Anti-VEGFR1 antibodies and uses thereof

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 treating chronic kidney disease using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the invention patent with application date of August 12, 2022, application number 202280069328.1, and invention name “Anti-VEGFR1 antibodies and their uses”.

[0003] This application claims priority to U.S. Provisional Application No. 63 / 233,343, filed on August 16, 2021, and U.S. Provisional Application No. 63 / 322,273, filed on March 22, 2022, entitled “ANTI-VEGFR1 ANTIBODIES AND THEIR USES,” the disclosures of each of which are incorporated herein by reference in their entirety.

[0004] Reference sequence listing submitted electronically

[0005] This application contains a sequence listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML copy created on July 22, 2022 is named JBI6512WOPCT1_SL.xml and is 181,681 bytes in size. Technical Field

[0006] Provided are proteins, polynucleotides, vectors, host cells comprising an antigen binding domain or a fragment thereof that binds to vascular endothelial growth factor receptor 1 (VEGFR1), and methods of using the same to treat chronic kidney disease. Background Art

[0007] Chronic kidney disease (CKD) is a worldwide public health problem that continues to grow with the aging population and the prevalence of diabetes / obesity. CKD increases the risk of all-cause and cardiovascular mortality and the risk of end-stage renal disease requiring dialysis or kidney transplantation for survival. Patients with CKD stage 4-5 are at the highest risk of developing renal failure, with a 5-year mortality rate of approximately 40%. Diabetes (38%) and hypertension (25%) are the main causes of advanced and end-stage renal disease. Diabetic nephropathy, the most common CKD, develops in approximately 25% of patients with type 2 diabetes within 10 years of diagnosis. Despite standard care with renin-angiotensin-aldosterone system inhibitors (i.e., angiotensin-converting enzyme inhibitors or angiotensin receptor blockers), or the recent introduction of SGLT2 inhibitors, most "high-risk" patients or poor responders still progress to end-stage renal disease. In the United States alone, more than 100,000 patients undergo dialysis each year. 70% of patients undergoing dialysis die within 5 years.

[0008] There remains a high medical need for therapies to prevent or treat advanced renal disease by protecting or restoring kidney function in patients with advanced CKD.

[0009] In kidney, vascular endothelial growth factor A (VEGFA) produced by glomerular podocytes and tubular epithelial cells is essential to maintaining glomerular integrity, renal microvasculature and function. In CKD patients, mRNA microarray analysis of patient renal biopsy shows that VEGFA expression is reduced, and VGFA expression levels in glomeruli and tubulointerstitium are related to eGFR, proteinuria or vascular rarefaction (Bortoloso, Del Prete et al. 2004, Martini, Nair et al. 2014, Pan, Jiang et al. 2018). In healthy kidney, the VEGFA produced locally promotes endothelial health by the signal transduction of VEGFR2, thereby protecting the glomerular filtration barrier. In CKD, VEGFA lacks the isolation of its decoy receptor VEGFR1, causing VEGFR2 signal transduction to be suppressed. Anti-VEGFR1 blocking antibodies have been developed to block VEGFA isolation, increase local VEGFA availability, and can achieve endothelial protection, preservation of glomerular filtration barrier and renal function recovery. Summary of the invention

[0010] 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 the binding of VEGFA to VEGFR1.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 binds 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 that binds to VEGFR1 binds to an epitope on VEGFR1 having the amino acid sequence FPLDTL (SEQ ID NO: 143) and EIGL (SEQ ID NO: 144).

[0012] In some embodiments, the isolated antibody or antigen-binding fragment thereof binds human, mouse, rat and / or cynomolgus monkey VEGFR1.

[0013] 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 monkeys, mice, and rats. 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 monkeys, mice, and rats. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to human, cynomolgus monkeys, mice, and rat VEGFR1.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof is measured at 6×10 -8 M or smaller, especially 1×10 -8 M or smaller, more particularly 5×10 -9 M or less, 1×10-9M or less, 5×10 -10 M or smaller or 1×10 -10 Binds to human VEGFR1, mouse VEGFR1, and cynomolgus monkey VEGFR1 with a KD of M or less.

[0015] In some embodiments, the isolated antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region has a heavy chain complementarity determining region (HCDR) of VH comprising the amino acid sequence of SEQ ID NO: 31, 33, 34, 36, 38 or 39, and the light chain variable region has a light chain complementarity determining region (LCDR) of VL comprising the amino acid sequence of SEQ ID NO: 32, 35, 37 or 40.

[0016] In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises:

[0017] a. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 31 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 32;

[0018] b. The HCDR of VH has the amino acid sequence of SEQ ID NO:33 and the LCDR of VL has the amino acid sequence of SEQ ID NO:32.

[0019] c. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 34 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 35;

[0020] d. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 36 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 37;

[0021] e. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 38 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 37; or

[0022] f. The HCDR of VH has the amino acid sequence of SEQ ID NO:39 and the LCDR of VL has the amino acid sequence of SEQ ID NO:40.

[0023] Provided herein is an isolated antibody or antigen-binding fragment thereof, comprising the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3:

[0024] SEQ ID NOs: 7, 175, 9, 10, 11 and 12, respectively;

[0025] SEQ ID NOs: 7, 8, 9, 10, 11 and 12, respectively;

[0026] SEQ ID NOs: 13, 14, 15, 16, 17 and 18, respectively;

[0027] SEQ ID NOs: 19, 20, 21, 22, 23 and 24, respectively;

[0028] SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively;

[0029] SEQ ID NOs: 71, 176, 73, 74, 75 and 76, respectively;

[0030] SEQ ID NOs: 71, 72, 73, 74, 75 and 76, respectively;

[0031] SEQ ID NOs: 77, 78, 79, 80, 81 and 82, respectively;

[0032] SEQ ID NOs: 83, 84, 85, 86, 87 and 88, respectively;

[0033] SEQ ID NOs: 89, 90, 91, 92, 93 and 94, respectively;

[0034] SEQ ID NOs: 95, 177, 97, 98, 99 and 100, respectively;

[0035] SEQ ID NOs: 95, 96, 97, 98, 99 and 100, respectively;

[0036] SEQ ID NOs: 101, 102, 103, 104, 105 and 106, respectively;

[0037] SEQ ID NOs: 107, 108, 109, 110, 111 and 112, respectively;

[0038] SEQ ID NOs: 113, 114, 115, 116, 117 and 118, respectively;

[0039] SEQ ID NOs: 119, 178, 121, 122, amino acid sequence LNS and SEQ ID NO: 124, respectively;

[0040] SEQ ID NOs: 119, 120, 121, 122, amino acid sequence LNS and SEQ ID NO: 124, respectively;

[0041] SEQ ID NOs: 125, 126, 127, 128, amino acid sequence FNF and SEQ ID NO: 130, respectively;

[0042] SEQ ID NO: 131, 132, 133, 134, amino acid sequence YD and SEQ ID NO: 136, respectively; or

[0043] SEQ ID NOs: 137, 138, 139, 140, amino acid sequence FNS and SEQ ID NO: 142, respectively;

[0044] Wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0045] Also disclosed is an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, the isolated antibody or antigen-binding fragment thereof comprising:

[0046] VH of SEQ ID NO:31 and VL of SEQ ID NO:32;

[0047] VH of SEQ ID NO:33 and VL of SEQ ID NO:32;

[0048] VH of SEQ ID NO:34 and VL of SEQ ID NO:35;

[0049] VH of SEQ ID NO:36 and VL of SEQ ID NO:37;

[0050] VH of SEQ ID NO:38 and VL of SEQ ID NO:37; or

[0051] VH of SEQ ID NO:39 and VL of SEQ ID NO:40.

[0052] Also disclosed is 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.

[0053] Also disclosed is 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: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 VL of SEQ ID NO:32.

[0054] Disclosed is 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: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 VL of SEQ ID NO:32.

[0055] Also disclosed is 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: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 VL of SEQ ID NO:35.

[0056] Also disclosed is 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: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 VL of SEQ ID NO:37.

[0057] Also disclosed is 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: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 VL of SEQ ID NO:37.

[0058] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, the isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60.

[0059] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, wherein the isolated antibody or antigen-binding fragment thereof 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.

[0060] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, wherein the isolated antibody or antigen-binding fragment thereof 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.

[0061] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, wherein the isolated antibody or antigen-binding fragment thereof 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.

[0062] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, wherein the isolated antibody or antigen-binding fragment thereof 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.

[0063] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, wherein the isolated antibody or antigen-binding fragment thereof 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.

[0064] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, wherein the isolated antibody or antigen-binding fragment thereof 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.

[0065] In some embodiments, the isolated antibody of the present application comprises:

[0066] a. HC comprising the amino acid sequence of SEQ ID NO: 51 and LC comprising the amino acid sequence of SEQ ID NO: 52;

[0067] b. HC comprising the amino acid sequence of SEQ ID NO: 53 and LC comprising the amino acid sequence of SEQ ID NO: 52;

[0068] c. HC comprising the amino acid sequence of SEQ ID NO: 54 and LC comprising the amino acid sequence of SEQ ID NO: 55;

[0069] d. HC comprising the amino acid sequence of SEQ ID NO: 56 and LC comprising the amino acid sequence of SEQ ID NO: 57;

[0070] e. HC comprising the amino acid sequence of SEQ ID NO: 58 and LC comprising the amino acid sequence of SEQ ID NO: 57; or

[0071] f. HC comprising the amino acid sequence of SEQ ID NO:59 and LC comprising the amino acid sequence of SEQ ID NO:60.

[0072] Also disclosed is an isolated antibody or antigen-binding fragment thereof, the isolated antibody or antigen-binding fragment thereof comprising

[0073] HCDR1, HCDR2,

[0074] HCDR3, LCDR1, LCDR2 and LCDR3;

[0075] VH of SEQ ID NO:31 and VL of SEQ ID NO:32; and / or

[0076] HC of SEQ ID NO:51 and LC of SEQ ID NO:52;

[0077] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0078] Also disclosed is an isolated antibody or antigen-binding fragment thereof, the isolated antibody or antigen-binding fragment thereof comprising

[0079] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 7, 8, 9, 10, 11 and 12, respectively;

[0080] VH of SEQ ID NO:33 and VL of SEQ ID NO:32; and / or

[0081] HC of SEQ ID NO:53 and LC of SEQ ID NO:52;

[0082] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0083] Also disclosed is an isolated antibody or antigen-binding fragment thereof, the isolated antibody or antigen-binding fragment thereof comprising

[0084] HCDR1, HCDR2,

[0085] HCDR3, LCDR1, LCDR2 and LCDR3;

[0086] VH of SEQ ID NO:34 and VL of SEQ ID NO:35; and / or

[0087] HC of SEQ ID NO:54 and LC of SEQ ID NO:55;

[0088] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0089] Also disclosed is an isolated antibody or antigen-binding fragment thereof, the isolated antibody or antigen-binding fragment thereof comprising

[0090] HCDR1, HCDR2,

[0091] HCDR3, LCDR1, LCDR2 and LCDR3;

[0092] VH of SEQ ID NO:36 and VL of SEQ ID NO:37; and / or

[0093] HC of SEQ ID NO:56 and LC of SEQ ID NO:57;

[0094] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0095] Also disclosed is an isolated antibody or antigen-binding fragment thereof, the isolated antibody or antigen-binding fragment thereof comprising

[0096] HCDR1, HCDR2,

[0097] HCDR3, LCDR1, LCDR2 and LCDR3;

[0098] VH of SEQ ID NO:38 and VL of SEQ ID NO:37; and / or

[0099] HC of SEQ ID NO:58 and LC of SEQ ID NO:57;

[0100] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0101] Also disclosed is an isolated antibody or antigen-binding fragment thereof, the isolated antibody or antigen-binding fragment thereof comprising

[0102] HCDR1, HCDR2,

[0103] HCDR3, LCDR1, LCDR2 and LCDR3;

[0104] VH of SEQ ID NO:39 and VL of SEQ ID NO:40; and / or

[0105] HC of SEQ ID NO:59 and LC of SEQ ID NO:60;

[0106] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0107] In some embodiments, the isolated antibody or its antigen-binding fragment is conjugated to a half-life extension portion. Optionally, the half-life extension portion is an immunoglobulin (Ig), a fragment of the Ig, an Ig constant region, a fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin binding domain, or polyethylene glycol. Optionally, the fragment of the Ig constant region comprises an Fc region. Optionally, the antibody or antigen-binding fragment that binds to VEGFR1 is conjugated to the C-terminus of the Ig constant region or a fragment of the Ig constant region.

[0108] Optionally, the Ig constant region or a fragment of the Ig constant region is of IgG1, IgG2, IgG3 or IgG4 isotype. Optionally, the Ig constant region or a fragment of the Ig constant region is of IgG1 isotype. Optionally, the Ig constant region or a fragment of the Ig constant region is of IgG4 isotype. Optionally, the Ig constant region or a fragment of the 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 resulting in reduced binding of the protein 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, wherein the residues are numbered according to the EU index. Optionally, the mutation resulting in reduced binding of the protein to FcγR is L234A_L235A_D265S. Optionally, the Ig constant region or the fragment of the Ig constant region comprises at least one mutation regulating the half-life of the protein. Optionally, the at least one mutation regulating 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, wherein residue numbering is carried out according to the EU index.

[0109] The present disclosure also provides an immunoconjugate comprising an isolated antibody or antigen-binding fragment thereof conjugated to a therapeutic agent or an imaging agent.

[0110] The present disclosure also provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1 and a pharmaceutically acceptable carrier.

[0111] The present disclosure also provides a polynucleotide encoding an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1.

[0112] 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.

[0113] Optionally, the polynucleotide encoding the isolated antibody or antigen-binding fragment thereof that binds 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.

[0114] The present disclosure also provides a vector comprising the polynucleotide of the present disclosure.

[0115] The present disclosure also provides a host cell comprising the polynucleotide or vector of the present disclosure.

[0116] The present disclosure also provides a host cell expressing the isolated antibody or antigen-binding fragment thereof and / or comprising the polynucleotide or vector of the present disclosure.

[0117] The present disclosure also provides a method of preventing VEGFA from binding to VEGFR1 in a subject in need thereof, the method comprising administering to the subject an effective amount of any isolated antibody or antigen-binding fragment thereof of the present disclosure, any immunoconjugate of the present disclosure, any pharmaceutical composition of the present disclosure, any isolated polynucleotide or vector of the present disclosure, or any host cell of the present disclosure, thereby preventing VEGFA from binding to VEGFR1.

[0118] The present disclosure also provides a method for treating chronic kidney disease (CKD) in a subject and slowing down its progression, the method comprising administering to the subject a therapeutically effective amount of any isolated antibody or antigen-binding fragment thereof of the present disclosure, any immunoconjugate of the present disclosure, or any pharmaceutical composition of the present disclosure, any isolated polynucleotide or vector of the present disclosure, or any host cell of the present disclosure, for a period of time sufficient to treat CKD. Optionally, the subject suffers from late stage 4 or stage 5 chronic kidney disease. Optionally, the subject suffers from early stage chronic kidney disease.

[0119] The present disclosure also provides a kit comprising any isolated antibody or antigen-binding fragment thereof of the present disclosure, any immunoconjugate of the present disclosure, any pharmaceutical composition of the present disclosure, any isolated polynucleotide or vector of the present disclosure, or any host cell of the present disclosure.

[0120] The present disclosure also provides a method of treating CKD in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any isolated antibody or antigen-binding fragment thereof of the present disclosure, any immunoconjugate of the present disclosure, any pharmaceutical composition of the present disclosure, any isolated polynucleotide or vector of the present disclosure, or any host cell of the present disclosure, for a period of time sufficient to reduce proteinuria in the subject.

[0121] The present disclosure also provides a method of reducing proteinuria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any isolated antibody or antigen-binding fragment thereof of the present disclosure, any immunoconjugate of the present disclosure, any pharmaceutical composition of the present disclosure, any isolated polynucleotide or vector of the present disclosure, or any host cell of the present disclosure, for a period of time sufficient to reduce proteinuria in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings.It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0123] Figure 1 An amino acid sequence alignment of the ligand binding region (domain 2-3) of VEGFR1 from different species is shown. Residues identical to the human sequence are indicated by dots. Residues that are not identical are explicitly indicated. Figure 1 SEQ ID NOs: 179-184 are disclosed in order of appearance, respectively.

[0124] Figure 2Shown are representative FACS binding data depicting species cross reactivity of VEGFR1 antibodies (VGFB54, VGFB71, VGFB78 and VGFB82) with human, cynomolgus monkey and mouse VEGFR1 but retaining specificity to VEGFR1 relative to VEGFR2 or VEGFR3. Human VEGR1, cynomolgus monkey VEGFR1, mouse VEGFR1, human VEGFR2 and human VEGFR3 are overexpressed on the cell surface. Average fluorescence intensity is drawn using cell lines overexpressing the VEGFR protein shown.

[0125] Figure 3 An assessment of the relative frequency of SHM leucine (L) compared to glutamine (Q) at position 105 of VGFB78 VH is shown.

[0126] Figure 4A Shown are paratope maps of the heavy chain variable domains of four anti-VEGFR1 antibodies (VGFB54, VGFB71, VGFB78, and VGFB82) directed against VEGFR1 (VGFW1). Figure 4A SEQ ID NOs: 31, 34, 36 and 39 are disclosed in order of appearance, respectively.

[0127] Figure 4B Paratope maps of the light chain variable domains of four anti-VEGFR1 antibody light chains (VGFB54, VGFB71, VGFB78, and VGFB82) directed against VEGFR1 (VGFW1) are shown. The highlighted residues represent HCDRs and LCDRs based on IMTG depictions. Residues with a free energy change of -1.0 kcal / mol or less upon binding to VEGFR1 are shown in a gradient color from medium gray to dark gray, as indicated by the scale. Residues that are unstable upon binding to VEGFR1 are shown in light gray. Residues that exchange too quickly or too slowly to see perturbations upon binding are shown in gray. Residues without color indicate that HDX behavior was not monitored because there was no peptide covering the residue or the residue was the first two residues of the peptide. Figure 4B SEQ ID NOs: 32, 35, 37 and 40 are disclosed in order of appearance, respectively.

[0128] Figure 5 Shown are plasma concentrations (ng / mL) of total and free VGFB54 over time following a single IV / SC administration in cynomolgus monkeys.

[0129] Figure 6 Shown are plasma concentrations (ng / mL) of total and free VGFB78 over time following a single IV / SC administration in cynomolgus monkeys.

[0130] Figure 7Shown are plasma concentrations (ng / mL) of total and free VGFB82 over time following a single IV / SC administration in cynomolgus monkeys.

[0131] Figure 8 Shown are plasma concentrations (ng / mL) of total and free VGFB80 over time following a single IV / SC administration in cynomolgus monkeys.

[0132] Fig. 9 Shown are total PlGF plasma concentrations (pg / mL) over time following a single IV / SC administration of VGFB54 in cynomolgus monkeys.

[0133] Fig.10 Shown are total PlGF plasma concentrations (pg / mL) over time following a single IV / SC administration of VGFB78 in cynomolgus monkeys.

[0134] Fig.11 Shown are total PlGF plasma concentrations (pg / mL) over time following a single IV / SC administration of VGFB82 in cynomolgus monkeys.

[0135] Fig.12 Shown are total PlGF plasma concentrations (pg / mL) over time following a single IV / SC administration of VGFB80 in cynomolgus monkeys. DETAILED DESCRIPTION

[0136] definition

[0137] 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 specific embodiments in conjunction with the accompanying drawings forming a part of the present disclosure. It should be understood that the disclosed antibodies, antigen-binding domains, antibody fragments, polynucleotides, vectors, cells, compositions, kits and methods are not limited to those specifically described and / or shown herein, and that the terms used herein are only for the purpose of describing specific embodiments by way of example, and are not intended to limit the claimed antibodies, antigen-binding domains, antibody fragments, polynucleotides, vectors, cells, compositions, kits and methods.

[0138] Unless specifically stated otherwise, any description of possible mechanisms or modes of action or reasons for improvement is intended for exemplary purposes only, and the disclosed antibodies, antigen-binding domains, antibody fragments, polynucleotides, vectors, cells, compositions, kits and methods are not bound by the correctness or incorrectness of any such suggested mechanisms or modes of action or reasons for improvement.

[0139] In this article, the description relates to antibodies, antigen-binding fragments thereof, and methods of using the antigen-binding domains. When the disclosure describes or claims a feature or embodiment associated with an antigen-binding domain, such a feature or embodiment is equally applicable to methods of using the antigen-binding domain. Similarly, when the disclosure describes or claims a feature or embodiment associated with a method of using an antigen-binding domain, such a feature or embodiment is equally applicable to the antigen-binding domain.

[0140] When enumerating or establishing a numerical range herein, the range includes its end values ​​and all individual integers and fractions within the range, and also includes each of the narrower ranges formed by all various possible combinations of those end values ​​and internal integers and fractions, to form a subset of the larger numerical group within the range to the same extent, as if each of those narrower ranges were explicitly enumerated. When stating a numerical range greater than a specified value herein, the range is however limited, and its upper limit is limited by a value operable in the context of the present invention as described herein. When stating a numerical range less than a specified value herein, the lower limit of the range is however limited by a non-zero value. It is not intended that the scope of the present invention is limited to the specific values ​​listed when the range is limited. All ranges include end values ​​and can be combined.

[0141] When "about" is used above to express a value as an approximation, it should be understood that the specific value constitutes another embodiment. A reference to a specific value includes at least that specific value unless the context clearly dictates otherwise.

[0142] It should be understood that certain features of the present invention are described in the context of separate embodiments for the sake of clarity, but may also be provided in combination in a single embodiment. That is, unless clearly incompatible or explicitly excluded, each individual embodiment is considered to be combinable with any other embodiment, and such combination is considered another embodiment. Conversely, various features of the present invention are described in the context of a single embodiment for the sake of simplicity, and may also be provided separately or in any sub-combination. Finally, although an embodiment may be described as part of a series of steps or part of a more general structure, each of the steps may also be considered an independent embodiment that can be combined with other embodiments.

