Human FGF23-binding antibodies with improved affinity and efficacy

Abstract

The present disclosure provides improved human FGF23-binding antibodies that have increased affinity for human FGF23 compared to burosumab, allowing for less frequent administration, improving convenience of treatment, and achieving greater efficacy compared to burosumab without compromising safety.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 548,711, filed February 1, 2024, U.S. Provisional Patent Application No. 63 / 514,180, filed July 18, 2023, and U.S. Provisional Patent Application No. 63 / 489,458, filed March 10, 2023, the entire disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence table XML reference This application contains a Sequence Listing that has been submitted electronically in XML format. This Sequence Listing XML is incorporated herein by reference. The XML file (created on February 28, 2024) is named UXB023-06WO_SL.xml and is 2,280,142 bytes in size.

[0003] Technical Field of the Disclosure The present disclosure relates to human FGF23 binding antibodies with improved affinity and improved efficacy compared to burosumab. [Background technology]

[0004] Burosumab (i.e., burosumab-twza, commercially available as CRYSVITA®) is a fibroblast growth factor 23 (FGF23)-blocking antibody indicated for the treatment of X-linked hypophosphatemia (XLH) in adult and pediatric patients 6 months of age and older, and for the treatment of FGF23-associated hypophosphatemia in adults and pediatric patients 2 years of age and older in tumor-induced osteomalacia (TIO) when the tumor cannot be located or removed. Burosumab is a recombinant human IgG1κ monoclonal antibody. Burosumab consists of four polypeptide chains (two identical heavy chains, each with 447 amino acids, and two identical light chains, each with 213 amino acids) and has a molecular weight of 147 kDa. Burosumab specifically binds to FGF23 and blocks its interaction with the Klotho-FGF receptor complex.

[0005] FGF23 is a natural cytokine involved in phosphate and vitamin D metabolism. Excessive levels of circulating FGF23 can lead to increased urinary phosphate excretion, reduced vitamin D synthesis, and subsequent hypophosphatemia, resulting in defective bone mineralization and other tissue effects, including muscle. Burosumab binds to FGF23 and inhibits its ability to bind to fibroblast growth factor receptor 1 (FGFR1) and its obligate co-receptor Klotho. This inhibition restores intraluminal reabsorption of phosphate from the kidney and increases vitamin D production, which promotes intestinal absorption of calcium and phosphate. These combined actions improve serum phosphorus levels and bone mineralization.

[0006] Burosumab administration to pediatric patients is administered by subcutaneous injection every two weeks (Q2W), representing a significant treatment burden. Furthermore, both adult and pediatric patients must receive high-dose burosumab injections, up to a maximum dose of 3 ml, in three separate 1 ml injections (see, e.g., prescribing information for burosumab-twza, available at www.accessdata.fda.gov / scripts / cder / daf / index.cfm). Therefore, there is a need for human FGF23-binding antibodies with improved affinity and improved efficacy compared to burosumab. Summary of the Invention

[0007] The present disclosure provides improved human FGF23-binding antibodies that have increased affinity for human FGF23 compared to burosumab, allowing for less frequent administration, improving convenience of treatment, and achieving greater efficacy compared to burosumab without compromising safety.

[0008] The present disclosure provides FGF23-binding antibodies engineered to have improved affinity compared to burosumab by altering one or more amino acids within the heavy chain (SEQ ID NO: 1) and / or light chain (SEQ ID NO: 2) sequence(s) of burosumab. In some embodiments, the FGF23-binding antibodies are engineered to have improved affinity compared to burosumab by altering one or more amino acids within the heavy chain variable region (VH, SEQ ID NO: 3) and / or light chain variable region (VL, SEQ ID NO: 4) sequence(s) of burosumab.

[0009] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 3, but contains one or more amino acid changes relative to SEQ ID NO: 3, wherein the one or more amino acid changes relative to SEQ ID NO: 3 are T30E, T30F, T30Q, T30S, T30Y, N31D, N31E, N31F, N31Q, N31R, N31Y, H32E, H32F, H32Q, H32S, H32T, I50D, I50E, I50F, I50S, I50T, I50Y, N52E, N52F , N52Q, N52Y, I54E, I54F, I54L, I54Q, I54S, I54T, I54V, I54Y, S55E, S55F, S55Q, S55T, S5 5Y, S59D, S59E, S59F, S59I, S59L, S59Q, S59T, S59V, S59Y, D99E, D99Q, D99Y, I100D, I100E , I100F, I100L, I100Q, I100V, I100Y, V101F, V101I, V101L, V101T, V101Y, D102E, D102F, D102I, D102L, D102Q, D102Y, A103D, A103E, A103F, A103Q, A103S, A103T, and A103Y.

[0010] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 4, but contains one or more amino acid changes relative to SEQ ID NO: 4, wherein the one or more amino acid changes are A25L, A25V, I29F, I29L, I29V, S30D, S30E, S30F, S30I, S30L, S30Q, S30T, S30V, S30Y, S31D, S31E, S31F, S31I, S31L, S31Q, S31T, S31V, S31Y, A32F, A32I, A32L, A32T, A32V, A32Y , V34F, V34I, V34L, V34Y, D50E, D50F, D50I, D50L, D50Q, D50R, D50S, D50T, D50V, A51F, A51I, A 51L, A51S, A51T, A51V, S52D, S52E, S52F, S52Q, S52T, S52Y, S53D, S53E, S53F, S53I, S53L, S53Q , S53R, S53T, S53V, S53Y, Q90F, Q90Y, F91K, F91R, F91Y, N92D, N92E, N92F, N92Q, N92S, N92T, N92Y, D93E, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94D, Y94E, Y94F, Y94Q, Y94S, and Y94T.

[0011] In some embodiments, the disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a VH amino acid sequence according to SEQ ID NO: 3 and a VL amino acid sequence according to SEQ ID NO: 4, wherein the VH amino acid sequence is any of the following: T30E, T30F, T30Q, T30S, T30Y, N31D, N31E, N31F, N31Q, N31R, N31Y, H32E, H32F, H32Q, H32S, H32T, I50D, I50E, I50F, I50S, I50Y 50T, I50Y, N52E, N52F, N52Q, N52Y, I54E, I54F, I54L, I54Q, I54S, I54T, I54 V, I54Y, S55E, S55F, S55Q, S55T, S55Y, S59D, S59E, S59F, S59I, S59L, S59Q, S 59T, S59V, S59Y, D99E, D99Q, D99Y, I100D, I100E, I100F, I100L, I100Q, I10 0V, I100Y, V101F, V101I, V101L, V101T, V101Y, D102E, D102F, D102I, D102L, and / or wherein the VL amino acid sequence comprises one or more amino acid changes selected from A25L, A25V, I29F, I29L, I29V, S30D, S30E, S30F, S30I, S30L, S30Q, S30T, S30V, S30Y, S31D, S31E, S31F, S31I, S31L, S31Q, S31T, S31V, S31Y, A32F, A32I, A32L, A32T, A32V, A32Y, V34F, V34I, V34L, V34Y, D50E, D50F, D50I, D50L, D50Q, D50R, D50S, D50T, D50V, A5 1F, A51I, A51L, A51S, A51T, A51V, S52D, S52E, S52F, S52Q, S52T, S52Y, S53D, S53E, S53F, S53I, S53L, S53Q, S53R, S53T, S53V, S53Y, Q90F, Q90Y, F91K, F9 1R, F91Y, N92D, N92E, N92F, N92Q, N92S, N92T, N92Y, D93E, D93F, D93Q, D93R,and one or more amino acid changes selected from D93S, D93T, D93Y, Y94D, Y94E, Y94F, Y94Q, Y94S, and Y94T.

[0012] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO:3 and a light chain variable region (VL) according to SEQ ID NO:4, wherein the VH and VL sequences comprise at least two amino acid changes, the at least two amino acid changes being VL:S31E / VL:D50F, VH:N31F / VH:S59F, VL:D50F / VL:S53E, VL:S31F / VL:S52E, VL:D50F / VH:V101I, VH:S59D / VH:V101I, VL:S31F / VL:S53E, VL:N 92E / VL:D93F, VL:S31E / VL:S53E, VL:S31F / VL:D50F, VH:N31E / VH:S59E, VH:S57E / VH:S59D, VH :N31E / VH:I54F, VH:S59T / VH:V101L, VH:I54Y / VH:V101F, VH:N52F / VH:V101I, VL:N92D / VL:D9 3F, VH:N52F / VH:V101L, VL:S31F / VL:D50E, VL:Y94D / VH:S59D, VL:Y94D / VH:S59E, VL:N92F / VH:S59E, VL:N92Y / VH:S59E, VL:Y94E / VH:S59E, VL:S52D / VH:S59E, and VL:S52E / VH:S59E.

[0013] Also provided herein are FGF23 binding antibodies that have one or more unique complementarity determining regions (CDRs) compared to burosumab.

[0014] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain complementarity-determining region 1 (HCDR1) according to an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NOs: 22-32.

[0015] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain complementarity-determining region 2 (HCDR2) according to an amino acid sequence selected from SEQ ID NO: 12 and SEQ ID NOs: 33-65.

[0016] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain complementarity-determining region 3 (HCDR3) according to an amino acid sequence selected from SEQ ID NO: 13 and SEQ ID NOs: 66-93.

[0017] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain complementarity-determining region 1 (LCDR1) according to an amino acid sequence selected from SEQ ID NO: 14 and SEQ ID NOs: 94-126.

[0018] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain complementarity-determining region 2 (LCDR2) according to an amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NOs: 127-158.

[0019] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain complementarity-determining region 3 (LCDR3) according to an amino acid sequence selected from SEQ ID NO: 16 and SEQ ID NOs: 159-185.

[0020] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: the HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NOs: 22-32; the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 12 and SEQ ID NOs: 33-65; the HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 13 and SEQ ID NOs: 66-93; the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 14 and SEQ ID NOs: 94-126; the LCDR2 comprises an amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NOs: 127-158; and the LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 16 and SEQ ID NOs: 159-185.

[0021] Also provided herein are FGF23 binding antibodies that have one or more unique variable region domains compared to burosumab.

[0022] For example, the isolated antibody or fragment thereof may comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, or the VL comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0023] The isolated antibody or fragment thereof may comprise a VH comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, or the antibody or fragment thereof may comprise a VL comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0024] The isolated antibody or fragment thereof may comprise a VH comprising an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, or the antibody or fragment thereof may comprise a VL comprising an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0025] The isolated antibody or fragment thereof may comprise a VH and a VL, wherein the VH may comprise an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 3, and SEQ ID NOs: 186 to 270, and the VL may comprise an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0026] The isolated antibody or fragment thereof may comprise a VH and a VL, wherein the VH may comprise an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, and the VL may comprise an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0027] The isolated antibody or fragment thereof may comprise a VH and a VL, wherein the VH may comprise an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, and the VL may comprise an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0028] The isolated antibody or fragment thereof may comprise a VH and a VL, and the VH may comprise an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, or the VL may comprise an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0029] The isolated antibody or fragment thereof may comprise a VH and a VL, wherein the VH may comprise an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, and the VL may comprise an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0030] In some aspects, the disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a VH amino acid sequence according to SEQ ID NO: 3 and a VL amino acid sequence according to SEQ ID NO: 4, wherein the VH amino acid sequence comprises one or more amino acid changes selected from I54F, I54Y, S59D, S59E, V101I, and A103S compared to SEQ ID NO: 3.

[0031] In some aspects, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a VH amino acid sequence according to SEQ ID NO: 3 and a VL amino acid sequence according to SEQ ID NO: 4, wherein the VL amino acid sequence comprises one or more amino acid changes selected from S52D, S52E, N92F, N92Y, Y94D, and Y94E compared to SEQ ID NO: 4.

