human VEGF-A and PlGF were used as ligands for the cynomolgus VEGFR1 competition assay. Bound ligand was detected by adding 1 μg / ml sulfo-tagged streptavidin. Signal was generated and detected with an MSD Imager, SECTOR S 600 (Meso Scale C Diagnostics, serial number 1201180626620) in the presence of 35 μl / well of 2×MSD Read Buffer T. The ECL readings were used to calculate % binding, IC50 and % maximum inhibition. % Binding = (((Sample - Avg(Background)) ÷ (Avg(Total Binding) - Avg(Background))) x 100 Total binding = biotin ligand only ○Background = no blocker or biotin ligand • Nonlinear regression log(inhibitor) vs response - curves were fitted using variable slope (four parameters). Formula: Y = bottom + (peak - bottom) / (1 + 10^((LogIC50-X) x Hill slope)) ○IC50 was extracted from Prism 8 software. Maximum inhibition % = 100 - average (% binding at highest concentration)

[0514] The in vitro efficacy (% maximum inhibition) and potency (IC50) of VEGFR1 mAbs in displacing VEGFA / PlGF from binding to human, cynomolgus, and mouse VEGFR1 are summarized in Tables 23 and 24. The anti-VEGFR1 antibodies showed potent ligand blocking activity in binding to human and cynomolgus VEGFR1 with maximal inhibition of approximately 100%. The IC50 values ​​were in the sub-nanomolar range and were comparable to their ligands. VGFB80 had no binding or ligand competition to mouse VEGFR1, while other VEGFR1 mAbs showed high blocking potency and efficacy against mouse VEGFR1 in this assay.

[0515] [Table 23]

[0516] [Table 24]

[0517] Ligand blockade on cells was determined as follows: Cells stained with Cell Trace Violet CSFE (ThermoFisher Scientific) were stained in FACS staining buffer at 15,000 cells per well (30 μL / well, parental CHO cells or CHO cells engineered to overexpress human VEGFR1). Equal volumes of serially diluted test antibodies were applied to the cells and incubated for 1 h at 4° C. Cells were washed twice with 200 μL / well staining buffer, followed by the addition of 100 μL / well biotinylated VEGF A ligand (500 pM). Cells were incubated for an additional hour at 4° C. before the addition of 50 μL / well AF647-conjugated streptavidin (3 nM). Plates were incubated for 30 min at 4 °C protected from light, washed twice with 200 μL / well staining buffer, collected by centrifugation, resuspended in running buffer containing Sytox Blue live / dead stain, and then read on an Intellicyt iQue Plus flow cytometer. Data were fitted to obtain IC 50 Percentage of ligand blockade was calculated for each concentration of each antibody tested and IC50 values ​​were determined (Table 25).

[0518] [Table 25]

[0519] Determination of thermal stability The thermal stability of each antibody was determined by the NanoDSF method using an automated Prometheus instrument from Nanotemper Technologies. Measurements were performed by loading samples from a 384-well sample plate into a 24-well capillary at a concentration of 0.25 mg / mL. Duplicate measurements are performed for each sample. The Prometheus NanoDSF user interface (Melting Scan tab) is used to set the experimental parameters for the run. A thermal scan of a typical IgG sample is performed from 20°C to 95°C at a rate of 1.0°C / min. NIST mAb (and / or CNTO3930, CNTO5825) was included as a control and run in duplicate. Unfolding during the temperature ramp is monitored using molecular intrinsic fluorescence at 330 and 350 nm and recorded as a change in fluorescence intensity over time. T 開始 is the temperature at which unfolding begins.

[0520] [Table 26]

[0521] The thermal stability of selected candidates was also performed by differential scanning calorimetry (DSC). A MicroCal VP-Capillary DSC instrument was used. Samples were dialyzed overnight into 1xDPBS or another appropriate buffer. Thermal scans were performed by increasing the temperature of sample and reference cells from 25 to 95°C at a rate of 60°C / h. Thermograms were fitted using "non-two-state" parameters. Tm1 for all samples may represent Fab unfolding and ranges from 68 to 73°C. Tm2 and Tm3 are associated with variant IgG1 Fc unfolding.