[0143] Unless specifically stated otherwise, any description of possible mechanisms or modes of action or reasons for improvement is intended for exemplary purposes only, and the methods disclosed herein are not bound by the correctness or incorrectness of any such suggested mechanisms or modes of action or reasons for improvement.

[0144] Various terms related to various aspects of the specification are used throughout the specification and claims. Unless otherwise indicated, such terms are given the ordinary meaning of the art. Other specifically defined terms should be understood in a manner consistent with the definitions provided herein.

[0145] 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 so forth.

[0146] The transitional terms "comprising," "consisting essentially of," and "consisting of" are intended to suggest their recognized meanings in patent parlance; that is, (i) "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, 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 disclosure protected by the claims." Embodiments described with the phrase "comprising" (or its equivalent) are also provided, such as those embodiments described independently with "consisting of" and "consisting essentially of." Embodiments described with the phrase "consisting essentially of" (or its equivalent) are also provided, such as those embodiments described independently with "consisting of."

[0147] As used in this specification and 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, so that, for example, the phrase "the group consisting of peptides A, B, and C and fragments thereof" specifies or lists a Markush group that includes A, B, C, a fragment of A, a fragment of B, and / or a fragment of C.

[0148] "Isolated" refers to a homogenous population of a molecule that has been substantially separated and / or purified from other components of a system (such as a recombinant cell) in which the molecule (such as a synthetic polynucleotide or polypeptide) is produced, as well as a protein that has been subjected to at least one purification or separation step. "Isolated" refers to a molecule that is substantially free of other cellular material and / or chemicals, and encompasses molecules isolated to higher purities (such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity).

[0149] "Polynucleotide" refers to a synthetic molecule comprising a chain of nucleotides covalently linked to a phosphate sugar backbone or other equivalent covalent chemicals. cDNA is a typical example of a polynucleotide.

[0150] "Polypeptide" or "protein" refers to a molecule comprising 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."

[0151] As used herein, "Tagg" refers to the temperature at which a protein begins to aggregate by dimerization or oligomerization. The aggregation temperature detects the beginning of aggregation, i.e., the temperature at which the protein will show a tendency to aggregate. Tagg can be measured by differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF) or by circular dichroism (CD). These techniques can detect slight changes in protein conformation, and therefore detect the starting point of aggregation. The Tagg value can be lower or higher than Tm. When Tagg is lower than Tm, the protein first dimerizes and / or oligomerizes, and then begins to unfold later at a temperature higher than Tagg. When Tagg is higher than Tm, the protein first begins to unfold, and then aggregates at a temperature higher than Tm. Both events are common and depend on amino acid composition and protein conformation.

[0152] As used herein, "Tm" or "midpoint temperature" is the temperature midpoint of the thermal unfolding 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 unfolding curves are usually plotted as a function of temperature. Tm is used to measure protein stability. Generally, the higher the Tm, the more stable the protein. Tm can be easily determined using methods well known to those skilled in the art, such as circular dichroism spectroscopy, differential scanning calorimetry, differential scanning fluorimetry (based on both intrinsic and extrinsic dyes), UV spectroscopy, FT-IR, and isothermal calorimetry (ITC).

[0153] "Complementarity determining regions" (CDRs) are regions of antibodies that bind antigen. There are three CDRs in VH (HCDR1, HCDR2, HCDR3) and three CDRs in VL (LCDR1, LCDR2, LCDR3). CDRs can be defined using various depictions, 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). The correspondence between various delineations and variable region numbers is described (see, 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 of UCL Business PLC) can be used to delineate CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include CDRs defined by any of the above methods (Kabat, Chothia, IMGT or AbM). The correspondence between numbering systems, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, for example, Kabat, supra; Chothia, supra; Martin, supra; Lefranc et al., supra).

[0154] Table 1.

[0155] IMGT Kabat AbM Chothia <![CDATA[V H CDR1]]> 27-38 31-35 26-35 26-32 <![CDATA[V H CDR2]]> 56-65 50-65 50-58 53-55 <![CDATA[V H CDR3]]> 105-117 95-102 95-102 96-101 <![CDATA[V L CDR1]]> 27-38 24-34 24-34 26-32 <![CDATA[V L CDR2]]> 56-65 50-56 50-56 50-52 <![CDATA[V L CDR3]]> 105-117 89-97 89-97 91-96

[0156] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain involved in binding of an antibody to an antigen. The variable domain of a heavy or light chain (VH and VL, respectively) comprises four framework regions (FR) and three complementarity determining regions (CDR).

[0157] "Subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, 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" are used interchangeably herein.

[0158] The terms "kit" and "article of manufacture" are used synonymously.

[0159] All publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if fully set forth herein.

[0160] Antibodies that bind to VEGFR1

[0161] 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 binds to VEGFR1 and comprises at least one binding domain that specifically binds to VEGFR1.

[0162] The antibodies of the present disclosure have one or more functional properties, including but not limited to high affinity binding to VEGFR1, blocking binding of VEGFA to VEGFR1, and the ability to treat chronic kidney disease (CKD).In some embodiments, the invention relates to isolated antibodies or antigen-binding fragments thereof that specifically bind to VEGFR1.

[0163] As used herein, the term "antibody" refers broadly to and includes immunoglobulin molecules, including monoclonal antibodies, including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies; antigen-binding fragments; multispecific antibodies, such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies; dimeric antibodies, tetrameric antibodies, or multimeric antibodies; single-chain antibodies; domain antibodies; and any other modified configuration of immunoglobulin molecules that contain an antigen-binding site with the desired specificity. The term antibody includes full-length antibodies, whole antibodies, intact antibodies, antibody fragments, antigen-binding fragments, and antigen-binding domains.

[0164] Generally speaking, an antibody is a protein or peptide chain that exhibits binding specificity for a specific antigen. Antibody structure is well known. According to the heavy chain constant domain amino acid sequence, immunoglobulins can be designated as five major classes (i.e., IgA, IgD, IgE, IgG, and IgM). IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibody of the present invention can be any one of the five main classes or corresponding subclasses. Preferably, the antibody of the present invention is IgG1, IgG2, IgG3, or IgG4. Based on the amino acid sequence of its constant domain, the antibody light chain of a vertebrate species can be designated as one of two completely different types, i.e., κ and λ. Therefore, the antibody of the present invention can contain κ or λ light chain constant domains. According to some embodiments, the antibody of the present invention includes heavy chain and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light chain constant domains, the antibody also contains an antigen binding region consisting of a light chain variable region and a heavy chain variable region, wherein each variable region contains three domains (i.e., complementary 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.

[0165] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to VEGFR1 is substantially free of antibodies that do not bind to VEGFR1). In addition, the isolated antibody may be substantially free of other cellular material and / or chemical substances. "Isolated antibody" encompasses antibodies that are separated to a higher purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure antibodies.

[0166] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical except for possible naturally occurring mutations that may be present in small amounts. The monoclonal antibodies of the present invention can be prepared by hybridoma methods, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods. For example, a monoclonal antibody can be produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal such as a transgenic mouse or rat, the transgenic non-human animal having a genome comprising a human heavy chain transgene and a light chain transgene.

[0167] 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 indicated, VEGFR1 used herein refers to human VEGFR1. The amino acid sequence of human VEGFR1 can be obtained 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 (amino acid residues 827-1158) separated by a kinase insert, 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 as a membrane receptor and a soluble protein. Soluble VEGFR1 acts as a negative regulator of VEGFA activity by binding this factor and preventing its interaction with its membrane receptor.

[0168] SEQ ID NO: 170, human VEGFR1 (Uniprot accession number P17948)

[0169] MVSYWDTGVLLCALLSCLLLTGSSSSGSKLKDPELSLKGTQHIMQAGQTL

[0170] HLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQ

[0171] ANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTE

[0172] GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIG

[0173] LLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCT

[0174] ATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQ

[0175] NKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKR

[0176] SYRLSMKVKAFPSPEVVWLKDGLPATEXARYLTRGYSLIIKDVTEEDAG

[0177] NEWLY SIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILT

[0178] CTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIE

[0179] SITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGRNISFYITDV

[0180] PNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSI

[0181] SKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITI

[0182] RDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEP

[0183] GIILGPGSSTLFIERVTEEDEGVYHCKATNQKGSVESSAYLTVQGTSDKSN

[0184] LELITLTCTCVAATLFWLLLTLFIRKMKRSSSEIKTDYLSIIMDPDEVPLDE

[0185] QCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRT

[0186] VAVKMLKEGATASEYKALMTELKILTHIGHHLNVVNLLGACTKQGGPL

[0187] MVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQGK

[0188] KPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQ

[0189] VARGMEFLSSRKCIHRDLAARNILLSENNVVKICDFGLARDIYKNPDYVR

[0190] KGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPGVQ

[0191] MDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEK

[0192] LGDLLQANVQQDGKDYIPINAILTGNSGFTYSTPAFSEDFFKESISAPKFNS

[0193] GSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLLASPML

[0194] KRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKR

[0195] RFTYDHAELERKIACCSPPPDYNSVVLYSTPPI

[0196] SEQ ID NO:171(Ig-like D2 domain)

[0197] GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIG

[0198] LLTCEATVNGH

[0199] SEQ ID NO:172(Ig-like D3 domain)

[0200] IDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVR

[0201] RRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVH

[0202] SEQ ID NO: 173 (Ig-like D2-D3)

[0203] GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIG

[0204] LLTCEATVNGHIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSY

[0205] PDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPS

[0206] FKSVNTSVH

[0207] "Specific binding" or "binding" refers to the binding of a protein molecule to an antigen or an epitope within an antigen with a greater affinity than to other antigens. Typically, a protein molecule binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (K D ) is about 1×10 -7 M or less, for example, about 5×10 -8 M or less, about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less or about 1×10 -12 M or lower, usually K D Compared with its K binding to nonspecific antigens (such as BSA, casein) D At least one hundred times lower. The term “K D " refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). According to the present disclosure, the K of an antibody is D The K value can be determined using methods known in the art. For example, the K value of an antibody D This can be accomplished by using surface plasmon resonance (SPR), such as by using a biosensor system (e.g. The K of the antibody can be determined by using biolayer interferometry techniques such as the Octet RED96 system. D The smaller the value, the higher the affinity of the antibody for binding to the target antigen.

[0208] As used herein, an antibody that "binds to VEGFR1" or "specifically binds to VEGFR1" refers to an antibody that binds to VEGFR1 at a concentration of 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M or smaller K D An antibody that binds to VEGFR1, preferably human VEGFR1.

[0209] In some embodiments, an antibody or antigen-binding fragment thereof that "binds to VEGFR1" or "specifically binds to VEGFR1" refers to an antibody or antigen-binding fragment thereof that is expressed in 1×10 -7 M or smaller K D Antibodies or antigen-binding fragments thereof that bind to VEGFR1. In certain embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof is preferably 1×10 -8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M or smaller K D Binds specifically to human VEGFR1.

[0210] The anti-VEGFR1 antibodies disclosed herein include whole antibodies, antibody fragments that specifically bind to VEGFR1, and antigen-binding fragments thereof that specifically bind to VEGFR1.

[0211] 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.

[0212] In some embodiments, the anti-VEGFR1 antibodies of the present disclosure are antibody fragments or antigen binding domains that specifically bind to VEGFR1.

[0213] 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 natural antibodies. "Intact antibodies" are composed of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, and their multimers (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed 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, which are called complementarity determining regions (CDRs) and are interspersed with framework regions (FRs). Each VH and VL is composed of three CDRs and four FR segments, and are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chains of any vertebrate species can be assigned to one of two distinct types, kappa and lambda, based on the amino acid sequence of their constant domains.

[0214] As used herein, the terms "antibody fragment" and "antigen-binding fragment" refer to molecules other than intact antibodies. Antigen-binding fragments can 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 (dsdiabodies), single-chain antibody molecules (scFv), single-domain antibodies (sdab), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, 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 that mimic the CDRs of antibodies (such as the FR3-CDR3-FR4 portion), HCDR1, HCDR2 and / or HCDR3 and LCDR1, LCDR2 and / or LCDR3, alternative scaffolds that bind to antigens, bivalent domain antibodies, multispecific proteins comprising antigen-binding fragments or any other antibody fragment that binds to an antigen but does not comprise a full antibody structure.

[0215] "dAb" or "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544546 (1989)).

[0216] "Fab" or "Fab fragment" refers to an antibody fragment consisting of the VH domain, CH1 domain, VL domain and CL domain.

[0217] "F(ab')2" or "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by a disulfide bridge in the hinge region.

[0218] "Fd" or "Fd fragment" refers to an antibody fragment consisting of a VH domain and a CH1 domain.

[0219] "Fv" or "Fv fragment" refers to an antibody fragment consisting of the VH domain and VL domain from a single arm of an antibody. The Fv fragment lacks the constant regions of the Fab (CH1 and CL) region. The VH and VL in the Fv fragment are held together by non-covalent interactions.

[0220] Antigen binding fragments (such as VH and VL) can be linked together via synthetic joints to form various types of single antibody designs, wherein the VH / VL domains can be paired intramolecularly or intermolecularly to form monovalent antigen binding domains, such as single-chain Fv (scFv) or double-chain antibodies. In recombinant expression systems, the joint is a peptide joint and can include any naturally occurring amino acid. Exemplary amino acids that can be included in the joint are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His and The. The length of the joint should be sufficient to allow VH and VL to form the correct conformation relative to each other, so that they maintain the desired activity (such as binding to VEGFR1) mode of connection. The length of the joint can be about 5 to 50 amino acids.

[0221] "Single-chain Fv" or "scFv" is a fusion protein comprising at least one antibody fragment containing a light chain variable region (VL) and at least one antibody fragment containing a heavy chain variable region (VH), wherein VL and VH are continuously connected via a polypeptide linker and can be expressed as a single-chain polypeptide. The scFv can have the VL variable region and the VH variable region in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0222] A divalent or bivalent single-chain variable fragment (di-scFv, bi-scFv) can be engineered by connecting two scFvs. A "(scFv)2" or "tandem scFv" or "double scFv" fragment refers to a fusion protein comprising two light chain variable regions (VL) and two heavy chain variable regions (VH), wherein the two VL regions and the two VH regions are continuously connected via a polypeptide linker and can be expressed as a single-chain polypeptide. The two VL regions and the two VH regions fused by a peptide linker form a divalent molecule VL A -Connector-VH A -Connector-VL B -Connector-VH B , to form two binding sites that can simultaneously bind to two different antigens or epitopes. (ScFv)2 can be expressed as a single-chain polypeptide.

[0223] Any one of the VH domains and VL domains identified herein in conjunction with VEGFR1 can be engineered into a scFv format oriented to VH-joint-VL or VL-joint-VH. Any one of the VH domains and VL domains identified herein can also be used to generate sc(Fv)2 structures, such as VH-joint-VL-joint-VL-joint-VH, VH-joint-VL-joint-VH-joint-VL, VH-joint-VH-joint-VL-joint-VL, VL-joint-VH-joint-VH-joint-VL, VL-joint-VH-joint-VH-joint-VL, VL-joint-VH-joint-VL, VL-joint-VH-joint-VH-joint-VL, VL-joint-VH-joint-VL-joint-VH or VL-joint-VL-joint-VH-joint-VH.

[0224] "Diabody" is a bivalent dimer formed by two chains, each chain containing a VH domain and a VL domain. The two domains in the 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 arrangement. The linker can be a glycine-rich pentamer linker (G4S).

[0225] "VHH" refers to a single domain antibody or nanobody consisting only of the antigen binding domain of the heavy chain. VHH single domain antibodies lack the light chain and heavy chain CH1 domains of the conventional 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, double-chain antibodies, VHH, dAb, Fd, Fv or other single-chain antibodies.

[0226] The anti-VEGFR1 antibodies disclosed herein include chimeric antibodies, humanized antibodies, or fully human antibodies that specifically bind to VEGFR1.

[0227] "Human antibody" refers to an antibody optimized to have a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody comprises a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained by a system using human germline immunoglobulins or rearranged immunoglobulin genes, the human antibody comprises a heavy chain variable region and a light chain variable region "derived from" a sequence of human origin. Such exemplary systems are human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci. Because of the differences between the systems for obtaining human antibodies and human immunoglobulin loci, the introduction of somatic mutations or the intentional introduction of substitutions into the framework or CDR or both, "human antibodies" generally comprise amino acid differences compared to immunoglobulins expressed in humans.

[0228] Typically, the amino acid sequence of 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 to an amino acid sequence encoded by a human germline immunoglobulin gene or a rearranged immunoglobulin gene. In some cases, a "human antibody" may comprise a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86), or a synthetic HCDR3 bound to a human immunoglobulin gene library displayed on a phage (e.g., as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication No. WO2009 / 085462). Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody".

[0229] Transgenic animals (such as mice, rats or chickens) carrying human immunoglobulin (Ig) loci in their genomes can be used to generate antigen-binding fragments that bind to VEGFR1 and are described, for example, in U.S. Pat. No. 6,150,584, International Patent Publication No. WO1999 / 45962, International Patent Publication No. WO2002 / 066630, WO2002 / 43478, WO2002 / 043478 and WO1990 / 04036. The endogenous immunoglobulin loci in such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the genome of the animal by homologous or nonhomologous recombination using a transchromosome or 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 involved in providing human antibodies against the selected antigen.

[0230] Antibodies or antigen-binding fragments thereof that bind to VEGRF1 generated by immunizing non-human animals can be humanized. Exemplary humanization techniques that include selecting a human acceptor framework include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28: 489-499), resurfacing of specificity-determining residues (U.S. Pat. Publication No. 2010 / 0261620), human framework remodeling (U.S. Pat. No. 8,748,356), or superhumanization (U.S. Pat. No. 7,709,226). In these methods, a subset of the CDRs or CDR residues of a parent antibody is transferred to a human framework, which can be selected based on its overall homology to the parent framework, based on similarity in CDR length or canonical structural identity, or a combination thereof.

[0231] The humanized antigen binding domain can be further optimized to improve its selectivity or affinity for the desired antigen by incorporating altered framework support residues to maintain binding affinity (back mutations) using techniques such as those described in International Patent Publication Nos. WO1090 / 007861 and WO1992 / 22653, or by introducing variations in any CDR to improve the affinity of the antigen binding domain.

[0232] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, the isolated antibody or antigen-binding fragment thereof comprising the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3:

[0233] a. SEQ ID NO: 7, 175, 9, 10, 11 and 12 respectively;

[0234] b. SEQ ID NO: 7, 8, 9, 10, 11 and 12 respectively;

[0235] c. SEQ ID NOs: 13, 14, 15, 16, 17 and 18, respectively;

[0236] d. SEQ ID NOs: 19, 20, 21, 22, 23 and 24, respectively;

[0237] e. SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively;

[0238] f. SEQ ID NOs: 71, 176, 73, 74, 75 and 76, respectively;

[0239] g. SEQ ID NOs: 71, 72, 73, 74, 75 and 76, respectively;

[0240] h. SEQ ID NOs: 77, 78, 79, 90, 81 and 82, respectively;

[0241] i. SEQ ID NOs: 83, 84, 85, 86, 87 and 88, respectively;

[0242] j. SEQ ID NOs: 89, 90, 91, 92, 93 and 94, respectively;

[0243] k. SEQ ID NO:95, 177, 97, 98, 99 and 100, respectively;

[0244] l. SEQ ID NO:95, 96, 97, 98, 99 and 100 respectively;

[0245] m. SEQ ID NOs: 101, 102, 103, 104, 105 and 106, respectively;

[0246] n. SEQ ID NO: 107, 108, 109, 110, 111 and 112 respectively;

[0247] o. SEQ ID NOs: 113, 114, 115, 116, 117 and 118, respectively;

[0248] p. SEQ ID NOs: 119, 178, 121, 122, amino acid sequence LNS and SEQ ID NO: 124, respectively;

[0249] q. SEQ ID NOs: 119, 120, 121, 122, amino acid sequence LNS and SEQ ID NO: 124, respectively;

[0250] r. SEQ ID NOs: 125, 126, 127, 128, amino acid sequence FNF and SEQ ID NO: 130, respectively;

[0251] s. SEQ ID NO: 131, 132, 133, 134, amino acid sequence YD and SEQ ID NO: 136, respectively; or

[0252] t. SEQ ID NOs: 137, 138, 139, 140, amino acid sequence FNS and SEQ ID NO: 142, respectively.

[0253] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 7, 175, 9, 10, 11, and 12, respectively.

[0254] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively.

[0255] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively.

[0256] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, 21, 22, 23, and 24, respectively.

[0257] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0258] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to VEGFR1, comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively.

[0259] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof, the isolated antibody or antigen-binding fragment thereof comprising

[0260] Heavy chain complementarity determining region (HCDR) of the heavy chain variable region (VH) of SEQ ID NO:31

[0261] 1, HCDR2 and HCDR3 and the light chain complementary determining region (LCDR) 1, LCDR2 and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 32; or HCDR1, HCDR2 and HCDR3 of the VH of SEQ ID NO: 33 and LCDR1,

[0262] LCDR2 and LCDR3; or HCDR1, HCDR2 and LCDR3 of VH of SEQ ID NO: 34;

[0263] HCDR3 and LCDR1, LCDR2 and LCDR3 of the VL of SEQ ID NO: 35; or

[0264] HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO:36 and SEQ ID NO:

[0265] 37 of the LCDR1, LCDR2 and LCDR3 of the VL; or SEQ ID NO: 38 of the VH of LCDR1, LCDR2 and LCDR3 of the VL of SEQ ID NO: 37; or SEQ ID NO: 39 of the HCDR1, HCDR2 and LCDR3 of the VH of LCDR1, LCDR2 and LCDR3 of the VL of LCDR2, LCDR3;

[0266] HCDR3 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.