[0032] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-001 and a light chain variable region (VL) according to VL-027 (i.e., an antibody with antibody structure ID number UGX126).

[0033] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-001 and a light chain variable region (VL) according to VL-066 (i.e., an antibody with antibody structure ID number UGX144).

[0034] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-001 and a light chain variable region (VL) according to VL-067 (i.e., an antibody with antibody structure ID number UGX145).

[0035] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-028 and a light chain variable region (VL) according to VL-001 (i.e., an antibody with antibody structure ID number UGX156).

[0036] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-029 and a light chain variable region (VL) according to VL-001 (i.e., an antibody with antibody structure ID number UGX157).

[0037] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-061 and a light chain variable region (VL) according to VL-001 (i.e., an antibody with antibody structure ID number UGX175).

[0038] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-055 and a light chain variable region (VL) according to VL-001 (i.e., an antibody according to antibody structure ID number UGX182).

[0039] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-050 (i.e., an antibody with antibody structure ID number UGX201).

[0040] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-054 (i.e., an antibody with antibody structure ID number UGX202).

[0041] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-027 (i.e., an antibody with antibody structure ID number UGX203).

[0042] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-030 (i.e., an antibody with antibody structure ID number UGX204).

[0043] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-066 (i.e., an antibody with antibody structure ID number UGX205).

[0044] Also provided is an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-067 (i.e., an antibody with antibody structure ID number UGX206).

[0045] In another aspect, the disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain (HC) amino acid sequence according to SEQ ID NO: 1 and a light chain (LC) amino acid sequence according to SEQ ID NO: 2, wherein the antibody or fragment thereof comprises one or more HC amino acid changes relative to SEQ ID NO: 1 and / or one or more LC amino acid changes relative to SEQ ID NO: 2, wherein the one or more HC amino acid changes are T28Y, T30F, T30K, N31D, N31E, N31F, N31Q, N31R, H32K, H32Y, Y33D, Y33L, Y33R, N52E, N52F, N52Q, N52R, N52Y, I54A, I54D, I54F, I54G, I54H, I54K, I54L, I54M, I54N, I54P, I54Y ... 54R, I54V, I54W, I54Y, S55E, S55F, S55I, S55Q, S55R, S55Y, S57E, S57F, S57 R, S57Y, T58F, T58R, S59A, S59D, S59E, S59F, S59G, S59H, S59I, S59K, S59L, S 59M, S59N, S59P, S59Q, S59R, S59T, S59V, S59W, S59Y, N60E, N60R, N60Y, A61 F, A61R, A61Y, Q62L, Q62R, Q62Y, Q65E, Q65R, Q65Y, D99F, D99R, D99Y, I100D, I100E, I100F, I100Y, V101A, V101D, V101E, V101F, V101G, V101H, V101I, V1 01K, V101L, V101M, V101N, V101P, V101Q, V101R, V101S, V101W, D102E, D102F , D102K, D102R, D102Y, A103D, A103F, A103G, A103H, A103I, A103K, A103L, A103M, A103N, A103P, A103R, A103S, A103T, A103V, and A103W, and the one or more LC amino acid changes are selected from Q27F, Q27R, G28D, G28Q, G28R, G28Y, I29F, I29L, S30D, S30E, S30F, S30L, S30Q, S30R, S30Y, S31D, S31E, S31F, S31Q, S31Y, A32F, A32V, V34F,V34I, D50E, D50F, D50L, D50Q, D50R, D50S, D50T, D50V, D50Y, A51V, S52A, S52D, S52E, S52F, S52G, S52H, S52I, S52K, S52L, S52M, S52N , S52P, S52Q, S52R, S52V, S52W, S52Y, S53D, S53E, S53F, S53R, S53Y, L54E, L54Y, E55R, E55Y, S67E, S67R, S67Y, T69R, T69Y, F91E, F91R , F91Y, N92A, N92D, N92E, N92F, N92G, N92H, N92I, N92K, N92L, N92M, N92P, N92Q, N92R, N92S, N92T, N92V, N92W, N92Y, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94A, Y94D, Y94E, Y94F, Y94G, Y94H, Y94I, Y94K, Y94L, Y94M, Y94N, Y94P, Y94Q, Y94R, Y94S, Y94V, and Y94W.

[0046] In another aspect, the disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 3 and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 4, wherein the antibody or fragment thereof comprises one or more VH amino acid changes relative to SEQ ID NO: 3 and / or one or more VL amino acid changes relative to SEQ ID NO: 4, wherein the one or more VH amino acid changes are T28Y, T30F, T30K, N31D, N31E, N31F, N31Q, N31R, H32K, H32Y, Y33D, Y33L, Y33R, N52E, N52F, N52Q, N52R, N52Y, I54A, I54D, I54F, I54G, I54H, I54K, I54L, I54M, I54R ... 4N, I54P, I54R, I54V, I54W, I54Y, S55E, S55F, S55I, S55Q, S55R, S55Y, S57E , S57F, S57R, S57Y, T58F, T58R, S59A, S59D, S59E, S59F, S59G, S59H, S59I, S5 9K, S59L, S59M, S59N, S59P, S59Q, S59R, S59T, S59V, S59W, S59Y, N60E, N60R , N60Y, A61F, A61R, A61Y, Q62L, Q62R, Q62Y, Q65E, Q65R, Q65Y, D99F, D99R, D9 9Y, I100D, I100E, I100F, I100Y, V101A, V101D, V101E, V101F, V101G, V101H , V101I, V101K, V101L, V101M, V101N, V101P, V101Q, V101R, V101S, V101W, D1 and the one or more VL amino acid changes are selected from Q27F, Q27R, G28D, G28Q, G28R, G28Y, I29F, I29L, S30D, S30E, S30F, S30L, S30Q, S30R, S30Y, S31D, S31E, S31F, S31Q, S31Y, A32F,A32V, V34F, V34I, D50E, D50F, D50L, D50Q, D50R, D50S, D50T, D50V, D50Y, A51V, S52A, S52D, S52E, S52F, S52G, S52H, S52I, S52K, S52L, S5 2M, S52N, S52P, S52Q, S52R, S52V, S52W, S52Y, S53D, S53E, S53F, S53R, S53Y, L54E, L54Y, E55R, E55Y, S67E, S67R, S67Y, T69R, T69Y, F91E , F91R, F91Y, N92A, N92D, N92E, N92F, N92G, N92H, N92I, N92K, N92L, N92M, N92P, N92Q, N92R, N92S, N92T, N92V, N92W, N92Y, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94A, Y94D, Y94E, Y94F, Y94G, Y94H, Y94I, Y94K, Y94L, Y94M, Y94N, Y94P, Y94Q, Y94R, Y94S, Y94V, and Y94W.

[0047] Also provided herein are FGF23 binding antibodies or fragments thereof that have increased affinity for human FGF23 compared to burosumab.

[0048] For example, the isolated antibody or fragment thereof may have about a 2-fold to about 10-fold increased binding affinity for human FGF23 compared to burosumab.

[0049] In some embodiments, the isolated antibody or fragment thereof has an antibody activity against human FGF23 of about 1 x 10 -12 M ~ approx. 7×10 -12 Binding affinity (K D )

[0050] In some embodiments, the isolated antibody or fragment provided herein is for use in a method of lowering serum FGF23 in a subject in need thereof.

[0051] In some embodiments, the isolated antibody or fragment provided herein is for use in a method of increasing the renal threshold maximal value (TmP) of phosphorus reabsorption in a subject in need thereof.

[0052] In some embodiments, the isolated antibody or fragment provided herein is for use in a method of increasing the renal maximal threshold for phosphorus reabsorption (TmP) in a subject in need of increasing serum inorganic phosphorus (Pi).

[0053] In some embodiments, the isolated antibody or fragment provided herein is for use in a method of increasing serum 1,25 dihydroxyvitamin D (1,25[OH]2D) concentrations in a subject in need thereof.

[0054] In some embodiments, the isolated antibody or fragment provided herein is for use in a method of treating a hypophosphatemic condition in a subject in need thereof.

[0055] In some embodiments, the subject has been diagnosed with X-linked hypophosphatemia (XLH) or tumor-induced osteomalacia (TIO).

[0056] Also provided herein is a method for lowering serum FGF23 in a subject in need thereof, the method comprising administering to the subject an isolated antibody or fragment thereof described herein.

[0057] In another aspect, the present disclosure provides a method in a subject in need of increasing the renal maximal threshold for phosphorus reabsorption (TmP), comprising administering to the subject an isolated antibody or fragment thereof described herein.

[0058] Also provided herein is a method in a subject in need of increasing serum inorganic phosphorus (Pi), comprising administering to the subject an isolated antibody or fragment thereof described herein.

[0059] Also provided is a method in a subject in need of increasing serum 1,25 dihydroxyvitamin D (1,25[OH]2D) levels, comprising administering to the subject an isolated antibody or fragment thereof described herein.

[0060] Also provided is a method of treating a hypophosphatemic condition in a subject in need thereof, comprising administering to the subject an isolated antibody or fragment thereof described herein.

[0061] In some embodiments, the subject to be treated by the methods described herein has been diagnosed with X-linked hypophosphatemia (XLH) or tumor-induced osteomalacia (TIO).

[0062] These and other aspects and features of the present disclosure are described in the following sections of this application.

[0063] The above and other objects, features, and advantages of the systems and methods of the present disclosure will be apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings, in which like-referenced elements identify common features of corresponding drawings. [Brief explanation of the drawings]

[0064] [Figure 1] 1 is a chart showing percent deuteration measured by HDX-MS analysis of FGF23 in the presence of burosumab. The three boxed regions identify amino acid residues potentially involved in antibody binding, as evidenced by increased protection from deuteration. [Figure 2]FIG. 1 is a diagram of protected region 1, protected region 2, and protected region 3 as defined herein from HDX-MS analysis superimposed on the 2P39 crystal structure of FGF23. [Figure 3] 1 is a chart showing percent deuteration measured by HDX-MS analysis of burosumab in the presence of FGF23. Six regions of low deuteration were identified, representing amino acid residues involved in antigen binding. [Figure 4] 1 shows the crystallographic data collection and refinement statistics for the FGF23(R179Q):burosumab Fab complex determined by molecular replacement using available structures (2P39.PDB and 2VEN.PDB). [Figure 5] Two molecules of the hFGF23(R179Q):burosumab Fab complex in the asymmetric unit at 3 Å. The two complex molecules are held together by an FGF23 dimer formed by residues known to be involved in FGFR1c binding. [Figure 6] Ribbon model of the FGF23:Fab complex (only variable regions shown) with FGF23 epitope residues (C-alpha atoms) identified as colored purple spheres and FGF23-dimer / FGFR1c binding residues (C-alpha atoms) as colored cyan, burosumab light chain paratope residues as green colored spheres, and burosumab heavy chain paratope residues as yellow colored spheres. [Figure 7] Previously reported epitopes identified in related systems (Yamasaki et al. JBMR. 2008 and Kanhasut et al. Sci Report. 2022) have been mapped to the FGF23:burosumab crystal structure, demonstrating that these epitopes are distinct from those identified in this study (Figure 6). [Figure 8] The FGF23:Fab complex is shown in ribbon model (left) and surface model (right panel), with rotated views of the FGF23 and burosumab binding interfaces, with detailed amino acid positions overlaid as stick models (far right panel). [Figure 9A]Figure 1 shows the FGF23 surface model interface with burosumab shown as a ribbon and stick model. Panel A shows the FGF23:Fab complex with the FGF23 moiety rotated 90 degrees to show all burosumab residues in contact with FGF23 at the binding interface. [Figure 9B] Panel A shows the FGF23 surface model interface with burosumab shown as ribbon and stick models. Panel B shows the surface model of FGF23 (top) with burosumab hydrogen-bonding residues in stick models (top and bottom). Black arrows indicate examples of burosumab residues that are subject to structure-based rational design for affinity optimization. [Figure 9C] Panel A shows the FGF23 surface model interface with burosumab, shown as a ribbon and stick model. Panel B shows a more detailed view of the ionic interactions at the FGF23:Fab interface. Black arrows indicate examples of burosumab residues that are subject to structure-based rational design for affinity optimization. [Figure 9D] Panel A shows the FGF23 surface model interface with burosumab, shown as a ribbon and stick model. Panel B shows a more detailed view of the aromatic stacking interactions at the FGF23:Fab interface. Black arrows indicate examples of burosumab residues that are subject to structure-based rational design for affinity optimization. [Figure 10] 1 shows the relevant interface positions between the burosumab light chain and human FGF23 based on the crystal structure of the FGF23:Fab complex. [Figure 11] 1 shows the relevant interface positions between the burosumab heavy chain and human FGF23 based on the crystal structure of the FGF23:Fab complex. [Figure 12]Figure 6 shows a ribbon model of the FGF23:Fab complex shown in Figure 6 (leftmost column), highlighting specific amino acid substitutions hypothesized to improve the affinity of the variant antibody compared to the parent burosumab. Burosumab light chain residues are shown in green, and heavy chain residues are shown in yellow. FGF23 residues are shown in purple. The stick model interface between burosumab and FGF23 highlights selected paratope residues that, when modified alone or in combination, conferred increased affinity for FGF23 as measured by SPR. Potential newly formed interactions that these modified residues form with nearby FGF23 epitope residues are listed in each box. DETAILED DESCRIPTION OF THE INVENTION