[0522] [Table 27]

[0523] Affinity measurement by kinetic exclusion assay Kinetic Exclusion Assay (KEA, KinExA) was used to analyze the binding of anti-VEGFR1 antibodies to human, mouse and cynomolgus VEGFR1. KEA is a fluorescence-based solution method, described in detail in the literature (Darling and Brault, 2004). To measure the affinity of the interaction, a series of solutions with a fixed concentration of one interactor (A) and various concentrations of another interactor (B) are prepared and allowed to reach equilibrium. After equilibration, the concentration of free A is determined by running the equilibrated mixture through a column packed with beads modified with reactant B. Free A binds to the beads and is then detected using a fluorescently labeled polyclonal antibody. To ensure that the equilibrium is not disrupted (not displaced), i.e., B does not dissociate from the AB complex (kinetic exclusion), the sample is loaded onto the column at a fast flow rate to achieve a short contact time between the mixture and the beads.

[0524] Briefly, serial dilutions of antigen (2 nM to 0.52 pM, or 800 pM to 5.37 fM) were prepared using fixed concentrations of Ab (300 pM, 60 pM, 12 pM, 2.4 pM, or 0.5 pM). The complex titrations were incubated at room temperature to reach binding equilibrium for at least 2 days. After incubation, samples were run on a KinExA3200 or KinExA4000 instrument to assess free antibody in the reaction. During these studies, some of the data were rejected because the experiment failed or did not pass the acceptance criteria. For mouse VEGFR1, the 60 and 300 pM concentrations were rejected due to high titrant-related NSB. The n-curve analysis function of the KinExA Pro software was used to calculate the dissociation equilibrium constant (K D ) was determined. The program uses nonlinear least-squares regression analysis of the data globally fitted to a 1:1 binding model. (CI=confidence interval).

[0525] [Table 28] a Mouse VEGFR1 specific antibody. Does not bind to human, cynomolgus monkey or rat VEGFR1

[0526] HDX epitope mapping of VEGFR for five mAbs The epitopes of VEGFR1 for five mAbs, VGFB54, VGFB71, VGFB78, VGFB80, and VGFB82, were mapped by HDX-MS. VGFB80 was a reference molecule that was not discovered in the antibody generation efforts described herein. Segments that reduced the free energy by more than 2 kcal / mol upon binding to the mAb are "strong" epitopes as defined by HDX-MS. Segments that reduced the free energy by 1-2 kcal / mol upon binding to the mAb are considered "weak" epitopes. The epitopes of VEGFR1 for all mAbs tested are very similar. The strong epitopes of VEGFR1 for VGFB54 are segments 172-177 (FPLDTL, SEQ ID NO: 143) and 201-204 (EIGL, SEQ ID NO: 144). The strong epitopes of VEGFR1 for VGFB71 are segments 172-177 (FPLDTL, SEQ ID NO: 143), 200 (K), and 201-204 (EIGL, SEQ ID NO: 144). The strong epitopes of VEGFR1 for VGFB78 are segments 172-177 (FPLDTL, SEQ ID NO: 143), 200 (K), and 201-204 (EIGL, SEQ ID NO: 144). The strong epitope of VEGFR1 for VGFB82 is segment 201-204 (EIGL, SEQ ID NO: 144). The sequence coverage of VGFW1 was 88% (=285 / 326) when digested by pepsin / FPXIII mixed bed column after quenching with 0.4 mM FC-14, 8 M urea, 1 M TCEP, pH 3.0. Of the five potential glycosylation sites, N196 was covered by a non-glycopeptide, while the other four sites were not covered.

[0527] [Table 29]