[0267] LCDR2 and LCDR3, and wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0268] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0269] a. VH of SEQ ID NO:31 and VL of SEQ ID NO:32;

[0270] b. VH of SEQ ID NO:33 and VL of SEQ ID NO:32;

[0271] c. VH of SEQ ID NO:34 and VL of SEQ ID NO:35;

[0272] d. VH of SEQ ID NO:36 and VL of SEQ ID NO:37;

[0273] e. VH of SEQ ID NO: 38 and VL of SEQ ID NO: 37;

[0274] f. VH of SEQ ID NO:39 and VL of SEQ ID NO:40; or

[0275] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0276] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the VH of SEQ ID NO:31 and the VL of SEQ ID NO:32.

[0277] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the VH of SEQ ID NO:33 and the VL of SEQ ID NO:32.

[0278] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the VH of SEQ ID NO:34 and the VL of SEQ ID NO:35.

[0279] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the VH of SEQ ID NO:36 and the VL of SEQ ID NO:37.

[0280] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the VH of SEQ ID NO:38 and the VL of SEQ ID NO:37.

[0281] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises the VH of SEQ ID NO:39 and the VL of SEQ ID NO:40.

[0282] In some embodiments, the present disclosure also provides an isolated antibody or antigen-binding fragment thereof, which comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60.

[0283] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:51.

[0284] In some embodiments, the present disclosure also provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:52.

[0285] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:53.

[0286] In certain embodiments, the present disclosure also provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:54.

[0287] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:55.

[0288] In some embodiments, the present disclosure also provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:56.

[0289] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:57.

[0290] In certain embodiments, the present disclosure also provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:58.

[0291] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:59.

[0292] In certain embodiments, the present disclosure also provides an isolated antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 60.

[0293] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0294] a. HC comprising the amino acid sequence of SEQ ID NO: 51 and LC comprising the amino acid sequence of SEQ ID NO: 52;

[0295] b. HC comprising the amino acid sequence of SEQ ID NO: 53 and LC comprising the amino acid sequence of SEQ ID NO: 52;

[0296] c. HC comprising the amino acid sequence of SEQ ID NO: 54 and LC comprising the amino acid sequence of SEQ ID NO: 55;

[0297] d. HC comprising the amino acid sequence of SEQ ID NO: 56 and LC comprising the amino acid sequence of SEQ ID NO: 57;

[0298] e. HC comprising the amino acid sequence of SEQ ID NO: 58 and LC comprising the amino acid sequence of SEQ ID NO: 57; or

[0299] f. HC comprising the amino acid sequence of SEQ ID NO:59 and LC comprising the amino acid sequence of SEQ ID NO:60.

[0300] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0301] HCDR1, HCDR2,

[0302] HCDR3, LCDR1, LCDR2 and LCDR3;

[0303] VH of SEQ ID NO:31 and VL of SEQ ID NO:32; and / or

[0304] HC of SEQ ID NO:51 and LC of SEQ ID NO:52;

[0305] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0306] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0307] HCDR1, HCDR2,

[0308] HCDR3, LCDR1, LCDR2 and LCDR3;

[0309] VH of SEQ ID NO:33 and VL of SEQ ID NO:32; and / or

[0310] HC of SEQ ID NO:53 and LC of SEQ ID NO:52;

[0311] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0312] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0313] HCDR1, HCDR2,

[0314] HCDR3, LCDR1, LCDR2 and LCDR3;

[0315] VH of SEQ ID NO:34 and VL of SEQ ID NO:35; and / or

[0316] HC of SEQ ID NO:54 and LC of SEQ ID NO:55;

[0317] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0318] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0319] HCDR1, HCDR2,

[0320] HCDR3, LCDR1, LCDR2 and LCDR3;

[0321] VH of SEQ ID NO:36 and VL of SEQ ID NO:37; and / or

[0322] HC of SEQ ID NO:56 and LC of SEQ ID NO:57;

[0323] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0324] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0325] HCDR1, HCDR2,

[0326] HCDR3, LCDR1, LCDR2 and LCDR3;

[0327] VH of SEQ ID NO:38 and VL of SEQ ID NO:37; and / or

[0328] HC of SEQ ID NO:58 and LC of SEQ ID NO:57;

[0329] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0330] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0331] HCDR1, HCDR2,

[0332] HCDR3, LCDR1, LCDR2 and LCDR3;

[0333] VH of SEQ ID NO:39 and VL of SEQ ID NO:40; and / or

[0334] HC of SEQ ID NO:59 and LC of SEQ ID NO:60;

[0335] And wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0336] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0337] VH of SEQ ID NO:31, VL of SEQ ID NO:32,

[0338] HC of SEQ ID NO:51 and LC of SEQ ID NO:52;

[0339] And the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0340] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0341] VH of SEQ ID NO:33, VL of SEQ ID NO:32,

[0342] HC of SEQ ID NO:53 and LC of SEQ ID NO:52;

[0343] And the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0344] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0345] VH of SEQ ID NO:34, VL of SEQ ID NO:35,

[0346] HC of SEQ ID NO:54 and LC of SEQ ID NO:55;

[0347] And the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0348] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0349] VH of SEQ ID NO:36, VL of SEQ ID NO:37,

[0350] HC of SEQ ID NO:56 and LC of SEQ ID NO:57;

[0351] And the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0352] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0353] VH of SEQ ID NO:38, VL of SEQ ID NO:37,

[0354] HC of SEQ ID NO:58 and LC of SEQ ID NO:57;

[0355] And the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0356] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising

[0357] VH of SEQ ID NO:39, VL of SEQ ID NO:40,

[0358] HC of SEQ ID NO:59 and LC of SEQ ID NO:60;

[0359] And the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0360] In some embodiments, the isolated antibody or its isolated antigen-binding fragment is a full-length antibody.In some embodiments, the isolated antibody or its isolated antigen-binding domain is an antibody fragment or antigen-binding fragment.

[0361] Homologous antibodies and antigen-binding fragments thereof having conservative substitutions

[0362] Derivatives, homologous antigen binding domains, functional equivalents or variants of the antibodies or antigen binding fragments thereof are also the objects of the present disclosure. Antibodies of the present disclosure also include homologous antibodies, homologous antigen binding domains, functional equivalents or variants of the disclosed antibodies or antigen binding fragments thereof that bind to VEGFR1, which include polypeptides having amino acid sequences substantially identical to the amino acid sequences of the variable domains or hypervariable domains of the antibodies of the present disclosure or polypeptides having conservative substitutions. Homologous antibodies and antigen binding domains, functional equivalents or variants of the present disclosure have sufficient homology to the sequences of the antibodies or antigen binding fragments thereof that bind to VEGFR1, and are functionally similar to unmodified anti-VEGFR1 antibodies to retain binding to VEGFR1 or retain at least one activity of the unmodified antibody.

[0363] 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 at any position in the molecule. In case several amino acids have been added, substituted or deleted, any combination of additions, substitutions or deletions is contemplated, provided that the resulting antibody still has at least the advantageous properties of the antibodies of the present invention.

[0364] The sequences of the present disclosure may comprise amino acid sequences having at least 80% identity or homology with the above sequences. In some embodiments, the sequence identity with the antigen binding domains of the present disclosure that bind to VEGFR1 may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Variants of the antigen-binding domains of VEGFR1 that contain 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 are within the scope of the present disclosure, as long as they retain or have improved functional properties compared to the parent antigen-binding domain. Functional equivalents or variants of the antigen-binding domains of VEGFR1 include one or more deletions and / or additions of one or more amino acid residues. Such additions, substitutions or deletions can be located at any position in the molecule. In the case of having added, substituted or deleted several amino acids, any combination of additions, substitutions or deletions can be considered, provided that the resulting antibody still has at least the favorable properties of the antibody of the present invention.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] 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.

[0370] 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.

[0371] The present disclosure also 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.

[0372] 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.

[0373] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises 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.

[0374] 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.

[0375] 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.

[0376] 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.

[0377] 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.

[0378] 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 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs:25, 26, 27, 28, 29, and 30, respectively.

[0379] The present disclosure also 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 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0380] 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0381] 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs:25, 26, 27, 28, 29, and 30, respectively.

[0382] 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs:25, 26, 27, 28, 29 and 30, respectively.

[0383] 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs:25, 26, 27, 28, 29 and 30, respectively.

[0384] 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs:25, 26, 27, 28, 29 and 30, respectively.

[0385] 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs:25, 26, 27, 28, 29 and 30, respectively.

[0386] In some embodiments, the present disclosure also provides an isolated antibody or antigen-binding fragment thereof, which 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, and wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0387] In some embodiments, the present disclosure also provides an isolated antibody or antigen-binding fragment thereof, which 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:60; and wherein the antibody or antigen-binding fragment thereof binds to VEGFR1.

[0388] In some embodiments, the isolated antibody or antigen-binding fragment thereof of the present application comprises a heavy chain (HC) and a light chain (LC), wherein the heavy chain has 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 the light chain has 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.

[0389] The present disclosure also 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.

[0390] 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.

[0391] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 comprises 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%, or at least 99%) identical to the LC of SEQ ID NO:60.

[0392] 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.

[0393] 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.

[0394] In some embodiments, the antibody or antigen-binding fragment thereof that binds 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.

[0395] 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.

[0396] The present disclosure also 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 25, 26, 27, 28, 29, and 30, respectively.

[0397] 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, 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.

[0398] 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, 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.

[0399] 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, 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.

[0400] 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, 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.

[0401] 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, 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.

[0402] In some embodiments, the 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, 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.

[0403] The present disclosure also 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 the VH of SEQ ID NO:39 and the VL of SEQ ID NO:40.

[0404] 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, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0405] 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, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0406] In some embodiments, the 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, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0407] In some embodiments, the 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, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0408] In some embodiments, the 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, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0409] In some embodiments, the 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, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40.

[0410] In the context of two or more nucleic acid or polypeptide sequences (e.g., anti-VEGFR1 antibodies and polynucleotides encoding them), the term "identical" or "identity" percentage refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The percentage (%) of amino acid sequence identity relative 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 percentage (%) identity between the 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 × 100), taking into account the number of gaps and the length of each gap, and these parameters need to be introduced for optimal alignment of the two sequences. The percentage of identity between two amino acid sequences can be determined using various algorithms within the skill of the art, using publicly available software such as BLAS, BLAST-2, ALIGN.Megalin (DNASTAR) or the GAP program available in the GCG software package.

[0411] The polypeptide is generally substantially identical to the second polypeptide, e.g., wherein the two peptides differ only in conservative substitutions. Antibodies disclosed herein also include those antibodies whose binding properties, functions, or physical properties have been improved by direct mutation. In some embodiments, a variant antigen-binding domain that binds to VEGFR1 comprises one or two conservative substitutions in any CDR region while maintaining the desired functional properties of the parent antigen-binding fragment that binds to VEGFR1.

[0412] "Conservative modifications" refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody containing the amino acid modification. Conservative modifications include amino acid substitutions, additions, and deletions. "Conservative amino acid substitutions" are substitutions 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 having the following side chains: 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). In addition, any native 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 for the antibodies of the invention may be made by known methods, such as by PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, a library of variants may be generated, for example, using random (NNK) or non-random codons (e.g., DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The characteristics of the resulting variants may be tested using the assays described herein.

[0413] Species Cross-Reactivity

[0414] In some embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof binds to human, mouse, rat and cynomolgus monkey VEGFR1. In some embodiments, the anti-VEGFR1 antibody or antigen-binding fragment thereof of the present disclosure is a species cross-reactive antibody that can bind to human, mouse, rat and cynomolgus monkey VEGFR1 with similar affinity, thereby providing significant value to the anti-VEGFR1 antibodies disclosed herein.

[0415] In some embodiments, the anti-VEGFR1 antibody or its antigen-binding fragment binds to human and mouse VEGFR1. In some embodiments, the anti-VEGFR1 antibody or its antigen-binding fragment binds to human and rat VEGFR1. In some embodiments, the anti-VEGFR1 antibody or its antigen-binding fragment binds to human, mouse and rat VEGFR1. In some embodiments, the anti-VEGFR1 antibody or its antigen-binding fragment binds to human, mouse and cynomolgus monkey VEGFR1. In some embodiments, the anti-VEGFR1 antibody or its antigen-binding fragment binds to human, rat and cynomolgus monkey VEGFR1. In some embodiments, the anti-VEGFR1 antibody or its antigen-binding fragment binds to human VEGFR1.

[0416] One way to quantify the extent of species cross-reactivity of an antibody is the fold difference in its affinity for an antigen of one species compared to its affinity for an antigen of another species, such as the fold difference in affinity for human VEGFR1 versus mouse VEGFR1 versus rat VEGFR1 or versus cynomolgus monkey VEGFR1. Affinity can be quantified as K D , refers to the equilibrium dissociation constant of an antibody-antigen reaction determined by SPR as described elsewhere herein. A species cross-reactive anti-VEGFR1 antibody may have a fold difference of 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 affinity for binding to human and mouse VEGFR1, human and rat VEGFR1, or human and cynomolgus monkey VEGFR1. In other words, the K for binding to human VEGFR1 is 500-fold or less, 250-fold or less, 10-fold or less, or 5-fold or less. D Can bind to mouse, rat or cynomolgus monkey VEGFR1 in K DIn some embodiments, the antibody that binds to human VEGFR1 can bind to mouse, rat or cynomolgus monkey with an affinity that is not weaker than 500 times weaker than the affinity for humans, for example, with an affinity that is not weaker than 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, the antibody that binds to human VEGFR1 can bind to mouse, rat or cynomolgus monkey VEGFR1 with an affinity that is not stronger than 500 times stronger than the affinity for humans, for example, with an affinity that is not stronger than 500 times, 250 times, 100 times, 50 times, 25 times, 10 times or 5 times stronger than the affinity for human VEGFR1. If the K of the antigens that bind to two different species is not weaker than 500 times weaker than the affinity for humans, for example, the K of the antigens that bind to two different species is not weaker than 500 times weaker than the affinity for humans. D If the threshold is reached, for example, if the K of binding to human VEGFR1 D and K binding to mouse, rat, or cynomolgus monkey VEGFR1 D If the concentration of K is 10 nM or less, 5 nM or less, or 1 nM or less, the antibody may also be considered cross-reactive. D It may be 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. D It 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.

[0417] Another measure of the cross-reactivity of binding to human VEGFR1 and mouse, rat or cynomolgus monkey VEGFR1 is the ability of the antibody to block the binding of ligand to VEGR1, such as VEGFA binding or PlGF binding. The IC50 of species cross-reactive anti-VEGFR1 antibodies to blocking the binding of VEGFA or PlGF to human VEGFR1 can be within 25 times, 20 times, 15 times, 10 times or 5 times of the IC50 of blocking the binding of VEGFA or PlGF to mouse, rat or cynomolgus monkey. As another option, species cross-reactive antibodies can have the ability to regulate the circulation or tissue VEGFA or circulation or tissue PlGF levels in animals, for example, antibodies binding to human VEGFR1 can regulate the circulation or tissue VEGFA or PlGF levels in mice, rats and / or cynomolgus monkeys.

[0418] Species cross-reactive antibodies enable translational studies across several species and can help demonstrate efficacy and pharmacodynamic properties in established preclinical disease models without the need to use surrogate antibodies during the development phase. Early reported anti-VEGFR1 antibodies were either human or mouse specific and showed poor species cross-reactivity.

[0419] It is challenging 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 disclosed herein are generated by a unique immunization method that includes (1) selecting human and mouse VEGFR1 antigens that contain the VEGFR1 D2 and D3 domains, (2) selecting chicken, a phylogenetically distant immunization species, (3) immunization boosts that alternate between human VEGFR1 D2-D3 and mouse VEGFR1 D2-D3, and (4) carefully monitoring and selecting serum titers that show reactivity to both human and mouse VEGFR1.

[0420] In some embodiments, the anti-VEGFR1 antibodies of the present disclosure are capable of binding to an epitope within the sequence of amino acid residues 130 to 331 of human VEGFR1 (Uniprot Accession No. P17948). In some embodiments, the anti-VEGFR1 antibodies of the present disclosure recognize and bind to an epitope within SEQ ID NO: 173. In some embodiments, the anti-VEGFR1 antibodies of the present disclosure recognize and bind to an epitope within SEQ ID NO: 4.

[0421] In some embodiments, the anti-VEGFR1 antibody binds to the 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 the epitope on VEGFR1 set forth in SEQ ID NO: 144.

[0422] In some embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds to human VEGFR1 and is also capable of specifically binding to VEGFR1 from at least one non-human species selected from the group consisting of cynomolgus monkeys, mice, and rats. In certain embodiments, the isolated antibody or antigen-binding fragment thereof of the present application is measured at 6×10 -8 M or smaller, especially 1×10 -8 M or smaller, more particularly 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M or smaller KD Binds specifically to human VEGFR1, mouse VEGFR1, and cynomolgus monkey VEGFR1.

[0423] "Epitope" refers to the part of an antigen that specifically binds to an antibody. An epitope is usually composed of chemically active (such as polar, nonpolar or hydrophobic) surface groups of a part (such as an amino acid or a polysaccharide side chain) and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be composed of continuous and / or discontinuous amino acids that form a conformational space unit. For a discontinuous epitope, amino acids from different parts of the linear sequence of an antigen are close in three-dimensional space due to the folding of the protein molecule.

[0424] "Paratope" refers to the part of an antibody that specifically binds to an antigen. The paratope may be linear or discontinuous in nature, formed by the spatial relationship between the non-adjacent amino acids of the antibody rather than a linear series of amino acids."Light chain paratope" and "heavy chain paratope" or "light chain paratope amino acid residues" and "heavy chain paratope amino acid residues" refer to the antibody light chain and heavy chain residues in contact with the antigen, respectively, or generally speaking, "antibody paratope residues" refer to those antibody amino acids in contact with the antigen.

[0425] The determination of the residues in hVEGFR1 that bind to the antibody can be determined by any technique known to those of ordinary skill in the art. In some embodiments, the residues in hVEGFR1 that bind to the antibody are determined by H / D exchange assay. In the H / D exchange assay, the recombinantly expressed hVEGFR1 is incubated in deuterated water for a predetermined time in the presence or absence of an antibody, thereby causing deuterium to be introduced at an exchangeable hydrogen atom that is not protected by the antibody, followed by protease digestion of the protein and analysis of the peptide fragments using LC-MS. The H / D exchange assay can be performed using known protocols. In some embodiments, the H / D exchange mixture is quenched by adding a quenching buffer (e.g., 8M urea, 1M TCEP, pH 3.0), followed by passing through a balanced fixed pepsin / FPXIII column (e.g., 600 μL / min) at room temperature. The peptide fragments are then loaded onto a reverse phase trap column (e.g., at 600 μL / min) and desalted (e.g., at 600 μL for 1 min), separated (e.g., on a C18 column) and analyzed by mass spectrometry (e.g., using LTQ TM Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) with capillary temperature of 275 °C, resolution of 150,000 and mass range (m / z) 300-1,800).

[0426] Antibodies that block the binding of VEGFA to VEGFR1

[0427] In some embodiments, the anti-VEGFR1 antibodies of the present disclosure are capable of recognizing and binding to an epitope in the D2-D3 domain of VEGFR1 and preventing the binding of vascular endothelial growth factor A (VEGFA).

[0428] In kidney, VEGFA produced by glomerular podocytes and tubular epithelial cells is necessary to maintain glomerular integrity, renal microvasculature and function. The critical role of VEGFA in kidney development, structure and function is shown in the severe phenotype observed in conditional or inducible VEGFA knockout (KO) mice. In chronic kidney disease (CKD) patients, mRNA microarray analysis of patient renal biopsy shows that VEGFA expression is reduced, and VGFA expression levels in glomeruli and tubulointerstitium are related to eGFR, proteinuria or vascular rarefaction (Bortoloso, Del Prete et al. 2004, Martini, Nair et al. 2014, Pan, Jiang et al. 2018). VEGFA plays a role through two endothelial restricted receptors: 1) main signal transduction receptor VEGFR2, and 2) decoy receptor VEGFR1, which isolates VEGFA from VEGFR2 with 10 times higher binding affinity, but has weak or undetectable tyrosine kinase activity (TK). The anti-VEGFR1 of the present disclosure aims to restore the impaired VEGFA levels and VEGFA activity in the kidney by blocking the sequestration of VEGFA by its decoy receptor VEGFR1. The anti-VEGFR1 antibodies of the present disclosure will block VEGFA sequestration and increase local VEGFA availability, which in turn will enable endothelial protection, preservation of the glomerular filtration barrier (GFB) and restoration of renal function.

[0429] Conjugation and Fc engineering

[0430] In addition to the above modifications, the anti-VEGFR1 antibodies or antigen-binding fragments thereof and their functional equivalents of the present disclosure may be conjugated to other antibodies, proteins, antigen-binding fragments or alternative scaffolds.

[0431] Anti-VEGFR1 antibodies, antigen-binding fragments thereof and functional equivalents of the present disclosure can be conjugated to a half-life extension moiety. Exemplary half-life extension moieties are albumin, albumin variants, albumin binding proteins and / or domains, transferrin and its fragments and analogs, immunoglobulins (Ig) or fragments thereof (such as Fc regions). The amino acid sequences of the aforementioned half-life extension moieties are known. Additional half-life extension moieties that can be conjugated to 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, myristate, stearate, arachidate, behenate, oleate, arachidonic acid, suberic acid, tetradecanedioic acid, octadecanedioic acid, docosane dioic acid, etc., polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides or polysaccharides) to obtain the desired properties. These moieties can be fused directly to the antibodies or antigen-binding fragments of the present disclosure and can be produced by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods can be used to attach these moieties to the recombinantly produced antibodies or antigen-binding fragments of the present disclosure.

[0432] The polyethylene glycol moiety can be conjugated to the antibody or antigen-binding fragment thereof that binds VEGFR1, for example, by incorporating a cysteine ​​residue into the C-terminus of the antibody or antigen-binding fragment that binds VEGFR1 or engineering a cysteine ​​into a residue position facing away from the VEGFR1 binding site and attaching a polyethylene glycol group to the cysteine ​​using well-known methods.

[0433] In some embodiments, the half-life extending moiety is albumin.

[0434] In some embodiments, the half-life extending moiety is an albumin binding domain.