[0065] The present disclosure provides a human FGF23-binding antibody that has increased affinity for human FGF23 compared to burosumab. The antibody of the present disclosure may enable reduced administration frequency, increased convenience and patient compliance, and improved efficacy compared to burosumab, while maintaining an acceptable toxicity and safety profile. To improve the affinity of burosumab for human FGF23, antibody variants were rationally designed to have increased human FGF23 binding affinity compared to burosumab.

[0066] I. Definition Throughout this specification, numerical ranges are provided for specific quantities. These ranges should be understood to include endpoints and all subranges therein, including each integer within and between the disclosed ranges. Thus, a range of "50 to 80" includes all possible ranges therein (e.g., 51 to 79, 52 to 78, 53 to 77, 54 to 76, 55 to 75, 60 to 70, etc.), and individual integers between 50 and 80 (e.g., 50, 51, 52, 53, 54, etc.). When ranges are provided in fractional, percentage, decimal, or other form, such ranges similarly include all possible subranges therein and individual fractional, percentage, decimal, etc., within and between the disclosed ranges. For example, the range "0.1 to 1.0" includes all possible ranges therein (e.g., 0.2 to 0.9, etc.) and individual tenths of a decimal point between 0.1 and 1.0 (e.g., 0.1, 0.2, 0.3, 0.4, etc.). Furthermore, all values ​​within a given range may be endpoints of the range encompassed thereby (e.g., the range 50 to 80 includes ranges having endpoints such as 55 to 80, 50 to 75, etc.).

[0067] The terms "a" or "an" refer to one or more of that entity; for example, "androgen receptor modulator" refers to one or more androgen receptor modulators or at least one androgen receptor modulator. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. Furthermore, reference to an "inhibitor" by the indefinite article "a" or "an" does not exclude the possibility that more than one inhibitor is present, unless the context clearly requires that only one of that inhibitor be present.

[0068] The term "affinity" (or "binding affinity") refers to the strength of binding of a molecule or complex of molecules to a binding partner, e.g., a ligand, or epitope, or antigen. Affinity is measured by the dissociation constant (K), which describes the rate of dissociation of a molecule or complex and its binding partner at equilibrium. D ) can be expressed in terms of K Dis the ligand association rate constant (k on ) and dissociation rate constant (k off ) ratio, or K D =k off / k on With respect to antibody / antigen binding affinity, the rate of antibody / antigen complex formation at equilibrium is equal to the rate of dissociation of the complex to unbound antibody and antigen, and the affinity of the antibody can be expressed as 1 / K D and so that a small K D A lower value reflects a higher affinity of the antibody for its target antigen. Methods for determining binding affinity are known and include, for example, enzyme-linked immunosorbent assay (ELISA) approaches and surface plasmon resonance (SPR)-based approaches.

[0069] As used herein, the term "Antibody Structure Numbering System" or "ASN#" refers to a numbering scheme for structural alignment and classification of complete sequences derived from antibodies and antibody-like structures. A description of the ASN# system is provided at www.just-evotecbiologics.com / white-papers-application-notes / coreab-sequence-classification / (last visited July 5, 2023; incorporated herein by reference in its entirety).The ASN# system is a variation of the Honneger numbering scheme, i.e., the "AHo" numbering scheme (e.g., Honnegger, Annemarie, and Andreas PluEckthun, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," Journal of molecular biology 309.3 (2001):657-670; incorporated herein by reference in its entirety), which "numbers the variable domains of the immunoglobulin superfamily in a homogenized format ... based on structural alignments of the 3D structures of the immunoglobulin variable regions covering the observed length variation" (Dondelinger, Mathieu, et al.), "Understanding the significance and implications of antibody numbering and antigen-binding surface / residue The definition is based on "Understanding the significance and impact of antibody numbering and antigen-binding surface / residue definitions." Frontiers in immunology 9(2018):2278) and is substantially identical.

[0070] The verb "comprise" as used in this specification and claims and in its conjugations is used in an open-ended sense to mean that the items following the word are included but not to exclude items not specifically mentioned.

[0071] The terms "treating" or "treatment" refer to one or more of relieving, alleviating, delaying, alleviating, improving, or managing at least one symptom of a condition in a subject. Treating can also mean one or more of halting a condition, delaying the onset of a condition (i.e., the period before clinical symptoms of the condition), or reducing the risk of developing or worsening a condition.

[0072] The term "subject" can refer to humans, non-human primates, mammals, rats, mice, cows, horses, pigs, sheep, goats, dogs, cats, etc. The subject may be suspected of having, or at risk for, a hypophosphatemic condition such as XLH or TIO, or may be diagnosed with a hypophosphatemic condition such as XLH or TIO.

[0073] This disclosure includes information that may be useful in understanding the subject matter of the present disclosure. None of the information provided herein is an admission that it is prior art or relevant to the presently claimed invention, or that any specifically or implicitly referenced publication is prior art.

[0074] II. Overview Burosumab therapy effectively improves the clinical symptoms of hypophosphatemia. Numerous clinical trials have established burosumab as a safe and effective treatment for adults and children with X-linked hypophosphatemia (XLH) (Schindler, et al., "Clinical evidence for the benefits of burosumab therapy for X-linked hypophosphatemia (XLH) and other conditions in adults and children," Frontiers in Endocrinology 11 (2020):338). Further studies have confirmed the safe and effective treatment of tumor-induced osteomalacia (TIO) with burosumab (Jan de Beur, et al., "Burosumab for the treatment of tumor-induced osteomalacia," Journal of Bone and Mineral Research 36.4 (2021):627-635).

[0075] However, treatment with burosumab only restored serum phosphorus levels to 3.3 mg / dL after 64 weeks, close to the lower limit of normal (3.2 mg / dL) seen in healthy populations (see, e.g., Figures 1 and 2 in the Health Sciences Authority of Singapore's Summary Report of the Benefit-Risk Assessment Related to the Novel Drug Application of Crysvita, available at www.hsa.gov.sg / docs / default-source / hprg-tpb / summary-reports / crysvita_summary-report_2021.pdf). Furthermore, treatment with burosumab requires subcutaneous injections every two weeks (Q2W) in pediatric patients, and both adult and pediatric patients must receive high-dose burosumab injections—up to 3 ml administered in three separate 1 ml injections—which represents a significant treatment burden for patients.

[0076] Thus, provided herein are human FGF23-binding antibodies that have improved binding affinity to human FGF23 compared to burosumab. Improving the binding affinity of burosumab increases the efficacy of the antibody, reduces the amount of bioavailable FGF23 in the serum, and allows for reduced dosage and / or administration frequency, thereby easing the treatment burden for patients requiring human FGF23-binding antibody therapy without significantly adversely affecting the safety profile of the treatment.

[0077] Burosumab is believed to bind to intact human FGF23 with high affinity. However, various assays using different raw materials to measure burosumab affinity have yielded different values. For example, the European Medicines Agency's Crysvita evaluation report states, "In a Biacore competitive binding study [burosumab] was shown to bind with similar binding affinity (K D about 10 -11 M) to human, cynomolgus monkey, and rabbit FGF23 (see Assessment Report: Crysvita, 14 December 2017 (EMA / 148319 / 2018), available at www.ema.europa.eu / en / documents / assessment-report / crysvita-epar-public-assessment-report_en.pdf). In our study, we used a surface plasmon resonance (SPR) approach to determine burosumab affinity using recombinant human FGF23 (R179Q) derived from HEK293S cell supernatant and burosumab produced using CHO cells, resulting in a binding affinity of approximately 1 × 10 -11 ~3×10 -11 The estimated binding affinity (K) of the magnitude (data not shown) D ) found a 4.7 × 10 antibody response to FN1, a mouse monoclonal anti-FGF23 antibody that the authors reported binds to human FGF23 in a manner similar to burosumab. -11(Yamazaki, Yuji, et al. "Anti-FGF23 neutralizing antibodies show the physiological role and structural features of FGF23." Journal of Bone and Mineral Research 23.9 (2008): 1509-1518.) Another group used ELISA to determine the affinity of burosumab using recombinant human FGF23 expressed in E. coli and burosumab expressed in HEK293 cells, and found that the affinity was approximately 0.5 × 10 -9 or 0.77 × 10 -9 (Kanhasut, et al., “Prediction of the structural interface between fibroblast growth factor 23 and burosumab using alanine scanning and molecular docking,” 16 May 2022, PREPRINT (Version 1) (available at Research Square [doi.org / 10.21203 / rs.3.rs-1630525 / v1]). The inconsistent results from studies evaluating burosumab affinity highlight the need for comprehensive studies of the binding kinetics of this monoclonal antibody to human FGF23.

[0078] Kanhasut et al. further explored approaches to improve burosumab's binding affinity for FGF23. Using alanine scan data and molecular docking, the authors modeled the interaction between homology-modeled burosumab and the crystal structure of FGF23 and identified four amino acid changes on the burosumab light chain variable domain (VL) that were predicted to enhance the interaction with the epitope: A32S, S52D, S67Y, and T69D (Kabat numbering scheme). Based on the original alanine scan data, the authors also investigated a fifth change on the heavy chain variable domain (VH), V97A (Kabat numbering scheme). This study demonstrated that the K in burosumab variant antibodies with both the amino acid changes VH:V97A (i.e., V101A according to the linear numbering scheme) and VL:A32S (i.e., A32S according to both the Kabat and linear numbering schemes) was improved. D =0.21×10 -9 Data were provided supporting an approximately three-fold enhancement in FGF23 binding affinity of .