[0528] HDX paratope mapping of four mAbs against VEGFR1 The paratopes of four mAbs against VEGFR1 were mapped by HDX-MS (Figures 4A and 4B). Segments that decreased the free energy by more than 1 kcal / mol upon binding to the mAb are HDX-MS-defined epitopes. The paratopes of VGFB54 for VEGFR1 are heavy chain segments 32-35 (YGMN, HCDR1, SEQ ID NO: 145), 38-46 (RQAPGKGLD, SEQ ID NO: 146), 49-54 (SSINRN, HCDR2, SEQ ID NO: 147), 55-59 (GDIST, HCDR2, SEQ ID NO: 148), 69-77 (FTISRDNSK, SEQ ID NO: 149), and 97-104 (CTKNHDYH, HCDR3, SEQ ID NO: 150) and light chain segments 1-32 (QSVLTQPPSASGTPGQRVTISCSGSRSNIGNN, LCDR1, SEQ ID NO: 151) and 51-74 (LNSQRPSGVPDRFSGSKSGTSASL, LCDR2, SEQ ID NO: 152). The paratopes of VGFB71 for VEGFR1 are heavy chain segments 30-34 (RDYNM, HCDR1, SEQ ID NO: 153), 39-47 (QAPGKGEW, SEQ ID NO: 154), and 49-68 (SIITNDGSSTAYSDSVKGRF, HCDR2, SEQ ID NO: 155), and light chain segments 1-34 (QSVLTQPPSASGTPGQRVTISCSGGSSNIGSNYV, LCDR1, SEQ ID NO: 156), 50-51 (YF, LCDR2), 54-74 (QRPSGVPDRFSGSKSGTSASL, SEQ ID NO: 158), and 94-98 (DRVNV, LCDR3, SEQ ID NO: 159). The paratopes of VGFB78 for VEGFR1 are light chain segments 50-73 (YYD-QRPSGVPDRFSGSKSGTSASL, LCDR2, SEQ ID NO: 160).The paratope of VGFB82 for VEGFR1 is heavy chain segments 7 to 18 (SGGGLVQPGGSL, SEQ ID NO: 161), 23 to 28 (EASGFD, HCDR1, SEQ ID NO: 162), 31 to 33 (TYA, HCDR1), 38 to 46 (RQAPGKGLE, SEQ ID NO: 163), 51 to 68 (INSEGTITSHAPAVKGRFT, HCDR2, SEQ ID NO: 164), and 96 to 116 (CSSTTGTTHGMDVWG, HCDR3, SEQ ID NO: 165) and light chain segments 1 to 34 (QSVLTQPPSA SGTPGQRVTISCSGSSSNIQNNYV, LCDR1, sequence number 166), 50 to 51 (YF, LCDR2), 52 to 74 (NSQRPSGVPDRFSGSKSGTSASL, LCDR2, sequence number 167), and 95 to 100 (SLNGWV, LCDR3, sequence number 168).

[0529] Tyrosine kinase activation assay Anti-VEGFR1 antibodies were tested to ensure that they could not activate VEGFR1 signaling. Due to the inherently weak signaling activity of VEGFR1, a cell-based bioassay previously described was used (Makinen, T. et al. EMBO Journal, 20(17), 4762-4773, 2001). In Ba / F3 cells stably expressing a chimeric receptor, the extracellular domain of human VEGFR1 (residues 1-758, Uniprot accession number P17948) was fused to the transmembrane and intracellular domains of the mouse erythropoietin receptor (EpoR, residues 250-507, Uniprot accession number P14753) and the IL-3-independent proliferation of these cells was determined in the presence or absence of anti-VEGFR1 antibodies or hVEGFR1 as a positive control. Briefly, approximately 20,000 cells / well were plated in medium lacking mouse IL-3 (RPMI + 10% FBS + 0.5 μg / ml puromycin). Mouse IL-3 was added to control wells at a final concentration of 10 ng / mL. Other wells received either anti-VEGFR1 antibody (final concentration 10 nM or 50 nM), hVEGFA165 (5 nM, R&D Systems), or basal medium (without mIL-3). Cells were incubated at 37°C, 5% CO2 for 72 hours. Proliferation was measured by addition of 50 μL / well CellTiter Glo (Promega) and incubation at room temperature for 10 minutes. Finally, plates were read on an Envision 2105 plate reader using the Ultrasensitive Luminescence protocol. Proliferation of BaF3\hVEGFR1-mEpoR cells was compared to mIL-3-dependent conditions. Unlike hVEGF (5 nM), which was demonstrated to rescue cell viability in the absence of mIL-3, none of the anti-VEGFR1 antibodies tested were able to promote IL-3-independent proliferation at any of the concentrations tested (Table 30). This result is consistent with the inability of the anti-VEGFR1 antibodies to stimulate tyrosine kinase activation or further downstream signaling.

[0530] [Table 30]

[0531] specificity cell surface-ome One risk associated with broad species cross-reactive antibodies is the increased off-target damage. Cell microarray technology was performed (Retrogenix Ltd.) to confirm the specificity of the anti-VEGFR1 antibody to VEGFR1 and the absence of any "off-target" damage.