[0435] In some embodiments, the half-life extending moiety is transferrin.

[0436] In some embodiments, the half-life extending moiety is polyethylene glycol.

[0437] In some embodiments, conjugation also includes, but is not limited to, conjugation to a detectable receptor molecule.

[0438] In some embodiments, the half-life extending moiety is an Ig constant region or a fragment of an Ig constant region.

[0439] In some embodiments, the half-life extending moiety is Ig.

[0440] In some embodiments, the half-life extending moiety is a fragment of Ig.

[0441] In some embodiments, the half-life extending moiety is an Ig constant region.

[0442] In some embodiments, the half-life extending moiety is a fragment of an Ig constant region.

[0443] In some embodiments, the half-life extending moiety is an Fc region.

[0444] 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 present disclosure, can be of any allotype or isotype, i.e., IgGl, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0445] In some embodiments, the Ig constant region or fragment of an Ig constant region is of the IgG1 isotype.

[0446] In some embodiments, the Ig constant region or fragment of the Ig constant region is of the IgG2 isotype.

[0447] In some embodiments, the Ig constant region or fragment of the Ig constant region is of the IgG3 isotype.

[0448] In some embodiments, the Ig constant region or fragment of the Ig constant region is of the IgG4 isotype.

[0449] It is expected that allotypes have no effect on the properties of the Ig constant region, such as binding or Fc-mediated effector functions. The immunogenicity of therapeutic proteins comprising Ig constant regions or fragments thereof is associated with increased risk of infusion reactions and reduced duration of therapeutic response (Baert et al., (2003) N Engl J Med 348: 602-08). The extent to which therapeutic proteins comprising Ig constant regions or fragments thereof induce immune responses in hosts can be determined in part by the allotypes of the Ig constant region (Stickler et al., (2011) Genes and Immunity 12: 213-21). Ig constant region allotypes are related to amino acid sequence variations at specific positions in the constant region sequence of antibodies.

[0450] The antibodies or antigen-binding fragments thereof disclosed herein and their functional equivalents can be conjugated to an Ig constant region or a fragment of an Ig constant region to modulate antibody or antigen-binding fragment effector functions, such as ADCC, ADCP and / or ADCP and / or pharmacokinetic properties. This can be achieved by introducing mutations into Fc that modulate the binding of the mutated Fc to activating FcγR (FcγRI, FcγRIIa, FcγRIII), inhibitory FcγRIIb and / or FcRn.

[0451] 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 comprising at least one mutation.

[0452] In some embodiments, the at least one mutation is in the Fc region.

[0453] In some embodiments, the antibody or antigen-binding fragment that binds to VEGFR1 conjugated to an Ig constant region or a fragment of an Ig constant region comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen mutations in the Fc region.

[0454] Neonatal Fc receptor (FcRn) plays a central role in the 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 comprises at least one mutation in the Fc region that modulates the binding of the antibody or antigen-binding fragment to FcRn and modulates the half-life of the antibody or antigen-binding fragment.

[0455] In some embodiments, the Ig constant region or fragment of the Ig constant region comprises at least one mutation that modulates the half-life of the isolated antibody or antigen-binding fragment thereof.

[0456] Fc positions 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 mutations T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R. Exemplary single mutations or combined mutations that can be generated to increase half-life are mutations M428L / N434S, M252Y / S254T / T256E, T250Q / M428L, N434A, and T307A / E380A / N434A. In some embodiments, at least one mutation that modulates an antibody or antigen-binding fragment thereof of the present disclosure, and their functional equivalents, is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, wherein residue numbering is performed according to the EU index.

[0457] In some embodiments, an antibody or antigen-binding fragment thereof that binds VEGFR1 is conjugated to an Ig constant region or a fragment of an Ig constant region comprising the M252Y / S254T / T256E mutations.

[0458] In some embodiments, the antibodies or antigen-binding fragments of the present disclosure and their functional equivalents are conjugated to an Ig constant region or a fragment of an Ig constant region comprising at least one mutation in the Fc region that reduces binding of the protein to an activating Fcγ receptor (FcγR) and / or reduces Fc effector functions such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).

[0459] 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 can be made alone or in combination are mutations K214T, E233P, L234V, L234A, G236 deletion, 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 that produce proteins with reduced ADCC are mutations L234A / L235A on IgG1, mutations L234A / L235A / D265S on IgG1, mutations V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, mutations F234A / L235A on IgG4, mutations S228P / F234A / L235A on IgG4, N297A on all Ig isotypes, mutations V234A / G237A on IgG2, mutations K214T / E233P / L234V / L235A / G236-deficient on IgG1. Loss / A327G / P331A / D365E / L358M on IgG2, mutations H268Q / V309L / A330S / P331S on IgG2, mutations S267E / L328F on IgG1, mutations L234F / L235E / D265A on IgG1, mutations L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, mutations S228P / F234A / L235A / G237A / P238S on IgG4, and mutations S228P / F234A / L235A / G236-deletion / G237A / P238S on 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.

[0460] 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, at least one mutation that results in reduced binding of the antibody to 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 a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S. In some embodiments, the FcγR is FcγRI, FcγRIIA, FcγRIIB or FcγRIII or any combination thereof.

[0461] In some embodiments, the antibodies or antigen-binding fragments of the present disclosure and their functional equivalents are conjugated to an Ig constant region or a fragment of an Ig constant region comprising at least one mutation in the Fc region that enhances binding of the protein to an Fcγ receptor (FcγR) and / or enhances Fc effector functions, such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and / or phagocytosis (ADCP).

[0462] Fc positions that can be mutated to increase binding of the protein to activating FcγRs 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 can be made alone or in combination are G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A, A330K, I332E, E333A, K334A, A339T, and P396L. Exemplary combination mutations that produce 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.

[0463] Fc positions that can be mutated to enhance CDC include positions 267, 268, 324, 326, 333, 345, and 430. Exemplary mutations that can be produced individually or in combination are S267E, F1268F, S324T, K326A, K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430F, and E430T. Exemplary combined mutations that produce proteins with increased CDC are K326A / E333A, K326W / E333A, H268F / S324T, S267E / H268F, S267E / S324T, and S267E / H268F / S324T.

[0464] 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, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S.

[0465] In some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising the VH of SEQ ID NO: 39 and the VL of SEQ ID NO: 40, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S.

[0466] 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 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 VL of SEQ ID NO: 40, NO:25, 26, 27, 28, 29 and 30 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S.

[0467] The present disclosure also 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 comprises 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 VL of SEQ ID NO: 40, NO:25, 26, 27, 28, 29 and 30 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S.

[0468] 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 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 is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0469] 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 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 is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0470] 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 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 is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0471] 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 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 is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0472] 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 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 is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0473] 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 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 is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0474] In some embodiments, the present disclosure also 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, and wherein the antibody or antigen-binding fragment thereof binds to VEGFR1, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S.

[0475] In some embodiments, the present disclosure also 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; and wherein the antibody or antigen-binding fragment thereof binds to VEGFR1, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S.

[0476] The present disclosure also 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, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S.

[0477] 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, and wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0478] 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, and wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S.

[0479] 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, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0480] 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, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0481] 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, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A L235A D265S.

[0482] 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, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0483] The present disclosure also 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 SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 90, 91, 92, 93 ...9, 90, 91, 92, 93, 99, 90, 91, 92, 93, 99, 90, 91, 92 NO:25, 26, 27, 28, 29 and 30 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprises the following mutation: L234A_L235A_D265S.

[0484] 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 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 is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0485] 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 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 is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0486] 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 95% identical to the LC of SEQ ID NO: 60, 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 wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0487] 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 comprises 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 is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0488] In some embodiments, an 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, 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 wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0489] In some embodiments, an 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, wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 26, 27, 28, 29 and 30, respectively, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0490] The present disclosure also 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 VH of SEQ ID NO: 40. NO:40 VL, and wherein the antibody or antigen-binding fragment is IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0491] 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 wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0492] 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 a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40, and wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0493] 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 95% 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, and wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0494] 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 comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40, and wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0495] In some embodiments, an 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, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40, and wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0496] In some embodiments, an 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, wherein the antibody or antigen-binding fragment comprises a VH of SEQ ID NO:39 and a VL of SEQ ID NO:40, and wherein the antibody or antigen-binding fragment is an IgG1 (e.g., IgG1λ), optionally wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S, such as wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265S.

[0497] Polynucleotide

[0498] Also provided are polynucleotides encoding the anti-VEGFR1 antibodies or antigen-binding fragments of the disclosure and their functional equivalents.The present disclosure provides isolated polynucleotides encoding any of the anti-VEGFR1 antibodies or antigen-binding fragments thereof of the disclosure.

[0499] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:31.

[0500] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:33.

[0501] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:34.

[0502] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:36.

[0503] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:38.

[0504] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VH of SEQ ID NO:39.

[0505] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VL of SEQ ID NO:32.

[0506] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VL of SEQ ID NO:35.

[0507] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VL of SEQ ID NO:37.

[0508] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the VL of SEQ ID NO:40.

[0509] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:51.

[0510] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:53.

[0511] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:54.

[0512] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:56.

[0513] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:58.

[0514] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO:59.

[0515] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the light chain of SEQ ID NO:52.

[0516] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the light chain of SEQ ID NO:55.

[0517] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the light chain of SEQ ID NO:57.

[0518] In some embodiments, the present disclosure provides an isolated polynucleotide encoding the light chain of SEQ ID NO:60.

[0519] In some embodiments, the present disclosure provides isolated polynucleotide sequences encoding the polypeptide sequences of SEQ ID NOs: 59 and 60.

[0520] 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.

[0521] In some embodiments, the present disclosure provides an isolated polynucleotide comprising a polynucleotide sequence that is at least 80% (such as 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.

[0522] The polynucleotides encoding the anti-VEGFR1 antibodies or antigen-binding fragments of the present disclosure include polynucleotides having nucleic acid sequences substantially identical to the nucleic acid sequences of the polynucleotides of the present disclosure. "Substantially identical" nucleic acid sequences are defined herein as sequences that have at least 80% identity with another nucleic acid sequence when the two sequences are aligned. If a polypeptide encoded by a first nucleic acid is immunologically cross-reactive with a polypeptide encoded by a second nucleic acid, the two nucleic acid sequences are substantially identical. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0523] Modified nucleotides can be used to generate the polynucleotides disclosed herein. Exemplary modified nucleotides are 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, β-D-mannosyl quercetin, 5″-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-Isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queuosine, β-D-galactosylqueuosine, inosine, N 6 -isopentenyl adenine, 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-oxyacetate, 3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine.

[0524] Vector comprising a polynucleotide encoding an anti-VEGFR1 antibody

[0525] Also provided are vectors containing DNA encoding the anti-VEGFR1 antibodies or antigen-binding fragments of the present disclosure. The disclosed vectors can be used, for example, to produce any of the above disclosed anti-VEGFR1 antibodies or antigen-binding fragments thereof. Standard molecular biology methods can be used to incorporate polynucleotides encoding any of the anti-VEGFR1 antibodies or antigen-binding fragments of the present disclosure into the vector.

[0526] In some embodiments, the disclosure provides expression vectors comprising polynucleotides of the present invention. Such vectors can be plasmid vectors, viral vectors, vectors for baculovirus expression, vectors based on transposons, or any other vectors suitable for introducing the synthetic polynucleotides of the present invention into a given organism or genetic background by any means. The vectors of the present disclosure can be expression vectors for effectively synthesizing VEGFR1 antibody polypeptides and expressing VEGFR1 antibody polypeptides of the present disclosure in prokaryotic and eukaryotic systems (including but not limited to yeast and mammalian cell cultures).

[0527] Exemplary vectors that can be used are bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBsKS, 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.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Phage 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, such as a retroviral vector, such as a γ retroviral vector.

[0528] The carrier of the present disclosure may contain promoter and enhancer sequences. The polynucleotide encoding VEGFR1 binding proteins of the present disclosure may be operably connected to the control sequence of the expression of VEGFR1 binding proteins guaranteed in the expression vector. Such regulatory elements may include a transcription promoter, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. The expression vector may also include one or more non-transcription elements, such as a replication origin, a suitable promoter and enhancer connected to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (such as necessary ribosome binding sites), polyadenylation sites, splicing donors and acceptor sites or transcription termination sequences. The replication origin conferring replication ability in the host may also be incorporated.

[0529] 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 the expression of some proteins. In some embodiments, the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the above-mentioned nucleic acid sequences. The vector components may be connected continuously, or arranged in a manner that provides the best spacing for expressing the gene product (i.e., by introducing a "spacer" nucleotide between the ORFs), or positioned in another manner. Regulatory elements such as the IRES motif may also be arranged to provide the best spacing for expression.

[0530] The vectors of the present disclosure may be circular or linear. They may be prepared to contain a replication system that functions in a prokaryotic or eukaryotic host cell. The replication system may be derived from, for example, ColE1, SV40, 2μ plasmid, lambda, bovine papilloma virus, and the like.

[0531] The recombinant expression vector can be designed for transient expression, for stable expression, or for both. Moreover, the recombinant expression vector can be prepared for constitutive expression or for inducible expression.

[0532] The vector may also include a selection marker well known in the art. The selection marker includes positive and negative selection markers. The marker gene includes biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementary effects of prototrophy provided 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 synthase gene, HSV-TK, HSV-TK derivatives selected for ganciclovir, or bacterial purine nucleoside phosphorylase genes (Gadi et al., 7Gene Ther. 1738-1743 (2000)) selected for 6-methylpurine. The nucleotide sequence encoding the selection marker or cloning site may be upstream or downstream of the nucleotide sequence encoding the polypeptide of interest or the cloning site.

[0533] Host cells

[0534] The present disclosure also provides host cells comprising any vector of the present disclosure. "Host cell" refers to a cell into which a vector has been introduced. It should be understood that the term host cell is intended not only to refer to a specific subject cell, but also to the progeny of such cells, and also to the stable cell line produced by a specific subject cell. Because certain modifications may occur in the offspring due to mutation or due to environmental influences, such progeny may be different from the parent cell, but are still included in the scope of the term "host cell" used herein. Such host cells can be eukaryotic cells, prokaryotic cells, plant cells or archaeal cells. Examples of prokaryotic host cells are Escherichia coli, bacilli (such as Bacillus subtilis and other enterobacteriaceae (such as Salmonella, Serratia)) and various Pseudomonas species. Other microorganisms such as yeast can also be used for expression. Saccharomyces (e.g., Saccharomyces cerevisiae) and Pichia pastoris are examples of suitable yeast host cells. Exemplary eukaryotic cells can be mammals, insects, avian or other animal sources. 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 Cell Culture Collection (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.

[0535] The present disclosure provides a recombinant host cell comprising any expression vector of the present disclosure. Nucleic acids encoding any VEGFR1 binding protein or fragments thereof can be used to transform suitable mammalian host cells. Host cell transformation, culture, antibody expression and purification are accomplished using well-known methods.

[0536] Cell lines can be selected based on high-level expression of the target VEGFR1 antibody and minimal contamination from host cell proteins. Mammalian cell lines that can be used as expression host cells are well known in the art, including but not limited to cells from Chinese hamster ovary (CHO) cells such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61) or CHO DG44, and baby hamster kidney (BHK) cells. These cell lines can be used to produce any anti-VEGFR1 antibody or antibody fragment of the present disclosure by culturing cells under conditions suitable for expressing the antibody and purifying the antibody from the host cell or the culture medium surrounding the host cell.

[0537] The present disclosure also provides a method for producing an anti-VEGFR1 binding protein of the present disclosure, the method comprising culturing a host cell of the present disclosure under conditions that express an anti-VEGFR1 binding protein, and recovering the anti-VEGFR1 antibody binding protein produced by the host cell using methods well known in the art. The subject protein can be substantially pure, for example, at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% pure, or more pure, for example, free of contaminants other than the subject protein, such as cell debris, macromolecules, etc.

[0538] In some embodiments, the present disclosure provides host cells that express any of the isolated antibodies or antigen-binding fragments thereof of the present disclosure.

[0539] In some embodiments, the present disclosure provides a host cell comprising a vector of the present disclosure.

[0540] Pharmaceutical composition

[0541] Also provided is the use of any disclosed antibody for the preparation of a medicament for treating chronic kidney disease.

[0542] Also provided is the use of any disclosed antibody for the preparation of a pharmaceutical composition for treating chronic kidney disease.

[0543] Also provided is a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present disclosure and a pharmaceutically acceptable carrier.

[0544] For therapeutic use, the anti-VEGFR1 antibodies and antigen-binding fragments thereof of the present disclosure can be prepared in the form of pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier.

[0545] "Carrier" refers to the diluent, adjuvant, excipient or vehicle with which the antibody of the present invention is administered. Such vehicles can be liquids, such as water and oils, including those derived from petroleum, animals, plants or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally do not contain particulate matter. They can be sterilized by conventional known sterilization techniques (e.g., filtration). The composition may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, stabilizers, thickeners, lubricants, and colorants, etc. The concentration of the antibody of the present invention in such pharmaceutical preparations can be changed from less than about 0.5% by weight, generally to at least about 1% to as much as 15% or 20%, and can be selected based on the selected mode of administration, mainly based on the desired dose, fluid volume, viscosity, etc. Suitable vehicles and formulations including other human proteins (e.g., human serum albumin) are described, for example, in "Remington: The Science and Practice of Pharmacy", 21st edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA, 2006, Part 5, "Pharmaceutical Manufacturing", pp. 691-1092 (see especially pp. 958-989).

[0546] Pharmaceutically acceptable carriers may include buffers, excipients, stabilizers or preservatives. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, such as salts, buffers, antioxidants, sugars, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers, or combinations thereof. The amount of pharmaceutically acceptable carriers in the pharmaceutical composition can be determined experimentally based on carrier activity and desired formulation characteristics (e.g., stability and / or minimum oxidation).

[0547] The pharmaceutical composition may include a buffer 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, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); an antibacterial and antifungal agent; and a preservative.

[0548] Pharmaceutical compositions of the present disclosure can be formulated for parenteral administration or the various modes of non-parenteral administration. In one embodiment, the composition can be formulated for infusion or intravenous administration. Pharmaceutical compositions disclosed herein can be provided, for example, as sterile liquid preparations (e.g., isotonic aqueous solutions, emulsions, suspensions, dispersions or viscous compositions), which can be buffered to the desired pH. Preparations suitable for oral administration can include liquid solutions, capsules, sachets, tablets, lozenges and lozenges, powders, and liquid suspensions in suitable liquids and emulsions.

[0549] The term "pharmaceutically acceptable," as used herein with respect to pharmaceutical compositions, means a material approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and / or humans.

[0550] Treatments and uses

[0551] Also provided is the use of any disclosed antibody, antigen-binding fragment thereof, or pharmaceutical composition for treating chronic kidney disease.

[0552] "Treatment" of a disease or condition (such as chronic kidney disease) refers to achieving one or more of the following results: reducing the severity of the condition and / or shortening the duration of the condition, delaying the progression of the condition, slowing the progression of the condition, inhibiting the worsening of the characteristic symptoms of the treated condition, limiting or preventing the recurrence of the condition in a subject previously suffering from the condition, or limiting or preventing the recurrence of symptoms in a subject previously having symptoms of the condition. As used herein, the terms "delaying the progression of" and "slowing the progression of" shall include: (a) delaying or slowing the development of one or more symptoms or complications of a disease, condition, or condition; (b) delaying or slowing the development of one or more new / additional symptoms or complications of a disease, condition, or condition; and / or (c) delaying or slowing the progression of a disease, condition, or condition to a subsequent stage or more severe form of the disease, condition, or condition.

[0553] "Subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, such as 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, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc. The terms "subject" and "patient" are used interchangeably herein. In some embodiments, the subject or patient is a human.

[0554] The separated antibody or antigenic fragment thereof of the present application can prevent the binding of VEGFA to VEGFR1 in a subject in need. Therefore, another general aspect of the present application relates to a method for preventing the binding of VEGFA to VEGFR1 in a subject in need, the method comprising administering to the subject an effective amount of the separated antibody or antigenic fragment thereof, immunoconjugate, pharmaceutical composition, isolated polynucleotide, vector or host cell of the present application, thereby preventing the binding of VEGFA to VEGFR1. The subject may need to treat a disease, disorder or medical condition associated with the binding of VEGFA to VEGFR1.

[0555] In some embodiments, the subject is in need of treatment for chronic kidney disease (CKD), is in need of reduction in proteinuria, has advanced stage 4 or stage 5 CKD, or has CKD associated with proteinuria, albuminuria, or diabetes.

[0556] In some embodiments, a method of preventing VEGFA from binding to VEGFR1 in a subject in need thereof comprises administering to the subject an effective amount of any isolated antibody or antigenic fragment thereof disclosed herein, any immunoconjugate disclosed herein, any pharmaceutical composition disclosed herein, any isolated polynucleotide disclosed herein, any vector disclosed herein, or any host cell disclosed herein, thereby preventing VEGFA from binding to VEGFR1.

[0557] Also provided is a method for treating a medical condition by administering a therapeutically effective amount of an anti-VEGFR1 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 that is effective in achieving the desired therapeutic outcome within the desired dosage and time period. A therapeutically effective amount may vary according to factors such as the individual's disease state, age, sex, and weight.

[0558] In some embodiments, the medical condition is chronic kidney disease.In some embodiments, the present disclosure provides methods of treating chronic kidney disease in a subject, the methods comprising administering to the subject a therapeutically effective amount of a disclosed pharmaceutical composition.

[0559] "Chronic kidney disease" is intended to include all types of chronic kidney disease (CKD) of any etiology or caused by any type of condition associated with chronic kidney disease and producing all types of symptoms associated with chronic kidney disease. CKD includes primary glomerular diseases (including but not limited to nephropathy and focal segmental glomerulosclerosis), secondary glomerular diseases (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).

[0560] In some embodiments, the present disclosure provides a method of treating diabetic nephropathy in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of any anti-VEGFR1 antibody or antigen-binding fragment thereof of the present disclosure or any disclosed pharmaceutical composition of the present disclosure.