[0079] However, the Kanhasut et al. study has several flaws that call into question the validity and biological significance of its conclusions. For example, Kanhasut et al. used FGF23 expressed in E. coli cells, which means that the antigenic protein was not glycosylated in a manner similar to that found in mammalian cell-based systems. Furthermore, K D For the determination, Kanhasut et al. utilized a titration ELISA approach, which is recognized as having high variability and not the most accurate assay for affinity measurement. DThis lack of consistency in the assays supports a high degree of variability and calls into question the ultimate accuracy and significance of the study. Finally, structural evaluation of the burosumab:FGF23 complex described herein suggests that the epitope residues identified by Kanhasut et al. do not match those identified in the crystal structure. Indeed, we performed surface plasmon resonance analysis of amino acid substitutions in VH:V97A and VL:A32S (i.e., heavy chain V101A and light chain A32S, respectively, according to the linear numbering scheme) and demonstrated that these changes impair function rather than improve burosumab affinity (data not shown).

[0080] These and other studies have demonstrated that the K D It is clear that more detailed investigation of the binding kinetics of burosumab, including the determination of the epitope, and precise epitope mapping are needed, along with new approaches to improve the binding affinity of burosumab. Importantly, when designing antibodies with improved binding affinity compared to burosumab, it is important to fine-tune the affinity for human FGF23 to improve efficacy without compromising safety. For example, if the affinity is excessively increased, a decrease in the subject's serum FGF23 concentration may lead to unsafe serum phosphorus levels and symptoms of hyperphosphatemia. On the other hand, if the affinity is only slightly improved, no clinical benefit may be achieved compared to treatment with burosumab. Therefore, rationally designed anti-human FGF23 antibodies with fine-tuned binding affinity to increase efficacy without causing unsafe serum phosphorus levels or clinical symptoms of hyperphosphatemia are needed.

[0081] The present disclosure provides rationally designed human FGF23 binding antibodies that have increased affinity for human FGF23 compared to burosumab. The rational design of human FGF23-binding antibodies described herein can include, for example, crystallographic analysis of the burosumab-human FGF23 interaction and / or in silico modeling involving any of the following: conformational sampling using molecular dynamics and selection of an ensemble of target conformations representing docking templates; generation and energy minimization of homology models of lead antibody(ies) (selecting likely conformational states); guided molecular docking of the lead antibody model to the FGFR23 structure (e.g., epitopes selected based on HDX data); determination of paratopes and epitopes based on the docking results; evaluation and ranking of antigen-antibody binding interactions (e.g., first shell paratope:epitope and second shell); comparison with recently published results describing burosumab:FGF23 binding (Kanhasut et al., Nature (2022) 12:14754); evaluation of any additional modifications required (i.e., stability, developability); and / or evaluation of post-translational modifications and related improvements. From these research studies, individual or multiple amino acid changes can be introduced into the burosumab backbone sequence and analyzed for binding properties. Variant antibody designs can be screened to identify burosumab variants with improved binding properties.

[0082] Thus, the present disclosure provides human FGF23 binding antibodies with increased affinity for human FGF23 compared to burosumab, which may allow for reduced dosing frequency, increased convenience and patient compliance, and improved efficacy compared to burosumab, while maintaining an acceptable toxicity and safety profile.

[0083] To improve the affinity of burosumab for human FGF23, antibody variants were designed that have increased human FGF23 binding affinity compared to burosumab.

[0084] These and other aspects of the antibodies of the present disclosure and their uses are further described below.

[0085] III. Composition The compositions of the present disclosure comprise one or more antibodies or antigen-binding fragments thereof that bind to human FGF23. In some embodiments, the human FGF23-binding antibody is a variant of burosumab (i.e., an engineered human FGF23-binding antibody) that has been engineered to have improved affinity.

[0086] In some embodiments, the engineered human FGF23 binding antibodies provided herein may be administered to a subject for use in reducing serum FGF23 in the subject.

[0087] In some embodiments, the engineered human FGF23 binding antibodies provided herein may be administered to a subject for use in lowering the renal maximum threshold (Tmp) for phosphorus reabsorption in the subject.

[0088] In some embodiments, the engineered human FGF23 binding antibodies provided herein may be administered to a subject for use in increasing serum inorganic phosphorus (Pi) in the subject.

[0089] In some embodiments, the engineered human FGF23 binding antibodies provided herein may be administered to a subject for use in increasing serum 1,25 dihydroxyvitamin D (1,25[OH]2D) concentrations in the subject.

[0090] In some embodiments, the engineered human FGF23 binding antibodies provided herein are useful for treating hypophosphatemic conditions in subjects with osteomalacia, such as XLH or tumor-induced osteomalacia (TIO).

[0091] In some embodiments, the engineered human FGF23 binding antibodies provided herein have increased affinity for human FGF23 compared to burosumab.

[0092] In some embodiments, the binding affinity of the antibodies provided herein for human FGF23 is determined by the K D , approximately 1 x 10 -12 ~Approx. 7×10 -12 This is increased compared to burosumab by reducing

[0093] In some embodiments, the binding affinity of the antibodies provided herein for human FGF23 is increased by about 2-fold to about 10-fold compared to burosumab. For example, the binding affinity of the antibodies provided herein for human FGF23 can be increased by about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, or about 10-fold compared to burosumab.

[0094] In some embodiments, the increased binding affinity of the antibodies provided herein for human FGF23 compared to burosumab results in a reduction in free FGF23 in a subject compared to the reduction in free FGF23 observed with burosumab treatment in a subject. Free FGF23 refers to bioavailable FGF23. The binding of FGF23 by the antibodies provided herein can reduce the amount of free FGF23 in a subject by at least about 5% or at least about 10% compared to burosumab.

[0095] The engineered human FGF23-binding antibodies described herein may have one or more amino acid substitutions compared to the amino acid sequence of burosumab. For example, the engineered FGF23-binding antibodies described herein may include one or more amino acid substitutions in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1). The engineered FGF23-binding antibodies described herein may include one or more amino acid substitutions in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2). For example, the engineered FGF23-binding antibodies described herein may include one or more amino acid substitutions in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1) and one or more amino acid substitutions in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2).

[0096] The engineered human FGF23 binding antibodies described herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions compared to the amino acid sequence of burosumab. For example, the engineered FGF23 binding antibodies described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1). The engineered FGF23 binding antibodies described herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2). The engineered FGF23 binding antibodies described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1), and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2).

[0097] The engineered human FGF23 binding antibodies described herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid deletions compared to the amino acid sequence of burosumab. For example, the engineered FGF23 binding antibodies described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid deletions in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1). The engineered FGF23 binding antibodies described herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid deletions in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2). The engineered FGF23 binding antibodies described herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid deletions in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1), and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid deletions in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2).

[0098] The engineered human FGF23 binding antibodies described herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid insertions compared to the amino acid sequence of burosumab. For example, the engineered FGF23 binding antibodies described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid insertions in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1). The engineered FGF23 binding antibodies described herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid insertions in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2). The engineered FGF23 binding antibodies described herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid insertions in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1), and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid insertions in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2).

[0099] The engineered human FGF23 binding antibodies described herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid changes compared to the amino acid sequence of burosumab, where the changes include a combination of substitutions, deletions, and / or insertions compared to the amino acid sequence of burosumab. For example, the engineered FGF23 binding antibodies described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid changes in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1), where the changes include a combination of substitutions, deletions, and / or insertions compared to the heavy chain sequence of burosumab (SEQ ID NO: 1). The engineered human FGF23 binding antibodies described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid changes in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2), where the changes include a combination of substitutions, deletions, and / or insertions compared to the light chain sequence of burosumab (SEQ ID NO: 2). The engineered FGF23 binding antibodies described herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid changes in the heavy chain sequence compared to the heavy chain sequence of burosumab (SEQ ID NO: 1), and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid changes in the light chain sequence compared to the light chain sequence of burosumab (SEQ ID NO: 2), where the changes include a combination of substitutions, deletions, and / or insertions compared to the heavy and light chain sequences of burosumab (SEQ ID NO: 1 and SEQ ID NO: 2, respectively).

[0100] In some embodiments, the engineered human FGF23 binding antibodies described herein contain one or more amino acid substitutions, deletions, and / or insertions (collectively, "alterations") compared to the burosumab heavy chain sequence and / or the amino acid sequence of burosumab, wherein the one or more amino acid changes affect one or more burosumab paratope residues and / or one or more residues adjacent to a burosumab paratope residue. Exemplary burosumab paratope regions and paratope residues are shown in Table 4 herein. The burosumab residues adjacent to a burosumab paratope residue may include residues within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the burosumab paratope residue.

[0101] In some embodiments, the engineered human FGF23 binding antibodies described herein comprise one or more light chain variable region amino acid changes shown in Table 13.

[0102] In some embodiments, the engineered human FGF23 binding antibodies described herein comprise one or more heavy chain variable region amino acid changes shown in Table 14.

[0103] In some embodiments, the engineered human FGF23 binding antibodies described herein contain two amino acid changes according to those shown in Table 15.

[0104] In some embodiments, the engineered human FGF23 binding antibody provided herein is one of UGX101-UGX196 listed in Tables 16 and 17.

[0105] Amino acid changes in specific engineered FGF23-binding antibodies The engineered FGF23 binding antibodies described herein may comprise one or more amino acid changes in the heavy chain variable region (VH) compared to SEQ ID NO: 3, wherein the one or more amino acid changes are T30E, T30F, T30Q, T30S, T30Y, N31D, N31E, N31F, N31Q, N31R, N31Y, H32E, H32F, H32Q, H32S, H32T, I50D, I50E, I50F, I50S, I50T, I50Y, N52E, N52F, N52Q, N52Y, I54E, I54F, I54L, I54Q, I54S, I54T, I54V ... 54Y, S55E, S55F, S55Q, S55T, S55Y, S59D, S59E, S59F, S59I, S59L, S59Q, S59T, S59V, S59Y, D99E, D99Q, D99Y, I100D, I100E, I100F, I100L, I100Q, I100V, I100Y, V101F, V101I, V101L, V101T, V101Y, D102E, D102F, D102I, D102L, D102Q, D102Y, A103D, A103E, A103F, A103Q, A103S, A103T, and A103Y.

[0106] The engineered FGF23 binding antibodies described herein may comprise one or more amino acid changes in the light chain variable region (VL) compared to SEQ ID NO: 4, wherein the one or more amino acid changes are A25L, A25V, I29F, I29L, I29V, S30D, S30E, S30F, S30I, S30L, S30Q, S30T, S30V, S30Y, S31D, S31E, S31F, S31I, S31L, S31Q, S31T, S31V, S31Y, A32F, A32I, A32L, A32T, A32V, A32Y, V34F, V34I, V34L, V34Y, D50E, D50F, D50I, D50L, D50Q, D50R, D50S, D50T, D50V, A51F, A51I, A51L, A51S, A51T, A51V, S52D, S52E, S5 2F, S52Q, S52T, S52Y, S53D, S53E, S53F, S53I, S53L, S53Q, S53R, S53T, S53V, S53Y , Q90F, Q90Y, F91K, F91R, F91Y, N92D, N92E, N92F, N92Q, N92S, N92T, N92Y, D93E, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94D, Y94E, Y94F, Y94Q, Y94S, and Y94T.