[0532] This human cell microarray methodology has been previously described (Freeth, J. et al, SLAS Discovery, 25(2), 223-230, 2020). The primary screen was performed on slides arrayed with fixed HEK293 cells, where each spot of HEK293 cells expressed either a unique protein from the cell surface-ome, a cell surface-tethered human secreted protein, or a heterodimeric human cell surface receptor. In total, 5,528 unique gene products were represented in this primary screen (4,103 human plasma membrane proteins (+ some non-tethered secreted proteins) + 1,425 cell surface-tethered human secreted proteins). Briefly, slides containing fixed transfected HEK293 cells were overlaid with a solution of test antibody (2 μg / mL), incubated, and washed. Binding was assessed using an AlexaFluor647-conjugated anti-human IgG Fc detection antibody and fluorescence quantification in ImageQuant. As expected, all anti-VEGFR1 molecules tested showed binding to isoforms of human VEGFR1 (FLT1 isoforms 1, 2 and 3) (Tables 31-34). VGFB54, VGFB80, and VGFB82 did not show binding to any other surface-ome proteins captured by this screen. Two of the anti-VEGFR1 antibodies (VGFB71 and VGFB78) showed unexpected binding to unrelated cell surface proteins (Table 32) Nectin-1, FGFR2 (fibroblast growth factor receptor 2), ISLR (immunoglobulin superfamily containing leucine-rich repeat proteins) and CRELD2 (protein disulfide isomerase CRELD2). Located in the ER, off-target binding to CRELD2 was less significant. On the other hand, Nectin-1, FGFR2, and ISLR present a broad tissue expression profile. Cross-reactivity to these proteins is undesirable as binding to these targets can significantly affect the pharmacokinetic and biodistribution profiles of the molecule.This off-target binding was then confirmed by flow cytometry on live HEK293 cells transiently expressing these "off-target" proteins identified in the primary screen. These data were used to further select anti-VEGFR1 antibodies with unknown off-target impairments.

[0533] [Table 31]

[0534] [Table 32]

[0535] [Table 33]

[0536] [Table 34]

[0537] Example 4. Systemic pharmacokinetics and target-associated biomarker profiles of anti-VEGFR1 antibodies in cynomolgus monkeys In addition to biophysical and in vitro activity profiling of anti-VEGFR1 antibodies, systemic pharmacokinetic (PK) and target engagement (TE) biomarker profiles were characterized in cynomolgus monkeys.

[0538] Naive male cynomolgus monkeys (Macaca Fascicularis, ages 2-7 years, Envigo RMS) were randomized into groups of two animals each by body weight. VGFB54, VGFB78, VGFB82 and VGFB80 at 0.3 mg / kg, 2 mg / kg and 10 mg / kg were administered intravenously (IV) over approximately 2 minutes on day 1, then subcutaneously (SC) on day 15. Blood samples (0.5-2 mL total) were collected via the femoral vein into tubes containing K2 EDTA from each animal at pre-dose and 1, 6, 24, 48, 96, 168 and 336 hours after IV dosing and SC dosing. Blood samples were kept at 2-8°C and then centrifuged to obtain plasma (within 1 hour of collection). Plasma samples were collected in PK and TE aliquots and stored at -70°C until sample analysis.

[0539] Immunoassay bioanalysis of anti-VEGFR1 antibodies The "total" and "free" anti-VEGFR1 mAb concentrations in cynomolgus monkey plasma were determined using an immunoassay method using a typical sandwich format with electrochemiluminescence detection. Briefly, MSD Gold streptavidin plates (Meso Scale Discovery) were pre-wetted with blocking buffer and tapped dry. A master mix solution containing the final working concentrations of capture and detection reagents was added to the plate (35 μL / well) along with diluted standards, quality controls (QCs) and samples (15 μL / well) and incubated for 60 min. After washing, 1× MSD-T read buffer was added to all wells. Immediately thereafter, the plate was read on an MSD Sector S600 Imager. The quantifiable ranges for "total" and "free" VEGFR1 mAb were defined as 0.010-2560 μg / mL and 0.020-5120 μg / mL, respectively, with a 10× minimum required dilution for plasma samples. For the measurement of "total" mAb, biotinylated and ruthenium-labeled anti-human Fc mAb (R10Z8E9) were used as capture / detection reagents. For the measurement of "free" mAb, biotinylated recombinant cynomolgus VEGFR1[D1-D3]-Avi6xHis and ruthenium-labeled anti-human Fc mAb (R10Z8E9) were used as capture / detection reagents.

[0540] The MSD output files containing the raw ECL counts were swept into Cyberlab Content Manager (Agilent) and imported into Watson LIMS (Thermo Scientific) regression software for analysis. The Watson study regression was predefined during assay qualification and used a 5-parameter logistic (auto-estimated) fit with a 1 / Y2 weighting factor.

[0541] Plasma drug concentration-time profiles are illustrated in Figures 5-8. Total and free concentrations were similar across all time points for all four molecules. Plasma PK parameters estimated using non-compartmental analysis are shown in Table 35. Terminal half-life (T 1 / 2 , maximum plasma drug concentration (Cmax ), and area under the drug serum concentration-time curve (AUC) increased with dose in a greater than dose-proportional manner. The nonlinear PK profiles and shorter half-lives at lower doses suggest target-mediated pharmacokinetics (TMDD). The mean half-lives of VGFB54, VGFB82, and VGFB80 at 10 mg / kg IV were approximately 4.9, 5.7, and 4.5 days, respectively (based on noncompartmental analysis of total PK). However, due to significant TMDD, short sampling times, and the possibility that the terminal phase has not been fully characterized, T 1 / 2 The mean mean and clearance (CL) should be interpreted with caution. The monkey PK profiles of VGFB82, VGFB54, and VGFB80 were further characterized using a minimal physiologically based PK model. The SC bioavailability of VGFB54, VGFB82, and VGFB80 was approximately 83.7%, 95.4%, and 87.7%.