[0561] In some embodiments, the present disclosure provides a method of treating 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 present disclosure, wherein the chronic kidney disease is diabetic nephropathy.

[0562] In some embodiments, the present disclosure provides a method of 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 present disclosure, wherein the chronic kidney disease is diabetic nephropathy.

[0563] In some embodiments, the present disclosure provides a method of treating 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 of the present disclosure, wherein the chronic kidney disease is focal segmental glomerulosclerosis.

[0564] In some embodiments, the present disclosure provides a method of delaying or slowing 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 of the present disclosure, wherein the chronic kidney disease is focal segmental glomerulosclerosis.

[0565] In some embodiments, the present disclosure provides a method of treating 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 present disclosure, wherein the chronic kidney disease is hypertensive nephropathy.

[0566] In some embodiments, the present disclosure provides a method of 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 present disclosure, wherein the chronic kidney disease is hypertensive nephropathy.

[0567] In some embodiments, the subject suffers from chronic kidney disease (CKD). Chronic kidney disease is divided into five nephropathy stages. The stages of nephropathy are classified according to the speed at which the kidneys can filter waste and excess fluid from the blood. The estimated glomerular filtration rate (eGFR) value is based on the level of creatine and waste in the blood, and has been used to classify the five stages of renal damage, from very mild damage in stage 1 to renal failure in stage 5.

[0568] 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 isolated antibody or antigenic fragment thereof of the disclosure, any immunoconjugate of the disclosure, any pharmaceutical composition of the disclosure, any isolated polynucleotide of the disclosure, any vector of the disclosure, or any host cell of the disclosure, for a period of time sufficient to treat the CKD.

[0569] In some embodiments, the present disclosure provides VEGFR1 for reducing glomerular filtration rate (GFR) loss.

[0570] In some embodiments, the present disclosure provides methods for reducing glomerular filtration rate (GFR) loss, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment of the present disclosure.

[0571] In some embodiments, the subject has advanced stage 3, 4, or 5 chronic kidney disease, wherein the eGFR (estimated glomerular filtration rate) is less than 60 ml / min / 1.73 m 2 In some embodiments, the subject has advanced stage 4 or stage 5 chronic kidney disease, wherein the measured eGFR is equal to or less than 30 ml / min / 1.73 m 2 In some embodiments, the subject has stage 3 chronic kidney disease, wherein the measured eGFR is 30 ml / min / 1.73 m 2 Up to 60ml / min / 1.73m 2 In some embodiments, the subject has stage 3b chronic kidney disease, wherein the measured eGFR is 30 ml / min / 1.73 m 2 Up to 45ml / min / 1.73m 2 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.

[0572] In some embodiments, the present disclosure provides a method for treating chronic kidney disease in a subject, the method comprising administering a therapeutically effective amount of an anti-VEGFR1 antibody or an antigen-binding fragment thereof of the present disclosure to a subject in need thereof, wherein the subject has stage 4 or stage 5 chronic kidney disease and a measured eGFR is equal to or less than 30 ml / min / 1.73 m 2 .

[0573] In some embodiments, the present disclosure provides a method for delaying the progression of chronic kidney disease in a subject or slowing its progression, the method comprising administering a therapeutically effective amount of an anti-VEGFR1 antibody or an antigen-binding fragment thereof of the present disclosure to a subject in need thereof, wherein the subject has stage 4 or stage 5 chronic kidney disease, and the measured eGFR is equal to or lower than 30 ml / min / 1.73 m 2 .

[0574] In some embodiments, the present disclosure provides a method for treating chronic kidney disease in a subject, the method comprising administering a therapeutically effective amount of an anti-VEGFR1 antibody or an antigen-binding fragment thereof of the present disclosure to a subject in need thereof, wherein the subject has stage 3 chronic kidney disease and a measured eGFR of 30 ml / min / 1.73 m 2 Up to 60ml / min / 1.73m 2 .

[0575] In some embodiments, the present disclosure provides a method for delaying the progression of chronic kidney disease in a subject or slowing its progression, the method comprising administering a therapeutically effective amount of an anti-VEGFR1 antibody or an antigen-binding fragment thereof of the present disclosure to a subject in need thereof, wherein the subject has stage 3 chronic kidney disease and a measured eGFR of 30 ml / min / 1.73 m 2 Up to 60ml / min / 1.73m 2 .

[0576] In some embodiments, the subject has chronic kidney disease with proteinuria. In some embodiments, the subject has chronic kidney disease with albuminuria. In some embodiments, the subject has chronic kidney disease with diabetes.

[0577] In some embodiments, the present disclosure provides a method of treating 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 present disclosure, wherein the subject has chronic kidney disease with proteinuria.

[0578] In some embodiments, the present disclosure provides a method of delaying or slowing the progression of chronic kidney disease in a subject, the method comprising administering a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the present disclosure to a subject in need thereof, wherein the subject has chronic kidney disease with proteinuria.

[0579] In some embodiments, the present disclosure provides a method of reducing proteinuria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any isolated antibody or antigenic fragment thereof of the present disclosure, any immunoconjugate of the present disclosure, any pharmaceutical composition of the present disclosure, any isolated polynucleotide of the present disclosure, any vector of the present disclosure, or any host cell of the present disclosure, for a period of time sufficient to reduce proteinuria in the subject.

[0580] In some embodiments, the present disclosure provides a method of treating 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 present disclosure, wherein the subject has chronic kidney disease with albuminuria.

[0581] In some embodiments, the present disclosure provides a method of delaying or slowing the progression of chronic kidney disease in a subject, the method comprising administering a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the present disclosure to a subject in need thereof, wherein the subject has chronic kidney disease with albuminuria.

[0582] In some embodiments, the present disclosure provides a method of treating 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 present disclosure, wherein the subject has chronic kidney disease associated with diabetes.

[0583] In some embodiments, the present disclosure provides a method of delaying or slowing the progression of chronic kidney disease in a subject, the method comprising administering a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the present disclosure to a subject in need thereof, wherein the subject has chronic kidney disease associated with diabetes.

[0584] "Proteinuria" refers to the presence of increased amounts of protein in the urine. Proteinuria may reflect abnormal loss of plasma protein due to a) increased glomerular permeability to large 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 of proteinuria, such as immunoglobulin light chains). Proteinuria may also reflect abnormal loss of protein from the kidneys (tubular cell components due to tubular damage) and the lower urinary tract.

[0585] "Albuminuria" is the condition in which albumin is present in the urine. The kidneys of healthy individuals filter albumin. When the kidneys are unable to filter macromolecules (such as albumin) from the urine normally, albumin is secreted into the urine and is often a sign of kidney damage. Albuminuria is a common but inconsistent condition in patients with CKD. It is the earliest marker of glomerular disease (including diabetic glomerulosclerosis), which usually appears before the eGFR decreases. It is a marker for hypertensive nephrosclerosis, but may not appear until after the eGFR decreases.

[0586] "Diabetic nephropathy" is one of the microvascular complications of diabetes and is characterized by the persistence of albuminuria and a gradual decline in renal function.

[0587] In some embodiments, the present disclosure provides a method of reducing proteinuria in a patient with chronic kidney disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the present disclosure.

[0588] In some embodiments, the present disclosure provides a method for reducing proteinuria in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment of the present disclosure. In some embodiments, proteinuria is caused by chronic kidney disease. In other embodiments, proteinuria is caused by diabetic nephropathy.

[0589] In some embodiments, the present disclosure provides a method of reducing albuminuria in a patient with chronic kidney disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of an anti-VEGFR1 antibody or antigen-binding fragment thereof of the present disclosure.

[0590] Also provided is a method of detecting VEGFR1 in a sample, the method 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.

[0591] In some embodiments, the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, synovial fluid, circulating cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically removed tissue, biopsy tissue, including fine needle aspirate tissue), histological preparations, etc. VEGFR1 can be detected using known methods. Exemplary methods include directly labeling antibodies with fluorescent or chemiluminescent labels, or radioactive labels, or attaching easily detectable moieties (such as biotin, enzymes, or epitope tags) to the antibodies of the invention. Exemplary labels and moieties are ruthenium, 111 In-DOTA, 111In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and β-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes and dye.

[0592] The antibodies and antigen-binding fragments thereof disclosed herein can 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.

[0593] Reagent test kit

[0594] Described herein are kits comprising the antibodies or antigen-binding fragments of the disclosure, or their functional equivalents.

[0595] The present disclosure provides kits comprising antibodies or antigen-binding fragments thereof that bind to VEGFR1.

[0596] The kits can be used for therapeutic applications and as diagnostic kits.

[0597] The kit can be used to detect the presence of VEGFR1 in a sample.

[0598] In some embodiments, the kit comprises an anti-VEGFR1 antibody or antigen-binding fragment of the present disclosure and a reagent for detecting VEGFR1 binding proteins. The kit may comprise one or more other elements, including: instructions for use; other reagents, such as labels, therapeutic agents, or agents that can be used to chelate or otherwise couple the antibody to a label or therapeutic agent, or a radiation protection composition; a device or other material for preparing the antibody for administration; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject.

[0599] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to VEGFR1 in a container and instructions for use of the kit.

[0600] In some embodiments, the present disclosure comprises a kit comprising any isolated antibody or antigen-binding fragment of the present disclosure, any immunoconjugate of the present disclosure, any pharmaceutical composition of the present disclosure, any isolated polynucleotide of the present disclosure, any vector of the present disclosure, any host cell of the present disclosure.

[0601] In some embodiments, the antibody or antigen-binding fragment thereof that binds to VEGFR1 in the kit is labeled.

[0602] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to VEGFR1, the antibody or antigen-binding fragment thereof comprising:

[0603] VH of SEQ ID NO:31 and VL of SEQ ID NO:32;

[0604] VH of SEQ ID NO:33 and VL of SEQ ID NO:32;

[0605] VH of SEQ ID NO:34 and VL of SEQ ID NO:35;

[0606] VH of SEQ ID NO:36 and VL of SEQ ID NO:37;

[0607] VH of SEQ ID NO:38 and VL of SEQ ID NO:37; or

[0608] VH of SEQ ID NO:39 and VL of SEQ ID NO:40.

[0609] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to VEGFR1, the antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO:39 and the VL of SEQ ID NO:40.

[0610] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that binds to VEGFR1, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:51, 52, 53, 54, 55, 56, 57, 59 or 60.

[0611] In some embodiments, the kit comprises an antibody or an antigen-binding fragment thereof that binds to VEGFR1, wherein the antibody or the antigen-binding fragment thereof 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; (c) SEQ ID NO:54 and SEQ ID NO:55; (d) SEQ ID NO:56 and SEQ ID NO:57; (e) SEQ ID NO:58 and SEQ ID NO:57; and SEQ ID NO:59 and SEQ ID NO:60.

[0612] Implementation

[0613] The following list of embodiments is intended to supplement, not replace or supersede, the preceding description.

[0614] 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, and wherein the antibody or antigen-binding fragment thereof prevents the binding of VEGFA to VEGFR1.

[0615] 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, and wherein the antibody or antigen-binding fragment thereof prevents the binding of VEGFA to VEGFR1.

[0616] 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, and wherein the antibody or antigen-binding fragment thereof prevents binding of VEGFA to VEGFR1.

[0617] Embodiment 4. An isolated antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 3, 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 sequence FPLDTL (SEQ ID NO: 143) and EIGL (SEQID NO: 144).

[0618] Embodiment 5. An isolated antibody or antigen-binding fragment thereof according to 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).

[0619] Embodiment 6. The isolated antibody or antigen-binding fragment thereof according to 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).

[0620] Embodiment 7. An isolated antibody or antigen-binding fragment thereof according to Embodiment 4, wherein the antibody or antigen-binding fragment thereof binds to human, mouse, rat and / or cynomolgus monkey VEGFR1.

[0621] Embodiment 8. The isolated antibody or antigen-binding fragment thereof according to Embodiment 7, wherein the antibody or antigen-binding fragment thereof binds to human VEGFR1.

[0622] Embodiment 9. The isolated antibody or antigen-binding fragment thereof according to Embodiment 8, wherein the 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.

[0623] Embodiment 10. The isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human and mouse VEGFR1.

[0624] Embodiment 11. The isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human and rat VEGFR1.

[0625] Embodiment 12. An isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human, mouse and rat VEGFR1.

[0626] Embodiment 13. The isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human and cynomolgus monkey VEGFR1.

[0627] Embodiment 14. The isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof binds to human, mouse, rat and cynomolgus monkey VEGFR1.

[0628] Embodiment 15. The isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof is quantified at 6×10 -8 M or smaller, especially 1×10 -8 M or smaller, more particularly 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M or smaller K D Binds to human VEGFR1, mouse VEGFR1, rat VEGFR1, and cynomolgus monkey VEGFR1.

[0629] Embodiment 16. The isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof is quantified at 6×10 -8 M or smaller, especially 1×10 -8 M or smaller, more particularly 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10-10 M or smaller or 1×10 -10 M or smaller K D Binds to human VEGFR1, mouse VEGFR1, and cynomolgus monkey VEGFR1.

[0630] Embodiment 17. The isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof is quantified at 6×10 -8 M or smaller, especially 1×10 -8 M or smaller, more particularly 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M or smaller K D Binds to human VEGFR1 and mouse VEGFR1.

[0631] Embodiment 18. The isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof is quantified at 6×10 -8 M or smaller, especially 1×10 -8 M or smaller, more particularly 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M or smaller K D Binds to human VEGFR1 and rat VEGFR1.

[0632] Embodiment 19. The isolated antibody or antigen-binding fragment thereof according to Embodiment 9, wherein the antibody or antigen-binding fragment thereof is quantified at 6×10 -8 M or smaller, especially 1×10 -8 M or smaller, more particularly 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M or smaller K D Binds to human VEGFR1 and cynomolgus monkey VEGFR1.

[0633] 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.

[0634] Embodiment 21. The isolated antibody or antigen-binding fragment thereof according to Embodiment 20, comprising:

[0635] a. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 31 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 32;

[0636] b. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 33 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 32;

[0637] c. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 34 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 35;

[0638] d. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 36 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 37;

[0639] e. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 38 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 37; or

[0640] f. The HCDR of VH has the amino acid sequence of SEQ ID NO:39 and the LCDR of VL has the amino acid sequence of SEQ ID NO:40.

[0641] Embodiment 22. An isolated antibody or antigen-binding fragment thereof according to Embodiment 21, comprising a VH having HCDR1, HCDR2 and HCDR3 and a VL having LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the following amino acid sequence:

[0642] a. SEQ ID NO: 7, 175, 9, 10, 11 and 12 respectively;

[0643] b. SEQ ID NO: 7, 8, 9, 10, 11 and 12 respectively;

[0644] c. SEQ ID NOs: 13, 14, 15, 16, 17 and 18, respectively;

[0645] d. SEQ ID NOs: 19, 20, 21, 22, 23 and 24, respectively;

[0646] e. SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively;

[0647] f. SEQ ID NOs: 71, 176, 73, 74, 75 and 76, respectively;

[0648] g. SEQ ID NOs: 71, 72, 73, 74, 75 and 76, respectively;

[0649] h. SEQ ID NOs: 77, 78, 79, 80, 81 and 82, respectively;

[0650] i. SEQ ID NOs: 83, 84, 85, 86, 87 and 88, respectively;

[0651] j. SEQ ID NOs: 89, 90, 91, 92, 93 and 94, respectively;

[0652] k. SEQ ID NO: 95, 177, 97, 98, 99 and 100, respectively.

[0653] l. SEQ ID NO:95, 96, 97, 98, 99 and 100 respectively;

[0654] m. SEQ ID NOs: 101, 102, 103, 104, 105 and 106, respectively;

[0655] n. SEQ ID NO: 107, 108, 109, 110, 111 and 112 respectively;

[0656] o. SEQ ID NOs: 113, 114, 115, 116, 117 and 118, respectively;

[0657] p. SEQ ID NOs: 119, 178, 121, 122, amino acid sequence LNS and SEQ ID NO: 124, respectively;

[0658] q. SEQ ID NOs: 119, 120, 121, 122, amino acid sequence LNS and SEQ ID NO: 124, respectively;

[0659] r. SEQ ID NOs: 125, 126, 127, 128, amino acid sequence FNF and SEQ ID NO: 130, respectively;

[0660] s. SEQ ID NO: 131, 132, 133, 134, amino acid sequence YD and SEQ ID NO: 136, respectively; or

[0661] t. SEQ ID NOs: 137, 138, 139, 140, amino acid sequence FNS and SEQ ID NO: 142, respectively.

[0662] Embodiment 23. An isolated antibody or antigen-binding fragment thereof according to Embodiment 22, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 175, 9, 10, 11 and 12, respectively.

[0663] Embodiment 24. An isolated antibody or antigen-binding fragment thereof according to Embodiment 22, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 14, 15, 16, 17 and 18, respectively.

[0664] Embodiment 25. An isolated antibody or antigen-binding fragment thereof according to Embodiment 22, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively.

[0665] Embodiment 26. An isolated antibody or antigen-binding fragment thereof according to Embodiment 22, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 8, 9, 10, 11 and 12, respectively.

[0666] Embodiment 27. An isolated antibody or antigen-binding fragment thereof according to Embodiment 22, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 20, 21, 22, 23 and 24, respectively.

[0667] 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) and a light chain variable domain (VL), wherein the heavy chain variable domain has 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 39, and the light chain variable domain has 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.

[0668] Embodiment 29. The isolated antibody or antigen-binding fragment thereof according to Embodiment 28, wherein the isolated antibody or antigen-binding fragment thereof comprises VH and VL, wherein VH and VL comprise the following amino acid sequences:

[0669] a. SEQ ID NO:31 and 32 respectively;

[0670] b. SEQ ID NO: 33 and 32, respectively;

[0671] c. SEQ ID NO: 34 and 35, respectively;

[0672] d. SEQ ID NO: 36 and 37, respectively;

[0673] e. SEQ ID NO: 38 and 37, respectively; or

[0674] f. SEQ ID NO: 39 and 40, respectively.

[0675] Embodiment 30. 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: 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.

[0676] Embodiment 31. An isolated antibody or antigen-binding fragment thereof according to Embodiment 30, wherein the isolated antibody or antigen-binding fragment thereof comprises a VH having an amino acid sequence of SEQ ID NO: 39 and a VL having an amino acid sequence of SEQ ID NO: 40.

[0677] Embodiment 32. An isolated antibody or antigen-binding fragment thereof according to Embodiment 29, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

[0678] Embodiment 33. An isolated antibody or antigen-binding fragment thereof according to Embodiment 32, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

[0679] Embodiment 34. An isolated antibody or antigen-binding fragment thereof according to Embodiment 29, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

[0680] Embodiment 35. An isolated antibody or antigen-binding fragment thereof according to Embodiment 34, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

[0681] Embodiment 36. An isolated antibody or antigen-binding fragment thereof according to Embodiment 29, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

[0682] Embodiment 37. An isolated antibody or antigen-binding fragment thereof according to Embodiment 36, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

[0683] 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.

[0684] Embodiment 39. An isolated antibody or antigen-binding fragment thereof according to Embodiment 38, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

[0685] 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.

[0686] Embodiment 41. An isolated antibody or antigen-binding fragment thereof according to Embodiment 40, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

[0687] Embodiment 42. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 41, comprising a heavy chain (HC) and a light chain (LC), wherein the heavy chain has 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 the light chain has 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.

[0688] Embodiment 43. An isolated antibody or antigen-binding fragment thereof according to Embodiment 42, wherein the isolated antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 59 and 60.

[0689] Embodiment 44. The isolated antibody according to embodiment 43, comprising

[0690] 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 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 100%) identical to the amino acid sequence of SEQ ID NO: 52;

[0691] 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;

[0692] 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;

[0693] 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;

[0694] 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

[0695] f. 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:59 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:60.

[0696] Embodiment 45. An isolated antibody according to 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.

[0697] Embodiment 46. An isolated antibody according to Embodiment 44, comprising a HC comprising the amino acid sequence of SEQ ID NO:53 and a LC comprising the amino acid sequence of SEQ ID NO:52.

[0698] Embodiment 47. An isolated antibody according to 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.

[0699] Embodiment 48. An isolated antibody according to 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.

[0700] Embodiment 49. An isolated antibody according to 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.

[0701] 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.

[0702] Embodiment 51. An isolated antibody or antigen-binding fragment thereof, comprising:

[0703] a. a HCDR of a VH comprising the amino acid sequence of SEQ ID NO: 33 and a LCDR of a VL comprising the amino acid sequence of SEQ ID NO: 32;

[0704] b. VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively, and VL comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively;

[0705] 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

[0706] d. HC having the amino acid sequence of SEQ ID NO:53 and LC having the amino acid sequence of SEQ ID NO:52.

[0707] Embodiment 52. An isolated antibody or antigen-binding fragment thereof, comprising:

[0708] a. a HCDR of a VH comprising the amino acid sequence of SEQ ID NO: 34 and a LCDR of a VL comprising the amino acid sequence of SEQ ID NO: 35;

[0709] b. VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 13, 14 and 15, respectively, and VL comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively;

[0710] 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.

[0711] Embodiment 53. An isolated antibody or antigen-binding fragment thereof, comprising:

[0712] a. A heavy chain variable region having a VH HCDR comprising the amino acid sequence of SEQ ID NO: 38

[0713] region (VH) and a light chain variable region (VL) having an LCDR of a VL comprising the amino acid sequence of SEQ ID NO: 37;

[0714] b. VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, and VL comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively;

[0715] 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

[0716] d. HC having the amino acid sequence of SEQ ID NO:58 and LC having the amino acid sequence of SEQ ID NO:57.

[0717] Embodiment 54. An isolated antibody or antigen-binding fragment thereof, comprising:

[0718] a. A heavy chain variable region having a VH HCDR comprising the amino acid sequence of SEQ ID NO: 39

[0719] region (VH) and a light chain variable region (VL) having an LCDR of a VL comprising the amino acid sequence of SEQ ID NO: 40;

[0720] b. VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively, and VL comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 28, 29 and 30, respectively;

[0721] 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

[0722] d. HC having the amino acid sequence of SEQ ID NO: 59 and LC having the amino acid sequence of SEQ ID NO: 60.