[0107] The engineered FGF23 binding antibodies described herein may comprise one or more amino acid changes in the heavy chain variable region (VH) and light chain variable region (VL) compared to SEQ ID NO:3 and SEQ ID NO:4, respectively, wherein the one or more amino acid changes in the VH are T30E, T30F, T30Q, T30S, T30Y, N31D, N31E, N31F, N31Q, N31R, N31Y, H32E, H32F, H32Q, H32S, H32T, I50D, I50E, I50F, I50S, I50T, I50Y, N52E, N52F, N52Q, N52Y, I54E, I54F, I54G, I54H ... 4F, I54L, I54Q, I54S, I54T, I54V, I54Y, S55E, S55F, S55Q, S55T, S55Y, S59D , S59E, S59F, S59I, S59L, S59Q, S59T, S59V, S59Y, D99E, D99Q, D99Y, I100D, I 100E, I100F, I100L, I100Q, I100V, I100Y, V101F, V101I, V101L, V101T, V10 1Y, D102E, D102F, D102I, D102L, D102Q, D102Y, A103D, A103E, A103F, A103Q, and one or more amino acid changes in the VL are selected from A103S, A103T, and A103Y, and wherein the one or more amino acid changes in the VL are selected from A25L, A25V, I29F, I29L, I29V, S30D, S30E, S30F, S30I, S30L, S30Q, S30T, S30V, S30Y, S31D, S31E, S31F, S31I, S31L, S31Q, S31T, S31V, S31Y, A32F, A32I, A32L, A32T, A32V, A32Y, V34F, V34I, V34L, V34Y, D50E, D50F, D50I, D50L, D50Q, D50R, D50S, D50T , D50V, A51F, A51I, A51L, A51S, A51T, A51V, S52D, S52E, S52F, S52Q, S52T, S52Y, S53D, S53E, S53F, S53I, S53L, S53Q, S53R, S53T, S53V, S53Y, Q90F, Q90Y, F91K, F91R, F91Y, N92D, N92E, N92F, N92Q, N92S, N92T, N92Y, D93E, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94D, Y94E, Y94F, Y94Q, Y94S, and Y94T.

[0108] The engineered FGF23 binding antibodies described herein may comprise at least two amino acid changes in the heavy chain variable region (VH) compared to SEQ ID NO:3, in the light chain variable region (VL) compared to SEQ ID NO:4, or in the VH compared to SEQ ID NO:3 and in the VL compared to SEQ ID NO:4, wherein the at least two amino acid changes are VL:S31E / VL:D50F, VH:N31F / VH:S59F, VL:D50F / VL:S53E, VL:S31F / VL:S52E, VL:D50F / VH:V101I, VH:S59D / VH:V101I, VL:S31F / VL:S53E, VL:N92E / VL:D93F, VL:S31E / VL:S 53E, VL:S31F / VL:D50F, VH:N31E / VH:S59E, VH:S57E / VH:S59D, VH:N31E / VH:I54F, VH:S 59T / VH:V101L, VH:I54Y / VH:V101F, VH:N52F / VH:V101I, VL:N92D / VL:D93F, VH:N52F / V VL:Y94D / VH:S59D, VL:Y94D / VH:S59E, VL:N92F / VH:S59E, VL:N92Y / VH:S59E, VL:Y94E / VH:S59E, VL:S52D / VH:S59E, and VL:S52E / VH:S59E.

[0109] In some embodiments, an engineered FGF23 binding antibody described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, the antibody or fragment thereof comprising a VH amino acid sequence according to SEQ ID NO:3 and a VL amino acid sequence according to SEQ ID NO:4, wherein the VH amino acid sequence comprises one or more amino acid changes selected from I54F, I54Y, S59D, S59E, V101I, and A103S compared to SEQ ID NO:3.

[0110] 21. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a VH amino acid sequence according to SEQ ID NO: 3 and a VL amino acid sequence according to SEQ ID NO: 4, wherein the VL amino acid sequence comprises one or more amino acid changes selected from S52D, S52E, N92F, N92Y, Y94D, and Y94E compared to SEQ ID NO: 4.

[0111] In some embodiments, the engineered FGF23 binding antibodies described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-001 and a light chain variable region (VL) according to VL-027 (UGX126).

[0112] In some embodiments, the engineered FGF23 binding antibodies described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-001 and a light chain variable region (VL) according to VL-066 (UGX144).

[0113] In some embodiments, the engineered FGF23 binding antibodies described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-001 and a light chain variable region (VL) according to VL-067 (UGX145).

[0114] In some embodiments, the engineered FGF23 binding antibodies described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-028 and a light chain variable region (VL) according to VL-001 (UGX156).

[0115] In some embodiments, the engineered FGF23 binding antibodies described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-029 and a light chain variable region (VL) according to VL-001 (UGX157).

[0116] In some embodiments, the engineered FGF23 binding antibodies described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-061 and a light chain variable region (VL) according to VL-001 (UGX175).

[0117] In some embodiments, an engineered FGF23 binding antibody described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-055 and a light chain variable region (VL) according to VL-001 (UGX182).

[0118] In some embodiments, the engineered FGF23 binding antibody described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-050 (UGX201).

[0119] In some embodiments, the engineered FGF23 binding antibody described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-054 (UGX202).

[0120] In some embodiments, the engineered FGF23 binding antibody described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-027 (UGX203).

[0121] In some embodiments, the engineered FGF23 binding antibody described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-030 (UGX204).

[0122] In some embodiments, the engineered FGF23 binding antibody described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-066 (UGX205).

[0123] In some embodiments, the engineered FGF23 binding antibody described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-067 (UGX206).

[0124] In some embodiments, an engineered FGF23 binding antibody described herein may comprise an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, the antibody or fragment thereof comprising a heavy chain (HC) amino acid sequence according to SEQ ID NO: 1 and a light chain (LC) amino acid sequence according to SEQ ID NO: 2, wherein the antibody or fragment thereof comprises one or more HC amino acid changes relative to SEQ ID NO: 1 and / or one or more LC amino acid changes relative to SEQ ID NO: 2, wherein the one or more HC amino acid changes are T28Y, T30F, T30K, N31D, N31E, N31F, N31Q, N31R, H32K, H32Y, Y33D, Y33L, Y33R, N52E, N52F, N52Q, N52R, N52Y, I54A, I54D, I54F, I54G, I54H ... 4K, I54L, I54M, I54N, I54P, I54R, I54V, I54W, I54Y, S55E, S55F, S55I, S55Q , S55R, S55Y, S57E, S57F, S57R, S57Y, T58F, T58R, S59A, S59D, S59E, S59F, S5 9G, S59H, S59I, S59K, S59L, S59M, S59N, S59P, S59Q, S59R, S59T, S59V, S59W , S59Y, N60E, N60R, N60Y, A61F, A61R, A61Y, Q62L, Q62R, Q62Y, Q65E, Q65R, Q6 5Y, D99F, D99R, D99Y, I100D, I100E, I100F, I100Y, V101A, V101D, V101E, V1 01F, V101G, V101H, V101I, V101K, V101L, V101M, V101N, V101P, V101Q, V101R , V101S, V101W, D102E, D102F, D102K, D102R, D102Y, A103D, A103F, A103G, A103H, A103I, A103K, A103L, A103M, A103N, A103P, A103R, A103S, A103T, A103V, and A103W, and the one or more LC amino acid changes are selected from Q27F, Q27R, G28D, G28Q, G28R, G28Y, I29F, I29L, S30D, S30E, S30F, S30L, S30Q, S30R, S30Y, S31D, S31E, S31F,S31Q, S31Y, A32F, A32V, V34F, V34I, D50E, D50F, D50L, D50Q, D50R, D50S, D50T, D50V, D50Y, A51V, S52A, S52D, S52E, S52F, S52G, S52H, S52I, S 52K, S52L, S52M, S52N, S52P, S52Q, S52R, S52V, S52W, S52Y, S53D, S53E , S53F, S53R, S53Y, L54E, L54Y, E55R, E55Y, S67E, S67R, S67Y, T69R, T6 9Y, F91E, F91R, F91Y, N92A, N92D, N92E, N92F, N92G, N92H, N92I, N92K, N92L, N92M, N92P, N92Q, N92R, N92S, N92T, N92V, N92W, N92Y, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94A, Y94D, Y94E, Y94F, Y94G, Y94H, Y94I, Y94K, Y94L, Y94M, Y94N, Y94P, Y94Q, Y94R, Y94S, Y94V, and Y94W.

[0125] 36. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, the antibody or fragment thereof comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 3 and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 4, wherein the antibody or fragment thereof comprises one or more VH amino acid changes relative to SEQ ID NO: 3 and / or one or more VL amino acid changes relative to SEQ ID NO: 4, wherein the one or more VH amino acid changes are T28Y, T30F, T30K, N31D , N31E, N31F, N31Q, N31R, H32K, H32Y, Y33D, Y33L, Y33R, N52E, N52F, N52Q, N 52R, N52Y, I54A, I54D, I54F, I54G, I54H, I54K, I54L, I54M, I54N, I54P, I54 R, I54V, I54W, I54Y, S55E, S55F, S55I, S55Q, S55R, S55Y, S57E, S57F, S57R, S57Y, T58F, T58R, S59A, S59D, S59E, S59F, S59G, S59H, S59I, S59K, S59L, S59 M, S59N, S59P, S59Q, S59R, S59T, S59V, S59W, S59Y, N60E, N60R, N60Y, A61F, A61R, A61Y, Q62L, Q62R, Q62Y, Q65E, Q65R, Q65Y, D99F, D99R, D99Y, I100D, I 100E, I100F, I100Y, V101A, V101D, V101E, V101F, V101G, V101H, V101I, V10 1K, V101L, V101M, V101N, V101P, V101Q, V101R, V101S, V101W, D102E, D102F, and the one or more VL amino acid changes are selected from D102K, D102R, D102Y, A103D, A103F, A103G, A103H, A103I, A103K, A103L, A103M, A103N, A103P, A103R, A103S, A103T, A103V, and A103W, and the one or more VL amino acid changes are selected from Q27F, Q27R, G28D, G28Q, G28R, G28Y, I29F, I29L, S30D, S30E, S30F, S30L, S30Q, S30R, S30Y, S31D, S31E, S31F, S31Q, S31Y, A32F, A32V, V34F,V34I, D50E, D50F, D50L, D50Q, D50R, D50S, D50T, D50V, D50Y, A51V, S52A, S52D, S52E, S52F, S52G, S52H, S52I, S52K, S52L, S52M, S52N , S52P, S52Q, S52R, S52V, S52W, S52Y, S53D, S53E, S53F, S53R, S53Y, L54E, L54Y, E55R, E55Y, S67E, S67R, S67Y, T69R, T69Y, F91E, F91R , F91Y, N92A, N92D, N92E, N92F, N92G, N92H, N92I, N92K, N92L, N92M, N92P, N92Q, N92R, N92S, N92T, N92V, N92W, N92Y, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94A, Y94D, Y94E, Y94F, Y94G, Y94H, Y94I, Y94K, Y94L, Y94M, Y94N, Y94P, Y94Q, Y94R, Y94S, Y94V, and Y94W.

[0126] Complementarity-determining regions of specific engineered FGF23-binding antibodies Burosumab has complementarity determining regions (CDRs) defined as follows: a heavy chain CDR1 having the amino acid sequence NHYMH (SEQ ID NO: 11) (referred to herein as HCDR1-001); a heavy chain CDR2 having the amino acid sequence IINPISGSTSNAQKFQG (SEQ ID NO: 12) (referred to herein as HCDR2-001); a heavy chain CDR3 having the amino acid sequence DIVDAFDF (SEQ ID NO: 13) (referred to herein as HCDR3-001); a light chain CDR1 having the amino acid sequence RASQGISSALV (SEQ ID NO: 14) (referred to herein as LCDR1-001); a light chain CDR1 having the amino acid sequence DASSLES (SEQ ID NO: 15) (referred to herein as LCDR2-001); and a light chain CDR1 having the amino acid sequence QQFNDYFT (SEQ ID NO: 16) (referred to herein as LCDR3-001).

[0127] The engineered FGF23-binding antibodies described herein may contain one or more unique complementarity-determining regions (CDRs) compared to those of burosumab, i.e., the engineered FGF23-binding antibodies may have one or more amino acid changes compared to SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16.