[0542] [Table 35]

[0543] C max , maximum (peak) concentration, AUC last , area under the concentration-time curve up to the last sampling point, AUC inf , area under the concentration-time curve extrapolated to infinity, CL, total clearance, T 1 / 2 , Half-life, NA, Not Applicable

[0544] Plasma PlGF in response to VEGFR1 mAb administration In addition to VEGFA, VEGFR1 also binds to placental growth factor (PlGF). Unlike VEGFA, which binds to both VEGFR1 and VEGFR2, PlGF is a selective ligand for VEGFR1. Because PlGF and VEGFA share overlapping binding domains on VEGFR1 (D2-D3) with comparable binding affinity, anti-VEGFR1 antibodies that block VEGFA binding competitively block PlGF binding to VEGFR1, resulting in increased levels of PlGF.

[0545] Total plasma PlGF levels, including both unbound and bound VEGFR1, in cynomolgus monkeys were determined using a stepwise electrochemiluminescence assay (Human PlGF V-plex kit, MSD K151MED-4). Plates coated with capture antibody were blocked and incubated for 1 h. PlGF calibrator (human PlGF, R&D Systems), quality control (QC), and plasma samples were diluted 1:10 in buffer containing anti-VEGFR1 antibody (167 μg / mL). Anti-VEGFR1 antibody binds to soluble VEGFR1, thus relieving signal suppression caused by VEGFR1 binding to PlGF. After incubation at room temperature for at least 15 min, the mixture was added to the blocked, washed MSD plate and incubated overnight at 4°C. After washing, detection antibody conjugated with SulfoTag was added and incubated for 2 h at room temperature. MSD plate was washed and 1× MSD-T read buffer was added to all wells. Plates were then immediately read on an MSD Sector S600 Imager.

[0546] The MSD output files containing the raw ECL counts were imported into Watson LIMS (Thermo Scientific) regression software for analysis. The Watson study regression was predefined during assay qualification and used a four-parameter logistic fit with a 1 / Y2 weighting factor.

[0547] Following IV or SC administration of VEGFR1 mAbs, systemic PlGF levels increased in a dose- and PK-related manner (Figures 9-12). The increase in PlGF levels was comparable between VGFB82 and VGFB54 and tended to be higher than VGFB80 and VGFB78.

[0548] Example 5. Target engagement biomarker profile of mouse reformatted anti-VEGFR1 leads in db / db mice To reduce the immunogenicity of human anti-VEGFR1 mAb in mice and allow long-term dosing and efficacy studies in mice, a mouse reformatted version of the human anti-VEGFR1 antibody was generated in which the variable regions of the human anti-VEGFR1 mAb (VH / VL) were grafted onto mouse constant regions composed of silent mouse IgG1_D265A (CH1, CH2, CH3) for the heavy chain and mouse lambda constant regions for the light chain. The control is a control antibody of the same isotype that does not bind to VEGFR1.

[0549] Mouse PlGF was measured using the Mouse PlGF-2 Quantikine ELISA Kit (R&D System Catalog MP200). Test plasma samples were diluted 1:4 to 1:15 in calibrator diluent RD 5-17 according to dose level and time point to keep the readout within the linear detection range of the assay (assay range of 23.4-1500 pg / mL with a sensitivity of 1.84 pg / mL). Plates were read on a SpectraMax plate reader (Molecular Devices Model Paradigm) set at 450 nm, and concentrations of PLGF-2 in mouse plasma samples were calculated using SoftMax® Pro 7.1.

[0550] When administered subcutaneously to db / db female mice (The Jackson laboratory, Stock No. 000642) at 1 mg / kg or 10 mg / kg on days 0, 7, and 14, once a week, mouse reformatted VGFB54, VGFB78, and VGFB82 induced different levels of PlGF response at the same dose level (Table 36 and Table 37). Reformatted VGFB78 induced the strongest increase in plasma PlGF among the three leads. Reformatted VGFB82 also led to an increase in PLGF. However, compared to VGFB78, VGFB82 induced a PlGF response at a significantly lower level in mice (approximately 4-fold lower than VGFB78 at 10 mg / kg). Reformatted VGFB54 caused minimal increases in TE biomarkers in mice.