[0723] Embodiment 55. An isolated antibody or antigen-binding fragment thereof, comprising:

[0724] a. A heavy chain variable region having a VH HCDR comprising the amino acid sequence of SEQ ID NO: 31

[0725] region (VH) and a light chain variable region (VL) having an LCDR of a VL comprising the amino acid sequence of SEQ ID NO: 32;

[0726] 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;

[0727] 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

[0728] d. HC having the amino acid sequence of SEQ ID NO:51 and LC having the amino acid sequence of SEQ ID NO:52.

[0729] Embodiment 56. An isolated antibody or antigen-binding fragment thereof, comprising:

[0730] a. a heavy chain variable region (VH) having a HCDR of VH comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable region (VL) having a LCDR of VL comprising the amino acid sequence of SEQ ID NO: 37;

[0731] b. comprising HCDR1 having the amino acid sequence of SEQ ID NO: 19, 20 and 21, respectively,

[0732] a VH of HCDR2 and HCDR3 and a VL comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively;

[0733] 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

[0734] d. HC having the amino acid sequence of SEQ ID NO:56 and LC having the amino acid sequence of SEQ ID NO:57.

[0735] Embodiment 57. An isolated antigen-binding fragment according to any one of Embodiments 1 to 56, wherein the antigen-binding fragment is a scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, VHH or a single-chain antibody.

[0736] 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.

[0737] Embodiment 59. The isolated antibody or antigen-binding fragment thereof according to Embodiment 58, wherein the antibody or antigen-binding fragment thereof is of the IgG1 isotype.

[0738] Embodiment 60. An isolated antibody or antigen-binding fragment thereof according to Embodiment 59, wherein the isolated antibody or antigen-binding fragment thereof comprises an Ig constant region or a fragment of the Ig constant region.

[0739] Embodiment 61. An isolated antibody or antigen-binding fragment thereof according to Embodiment 60, wherein said Ig constant region or said fragment of said Ig constant region comprises at least one mutation that results in reduced binding of said antibody or antigen-binding fragment thereof to an Fcγ receptor (FcγR).

[0740] Embodiment 62. An isolated antibody or antigen-binding fragment thereof according to Embodiment 61, wherein the at least one mutation that results in reduced binding of the antibody or antigen-binding fragment thereof to 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.

[0741] Embodiment 63. An isolated antibody or antigen-binding fragment thereof according to Embodiment 62, wherein the Ig constant region or the fragment of the constant region comprises the mutation of L234A_L235A_D265S.

[0742] Embodiment 64. The isolated antibody or antigen-binding fragment thereof according to Embodiment 62, wherein the FcγR is FcγRI, FcγRIIA, FcγRIIB or FcγRIII, or any combination thereof.

[0743] Embodiment 65. An isolated antibody or antigen-binding fragment thereof according to Embodiment 64, wherein said Ig constant region or said fragment of said Ig constant region further comprises at least one mutation that modulates the half-life of said antibody or antigen-binding fragment thereof.

[0744] Embodiment 66. An isolated antibody or antigen-binding fragment thereof according to Embodiment 65, wherein the 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, wherein the residues are numbered according to the EU index.

[0745] Embodiment 67. An immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 66 conjugated to a therapeutic agent or an imaging agent.

[0746] Embodiment 68. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 66 or the immunoconjugate according to Embodiment 67 and a pharmaceutically acceptable carrier.

[0747] Embodiment 69. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 66.

[0748] Embodiment 70. An isolated polynucleotide according to embodiment 69, comprising a polynucleotide sequence that is at least 80%, such as 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.

[0749] Embodiment 71. A vector comprising the polynucleotide according to embodiment 69 or 70.

[0750] Embodiment 72. A host cell expressing the isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 70, such as a host cell comprising the vector according to embodiment 71.

[0751] Embodiment 73. 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 antigenic fragment thereof according to any one of embodiments 1 to 66, the immunoconjugate according to embodiment 67, the pharmaceutical composition according to embodiment 68, the isolated polynucleotide according to embodiment 69 or 70, the vector according to embodiment 71, or the host cell according to embodiment 72, thereby preventing VEGFA from binding to the VEGFR1.

[0752] Embodiment 74. The method of embodiment 73, wherein the subject is in need of treatment for chronic kidney disease (CKD), is in need of reduction in proteinuria, has advanced stage 4 or stage 5 chronic kidney disease, or has CKD associated with proteinuria, albuminuria, or diabetes.

[0753] Embodiment 75. A method of treating chronic kidney disease (CKD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated antibody or antigenic 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, for a time sufficient to treat the CKD.

[0754] Embodiment 76. A method according to embodiment 75, wherein the subject has advanced stage 4 or stage 5 chronic kidney disease.

[0755] Embodiment 77. A method according to Embodiment 75, wherein the subject has stage 3 chronic kidney disease.

[0756] Embodiment 78. The method of embodiment 75, wherein the subject has CKD with proteinuria, albuminuria or diabetes.

[0757] Embodiment 79. A method of reducing proteinuria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated antibody or antigenic fragment thereof according to any one of embodiments 1 to 66, the immunoconjugate according to embodiment 67, the pharmaceutical composition according to embodiment 68, the isolated polynucleotide according to embodiment 69 or 70, the vector according to embodiment 71, or the host cell according to embodiment 72, for a period of time sufficient to reduce proteinuria in the subject.

[0758] Embodiment 80. A method of reducing albuminuria in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated antibody or antigenic fragment thereof according to any one of embodiments 1 to 66, the immunoconjugate according to embodiment 67, the pharmaceutical composition according to embodiment 68, the isolated polynucleotide according to embodiment 69 or 70, the vector according to embodiment 71, or the host cell according to embodiment 72, for a period of time sufficient to reduce proteinuria in the subject.

[0759] 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.

[0760] Example

[0761] Example 1. Antigen Generation

[0762] Vascular endothelial growth factor receptor 1 (VEGFR1) is a member of the V-type subfamily of receptor tyrosine kinases (RTKs) that consists of seven extracellular immunoglobulin (Ig)-like domains, a single transmembrane domain, and a cytoplasmic kinase domain. Previous structural studies have shown that ligand binding to VEGFR1 is mediated by Ig domains 2 and 3 (D2 and D3), which contribute to the binding interface, respectively. and (Markovic-Mueller, S. et al., Structure, 25(2), 341-352, 2017). This was confirmed by binding analysis, where 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 <45pM and <30pM, respectively. Domain 2 of hVEGFR1 alone exhibited a significantly lower affinity of 22nM (approximately 1,000-fold reduction in affinity), which is consistent with previously published biochemical data showing that D2 of VEGFR1 alone (Flt-1(2)-IgG) exhibited significantly reduced affinity for VEGF165 (Davis-Smyth, T. et al., EMBO Journal, 15(18), 4919-4927, 1996).

[0763] Therefore, to identify antibodies capable of blocking ligand binding to VEGFR1, truncated forms of the VEGFR1 extracellular domain (ECD) containing only D2-D3 or D1-D3 were used as primary immunogens during antibody discovery. An Fc chimera of the second and third immunoglobulin (Ig)-like domains of human VEGFR1 (hVEGFR1 D2–D3-Fc-6xHis) was generated by fusing amino acid residues 130-331 (UniProt Accession No. P17948) to residues 100-330 of human immunoglobulin heavy chain constant gamma 1 (UniProt Accession No. P01857) via the HRV3C protease recognition sequence 'LEVLFQGP' (SEQ ID NO: 174). A 6X His-tag was added to the C-terminus of the construct for flexible purification options (construct VGFW8; SEQ ID NO: 1). In addition, an Fc chimera of the first to third immunoglobulin (Ig)-like domains of murine VEGFR1 (mVEGFR1 D1–D3-Fc-6xHis) was generated by linking residues 23–332 (UniProt Accession No. P35969) to residues 100–330 of human immunoglobulin heavy chain constant γ1 (UniProt Accession No. P01857) via the HRV3C protease recognition sequence ‘LEVLFQGP’ (SEQ ID NO: 174).

[0764] 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). For flexibility in purification strategies, a 6X His-tag was also added to the C-terminus of the construct (construct VGFW9; SEQ ID NO: 2). Additional constructs were designed in which part or all of the VEGFR1 extracellular domain (ECD) from humans, mice or cynomolgus monkeys (Macaca fascicularis) was appended with an Avi-tag and a 6x His-tag sequence at the C-terminus. These include: Ig-like domain 2 of human VEGFR1 (residues 130-225 of Uniprot Accession No. P17948; construct VGFW2; SEQ ID NO: 3), Ig-like domains 2 and 3 of human VEGFR1 (residues 130-331 of Uniprot Accession No. P17948; construct VGFW3; SEQ ID NO: 4), Ig-like domains 1 to 3 of murine VEGFR1 (residues 23-332 of Uniprot Accession No. P35969; construct VGFW4; SEQ ID NO: 5), and the entire extracellular domain of monkey VEGFR1 (residues 27-758 of Accession No. XP_005585612.1; construct VGFW5; SEQ ID NO: 6).

[0765] VEGFR1 ECD expression constructs were transiently transfected into HEK 293-6E cells using polyethyleneimine. Cells were cultured in 20 L batch-fed Wave bags (Culti bags) [Sartorius, RM 20L optical catalog number DFO020L] was incubated at 37 ° C and 5% CO2 for six days and then harvested when the viability of the culture dropped to <80%. Cells were removed by centrifugation and immobilized metal affinity chromatography (IMAC) was used, using Ni Sepharose 6Fast Flow resin (GE Healthcare), and then preparative size exclusion chromatography (SEC) was performed using a Superdex200 column (GE Healthcare) balanced in Dubelcco phosphate buffer (1x DPBS) at pH 7.2, purifying soluble VEGFR1 proteins with His tags from culture medium. Alternatively, for VGFW8 and VGFW9, recombinant proteins were captured from the spent supernatant by being applied to MabSelectSuRe protein A chromatographic resin (GE Healthcare) and eluted therefrom. The amino acid sequence of the antigen generated is shown in Table 2.

[0766] Table 2.

[0767]

[0768]

[0769]

[0770] Example 2. Generation and preliminary characterization of VEGFR1 antibodies in scFv-Fc format

[0771] Immunization strategies

[0772] There is a strong preference for identifying ligand-blocking anti-VEGFR1 antibodies that exhibit broad cross-reactivity in a variety of species (in addition to humans), including cynomolgus monkeys and rodents, to enable nonclinical 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 onset of discovery activities eliminates the need to develop surrogate antibodies that, given the complexity of the biology, may not mimic the behavior of the antibody of interest.

[0773] Obtaining antibodies with broad species cross-reactivity, including rodents, can represent a stringent design requirement. Indeed, previously reported anti-VEGFR1 antibodies are either strictly mouse-specific or strictly human-specific and display poor to no species cross-reactivity. Although VGFB80 is a potent human VEGFR1 binder, VGFB80, used herein as a reference, does not exhibit binding to rodent-derived VEGFR1. It is well known that the generation of broadly cross-reactive antibodies is often hampered by immune tolerance to epitopes that are conserved in mammalian species commonly used for antibody discovery, such as mouse, rat, and rabbit. The conservation of the ligand-binding domain of VEGFR1 in typical antibody discovery species (i.e., mouse and rat) is due to the fact that the ligand-binding domain of VEGFR1 is conserved in rodent-derived VEGFR1. Figure 1 The amino acid sequences of the VEGFR1 domains 2 and 3 are shown in Table 3. Sequence identities of approximately 80% and 67% were observed between human and mouse / rat for VEGFR1 domains 2 and 3, respectively.

[0774] Table 3. Summary of VEGFR1 sequence identities for domain 2 or domains 2 and 3 between human, cynomolgus monkey, mouse, rat, rabbit or chicken. 2021.1.1 Calculate the identity percentage through alignment and analysis.

[0775]

[0776]

[0777] Alternatively, immunization of phylogenetically distant species, such as chicken, can sometimes overcome immune tolerance to "pan-mammalian" epitopes (Ching, KH et al., MAbs, 10(1), 71–80, 2018). Transgenic Omnichicken, which is phylogenetically more distant from humans than mice and rats, exhibits a robust immune response to conserved human proteins. However, the use of Omnichicken alone does not guarantee high-affinity species cross-reactive antibodies.

[0778] The high affinity and species cross-reactive anti-VEGFR1 antibodies described herein were generated using a unique antibody generation strategy 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 schedule, and (4) an elaborate serum titer screening process that identifies anti-VEGFR1 antibodies with broad species reactivity. Transgenic chickens (n=12) with 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 hVEGFR1 D2–D3-Fc-6xHis (VGFW8) or with alternating boosts of VGFW8 and mVEGFR1 D1–D3-Fc-6xHis (VGFW9). Serum titers of birds were monitored by ELISA using recombinant proteins purified from VGFW2 (hVEGFR1_D2-Avi-His), VGFW3 (hVEGFR1_D2-3 Avi His), VGFW4 (mVEGFR1_D1-3_Avi-6xHis) and VGFW5 (cynomolgus monkey VEGFR1_ECD_Avi-6xHis). Spleens were harvested from birds with the best titers for human and mouse VEGFR1 recombinant proteins. Lymphocytes were isolated from the spleen and interrogated for positive binding to two beads coated with VGFW4 and VGFW3 and / or VGFW2. Single cell reverse transcriptase PCR was performed on lymphocytes to recover the v gene sequence. VH and VL sequences were assembled into 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 expressions were screened for the presence of recombinant antibodies specific for VEGFR1 by ELISA using purified antigen. Reconfirmed positive clones were expanded into 6-well plates (2 mL) and serial dilutions of the supernatants were used to repeat the ELISA and FACS analysis to confirm binding to human, cynomolgus monkey, and mouse VEGFR1—both soluble purified recombinant proteins of the extracellular domain and full-length cell surface-bound receptors on HEK cells engineered to overexpress human, cynomolgus monkey, or mouse VEGFR1. In addition, amplified clones were counter-screened on HEK and K562 cells engineered to overexpress hVEGFR2 and hVEGFR3, respectively ( Figure 2 ).

[0779] A subset of clones that showed specific binding to human, cynomolgus monkey, and mouse VEGFR1 by ELISA and FACS were sequenced and reconstituted into human monoclonal IgG1 (L234A / L235A / D265S) molecules (2H:2L) to eliminate any Fc receptor effector function while retaining FcRn affinity. Of the approximately 800 lymphocytes initially interrogated, 43 unique molecules that met the above criteria were generated (5.6%). The remaining clones generally either lacked cross-reactivity with murine VEGFR1 or did not show specific binding to VEGFR1 (i.e., also bound to human VEGFR2 or human VEGFR3 on cells). In contrast, more conventional rodent immunization campaigns were performed in parallel (n=42 immunized animals), and of the approximately 2,200 MSD primary hits screened on cells, only 4 clones showed broad species cross-reactivity to Hu / Cy / Mo VEGFR1 (0.18% hit rate), and these molecules ultimately proved to be less effective in blocking ligand binding or to have undesirable sequence preferences.

[0780] 43 unique Omnichicken-derived clones of recombinant 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 (biochemical and cell-based), (3) thermal stability, (4) surface hydrophobicity, (5) nonspecific binding, and (6) lack of modification after stress / forced degradation studies. 28 of the 43 molecules (approximately 65%) were identified as potent ligand blockers representing 10-11 sequence families. Eight anti-VEGFR1 mAbs were evaluated in a single-dose PK-TE-PD study in db / db mice. VGFB54, VGFB71, VGFB78, and VGFB82 were evaluated in a single-dose PK-TE study in cynomolgus monkeys and subjected to extensive intrinsic properties and structural characterization.

[0781] Design of VGFB883 and VGFB400

[0782] VGFB54 was observed to contain an 'NG' deamidation sequence in the heavy chain CDR2, and it was observed by peptide mapping that it underwent significant deamidation (14.7% modification) when maintained at pH 8.5 (40°C) for one week. To address this risk, a small variant library was generated in which N54 or G55 was substituted with every possible alternative amino acid (except Cys or Trp). The variant library was then evaluated for retained binding to the target in biochemical ligand blocking assays and cell binding assays. Almost all variants exhibited retained or slightly improved binding to VEGFR1. The N54Q variant of VGFB54 (VGFB883) was selected for further development because it is one of the most chemically conservative amino acid substitutions, addresses the risk of post-translational modifications, and exhibits retained binding to the target (Table 4).

[0783] Table 4. Ligand Blockade and Cell Binding of VGFB883

[0784] Antibody Ligand blocking, IC50(M) Cell binding, IC50(M) VGFB54 <![CDATA[8.94095E -11 ]]> <![CDATA[1.97903E -10 ]]> VGFB883 <![CDATA[4.40442E -11 ]]> <![CDATA[6.54912E -11 ]]>

[0785] Antibody sequences obtained from transgenic animals may contain somatic hypermutations in the framework and CDR regions. Somatic hypermutations may result in abnormal or low-frequency residues in the human framework regions and affect the stability and immunogenicity of biotherapeutics. The parent anti-VEGFR1 antibody VGFB78 heavy chain variable region contains somatic hypermutations at position 110 (105 Chothia depiction) within the framework region. The leucine residue at position 110 (105 Chothia) is typically a low-frequency residue (frequency less than 1%) compared to the most common glutamine (Q) residue (frequency 76%) typically found at this position ( Figure 3 ). Therefore, the L11Q mutation was engineered in the heavy chain of VGFB78, resulting in VGFB400, which represents a reversal of germline identity at this position. This substitution was chosen to reflect the most common identity at this position.

[0786] ExpiCHO small-scale transfection and purification

[0787] The antibodies identified from the immunization campaign were cloned and expressed as IgG1-AAS (L234A / L235A / D265S) at 2 ml scale and purified. TM Cells (ThermoFisher Scientific) were cultured in an ExpiCHO incubator at 37°C, 7% CO2. TM When the density reaches 4×10 with 98%-99% viability, 6 to 6×10 6The cells were subcultured when the cells reached logarithmic growth phase with viable cells / mL. On the day of transfection, the viable cell density and percent viability were determined. The transfection protocol (ThermoFisher ExpiCHO Expression System protocol for 24- and 96-well plates and small bioreactor tubes) was followed by 6 × 10 6 Cells were transfected at a density of 10 viable cells / mL. On day 7 after transfection, culture supernatants were harvested by centrifugation at 850 × g for 15 minutes before purification. Antibodies were purified from the clarified supernatant using MabSelect Sure resin (GE Healthcare) and dialyzed into PBS. Protein concentration was determined by measuring the absorbance of the filtrate at 280 nm using a DropSense instrument (Trinean NV / SA).

[0788] Sequences of anti-VEGFR1 antibodies

[0789] Table 5 shows the Kabat HCDR1, HCDR2 and HCDR3 of the selected anti-VEGFR1 selected antibodies. Table 6 shows the Kabat LCDR1, LCDR2 and LCDR3 of the selected anti-VEGFR1 antibodies. Table 7 shows the Chothia HCDR1, HCDR2 and HCDR3 of the selected anti-VEGFR1 antibodies. Table 8 shows the Chothia LCDR1, LCDR2 and LCDR3 of the selected anti-VEGFR1 antibodies. Table 9 shows the ABM HCDR1, HCDR2 and HCDR3 of the selected anti-VEGFR1 antibodies. Table 10 shows the ABM LCDR1, LCDR2 and LCDR3 of the selected anti-VEGFR1 antibodies. Table 11 shows the IMTG HCDR1, HCDR2 and HCDR3 of the selected anti-VEGFR1 antibodies. Table 12 shows the IMTG LCDR1, LCDR2 and LCDR3 of the selected anti-VEGFR1 antibodies. Table 13 shows the VH and VL amino acid sequences of the 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.

[0790] Table 16 shows the HC amino acid sequences of the selected anti-VEGFR1 antibodies. Table 17 shows the LC amino acid sequences of the selected anti-VEGFR1 antibodies. Table 18 shows the HC nucleotide sequences of the selected anti-VEGFR1 antibodies. Table 19 shows the LC nucleotide sequences of the selected anti-VEGFR1 antibodies. Table 20 summarizes the SEQ ID NOs assigned to the selected anti-VEGFR1 antibodies.

[0791] Table 5. HCDRs of selected anti-VEGFR1 antibodies using Kabat delineation

[0792]

[0793] Table 6. LCDR of selected anti-VEGFR1 antibodies using Kabat profiling

[0794]

[0795] Table 7. HCDRs of selected anti-VEGFR1 antibodies profiled using Chothia

[0796]

[0797]

[0798] Table 8. LCDR of selected anti-VEGFR1 antibodies profiled using Chothia

[0799]

[0800] Table 9. HCDRs of selected anti-VEGFR1 antibodies profiled using ABM

[0801]

[0802] Table 10. LCDR of selected anti-VEGFR1 antibodies profiled using ABM

[0803]

[0804] Table 11. HCDRs of selected anti-VEGFR1 antibodies profiled using IMTG

[0805]

[0806] Table 12. LCDR of selected anti-VEGFR1 antibodies profiled using IMTG

[0807]

[0808] NA = Not applicable

[0809] Table 13. VH and VL amino acid sequences of selected anti-VEGF1 antibodies

[0810]

[0811]

[0812] Table 14. VH nucleic acid sequences of selected anti-VEGFR1 antibodies

[0813]

[0814]

[0815]

[0816] Table 15. VL nucleic acid sequences of selected anti-VEGFR1 antibodies

[0817]

[0818]

[0819] Table 16. HC amino acid sequences of selected anti-VEGFR1 antibodies

[0820]

[0821]

[0822]

[0823] Table 17. LC amino acid sequences of selected anti-VEGFR1 antibodies

[0824]

[0825]

[0826] Table 18. HC nucleotide sequences of selected anti-VEGFR1 antibodies 。

[0827]

[0828]

[0829]

[0830]

[0831]

[0832] Table 19. LC nucleotide sequences of selected anti-VEGFR1 antibodies 。

[0833]

[0834]

[0835] Table 20. Summary of anti-VEGFR1 antibody SEQ ID NOs

[0836]

[0837] Example 3. Biophysical Characterization and In Vitro Activity Distribution of Anti-VEGFR1 Antibodies

[0838] Confirmation of binding to cells overexpressing VEGFR1

[0839] 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 no staining, CSFE, CTV, or CTV+CSFE for 10 minutes at 4°C. The 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 cells per well of each cell type). Antibody samples (5 μL / well) were added to a final concentration of 10 nM and incubated at 4°C for 60 min in the dark. The cells were washed twice with 150 μL / well staining buffer. AlexaFluor647-conjugated anti-human IgG1 secondary antibody (100 μL / well) was added and incubated at 4°C for 30 minutes in the dark. The cells were washed twice with 150 μL staining buffer, finally resuspended in 30 μL running buffer, and read on an iQue flow cytometer. This single concentration assay was used to quickly confirm binding to human and mouse VEGFR1 on cells. Similar assays were performed to confirm binding of candidate antibodies to HEK293 cells that stably overexpressed cynomolgus monkey VEGFR1 at a single concentration of 10 nM. In order to demonstrate specificity for VEGFR1, similar counter-screening assays were also performed to confirm that anti-VEGFR1 antibodies lacked binding to HEK293 cells that stably expressed human VEGFR2 or K562 cells that stably expressed human VEGFR3 at a single high concentration (10 nM) (Table 21).