[0128] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain complementarity-determining region 1 (HCDR1) according to an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NOs: 22-32.

[0129] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain complementarity-determining region 2 (HCDR2) according to an amino acid sequence selected from SEQ ID NO: 12 and SEQ ID NOs: 33-65.

[0130] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain complementarity-determining region 3 (HCDR3) according to an amino acid sequence selected from SEQ ID NO: 13 and SEQ ID NOs: 66-93.

[0131] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain complementarity-determining region 1 (LCDR1) according to an amino acid sequence selected from SEQ ID NO: 14 and SEQ ID NOs: 94-126.

[0132] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain complementarity-determining region 2 (LCDR2) according to an amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NOs: 127-158.

[0133] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain complementarity-determining region 3 (LCDR3) according to an amino acid sequence selected from SEQ ID NO: 16 and SEQ ID NOs: 159-185.

[0134] In some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NOs: 22-32; the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 12 and SEQ ID NOs: 33-65; the HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 13 and SEQ ID NOs: 66-93; the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 14 and SEQ ID NOs: 94-126; the LCDR2 comprises an amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NOs: 127-158; and the LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 16 and SEQ ID NOs: 159-185.

[0135] Variable regions of specific engineered FGF23-binding antibodies Burosumab has a heavy chain variable region (VH) and a light chain variable region (VL) as follows: VH of amino acid sequence SEQ ID NO: 3 (referred to herein as VH-001), and VL of amino acid sequence SEQ ID NO: 4 (referred to herein as VL-001).

[0136] The FGF23-binding antibodies described herein may have one or more unique variable region domains compared to burosumab, i.e., the FGF23-binding antibodies may have one or more amino acid changes compared to the VH and / or VL sequences of burosumab.

[0137] For example, the isolated antibody or fragment thereof may comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, or the VL comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0138] The isolated antibody or fragment thereof may comprise a VH comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, or the antibody or fragment thereof may comprise a VL comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0139] The isolated antibody or fragment thereof may comprise a VH comprising an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, or the antibody or fragment thereof may comprise a VL comprising an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0140] The isolated antibody or fragment thereof may comprise a VH and a VL, wherein the VH may comprise an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 3, and SEQ ID NOs: 186 to 270, and the VL may comprise an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0141] The isolated antibody or fragment thereof may comprise a VH and a VL, wherein the VH may comprise an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, and the VL may comprise an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0142] The isolated antibody or fragment thereof may comprise a VH and a VL, wherein the VH may comprise an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, and the VL may comprise an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0143] The isolated antibody or fragment thereof may comprise a VH and a VL, and the VH may comprise an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, or the VL may comprise an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0144] The isolated antibody or fragment thereof may comprise a VH and a VL, wherein the VH may comprise an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186 to 270, and the VL may comprise an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

[0145] Specific engineered FGF23-binding antibodies with dual amino acid changes The FGF23 binding antibody described herein may have one or more amino acid changes compared to the amino acid sequence of burosumab.For example, the FGF23 binding antibody may have at least two amino acid changes compared to burosumab.In some embodiments, the FGF23 binding antibody has one amino acid change in the heavy chain variable region compared to burosumab and one amino acid change in the light chain variable region compared to burosumab; two amino acid changes in the heavy chain variable region compared to burosumab; or two amino acid changes in the light chain variable region compared to burosumab.

[0146] For example, in some embodiments, the present disclosure provides an isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO:3 and a light chain variable region (VL) according to SEQ ID NO:4, but the VH and VL sequences comprise at least two amino acid changes, the at least two amino acid changes in the VH and VL sequences being VL:S31E / VL:D50F, VH:N31F / VH:S59F, VL:D50F / VL:S53E, VL:S31F / VL:S52E, VL:D50F / VH:V101I, VH:S59D / VH:V101I, VL:S31F / VL:S5 3E, VL:N92E / VL:D93F, VL:S31E / VL:S53E, VL:S31F / VL:D50F, VH:N31E / VH:S59E, VH:S57E / VH:S5 9D, VH:N31E / VH:I54F, VH:S59T / VH:V101L, VH:I54Y / VH:V101F, VH:N52F / VH:V101I, VL:N92D / VL :D93F, VH:N52F / VH:V101L, VL:S31F / VL:D50E, VL:Y94D / VH:S59D, VL:Y94D / VH:S59E, VL:N92F / VH:S59E, VL:N92Y / VH:S59E, VL:Y94E / VH:S59E, VL:S52D / VH:S59E, and VL:S52E / VH:S59E.

[0147] Leader sequence The engineered FGF23 binding antibodies described herein may comprise a leader sequence at the N-terminus of the light chain and / or the N-terminus of the heavy chain. In some embodiments, the leader sequence is a self-cleaving leader sequence. In some embodiments, the leader sequence facilitates expression, transport, stability, secretion, and / or purification of the heavy and / or light chain when expressed in a cell. In some embodiments, the leader sequence is a secretion signal to improve extracellular transport of the heavy and / or light chain.

[0148] In certain embodiments, the engineered FGF23 binding antibodies described herein may comprise a leader sequence at the N-terminus of the heavy chain, which leader sequence comprises the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 369).

[0149] In certain embodiments, the engineered FGF23 binding antibodies described herein may comprise a leader sequence at the N-terminus of the light chain, which leader sequence comprises the amino acid sequence MVSSAQFLGLLLLCFQGTRC (SEQ ID NO: 370).

[0150] C-terminal lysine clipping The heavy chain of burosumab has a C-terminal lysine residue. It is known that the C-terminal lysine residue of antibody heavy chains is often clipped during in vivo production by carboxypeptidases present in cell culture medium or cell culture media. This phenomenon can result in process-related charge variants and variable amounts of non-uniform antibody preparations, thereby complicating attempts to manufacture uniform, safe, and effective antibody lots (see, e.g., Faid et al., Eur J Pharm Sci., 2021 Apr 1:159:105730). Therefore, in some embodiments, the antibody variants described herein are modified to remove the lysine residue at the C-terminus of the heavy chain. That is, any heavy chain antibody sequence provided herein, including the parent burosumab heavy chain sequence (SEQ ID NO: 1), may be further modified to remove the C-terminal lysine residue (SEQ ID NO: 371). Therefore, all variant heavy chain variable region (VH) sequences described in this application are intended to be incorporated into full-length heavy chain sequences, regardless of the presence or absence of a C-terminal lysine residue.

[0151] IV. Pharmaceutical Preparations The human FGF23-binding antibodies described herein may be provided in a pharmaceutical formulation. The pharmaceutical formulation may be administered locally or systemically. In certain embodiments, administration may be by any route, including intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, cutaneous, oral, inhalation, or nasal administration.

[0152] Pharmaceutical formulations of the present disclosure may include carriers, diluents, or excipients known in the art. Examples of pharmaceutical formulations and methods are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro ed. 1985), and Remington, The Science and Practice of Pharmacy, 21st Edition (2005).

[0153] Examples of excipients for pharmaceutical formulations include antioxidants, suspending agents, dispersing agents, preservatives, buffers, isotonicity agents, and surfactants.

[0154] In certain embodiments, the pharmaceutical formulation of the present disclosure is a solution for subcutaneous injection comprising a human FGF23 binding antibody provided herein and one or more pharmaceutically acceptable excipient(s) selected from L-histidine, D-sorbitol E420, polysorbate 80, L-methionine, hydrochloric acid (for pH adjustment), and / or water.

[0155] V. Method of Administration Burosumab is indicated for subcutaneous use only. In pediatric patients with XLH aged 6 months and older, the starting dose regimen is 1 mg / kg body weight, rounded to the nearest mg, administered every 2 weeks (Q2W) for patients weighing less than 10 kg. In patients with XLH aged 6 months and older who weigh 10 kg or more, the starting dose regimen is 0.8 mg / kg body weight, rounded to the nearest mg, administered every 2 weeks (Q2W). The minimum starting dose is 10 mg, and the maximum dose is 90 mg. The dose may be increased to approximately 2 mg / kg (maximum 90 mg), administered every 2 weeks (Q2W) until serum phosphorus normalizes. In adult patients with XLH, the dose regimen is 1 mg / kg body weight, rounded to the nearest 10 mg, administered every 4 weeks (Q4W) with a maximum dose of 90 mg. For pediatric TIO patients 2 years of age and older, the starting dose is 0.4 mg / kg body weight every 2 weeks (Q2W), rounded to the nearest 10 mg. The dose may be increased to 2 mg / kg every 2 weeks (Q2W) not to exceed 180 mg. For adult TIO patients, the starting dose is 0.5 mg / kg every 4 weeks (Q4W). The dose may be increased to 2 mg / kg every 2 weeks (Q2W) not to exceed 180 mg.

[0156] The antibodies of the present disclosure may be administered according to the indicated use of burosumab.

[0157] In some embodiments, an antibody of the present disclosure or a pharmaceutical formulation thereof may be administered at a therapeutically effective dose, which may be an amount of an agent or formulation sufficient to elicit a therapeutic effect in a subject receiving the therapeutically effective dose.

[0158] A therapeutically effective dose of a human FGF23 binding antibody provided herein, or a pharmaceutical formulation thereof, is useful for lowering serum FGF23 in a subject; increasing the renal maximal threshold for phosphorus reabsorption (TmP) in a subject; increasing serum inorganic phosphorus (Pi) in a subject; increasing serum 1,25 dihydroxyvitamin D (1,25[OH]2D) concentration in a subject; and / or treating a hypophosphatemic condition in a subject, including XLH or osteomalacia.

[0159] A therapeutically effective dose may be administered in one or more separate administrations and by different routes. As understood in the art, a therapeutically effective dose or amount is determined in large part based on the total amount of therapeutic agent contained in a pharmaceutical formulation. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit in a subject (e.g., treating, regulating, curing, preventing, and / or ameliorating a hypophosphatemia-related condition). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and / or prophylactic effect. Generally, the amount of a therapeutic agent (e.g., an antibody or functionally active fragment thereof of the present disclosure) administered to a subject in need thereof will depend on the subject's characteristics. Such characteristics include the subject's condition, disease severity, general health, age, sex, and weight. One of ordinary skill in the art will readily be able to determine appropriate dosages depending on these and other relevant factors. In addition, both objective and subjective assays may optionally be used to identify optimal dosage ranges.

[0160] The methods provided herein contemplate single and multiple administrations of therapeutically effective amounts of human FGF23-binding antibodies and pharmaceutical formulations thereof described herein. Pharmaceutical formulations containing human FGF23-binding antibodies may be administered at regular intervals depending on the nature, severity, and extent of the subject's condition (e.g., the severity of the subject's disease state and associated symptoms of hypophosphatemia). In some embodiments, therapeutically effective amounts of human FGF23-binding antibodies and pharmaceutical formulations thereof of the present disclosure may be administered periodically at regular intervals (e.g., once a year, once every six months, once every four months, once every three months, once every two months, or once a month), once every four weeks, once every three weeks, once every two weeks, weekly, daily, twice a day, three times a day, four times a day, five times a day, six times a day, or continuously. For example, therapeutically effective amounts of human FGF23-binding antibodies and pharmaceutical formulations thereof of the present disclosure may be administered weekly, once every two weeks, or monthly.