[0551] [Table 36]

[0552] [Table 37]

[0553] Example 6. Effect of VGFB82 and VGFB78 on reducing albuminuria in hypertensive unilateral nephrectomized db / db mice To evaluate the efficacy of anti-VEGFR1 lead mAbs, a severe progressive diabetic kidney disease mouse model named after ReninAAV uNx db / db model was used herein (Harlan SM, et al. J Am Soc Nephrol 2018;29:477-91; Harlan SM, et al. Am J Physiol Regul Integr Comp Physiol 2015;309:R467-74). In this model, persistent hypertension induced by delivery of renin-expressing adeno-associated virus further accelerates disease progression and renal injury in unilaterally nephrectomized db / db mice. ReninAAV uNx db / db mice exhibit severe renal dysfunction characterized by a significant increase in urinary albumin-to-creatinine ratio (UACR), reduced glomerular filtration rate (GFR), and increased serum creatinine. Histopathological characterization revealed similar features of advanced human diabetic kidney disease, including mesangial expansion, glomerular sclerosis, tubular degeneration and tubulointerstitial inflammation and fibrosis.

[0554] Female db / db mice were unilaterally nephrectomized at 7–8 weeks of age (Surg 3041 from Jackson Laboratory) and transfected with renin-expressing adeno-associated virus (1 × 10 10Mice were infected with 1000 ng / mL of IgG (genome copies) from 1000 mice. After 6 weeks, mice were randomized into groups of 9–15 animals each, using weights of 0.8, 0.1, and 0.1, respectively, in the IRINI software based on their UACR, systolic blood pressure, and body weight. Blood glucose >300 mg / dL and UACR >5000 mg / g were used as inclusion criteria for entering randomization. Mice were administered either vehicle, lisinopril (10 mg / kg in drinking water), isotype control, low or high doses of murine reformatted VGFB82 or VGFB78 by subcutaneous injection at the indicated dosing frequency for 4–6 weeks. Urine samples were collected 2 h before treatment and every 2 weeks after dosing by individually housing mice in metabolic cages (MMC100, Hatteras Instruments). Urinary concentrations of albumin and creatinine were measured using a Sekisui Diagnostics Microalbumin Assay Kit (Cat. No. 252-20), an Enzymatic Creatinine Assay Kit (Cat. No. 265-30) and a Vet Axcel Chemistry Analyzer (Alfa Wassermann). Plasma samples were collected for PlGF measurements.

[0555] Data were presented as mean ± SE. GraphPad Prism (Ver 8.0 GraphPad) and two-way repeated measures ANOVA or / and linear mixed effects models were used for treatment comparisons.

[0556] Mice were administered SC VGFB82 reformatted at 3 mg / kg and 30 mg / kg once weekly (QW) for 4 weeks, followed by 30 mg / kg three times weekly (TW) and 90 mg / kg once weekly for an additional 2 weeks, respectively. Table 38 shows UACR and percent change from baseline at 2, 4, and 6 weeks after dosing. Table 39 shows plasma PlGF levels at 4 and 6 weeks after dosing. The vehicle-treated group exhibited a continuous and sustained increase in UACR throughout treatment. Lisinopril, an angiotensin-converting enzyme inhibitor (ACEi, standard of care), significantly reduced UACR to about 26% from its baseline at week 4 compared to vehicle control. Mice treated with VGFB877 at 3 mg / kg QW for 4 weeks showed no discernible change in UACR when compared to the vehicle group. High dose VGFB877 at 30mg / kg / QW partially halted UACR progression when compared to vehicle-treated mice at week 4 (% change from baseline, +15% for VGFB877 vs. +83% for vehicle). When dose and dosing frequency were increased from 3mg / kg QW to 30mg / kg TW, UACR% change from baseline decreased from +63% to +28% at week 6. When dose was adjusted from 30mg / kg QW to 90mg / kg QW, UACR% change from baseline decreased from +15% to +6% at week 6. Consistent with improved UACR reduction, plasma PlGF levels increased when dose level or frequency of VGFB82 was increased.

[0557] [Table 38]

[0558] [Table 39]

[0559] For efficacy studies of mouse reformatted VGFB78 (VGFB876), ReninAAV uNx db / db mice were administered 3 mg / kg and 10 mg / kg TW SC for 4 weeks. Tables 40-41 showed UACR and percent change from baseline at 2 and 4 weeks after administration. Table 39 showed plasma PlGF levels at 4 weeks after administration. In contrast to the gradual increase in UACR in vehicle and isotype-treated mice, VGFB876 significantly reduced UACR at week 4 compared to the control group. A low dose of 3 mg / kg reduced UACR by about 9% from baseline, and a high dose of 10 mg / kg reduced UACR by about 20%, which is comparable to ACEi. Consistent with the significant PD effect (UACR reduction), VGFB876 induced a strong dose-dependent increase in plasma PlGF levels, consistent with its high binding potency to mouse VEGFR1.