[0840] Table 21. Binding to VEGFR1 / 2 / 3 on cells: cross-reactivity and specificity .

[0841] The positive threshold for CyVEGFR1 is S / B>3

[0842] ND = Not tested Goat anti-human VEGFR2 = R&D Systems Cat. No. AF357

[0843] Goat anti-human VEGFR2 = R&D Systems Cat. No. AF349

[0844] A full dose-response curve of the 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 was stained with CSFE as described above, and parental CHO-K1 cells remained unstained. The cells were washed and combined (approximately 0.5×10 6Cells were plated at approximately 40,000 cells per well (75 μL / well) at 40 μL / well. Serially diluted antibody samples were then added to the cells and incubated at 37°C for 1 hour. The cells were washed twice with staining buffer and then stained with AF647-conjugated secondary antibody for 30 min at 4°C. The plated cells were washed twice and then read with Sytox Blue (1:1000) in read buffer and read on an iQue flow cytometer. The data were fitted to obtain the EC 50 Values ​​(Table 22).

[0845] Table 22. Binding of anti-VEGFR1 antibodies to cells expressing human VEGFR1 .

[0846]

[0847]

[0848] Ligand Blockade: Biochemical and Cell-Based

[0849] The ability of anti-VEGFR1 antibodies to prevent binding of VEGFA or placental growth factor (PlGF) ligand to VEGFR1 was determined in a biochemical electrochemiluminescence (ECL) assay and a cell-based assay format.

[0850] Briefly, MSD MA6000 384 plates were coated with 0.5 μg / ml protein A / G (Thermo Fisher Scientific) or anti-His mAb (R&D) in TBS in a cold room for 16 hours. The plates were washed twice with 1×TBS and blocked with 35 μl / well of blocking agent A for 1 hour at room temperature. Recombinant human or mouse VEGFR1-Fc protein (R&D Systems) or His-tagged cynomolgus monkey VEGFR1 was added to the plates at 0.5 μg / ml and incubated for 1 hour at room temperature. Unbound VEGFR1 protein was washed away using 70 μl / well 1×MSD ​​Tris wash buffer. Unlabeled VEGFR1 ligands or anti-VEGFR1 antibodies in half-log serial dilutions were pre-incubated with bound VEGFR1 at room temperature for 30 minutes, followed by the addition of diluted biotinylated human VEGFA165 (0.2 nM), mouse VEGFA164 (0.2 nM) or PLGF (2 nM) for binding. It is noteworthy that the amino acid sequences of cynomolgus monkey and human VEGFA and PlGF are identical, and biotinylated human VEGF-A and PlGF are used as ligands for cynomolgus monkey VEGFR1 competition assays. The bound ligands were detected by adding 1 μg / ml sulfo-labeled streptavidin. Signals were generated and detected on an MSD imager, SECTOR S 600 (Meso Scale CDiagnostics, Serial No. 1201180626620), in the presence of 35 μl / well 2×MSD read buffer T. ECL readings were used to calculate % binding, IC50, and % maximum inhibition.

[0851] % binding = (((sample - mean (background)) ÷ (mean (total binding) - mean (background))) × 100

[0853] ○ Total binding = biotin ligand only

[0854] ○ Background = no blocker and no biotin ligand

[0855] • Curves were fitted using nonlinear regression log(inhibitor) vs. response - variable slope (four parameters).

[0856] ○Formula: Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0858] ○ IC50 extracted from Prism 8 software.

[0859] Maximum inhibition % = 100 - average (% binding at the highest concentration)

[0860] Tables 23 and 24 summarize the in vitro efficacy (maximum inhibition %) and potency (IC50) of VEGFR1 mAbs in replacing VEGFA / PlGF and binding to human, cynomolgus monkey, and mouse VEGFR1. Anti-VEGFR1 antibodies showed effective ligand blocking activity when binding to human and cynomolgus monkey VEGFR1, with a maximum inhibition rate of about 100%. The IC50 value is in the subnanomolar range, which is comparable to its ligand. Although VGFB80 has no binding or ligand competition to mouse VEGFR1, other VEGFR1 mAbs showed high blocking efficacy and potency to mouse VEGFR1 in this assay.

[0861] Table 23. Competition of VEGFA binding to human, cynomolgus monkey and mouse VEGFR1

[0862]

[0863] Table 24. Binding competition of PlGF with human, cynomolgus monkey and mouse VEGFR1

[0864]

[0865] Ligand blocking on cells was determined as follows: Cells stained with Cell Trace Violet CSFE (ThermoFisherScientific) were plated at 15,000 cells / well (30 μL / well; parental CHO or CHO cells engineered to overexpress human VEGFR1) in FACS staining buffer. An equal volume of serially diluted test antibodies was applied to the cells and incubated for one hour at 4°C. The cells were washed twice with 200 μL / well of staining buffer, and then 100 μL / well of biotinylated VEGF A ligand (500 pM) was added. The cells were incubated for another hour at 4°C, and then 50 μL / well of AF647-conjugated streptavidin (3 nM) was added. The plate was incubated at 4°C in the dark for 30 min, washed twice with 200 μL / well of staining buffer, collected by centrifugation, resuspended in running buffer containing Sytox Blue live / dead stain, and read on an Intellicyt iQue Plus flow cytometer. Data were fitted to obtain IC 50 The percentage of ligand blocking was calculated for each test antibody at each concentration and the IC50 value was determined (Table 25).

[0866] Table 25. Effects of VEGFA ligands on cells overexpressing human VEGFR1 (CHOK1) or cynomolgus monkey VEGFR1 (HEK) Blocking .

[0867]

[0868] Thermal stability determination

[0869] The thermal stability of each antibody was determined by the NanoDSF method using an automated Prometheus instrument from Nanotemper Technologies. The sample was loaded from a 384-well sample plate into a 24-well capillary at a concentration of 0.25 mg / mL for measurement. Each sample was run repeatedly. The Prometheus NanoDSF user interface (melting scan tab) was used to set the experimental parameters for the run. The thermal scan range for a typical IgG sample was 20°C to 95°C at a rate of 1.0°C / minute. NIST mAb (and / or CNTO3930, CNTO5825) was included as a control and run repeatedly. The intrinsic fluorescence of the molecule at 330nm and 350nm was used to monitor the development during the temperature ramp and was recorded as the change in fluorescence intensity over time. T 开始 is the temperature at which expansion begins.

[0870] Table 26. Thermal stability of anti-VEGFR1 antibodies measured by NanoDSF

[0871] Antibody <![CDATA[T 开始 (℃)]]> <![CDATA[T agg (℃)]]> Tm1(℃) Tm2(℃) VGFB54 63.99 72.27 71.75 - VGFB71 62.74 71.81 67.85 - VGFB78 62.57 68.48 67.86 - VGFB82 61.20 73.15 71.32 77.32

[0872] The thermal stability of the selected candidates was also determined by differential scanning calorimetry (DSC). A MicroCal VP-capillary DSC instrument was used. The samples were dialyzed into 1X DPBS or other suitable buffer overnight. Thermal scanning was performed by increasing the temperature of the samples and reference cells from 25°C to 95°C at a rate of 60°C / hour. The thermograms were fitted using "non-binary" parameters. The Tm1 of all samples can represent Fab development, ranging from 68°C to 73°C. Tm2 and Tm3 are related to variant IgG1 Fc development.

[0873] Table 27. Thermal stability of anti-VEGFR1 antibodies measured by DSC

[0874]

[0875] Kinetic exclusion assay to determine affinity

[0876] Kinetic exclusion assay (KEA; KinExA) is used to analyze the binding of anti-VEGFR1 antibodies to human, mouse and cynomolgus monkey VEGFR1. KEA is a fluorescence-based solution method and is described in detail in the literature (Darling and Brault, 2004). In order to measure the affinity of the interaction, a series of solutions with a fixed concentration of an interactor (A) and different concentrations of another interactor (B) are prepared and allowed to reach equilibrium. After equilibrium, the concentration of free A is determined by passing the balanced mixture through a column filled with beads modified with reactant B. Free A is bound to the beads and then detected with a fluorescently labeled polyclonal antibody. The sample is loaded into the column at a fast flow rate to achieve a short contact time between the mixture and the beads to ensure that the equilibrium is not destroyed (shifted), that is, and B does not dissociate from the AB complex (kinetic exclusion).

[0877] In brief, fixed concentrations (300pM, 60pM, 12pM, 2.4pM or 0.5pM) of Ab were used to prepare serial dilutions of antigen (2nM to 0.52pM, or 800pM to 5.37fM). Complex titration was incubated at room temperature to reach binding equilibrium for at least 2 days. After incubation, samples were run on KinExA3200 or KinExA4000 instruments to assess free antibodies in the reaction. During these studies, some data were rejected because the experiment failed or did not meet the acceptance criteria. For mouse VEGFR1, 60pM and 300pM concentrations were rejected due to NSBs associated with high titrants. The dissociation equilibrium constant (K) was determined using the n-curve analysis feature of KinExA Pro software. D The program uses a nonlinear least squares regression analysis of the global fit data to a 1:1 binding model (CI = confidence interval).

[0878] Table 28. Binding affinity of anti-VEGFR1 antibodies to human, mouse and cynomolgus monkey VEGFR1

[0879]

[0880] a Mouse VEGFR1 specific antibody. Does not bind to human, cynomolgus monkey or rat VEGFR1

[0881] HDX epitope mapping of VEGFR against five mAbs

[0882] The epitopes of VEGFR1 for five mAbs (VGFB54, VGFB71, VGFB78, VGFB80 and VGFB82) were mapped by HDX-MS. VGFB80 is a reference molecule not found in the antibody generation work described herein. Fragments that reduce the free energy by more than 2 kcal / mol when bound to mAb are "strong" epitopes defined by HDX-MS. Fragments that reduce the free energy by 1 kcal / mol to 2 kcal / mol when bound to mAb are considered "weak" epitopes. The epitopes of VEGFR1 for all tested mAbs are very similar. The strong epitopes of VEGFR1 for VGFB54 are fragments 172-177 (FPLDTL, SEQ ID NO: 143) and 201-204 (EIGL, SEQ ID NO: 144). The strong epitopes of VEGFR1 against VGFB71 are fragments 172-177 (FPLDTL, SEQ ID NO: 143), 200 (K) and 201-204 (EIGL, SEQ ID NO: 144). The strong epitopes of VEGFR1 against VGFB78 are fragments 172-177 (FPLDTL, SEQ ID NO: 143), 200 (K) and 201-204 (EIGL, SEQ ID NO: 144). The strong epitope of VEGFR1 against VGFB82 is fragment 201-204 (EIGL, SEQ ID NO: 144). After quenching with 0.4 mM FC-14, 8 M urea, 1 M TCEP, pH 3.0, the sequence coverage of VGFW1 was 88% (= 285 / 326) when digested with a pepsin / FPXIII mixed bed column. Among the five potential glycosylation sites, N196 was covered by a non-glycopeptide, while the other four sites were not covered.

[0883] Table 29. Free energy changes (ΔΔG) upon binding to anti-VEGFR1 mAbs (kcal / mol) .

[0884] Starting residue 141 147 172 178 200 201 211 218 terminal residues 147 153 177 199 200 204 215 228 VGFB71 -0.3 -1.2 -4.2 0 -2.8 -2.8 -0.1 -0.3 VGFB78 -0.3 -0.6 -2.7 -1.1 -2.9 -3.6 0.1 -0.2 VGFB54 -0.2 -0.4 -2.6 -0.3 -1.4 -3.6 -0.1 -0.7 VGFB82 -0.6 -0.1 -0.6 -1 -0.5 -3.3 -0.1 -0.3

[0885] HDX paratope mapping of four mAbs targeting VEGFR1

[0886] Paratope mapping of four mAbs against VEGFR1 by HDX-MS ( Figure 4A and Figure 4BFragments that decrease the free energy by more than 1 kcal / mol upon mAb binding are epitopes defined by HDX-MS. The paratopes of VGFB54 against VEGFR1 are heavy chain fragments 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 fragments 1-32 (QSVLTQPPSASGTPGQRVTISCSGSRSNIGNN, LCDR1, SEQ ID NO:151) and 51-74 (LNSQRPSGVPDRFSGSKSGTSASL, LCDR2, SEQ ID NO:152). The paratopes of VGFB71 against VEGFR1 are heavy chain fragments 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 fragments 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 paratope of VGFB78 against VEGFR1 is the light chain fragment 50-73 (YYD-QRPSGVPDRFSGSKSGTSASL, LCDR2, SEQ ID NO: 160).The paratopes of VGFB82 against VEGFR1 are heavy chain fragments 7-18 (SGGGLVQPGGSL, SEQ ID NO: 161), 23-28 (EASGFD, HCDR1, SEQ ID NO: 162), 31-33 (TYA, HCDR1), 38-46 (RQAPGKGLE, SEQ ID NO: 163), 51-68 (INSEGTITSHAPAVKGRFT, HCDR2, SEQ ID NO: 164) and 96-116 (CSSTTGTTHGMDVWG, HCDR3, SEQ ID NO: 165) and light chain fragments 1-34 (QSVLTQPPSASGTPGQRVTISCSGSSSNIQNNYV, LCDR1, SEQ ID NO: 166), 50-51 (YF, LCDR2), 52-74 (NSQRPSGVPDRFSGSKSGTSASL, LCDR2, SEQ ID NO:167) and 95-100 (SLNGWV, LCDR3, SEQ ID NO:168).

[0887] Tyrosine kinase activation assay

[0888] Anti-VEGFR1 antibodies were tested to confirm their inability to activate VEGFR1 signaling. Due to the inherently weak signaling activity of VEGFR1, a previously described cell-based bioassay was used ( T. et al. EMBO Journal, 20(17), 4762–4773, 2001). Ba / F3 cells stably expressing chimeric receptors in which the extracellular domain of human VEGFR1 (residues 1-758; Uniprot Accession No. P17948) was fused to the transmembrane and intracellular domains of the mouse erythropoietin receptor (EpoR; (residues 250-507; Uniprot Accession No. P14753)) were generated 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 seeded in medium lacking mouse IL-3 (RPMI+10% FBS+0.5 μg / ml puromycin). Mouse IL-3 was added to control wells to a final concentration of 10 ng / mL. Other wells received anti-VEGFR1 antibodies (final concentration 10nM or 50nM), hVEGFA165 (5nM; R&D Systems) or basal medium (without mIL-3). The cells were incubated at 37°C, 5% CO2 for 72 hours. Proliferation was measured by adding 50μL / well CellTiter Glo (Promega) and incubating for 10 minutes at room temperature. Finally, the plate was read on the Envision 2105 plate reader using an ultrasensitive luminescence protocol. The proliferation of BaF3\hVEGFR1-mEpoR cells was compared with mIL-3-dependent conditions. Unlike hVEGFA (5nM), which was shown to rescue the viability of cells in the absence of mIL-3, the anti-VEGFR1 antibodies tested could not promote IL-3-independent growth at any tested concentration (Table 30). This result is consistent with the inability of anti-VEGFR1 antibodies to stimulate tyrosine kinase activation or further downstream signaling.

[0889] Table 30. Anti-VEGFR1 Antibodies Activate Tyrosine Kinases

[0890]

[0891] Specific cell surface group

[0892] One risk associated with broadly species cross-reactive antibodies is increased off-target propensity. Cellular microarray technology (Retrogenix Ltd.) was performed to confirm the specificity of the anti-VEGFR1 antibodies for VEGFR1 and the absence of any "off-target" propensity.

[0893] This human cell microarray method has been previously described in the literature (Freeth, J. et al., SLAS Discovery, 25 (2), 223–230, 2020). A preliminary screen was performed on a slide arrayed with fixed HEK293 cells, where each spot of the HEK293 cells expressed a unique protein from the cell surface group, a cell surface-bound human secretory protein, or a heterodimeric human cell surface receptor. In this preliminary screen, a total of 5,528 unique gene products (4,103 human plasma membrane proteins (+ some unbound secretory proteins) + 1,425 cell surface-bound human secretory proteins) appeared. In brief, a slide containing fixed transfected HEK293 cells was covered with a test antibody solution (2 μg / mL), incubated and washed. Binding was assessed using AlexaFluor647-conjugated anti-human IgG Fc detection antibody and fluorescence quantification on ImageQuant. As expected, all tested anti-VEGFR1 molecules show binding to isoforms of human VEGFR1 (FLT1 isoforms 1, 2 and 3) (Tables 31-34). VGFB54, VGFB80 and VGFB82 do not show binding to any other surface histones captured by this screening. Two anti-VEGFR1 antibodies (VGFB71 and VGFB78) show unexpected binding to unrelated cell surface proteins (Table 32) Nectin1, FGFR2 (fibroblast growth factor receptor 2), ISLR (immunoglobulin superfamily containing leucine-rich repeat proteins) and CRELD2 (protein disulfide isomerase CRELD2). Located in the ER, the off-target binding to CRELD2 is not much concerned. On the other hand, Nectin-1, FGFR2 and ISLR show a wide range of tissue expression profiles. Cross-reactivity with these proteins is undesirable because the binding to these targets may have a significant impact on the pharmacokinetics and biodistribution characteristics of the molecule. This off-target binding was subsequently confirmed by flow cytometry on live HEK293 cells transiently expressing these "off-target" proteins identified in the initial screen. These data were used to further select anti-VEGFR1 antibodies without known off-target propensity.

[0894] Table 31. Retrogenix specificity data. VEGFR1 specific membrane hits above very weak intensity (expected hits Overview of .

[0895]

[0896] Table 32. Retrogenix specificity data. Non-VEGFR1 specific membrane hits with higher than very weak intensity (unexpected Overview of hit .

[0897]

[0898]

[0899] Table 33. Retrogenix specificity data. Common Fcγ receptor specific membrane hits above very weak intensity (expected Overview of hit .

[0900]

[0901] Table 34. Retrogenix specificity data. Common Fcγ receptor specific membrane hits above very weak intensity (expected Overview of hit .

[0902]

[0903] Example 4. Systemic Pharmacokinetics and Target Binding Biomarker Profiles of Anti-VEGFR1 Antibodies in Cynomolgus Monkeys

[0904] In addition to the biophysical and in vitro activity profiles of the anti-VEGFR1 antibodies, the systemic pharmacokinetic (PK) and target engagement (TE) biomarker profiles in cynomolgus monkeys were characterized.

[0905] Unexperimented male cynomolgus monkeys (Macaca fascicularis, age 2 to 7 years old, Envigo RMS) were randomly divided into groups of 2 animals per group according to body weight. VGFB54, VGFB78, VGFB82 and VGFB80 were administered intravenously (IV) at 0.3mg / kg, 2mg / kg and 10mg / kg for about 2 minutes on the first day, followed by subcutaneous (SC) administration on the 15th day. Before administration and at the following time points, i.e. 1 hour, 6 hours, 24 hours, 48 ​​hours, 96 hours and 168 hours after IV administration, and 1 hour, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours and 336 hours after SC administration, blood samples (a total of 0.5mL-2mL) of each animal were collected into test tubes containing K2 EDTA via the femoral vein. Before centrifugation to obtain plasma (within 1 hour after collection), blood samples were kept at 2°C-8°C. Plasma samples were collected into PK and TE aliquots and stored at -70 °C until sample analysis.

[0906] Immunoassay bioanalysis of anti-VEGFR1 antibodies

[0907] The “total” and “free” mAb concentrations of anti-VEGFR1 mAbs in cynomolgus monkey plasma were determined using an immunoassay method that employed a typical sandwich format with electrochemiluminescence detection. Briefly, MSD gold streptavidin plates (MesoScale Discovery) were pre-wetted with blocking buffer and tapped dry. A master mix solution (35 μL / well) containing final working concentrations of capture and detection reagents was added to the plate 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. The plate was immediately read on an MSD Sector S600 imager. The quantifiable ranges for “total” and “free” VEGFR1 mAbs were defined as 0.010 μg / mL–2560 μg / mL and 0.020 μg / mL–5120 ug / mL, respectively, with a 10-fold minimum required dilution for plasma samples. To measure "total" mAb, biotinylated and ruthenium-labeled anti-human Fc mAb (R10Z8E9) was used as capture / detection reagent. To measure "free" mAb, biotinylated recombinant cynomolgus monkey VEGFR1[D1-D3]-Avi6xHis and ruthenium-labeled anti-human Fc mAb (R10Z8E9) were used as capture / detection reagents.

[0908] MSD output files containing raw ECL counts were scanned into Cyberlab Content Manager (Agilent) and imported into Watson LIMS (Thermo Scientific) regression software for analysis. The Watson research regression was predefined during assay identification and used a 5-parameter logistic (automatically estimated) fit with a 1 / Y2 weighting factor.