[0161] Administering a therapeutically effective amount of a human FGF23-binding antibody or a formulation thereof provided herein to a subject means administering to the subject about 0.1 milligram of antibody per kilogram of subject body weight (mg / kg) to about 100 mg / kg (subject body weight), about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8mg / kg, about 1.9mg / kg, about 2mg / kg, about 2.1mg / kg, about 2.2mg / kg, about 2.3mg / kg, about 2.4mg / kg, about 2.5mg / kg, about 2 .6mg / kg, approx. 2.7mg / kg, approx. 2.8mg / kg, approx. 2.9mg / kg, approx. 3mg / kg, approx. 3.1mg / kg, approx. 3.2mg / kg, approx. 3.3mg / kg, approx. 3.4mg / kg , about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg, or about 5 mg / kg. In some embodiments, administering a therapeutically effective amount of a human FGF23 binding antibody or formulation thereof provided herein can include administering less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, or less than 1 mg / kg to the subject.

[0162] Throughout this specification, when compositions are described as having, including, or comprising specific ingredients, or when processes and methods are described as having, including, or comprising specific steps, it is additionally assumed that compositions of the disclosure exist that consist essentially of or consist of the recited ingredients, and that processes and methods of the disclosure exist that consist essentially of or consist of the recited steps.

[0163] In this application, an element or component is considered to be included in and / or selected from a list of recited elements or components, with the understanding that the element or component may be any one of the recited elements or components, or the element or component may be selected from a group consisting of two or more of the recited elements or components.

[0164] Furthermore, it will be understood that elements and / or features of the compositions or methods described herein, whether expressly or implied herein, can be combined in various ways without departing from the spirit and scope of the present disclosure. For example, when a particular compound is referenced, that compound can be used in various embodiments of the disclosed compositions and / or methods, unless otherwise understood from the context. In other words, within this application, while embodiments are described and illustrated in a manner that facilitates writing and drafting a clear and concise application, it is intended and understood that the embodiments may be combined or separated in various ways without departing from the present teachings and disclosure(s). For example, it will be understood that all features described and illustrated herein may be applicable to all aspects of the present disclosure(s) described and illustrated herein. All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with an alternative feature serving the same, equivalent, or similar purpose.

[0165] The phrase "at least one of" will be understood to include each of the listed items following the phrase individually and various combinations of two or more of the listed items, unless otherwise understood from context and usage. The phrase "and / or" in connection with more than two listed items will be understood to have the same meaning, unless otherwise understood from context.

[0166] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents, including, is generally understood to be open-ended and open-ended, e.g., not excluding additional, unrecited elements or steps unless otherwise specifically stated or understood from the context.

[0167] The use of any and all examples or exemplary language herein, such as "for instance," "such as," "for example," "eg," or "including," is solely for the purpose of better describing the disclosure and does not limit the scope of the disclosure unless otherwise asserted. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the subject matter of the present disclosure.

[0168] It is to be understood that this disclosure is not limited to the particular methodology, procedures, materials, and reagents described, as these may vary. Also, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure as encompassed by the appended claims.

[0169] All publications, patents, and patent applications (including any drawings, sequences, and appendices contained therein) referenced throughout this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, sequence, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. [Example]

[0170] The present disclosure, generally described herein, will be more readily understood by reference to the following examples, which are included for the purpose of illustrating certain aspects and embodiments of the present disclosure, without limiting the disclosure.

[0171] Example 1. Antibody Structure Numbering (ASN#) System Various numbering schemes are used to describe antibody amino acid sequences, including linear numbering, Kabat numbering, Chothia numbering, and the antibody structure numbering (ASN#) system. The antibody residues described herein are presented according to the ASN# system and / or the linear numbering system. To facilitate conversion between these numbering schemes, Table 1 shows the parent light chain amino acid sequence of burosumab in the linear, Kabat, and ASN# systems, and Table 2 shows the parent heavy chain amino acid sequence of burosumab in the linear, Kabat, and ASN# systems. In Tables 1 and 2, the "Residue" column lists the amino acid residues from the burosumab light chain sequence of SEQ ID NO:2 and the burosumab heavy chain sequence of SEQ ID NO:1, respectively. In the "Kabat #" column, the designation "L" refers to the light chain, and the designation "H" refers to the heavy chain. In the "ASN#" column, "KV" refers to the light chain variable region, "KCnst-Ig" refers to the light chain constant region, "HV" refers to the heavy chain variable region, "HCnst-Ig" refers to the heavy chain constant region, "Fc-N" refers to the CH2 region of Fc, "Fc-C" refers to the CH3 region of Fc, "HCnst-Po" refers to residues that may be attributable to the heavy chain constant region, and "hinge" refers to residues in the hinge region.

[0172] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] Table 1-5 Table 1-6 Table 1-7

[0173] Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 2-10 [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15]

[0174] Example 2: Burosumab-Human FGF23 Epitope Mapping Hydrogen-deuterium exchange mass spectrometry (HDX-MS) measures the rate of solvent exchange, i.e., the exchange of heavier hydrogen isotopes with solvent atoms in the backbone amide bonds of a protein. This approach allows for the determination of solvent uptake by a protein by exposing the protein to a deuterated solvent for a defined period, quenching the solvent exchange reaction, and then measuring the increase in protein mass by mass spectrometry. Ligand binding events typically result in a localized reduction in deuterium uptake at the ligand interaction site, allowing, for example, for the approximate mapping of regions involved in antibody / target epitope and paratope interactions.

[0175] In an attempt to map the burosumab epitope to human FGF23, HDX-MS was performed using FGF23(R179Q) produced in HEK293S cells. Burosumab was produced in CHO cells (while the FGF23-binding antibody engineered in later examples was expressed in HEK293S cells). Antigen-antibody complexes were formed by combining burosumab and FGF23(R179Q) in solution and then diluted 1:10 in ice-cold deuterated assay buffer for 60 seconds, 600 seconds, 1800 seconds, or 3600 seconds. Briefly, the HDX-MS assay utilized PBS at pH 7.4 as the assay buffer. A quench buffer containing 1 M glycine, 4 M guanidine HCl, and 400 mM TCEP at pH 2.5 was used. 100 pmol were injected in duplicate at a 1:1 ratio of assay buffer to quench buffer. The antigen concentration at labeling was 1.5 μM, and the antibody concentration at labeling was 1 μM. The deuteration reaction was quenched using quench buffer (pH 2.5) at a 1:1 ratio, after which the solution was loaded onto a liquid chromatography (LC) column for on-column proteolytic digestion, followed by LC separation and mass spectrometry.

[0176] The results of HDX-MS are shown in Figure 1. The y-axis represents the % deuteration (%D), i.e., the peptide-length-normalized difference in deuteration between unbound FGF23 and antibody-bound FGF23. Values ​​below zero (0) reflect decreased deuteration, i.e., protection from deuteration by antibody-antigen binding. Values ​​above zero (0) reflect increased deuteration, i.e., exposure to deuteration. The x-axis represents amino acid residues 1–251 of FGF23. Colored bars represent the deuterium labeling time points for each identified peptide (black = 60 s, blue = 600 s, orange = 1800 s, magenta = 3600 s). The C-terminal region of FGF23 is not shown due to a lack of sequence coverage of the region encompassing approximately amino acid residues 158–251. Endogenous glycosylation in this region of the protein would likely prevent accurate measurement of deuterium exchange in this assay. Repeated experiments after enzymatic deglycosylation of FGF23 support this hypothesis (data not shown). Overall, three regions of strong protection (red boxes) were identified (protected region 1, protected region 2, and protected region 3), each with a low % deuteration and / or a relatively short labeling time point. Protected region 1 (Y43-Y70) had the weakest protection of the three strong protected regions. Protected region 2 (A80-R92) had stronger protection than protected region 1. Protected region 3 (L135-F157) had the strongest protection of the three.

[0177] Based on the HDX-MS data, it was hypothesized that protected regions 1–3 on FGF23 are involved in antibody binding or are protected by conformational changes in the presence of antibody.

[0178] These three protected regions were mapped onto the crystal structure of FGF23(R179Q) (the "2P39" structure; see Goetz et al., "Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members," Mol Cell Biol (2007) 27, pp. 3417-3428) to visualize the likely regions of antibody binding interaction. Figure 2 shows the 23P9 crystal structure of FGF23 with the protected regions defined according to the findings from HDX-MS analysis: amino acids 43 to 70 (protected region 1) are shown in purple, amino acids 80 to 92 (protected region 2) are shown in green, and amino acids 135 to 157 (protected region 3) are shown in red.

[0179] In summary, HDX-MS identified three FGF23(R179Q) regions that are protected in the presence of burosumab, as shown in Table 3:

[0180] [Table 3]

[0181] HDX-MS data suggest that the most strongly protected regions (2 and 3) represent a potential burosumab epitope near residues 82-90 and 136-140 of FGF23 (R179Q). Within protected region 3, residues L135, V136, S137, L138, G139, R140, and A141 represent the most strongly protected region and may therefore be among the most important residues involved in the FGF23-burosumab interaction.

[0182] Comparing these findings with previous work mapping the burosumab epitope on human FGF23, we observed that protected region 1 contains residues from FN1-epitope 1 and FN1-epitope 2 identified in Yamazaki et al., "Anti-FGF23 neutralizing antibodies show the physiological role and structural features of FGF23," Journal of Bone and Mineral Research 23.9 (2008):1509-1518. However, FN1-epitope 3 and FN1-epitope 4 lie outside the protected regions 1, 2, and 3 identified in this study, suggesting that the FN1 antibody previously characterized by SiSi et al. recognizes a different surface on FGF23 compared to burosumab. Furthermore, Kanhasut et al. (2022) identified an epitope that is largely consistent with that previously reported by Yamazaki et al., further demonstrating the importance of the comprehensive study of the present invention, in which the hFGF23:burosumab binding surface is clearly defined by crystallographic analysis and supported by HDX-MS.

[0183] Example 3: Paratope mapping of Burosumab-human FGF23 The paratope residues / regions of burosumab for binding to human FGF23 were probed using HDX-MS in a manner similar to the FGF23 epitope mapping described in Example 2. FGF23(R179Q) was produced in HEK293S cells. burosumab was produced in CHO cells (whereas the FGF23-binding antibodies engineered in later examples were expressed in HEK293S cells). Antigen-antibody complexes were formed by combining burosumab and FGF23(R179Q) in solution and then diluted 1:10 in ice-cold deuterated assay buffer for 60 seconds, 600 seconds, 1800 seconds, or 3600 seconds. The deuteration reaction was quenched with quench buffer (pH 2.5) at a 1:1 ratio, after which the solution was loaded onto a liquid chromatography (LC) column for on-column proteolytic digestion, followed by LC separation and mass spectrometry.

[0184] Briefly, the HDX-MS assay utilized PBS at pH 7.4 as the assay buffer. A quench buffer containing 1 M glycine, 4 M guanidine HCl, and 400 mM TCEP at pH 2.5 was used. 100 pmol were injected in duplicate at a 1:1 ratio of assay buffer to quench buffer. Labeling (deuteration) was performed for 15 s, 60 s, 600 s, and 3600 s. The antigen concentration at labeling was 1.5 μM, and the antibody concentration at labeling was 1 μM.

[0185] The HDX-MS results are shown in Figure 3. The y-axis represents the % deuteration (%D), i.e., the peptide-length-normalized difference in deuteration between the unbound antibody and the FGF-bound antibody. Values ​​less than 0 reflect decreased deuteration, i.e., protection from deuteration by antibody-antigen binding. Values ​​greater than 0 reflect increased deuteration, i.e., exposure to deuteration. The x-axis represents the amino acid residues of burosumab (light chain on the left, heavy chain on the right, separated by vertical bars). Colored bars represent the deuterium-labeling time points for each identified peptide (black = 15 s, blue = 60 s, orange = 600 s, magenta = 3600 s).