[0560] [Table 40]

[0561] [Table 41]

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to vascular endothelial growth factor receptor 1 (VEGFR1), comprising: a heavy chain variable region (VH) comprising heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3); a light chain variable region (VL) comprising light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3); Including, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are (a) SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively; SEQ ID NOs: 89, 90, 91, 92, 93, 94, SEQ ID NOs: 113, 114, 115, 116, 117, 118, or SEQ ID NOs: 137, 138, 139, 140, amino acid sequence FNS, and SEQ ID NO: 142, respectively; (b) SEQ ID NOs: 7, 175, 9, 10, 11, and 12, respectively; SEQ ID NOs: 71, 176, 73, 74, 75, 76, SEQ ID NOs: 95, 177, 97, 98, 99, 100, or SEQ ID NOs: 119, 178, 121, 122, amino acid sequence LNS, and SEQ ID NO: 124, respectively; (c) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively; SEQ ID NOs: 71, 72, 73, 74, 75, 76, SEQ ID NOs: 95, 96, 97, 98, 99, 100, or SEQ ID NOs: 119, 120, 121, 122, amino acid sequence LNS, and SEQ ID NO: 124, respectively; (d) SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively; SEQ ID NOs: 77, 78, 79, 80, 81, 82, SEQ ID NOs: 101, 102, 103, 104, 105, 106, or SEQ ID NOs: 125, 126, 127, 128, amino acid sequence FNF, and SEQ ID NO: 130, respectively; (e) SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively; SEQ ID NOs: 83, 84, 85, 86, 87, 88, SEQ ID NOs: 107, 108, 109, 110, 111, 112, or SEQ ID NOs: 131, 132, 133, 134, amino acid sequence YD, and SEQ ID NO: 136, respectively. An isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence:

2. a. the antibody or binding fragment thereof: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 39, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 39; (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 40, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 40; Including, b. the antibody or binding fragment thereof is (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 31, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31; (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 32, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 32; Including, c. the antibody or binding fragment thereof is (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 33, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 33; (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 32, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 32; Including, d. the antibody or binding fragment thereof is (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 34, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 34; (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 35, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 35; Including, e. the antibody or binding fragment thereof is (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 36, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 36; (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 37, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 37; Contains, or f. the antibody or binding fragment thereof is (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 38, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 38; (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 37, or having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 37; 2. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising:

3. a. The antibody or binding fragment thereof is (i) a heavy chain (HC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 59; (ii) a light chain (LC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60; and Including, b. the antibody or binding fragment thereof is (i) a heavy chain (HC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51; (ii) a light chain (LC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52; and Including, c. the antibody or binding fragment thereof is (i) a heavy chain (HC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53; (ii) a light chain (LC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52; and Including, d. the antibody or binding fragment thereof is (i) a heavy chain (HC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54; (ii) a light chain (LC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55; and Including, e. the antibody or binding fragment thereof is (i) a heavy chain (HC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56; (ii) a light chain (LC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 57; and Including, f. the antibody or binding fragment thereof is (i) a heavy chain (HC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 58; (ii) a light chain (LC) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 57; and 3. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2, comprising: Claim 4: a. The antibody or binding fragment thereof comprises a heavy chain amino acid sequence of SEQ ID NO: 59 and a light chain amino acid sequence of SEQ ID NO:

60. b. the antibody or binding fragment thereof comprises the heavy chain amino acid sequence of SEQ ID NO: 51 and the light chain amino acid sequence of SEQ ID NO: 52; c. the antibody or binding fragment thereof comprises the heavy chain amino acid sequence of SEQ ID NO: 53 and the light chain amino acid sequence of SEQ ID NO: 52; d. The antibody or binding fragment thereof comprises the heavy chain amino acid sequence of SEQ ID NO: 54 and the light chain amino acid sequence of SEQ ID NO: 55; e. The antibody or binding fragment thereof comprises the heavy chain amino acid sequence of SEQ ID NO: 56 and the light chain amino acid sequence of SEQ ID NO:

57. f. The antibody or binding fragment thereof comprises the heavy chain amino acid sequence of SEQ ID NO: 58 and the light chain amino acid sequence of SEQ ID NO:

57.

3. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2.

5. An isolated antibody or antigen-binding fragment thereof that binds to an epitope within the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 173, and prevents VEGFA from binding to VEGFR1.

6. The isolated antibody or antigen-binding fragment thereof of claim 5, wherein the antibody or antigen-binding fragment thereof binds to an epitope on VEGFR1 having the amino acid sequence FPLDTL (SEQ ID NO: 143) or EIGL (SEQ ID NO: 144), or binds to an epitope having the amino acid sequences FPLDTL (SEQ ID NO: 143) and EIGL (SEQ ID NO: 144).

7. An isolated antibody or antigen-binding fragment thereof described in claim 5 or 6, wherein the antibody or antigen-binding fragment thereof binds to human, mouse, rat and / or cynomolgus monkey VEGFR1.

8. An isolated antibody or antigen-binding fragment thereof described in claim 7, wherein the antibody or antigen-binding fragment thereof binds to human VEGFR1.

9. The isolated antibody or antigen-binding fragment thereof described in claim 7, wherein the antibody or antigen-binding fragment thereof binds to human VEGFR1, and VEGFR1 derived from cynomolgus monkeys, mice, and rats.

10. The isolated antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment thereof binds to human VEGFR1, mouse VEGFR1, and cynomolgus VEGFR1 with a K D of 6×10 −8 M or less, as determined using surface plasmon resonance (SPR).

11. The antigen-binding fragment is an scFv, (scFv) ２ , Fv, Fab, F(ab') ２ 3. The isolated antigen-binding fragment of claim 1 or 2, which is a Fd, dAb, VHH or single-chain antibody.

12. 3. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is an IgG1, IgG2, IgG3, or IgG4 isotype. (i) the antibody or antigen-binding fragment thereof is an IgG1 isotype; (ii) the isolated antibody or antigen-binding fragment thereof comprises an Ig constant region or a fragment of an Ig constant region; (iii) the Ig constant region or fragment of the Ig constant region comprises at least one mutation that results in reduced binding of the isolated antibody or antigen-binding fragment thereof to an Fcγ receptor (FcγR). (iv) at least one mutation that results in reduced binding of the antibody or antigen-binding fragment thereof to an FcγR is selected from the group consisting of F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 / A32 7G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, wherein residue numbering is according to the EU index; (v) the Ig constant region or a fragment of the constant region comprises the following mutations: L234A_L235A_D265S; (vi) the FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof; (vii) the Ig constant region or fragment of the Ig constant region further comprises at least one mutation that modulates the half-life of the antibody or antigen-binding fragment thereof. (viii) at least one mutation that modulates the half-life of the antibody or antigen-binding fragment thereof is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, where residue numbering is according to the EU index; 13. The isolated antibody or antigen-binding fragment thereof of claim 12.

14. 10. An immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof of claim 1 or 2 conjugated to a therapeutic or imaging agent. (i) an isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, or an immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof according to claim 1 or 2 conjugated to a therapeutic agent or an imaging agent; and (ii) a pharmaceutically acceptable carrier; A pharmaceutical composition comprising:

16. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof of claim 1 or 2.

17. 17. The isolated polynucleotide of claim 16, comprising a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70.

18. A vector comprising the polynucleotide of claim 16.

19. A host cell expressing the isolated antibody or antigen-binding fragment thereof described in claim 1 or 2.

20. In the manufacture of a pharmaceutical for preventing the binding of VEGFA to VEGFR1, (i) an isolated antibody or antigen-binding fragment thereof according to claim 1 or 2; (ii) an immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof of claim 1 or 2; (iii) a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of claim 1 or 2 and a pharmaceutically acceptable carrier; (iv) a pharmaceutical composition comprising an immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof of claim 1 or 2, and a pharmaceutically acceptable carrier; (v) an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof of claim 1 or 2; (vi) a vector comprising an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof of claim 1 or 2; or (vii) A host cell expressing the isolated antibody or antigen-binding fragment thereof of claim 1 or 2. Use of.

21. (i) an isolated antibody or antigen-binding fragment thereof according to claim 1 or 2; (ii) an immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof of claim 1 or 2; (iii) a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of claim 1 or 2 and a pharmaceutically acceptable carrier; (iv) a pharmaceutical composition comprising an immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof of claim 1 or 2, and a pharmaceutically acceptable carrier; (v) an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof of claim 1 or 2; (vi) a vector comprising an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof of claim 1 or 2; or (vii) A host cell expressing the isolated antibody or antigen-binding fragment thereof of claim 1 or 2. Includes a kit.

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