[0909] The plasma drug concentration-time curve is shown in Figure 5-Figure 8 The total and free concentrations of all four molecules were similar at all time points. The plasma PK parameters estimated using non-compartmental analysis are shown in Table 35. The terminal half-life (T 1 / 2 ), maximum plasma drug concentration (C max ) and the area under the drug serum concentration-time curve (AUC) increased with dose in a greater than dose-proportional manner. Nonlinear PK profiles and shorter half-lives at lower doses suggest target-mediated drug disposition (TMDD). The mean half-lives of VGFB54, VGFB82, and VGFB80 at 10 mg / kg IV were approximately 4.9 days, 5.7 days, and 4.5 days, respectively (based on non-compartmental analysis of total PK). However, due to significant TMDD, short sampling time, and the terminal phase may not be fully characterized, T should be interpreted with caution. 1 / 2and clearance (CL). The monkey PK profiles of VGFB82, VGFB54, and VGFB80 were also characterized using a minimal physiologically based PK model. The SC bioavailabilities of VGFB54, VGFB82, and VGFB80 were approximately 83.7%, 95.4%, and 87.7%, respectively.

[0910] Table 35. Single IV administration of VEGFR1 mAbs VGFB82, VGFB54, VGFB78, and VGFB80 on Day 0 and subsequent Mean pharmacokinetic parameters after administration of 0.2 mg / kg, 3 mg / kg, or 10 mg / kg to cynomolgus monkeys on day 14 .

[0911]

[0912]

[0913] C max , maximum (peak) concentration; AUC last , the 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 available

[0914] Plasma PlGF in response to administration of VEGFR1 mAb

[0915] In addition to VEGFA, VEGFR1 also binds to placental growth factor (PlGF). Unlike VEGFA, which binds both VEGFR1 and VEGFR2, PlGF is a selective ligand for VEGFR1. Since PlGF and VEGFA share overlapping binding domains on VEGFR1 (D2-D3) with comparable binding affinity, anti-VEGFR1 antibodies that block VEGFA binding will competitively block the binding of PlGF to VEGFR1, leading to an increase in PlGF levels.

[0916] Total plasma PlGF levels, including unbound and bound to VEGFR1, were determined in cynomolgus monkeys using a stepwise electrochemiluminescence assay (human PlGF V plex kit, MSD K151MED-4). The plate coated with the capture antibody was blocked and incubated for 1 hour. The PlGF calibrator (human PlGF, R&D Systems), quality control (QC) and plasma samples were diluted 1:10 with a buffer containing an anti-VEGFR1 antibody (167 μg / mL), which binds to soluble VEGFR1, thereby alleviating signal inhibition caused by the binding of VEGFR1 to PlGF. After incubation at room temperature for at least 15 min, the mixture was added to the washed MSD plate after blocking and incubated overnight at 4°C. After washing, the detection antibody conjugated to SulfoTag was added and incubated for 2 hours at room temperature. The MSD plate was washed and 1×MSD-T reading buffer was added to all wells. The plate was read immediately on an MSD Sector S600 imager.

[0917] The MSD output files containing raw ECL counts were imported into Watson LIMS (Thermo Scientific) regression software for analysis. The Watson research regression was predefined during assay identification and used a 4-parameter logistic fit with a 1 / Y2 weighting factor.

[0918] Following IV or SC administration of VEGFR1 mAbs, systemic PlGF levels increased in a dose- and PK-related manner ( Figure 9-12 ). The increase in PlGF levels was comparable between VGFB82 and VGFB54, and tended to be higher than that of VGFB80 and VGFB78.

[0919] Example 5. Target binding biomarker profile of mouse recombinant anti-VEGFR1 leads in db / db mice

[0920] In order to reduce the immunogenicity of human anti-VEGFR1 mAb in mice, so that long-term administration and efficacy studies can be performed in mice, a mouse recombinant form of human anti-VEGFR1 antibody was generated, wherein the variable region (VH / VL) of human anti-VEGFR1 mAb was transplanted onto a mouse constant region consisting of a silent mouse IgG1_D265A (CH1, CH2, CH3) for the heavy chain and a constant region of mouse λ for the light chain. The control was a control antibody of the same isotype that did not bind to VEGFR1.

[0921] To measure mouse PlGF, a mouse PlGF-2 Quantikine ELISA kit (R&D SystemCatalog MP200) was used. Test plasma samples were diluted 1:4 to 1:15 with calibrator diluent RD 5-17, depending on the dose level and time point, to keep the reading within the linear detection range of the assay (assay range 23.4 pg / mL-1500 pg / mL, sensitivity 1.84 pg / mL). Plates were read on a SpectraMax plate reader (Molecular Devices Model Paradigm) set at 450 nm and quantified using Pro 7.1 calculated the concentration of PLGF-2 in mouse plasma samples.

[0922] After repeated subcutaneous administration to db / db female mice (TheJackson laboratory, stock number 000642) at 1 mg / kg or 10 mg / kg per week on days 0, 7 and 14, mouse recombinant VGFB54, VGFB78 and VGFB82 triggered different levels of PlGF responses at the same dose level (Tables 36 and 37). In 3 leads, recombinant VGFB78 induced the strongest increase in plasma PlGF. Recombinant VGFB82 also led to an increase in PLGF. However, compared with VGFB78, VGFB82 induced PlGF responses at significantly lower levels in mice (about 4 times lower than VGFB78 at 10 mg / kg). Recombinant VGFB54 had the smallest increase in TE biomarkers in mice.

[0923] Table 36. Mouse recombinant VGFB54 and VGFB78 were administered SC three times on days 0, 7 and 14 to db / db mice (N =3 / time point) in plasma PlGF concentration over time (pg / mL, mean ± SD)

[0924]

[0925] Table 37. Mouse recombinant VGFB82 was administered SC three times to db / db mice on days 0, 7, and 14 (N=3 / time PlGF plasma concentrations over time after 6-point (pg / mL, mean ± SD)

[0926]

[0927] Example 6. Effects of VGFB82 and VGFB78 on reducing proteinuria in hypertensive uninephrectomized db / db mice

[0928] A severe progressive diabetic nephropathy mouse model, designated herein as the ReninAAV uNx db / db model, was used to evaluate the efficacy of anti-VEGFR1 lead mAbs (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, sustained hypertension induced by delivery of adeno-associated viruses expressing renin further accelerated disease progression and renal damage in uninephrectomized db / db mice. ReninAAV uNx db / db mice showed severe renal dysfunction, characterized by a significant increase in the urine albumin to creatinine ratio (UACR), a decrease in the glomerular filtration rate (GFR), and an increase in serum creatinine. Histopathological features showed features similar to those of advanced human diabetic nephropathy, including mesangial expansion, glomerular sclerosis, tubular degeneration, and tubulointerstitial inflammation and fibrosis.

[0929] Female db / db mice were subjected to uninephrectomy at 7-8 weeks of age (Surg3041 from Jackson Laboratory) and infected with adeno-associated virus expressing renin (1×10 10 genomic copies). Six weeks later, based on their UACR, systolic blood pressure and body weight, mice were randomized using 0.8, 0.1, 0.1 weights on IRINI software, 9-15 animals per group. Blood glucose>300mg / dL and UACR>5000mg / g were used as inclusion criteria for randomization. Mice received vehicle lisinopril (Lisinopril) (10mg / kg in drinking water), isotype control, low-dose or high-dose mouse recombinant VGFB82 or VGFB78, subcutaneously injected at a specific dosing frequency for 4 weeks to 6 weeks. Urine samples were collected by placing mice individually in metabolic cages (MMC100, Hatteras Instruments) for 2 hours every two weeks before treatment and after administration. Urinary albumin and creatinine concentrations were measured using the Sekisui Diagnostics Microalbumin Assay Kit (Cat. No. 252-20), Enzymatic Creatinine Assay Kit (Cat. No. 265-30) and Vet Axcel Chemistry Analyzer (Alfa Wassermann). Plasma samples were collected for PlGF measurement.

[0930] Data are 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.

[0931] 3mg / kg and 30mg / kg of mouse recombinant VGFB82 (VGFB877) were administered SC (QW) weekly for 4 weeks, followed by 30mg / kg three times a week (TW) and 90mg / kg per week for another 2 weeks. Table 38 shows the UACR and the percentage change relative to baseline at weeks 2, 4, and 6 after administration. Table 39 shows the plasma PlGF levels at weeks 4 and 6 after administration. The vehicle-treated group showed continuous and sustained UACR increases throughout the treatment period. Lisinopril, an angiotensin-converting enzyme inhibitor (ACEi, standard treatment), significantly reduced UACR from its baseline to -26% in week 4 compared to the vehicle control group. Mice treated with 3mg / kg QW of VGFB877 for 4 weeks did not show discernible UACR changes when compared to the vehicle group. At week 4, a high dose of VGFB877 at 30 mg / kg / QW partially halted UACR progression compared to vehicle-treated mice (% change from baseline for VGFB877 was +15% vs. +83% for vehicle). At week 6, the % change from baseline in UACR decreased from +63% to +28% when the dose and dosing frequency increased from 3 mg / kg QW to 30 mg / kg TW. At week 6, the % change from baseline in UACR decreased from +15% to +6% when the dose was adjusted from 30 mg / kg QW to 90 mg / kg QW. Consistent with the improved UACR reduction, plasma PlGF levels increased when the dose level or frequency of VGFB82 was increased.

[0932] Table 38. Effects of recombinant VGFB82 (VGFB877) on UACR and changes relative to baseline in ReninAAVuNxdb / db mice The impact of %

[0933]

[0934] Table 39. Plasma PlGF levels at weeks 4 and 6 after administration of VGFB877 in ReninAAV uNx db / db mice Mean ± SEM

[0935]

[0936]

[0937] For the efficacy study of mouse recombinant VGFB78 (VGFB876), ReninAAV uNx db / db mice were administered 3mg / kg and 10mg / kg TW SC for 4 weeks. Tables 40-41 show the UACR and percentage changes relative to baseline at weeks 2 and 4 after administration. Table 39 shows the plasma PlGF levels at week 4 after administration. In contrast to the gradual increase in UACR in mice treated with vehicle and isotype, VGFB876 significantly reduced UACR at week 4 compared with the control group. The low dose of 3mg / kg reduced UACR by about -9% from baseline, and the high dose of 10mg / kg reduced UACR by about -20% from baseline, which is comparable to ACEi. Consistent with the significant PD effect (reduction in UACR), VGFB876 induced a strong, dose-related increase in plasma PlGF levels, consistent with its high binding potency to mouse VEGFR1.

[0938] Table 40. Effect of recombinant VGFB78 (VGFB876) on UACR and changes relative to baseline in ReninAAVuNxdb / db mice The impact of %

[0939]

[0940] Table 41. Plasma at Week 4 after VGFB876 Administration in ReninAAV uNx db / db Mice PlGF levels (pg / mL, mean ± SEM)

[0941]

Claims

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

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, 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 sequence FPLDTL (SEQ ID NO: 143) and EIGL (SEQ ID NO: 144).

3. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to human, mouse, rat and / or cynomolgus monkey VEGFR1. 4 . The isolated antibody or antigen-binding fragment thereof according to claim 3 , wherein the antibody or antigen-binding fragment thereof binds to human VEGFR1.

5. The isolated antibody or antigen-binding fragment thereof according to claim 4, wherein the 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.

6. The isolated antibody or antigen-binding fragment thereof according to claim 5, wherein the 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.

7. The isolated antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof binds to human VEGFR1 and VEGFR1 from cynomolgus monkey, mouse and rat.

8. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is expressed at a density of 6×10 -8 M or smaller, especially 1×10 -8 M or smaller, more particularly 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M or smaller K D Binds to human VEGFR1, mouse VEGFR1, and cynomolgus monkey VEGFR1.

9. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising a heavy chain complementarity determining region (HCDR) of a VH of SEQ ID NO: 31, 33, 34, 36, 38 or 39 and a light chain complementarity determining region (LCDR) of a VL of SEQ ID NO: 32, 35, 37 or 40.

10. The isolated antibody or antigen-binding fragment thereof according to claim 9, comprising: a. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 31 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 32; b. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 33 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 32; c. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 34 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 35; d. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 36 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 37; e. a HCDR of a VH having the amino acid sequence of SEQ ID NO: 38 and a LCDR of a VL having the amino acid sequence of SEQ ID NO: 37; or f. The HCDR of VH has the amino acid sequence of SEQ ID NO:39 and the LCDR of VL has the amino acid sequence of SEQ ID NO:

40.

11. The isolated antibody or antigen-binding fragment thereof according to claim 10, comprising a VH having HCDR1, HCDR2 and HCDR3 and a VL having LCDR1, LCDR2 and LCDR3, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprise the following amino acid sequence: a. SEQ ID NO: 7, 175, 9, 10, 11 and 12 respectively; b. SEQ ID NO: 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 NO: 95, 177, 97, 98, 99 and 100, respectively. l. SEQ ID NO:95, 96, 97, 98, 99 and 100 respectively; m. SEQ ID NOs: 101, 102, 103, 104, 105 and 106, respectively; n. SEQ ID NO: 107, 108, 109, 110, 111 and 112 respectively; o. 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. SEQ ID NOs: 137, 138, 139, 140, amino acid sequence FNS and SEQ ID NO: 142, respectively.

12. The isolated antibody or antigen-binding fragment thereof according to claim 10, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 175, 9, 10, 11 and 12, respectively.

13. The isolated antibody or antigen-binding fragment thereof according to claim 11, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 14, 15, 16, 17 and 18, respectively.

14. The isolated antibody or antigen-binding fragment thereof according to claim 11, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively.

15. The isolated antibody or antigen-binding fragment thereof according to claim 11, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 8, 9, 10, 11 and 12, respectively.

16. The isolated antibody or antigen-binding fragment thereof of claim 11, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 20, 21, 22, 23 and 24, respectively.

17. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the heavy chain variable domain has the same sequence as SEQ ID NO: 31, The light chain variable domain has 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: 33, 34, 36, 38 or 39, wherein the light chain variable domain has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 32, 35, 37 or 40. (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical amino acid sequences.

18. The isolated antibody or antigen-binding fragment thereof according to claim 17, comprising VH and VL, wherein the VH and the VL comprise the following amino acid sequences: a. SEQ ID NO:31 and 32 respectively; b. SEQ ID NO: 33 and 32, respectively; c. SEQ ID NO: 34 and 35, respectively; d. SEQ ID NO: 36 and 37, respectively; e. SEQ ID NO: 38 and 37, respectively; or f. SEQ ID NO: 39 and 40, respectively.

19. An isolated antibody or antigen-binding fragment thereof according to claim 18, 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.

20. The isolated antibody or antigen-binding fragment thereof according to claim 19, 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.

21. The isolated antibody or antigen-binding fragment thereof of claim 18, 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.

22. The isolated antibody or antigen-binding fragment thereof according to claim 21, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

23. The isolated antibody or antigen-binding fragment thereof of claim 18, 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.

24. The isolated antibody or antigen-binding fragment thereof according to claim 23, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

25. The isolated antibody or antigen-binding fragment thereof of claim 18, 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.

26. The isolated antibody or antigen-binding fragment thereof according to claim 25, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

27. An isolated antibody or antigen-binding fragment thereof according to claim 18, 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.

28. The isolated antibody or antigen-binding fragment thereof according to claim 27, wherein the isolated antibody or antigen-binding fragment thereof comprises 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.

29. An isolated antibody or antigen-binding fragment thereof according to claim 18, 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.

30. The isolated antibody or antigen-binding fragment thereof according to claim 29, 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.

31. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 30, comprising a heavy chain (HC) and a light chain (LC), wherein the heavy chain has 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 the light chain has 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: 56, SEQ ID NO: 57 or SEQ ID NO:

58. An amino acid sequence that is at least 80% (eg, at least 85%, at least 90%, at least 95% or at least 99%) identical to the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:

60.

32. The isolated antibody or antigen-binding fragment thereof of claim 31, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:51, 52, 53, 54, 55, 56, 57, 59 and 60.

33. The isolated antibody of claim 32, 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 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 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. 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:59 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:

60.

34. The isolated antibody of claim 33, 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.

35. The isolated antibody of claim 33, comprising a HC comprising the amino acid sequence of SEQ ID NO: 53 and a LC comprising the amino acid sequence of SEQ ID NO:

52.

36. The isolated antibody of claim 33, 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.

37. The isolated antibody of claim 33, 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.

38. The isolated antibody of claim 33, 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.

39. The isolated antibody of claim 33, 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.

40. An isolated antibody or antigen-binding fragment thereof, comprising: a. a HCDR of a VH comprising the amino acid sequence of SEQ ID NO: 33 and a LCDR of a VL comprising the amino acid sequence of SEQ ID NO: 32; b. VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively, and 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: 33 and a VL having the amino acid sequence of SEQ ID NO: 32; and / or d. HC having the amino acid sequence of SEQ ID NO:53 and LC having the amino acid sequence of SEQ ID NO:

52.

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

55.

42. An isolated antibody or antigen-binding fragment thereof, comprising: a. a heavy chain variable region (VH) having a HCDR of VH comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable region (VL) having a LCDR of VL comprising the amino acid sequence of SEQ ID NO: 37; b. VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, and 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. HC having the amino acid sequence of SEQ ID NO:58 and LC having the amino acid sequence of SEQ ID NO:

57.

43. An isolated antibody or antigen-binding fragment thereof, comprising: a. a heavy chain variable region (VH) having a HCDR of VH comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region (VL) having a LCDR of VL comprising the amino acid sequence of SEQ ID NO: 40; b. VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively, and 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. HC having the amino acid sequence of SEQ ID NO: 59 and LC having the amino acid sequence of SEQ ID NO:

60.

44. An isolated antibody or antigen-binding fragment thereof, comprising: a. a heavy chain variable region (VH) having a HCDR of VH comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable region (VL) having a LCDR of 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. HC having the amino acid sequence of SEQ ID NO:51 and LC having the amino acid sequence of SEQ ID NO:

52.

45. An isolated antibody or antigen-binding fragment thereof, comprising: a. a heavy chain variable region (VH) having a HCDR of VH comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable region (VL) having a LCDR of VL comprising the amino acid sequence of SEQ ID NO: 37; b. VH comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, and 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. HC having the amino acid sequence of SEQ ID NO:56 and LC having the amino acid sequence of SEQ ID NO:

57.

46. ​​The isolated antigen-binding fragment of any one of claims 1 to 45, wherein the antigen-binding fragment is a scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, VHH, or a single-chain antibody.

47. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 46, wherein the antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3 or IgG4 isotype.

48. The isolated antibody or antigen-binding fragment thereof according to claim 47, wherein the antibody or antigen-binding fragment thereof is of the IgGl isotype.

49. The isolated antibody or antigen-binding fragment thereof of claim 48, comprising an Ig constant region or a fragment of said Ig constant region.

50. The isolated antibody or antigen-binding fragment thereof of claim 49, wherein the Ig constant region or the fragment of the Ig constant region comprises at least one mutation that results in reduced binding of the antibody or antigen-binding fragment thereof to an Fcγ receptor (FcγR).

51. The isolated antibody or antigen-binding fragment thereof of claim 50, wherein the at least one mutation that results in reduced binding of the antibody or antigen-binding fragment thereof to 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 / L236 34V / 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.

52. The isolated antibody or antigen-binding fragment thereof of claim 51, wherein the Ig constant region or the fragment of the constant region comprises the mutations L234A_L235A_D265S.

53. The isolated antibody or antigen-binding fragment thereof of claim 52, wherein the FcγR is FcγRI, FcγRIIA, FcγRIIB or FcγRIII, or any combination thereof.

54. The isolated antibody or antigen-binding fragment thereof of claim 53, wherein the Ig constant region or the 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.

55. The isolated antibody or antigen-binding fragment thereof of claim 54, wherein the 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, wherein residue numbering is according to the EU index.

56. An immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 55 conjugated to a therapeutic agent or an imaging agent.

57. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 55 or the immunoconjugate according to claim 56 and a pharmaceutically acceptable carrier.

58. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 55.

59. An isolated polynucleotide according to claim 58, comprising a polynucleotide sequence that is at least 80%, such as 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.

60. A vector comprising the polynucleotide according to claim 58 or 59.

61. A host cell expressing the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 55, such as a host cell comprising the vector according to claim 60.

62. A method for 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 antigenic fragment thereof according to any one of claims 1 to 55, the immunoconjugate according to claim 56, the pharmaceutical composition according to claim 57, the isolated polynucleotide according to claim 58 or 59, the vector according to claim 60 or the host cell according to claim 61, thereby preventing VEGFA from binding to the VEGFR1.

63. The method of claim 62, wherein the subject is in need of treatment for chronic kidney disease (CKD), is in need of reduction in proteinuria, has advanced stage 4 or stage 5 chronic kidney disease, or has CKD with proteinuria, albuminuria, or diabetes.

64. A method of treating chronic kidney disease (CKD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated antibody or antigenic fragment thereof according to any one of claims 1 to 55, an immunoconjugate according to claim 56, a pharmaceutical composition according to claim 57, an isolated polynucleotide according to claim 58 or 59, a vector according to claim 60, or a host cell according to claim 61, for a time sufficient to treat the CKD.

65. The method of claim 64, wherein the subject has advanced stage 4 or stage 5 chronic kidney disease.

66. The method of claim 64, wherein the subject has stage 3 chronic kidney disease.

67. The method of claim 64, wherein the subject has CKD with proteinuria, albuminuria, or diabetes.

68. A method of 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 according to any one of claims 1 to 55, an immunoconjugate according to claim 56, a pharmaceutical composition according to claim 57, an isolated polynucleotide according to claim 58 or 59, a vector according to claim 60, or a host cell according to claim 59, for a time sufficient to reduce proteinuria in the subject.

69. A kit comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 55, the immunoconjugate according to claim 56, the pharmaceutical composition according to claim 57, the isolated polynucleotide according to claim 58 or 59, the vector according to claim 60, or the host cell according to claim 61.

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