[0186] Protection was observed on the burosumab antibody, which was believed to represent deuteration and / or conformational protection of regions protected from antigen (FGF23) binding. In particular, the following regions on the burosumab light and heavy chain sequences, shown in Table 4, were identified as exhibiting reduced deuteration by HDX-MS:

[0187] [Table 4]

[0188] Of these, burosumab heavy chain paratope region 1 (SEQ ID NO: 5) and light chain paratope region 1 (SEQ ID NO: 8) were hypothesized to be involved in antigen binding.

[0189] Example 4: Molecular docking to evaluate burosumab-FGF23 interactions Protein-protein docking was used to predict the three-dimensional structure of the burosumab-FGF23 complex and identify the antibody-antigen interaction surfaces and residues involved in binding. Even without a crystal structure of the Fab-antigen complex, computational docking was used to gain insight into the interaction of the burosumab Fab fragment with the FGF23 structure.

[0190] A crystal structure of FGF23 complexed with sucrose octasulfate is available (see "2P39," Goetz et al., "Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members," Mol Cell Biol (2007) 27 p. 3417-28, incorporated herein by reference). Additionally, a crystal structure of the FGF23-FGFR1c-aKloth ternary complex is also available (see "5W21," Chen et al., "alpha-Klotho is a non-enzymic molecular scaffold for FGF23 hormone signaling," (2018) Nature 553: 461-466, incorporated herein by reference). These available crystal structures were utilized to perform molecular docking studies of human FGF23 with the known crystal structure of burosumab Fab (see "7VEN"; Heo, YS "Structure of burosumab Fab" DOI: 10.2210 / pdb7ven / pdb; published September 14, 2022, incorporated herein by reference). The crystal structure of 2P39 FGF23 and the crystal structure of 7VEN burosumab were utilized for the docking studies herein.

[0191] Briefly, protein-protein docking was performed using an FFT-based search algorithm implemented in the Molecular Operating Environment "MOE" (2022.02). After molecular preparation (i.e., minimization, geometry, protonation, and capping), all docking studies were initially performed with the antibody and antigen molecules treated as rigid bodies. Conformational sampling of individual loops in the FGF23 antigen, which are expected to be more flexible, was performed using low-mode MD and conventional MD. First, the model was rendered into a coarse-grained (CG) residue-based representation, in which each residue consists of one to three "bonded atom" beads, following the scheme proposed by Basdevant et al. (doi:10.1021 / jp0727190). The CG representation includes the following energy components: van der Waals, electrostatic, and solvation in the generalized Born volume integral (GB / VI) formalism.

[0192] In the docking process, the antibody was considered the "ligand" and the antigen FGF23 was considered the "receptor." The system was paratope-biased to consider the published Kabat-defined CDRs of burosumab as the primary interaction sites: heavy and light chain CDR1, CDR2, and CDR3, respectively, represented by SEQ ID NOs: 11, 12, and 13 (HCDR1, HCDR2, and HCDR3), and SEQ ID NOs: 14, 15, and 16 (LCDR1, LCDR2, and LCDR3). In other words, the constrained ligand site was fixed to the burosumab CDRs, while epitopes were considered globally (i.e., biased) or specifically selected based on HDX data. The potential of hydrophobic patches was utilized throughout the search and refinement process. For pose selection, the thresholds for retaining poses as valid were set at 10,000 pre-positions, 1,000 placements, and 100 refinements.

[0193] For docking purposes, epitopes were selected based on empirical data from HDX-MS (Examples 2 and 3 herein) in combination with (i) comparative analysis of cross-reactivity studies using different species of burosumab and FGF23, (ii) kinetic studies using sandwich SPR analysis of different burosumab mutational variants (Yamazaki et al., 2008), and / or (iii) patch analysis to assess the alignment of hydrophobic and polar surfaces of FGF23 and burosumab that influence the binding interaction.

[0194] Regarding comparative analysis of burosumab binding across different species, burosumab showed similar high binding affinity (K D 10-11 mol / L)" (Study R-12-0326), and "cross-reactivity with recombinant forms of monkey and human FGF23, but not mouse FGF23 (Study R-13-0026), and with plasma FGF23 from humans and monkeys, but not dogs or rats (Study R-13-0027)." (Center for Drug Evaluation and Research Application Number 761068Orig1s000, Multidisciplinary Review for Biologics License Application, available at www.accessdata.fda.gov / drugsatfda_docs / nda / 2018 / 761068Orig1s000MultidisciplineR.pdf) Therefore, FGF23 amino acid sequences from human, cynomolgus monkey, rabbit, dog, and rat were aligned and overlaid with the HDX-MS data to identify potential epitope residues or regions that are consistent across burosumab cross-reactive species, but not necessarily across non-cross-reactive species.

[0195] Findings from these various analyses were used to guide different bias conditions in five separate docking searches (all FFT-based, and all antigen-biased) to generate diversity in poses and antigen-antibody contacts:

[0196] Search No. 1: Unbiased

[0197] Search No. 2: Biased with each HDX Protection Area 1, Protection Area 2, or Protection Area 3 considered separately

[0198] Search No. 3: All HDX protected areas (1, 2, and 3) combined and biased

[0199] Search No. 4: Biased using epitopes described in Yamazaki et al., 2008

[0200] Search No. 5: Biased using shared epitopes between HDX protected regions 1, 2, and 3 and Yamazaki et al. (2008)

[0201] Each search run resulted in five distinct clusters, all of which were further refined for energetics and validation against empirical data (e.g., mutational data). The primary requirement for a pose to be considered a valid hit was a correlation of approximately 30% to 40% with the available HDX data, i.e., at least approximately 30% to 40% of the total number of residues in the three HDX-suggested epitopes (based on contacts within a 4.5 Å sphere) had to be involved in at least one contact with any residue from the burosumab CDR. The clusters resulting from unbiased docking showed a lower overall correlation with the HDX data and the murine antibody-binding epitope found by Yamazaki et al. (doi:10.1359 / JBMR.080417). Biased runs were more successful in this regard, with the best hit (i.e., highest correlation with HDX data) resulting from the antigen-side biased run with the epitope reported from the murine Ab in Yamazaki et al. (i.e., search number 4). Two poses were selected as leads based on the criteria set out above: FGF23:Ab-Pose-1 (first-rank structure (1st-preferred.moe)) and FGF23:Ab-Pose-2 (second-rank structure (2nd-preferred.moe)). Several additional poses from docking trials 3, 4, and 5 were further evaluated.

[0202] Valid poses from all docking runs were inspected to identify common paratope positions. This information was combined with that learned from the epitope and paratope mapping studies described in Examples 2 and 3. Overall, 18 Burosumab heavy chain and 18 Burosumab light chain residues were identified as potential common paratope positions. The energies of the antigen-antibody contacts at these positions were evaluated for all positions. A summary of the modeling and docking runs is shown in Table 5.

[0203] [Table 5]

[0204] Example 5. Predicted mutagenesis of burosumab based on molecular docking, HDX-MS protection mapping, and comparative mutation analysis Burosumab mutation variants were designed using predictive mutagenesis performed at common interacting burosumab positions across the five averaged clusters. Considering valid poses from all docking searches that bound at least approximately 60% of the HDX epitope residues, common paratope positions (18 burosumab VH residues and 18 burosumab VL residues) were targeted for predictive mutagenesis. Positions and mutation variants were evaluated based on energy calculations and MM-based mutations and rotamer optimization (extension) within 4.5 Å of the mutation site.

[0205] An initial 1X scan using predictive mutagenesis yielded 520 engineered FGF23-binding antibody designs, each containing a single amino acid change to either the burosumab light chain (SEQ ID NO: 2) or the burosumab heavy chain (SEQ ID NO: 1). All 520 mutational variants were evaluated and screened according to the following criteria: dAffinity (ΔΔG (kcal / mol)) -4.5 kcal / mol to 0.4 kcal / mol, dStability (ΔΔS (kcal / mol)) < 3.0 kcal / mol; and redundant positions were re-evaluated based on dSASA (difference in solvent-accessible surface area) as an indicator of gain or loss of buried surface area due to mutation. ΔΔG is a metric for predicting how a single point mutation will affect protein stability. It is a measure of the change in energy between the folded and unfolded states and the change in folding ΔG when a point mutation is present. This metric is used as a predictor of whether a point mutation is advantageous with respect to protein stability. The more negative the value, the more stable the protein. A ΔΔG value >0.5 suggests that the mutation is destabilizing; values ​​of 0.5 > ΔΔG > -0.5 suggest that the change should be considered neutral or near-neutral; and ΔΔG < -0.5 suggest that the mutation results in a more stable protein. ΔΔS, on the other hand, is a measure of the difference in vibrational entropy. Positive values ​​indicate increased plasticity, while negative values ​​indicate a more rigid protein. It is used to predict how mutations will alter the entropy of a protein. The water-accessible surface area ("VdW SA"), the water-accessible surface area of ​​hydrophobic atoms of the protein ("hydrophobic SA"), and the water-accessible surface area of ​​hydrophilic atoms of the protein ("hydrophilic SA") were also calculated. Table 6 summarizes the full 1X scan predicted mutagenesis results for the burosumab light chain positions, and Table 7 summarizes the full 1X scan predicted mutagenesis results for the burosumab heavy chain positions.

[0206] From the results of the full 1X scan, 96 mutational variants were selected for further evaluation based on the above screening criteria to select for predicted improvements in affinity with minimal adverse effects on predicted stability or solvation. These 96 mutational variants are referred to herein as burosumab variants or engineered FGF23-binding antibody "Design Panel 1." Table 8 provides a summary of the 1X scan predicted mutagenesis results for selected burosumab light chain positions for Design Panel 1, and Table 9 provides a summary of the 1X scan predicted mutagenesis results for selected burosumab heavy chain positions for Design Panel 1.

[0207] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0208] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11]

[0209] [Table 8]

[0210] [Table 9-1] [Table 9-2]

[0211] Example 6. Rational Design Panel 1 of Engineered FGF23 Binding Antibodies Based on Molecular Docking Studies Table 10 shows the 96 engineered FGF23-binding antibody designs from the 1X scan. Each variant is labeled with a consecutive engineered FGF23-binding antibody structure ID numbered 001 to 096 and has a single amino acid change compared to the parent burosumab heavy chain amino acid sequence (SEQ ID NO: 1) or the parent burosumab light chain amino acid sequence (SEQ ID NO: 2). Design panel 1 did not contain combined amino acid changes compared to the parent sequence and did not include variants with multiple amino acid changes in the light or heavy chain. In Table 10, all amino acid numbering is presented according to the ASN# system.

[0212] [Table 10-1] [Table 10-2]

[0213] Example 7. Structural evaluation of burosumab-FGF23 interactions To further characterize the human FGF23-burosumab interaction, X-ray crystallography studies were performed using fully glycosylated human FGF23 (R179Q) derived from HEK293S cell supernatant complexed with the burosumab Fab fragment, which was also expressed in HEK293S cells. Following conventional methods (Pierce™ Fab Preparation Kit, Thermo Scientific™, catalog number 44985), the heavy and light chains of burosumab IgG were subjected to papain digestion to separate the Fc and Fab fragments, and the approximately 50 kDa Fab fragment was then isolated using protein A. Fully glycosylated human FGF23 (R179Q) and the burosumab Fab fragment were incubated on ice for >1 hour (1:0.8 molar ratio), and stable complexes were isolated by size-exclusion chromatography (SEC) followed by SDS-PAGE. The SEC fractions containing the complex were pooled, concentrated, and stored at -80°C.

[0214] Briefly, crystallization trials were performed in 96-well vapor diffusion sitting drop plates by mixing 200 nl of protein solution (4.5 mg / ml in 25 mM Hepes pH 7.5 and 150 mM NaCl) with 200 nl of well solution (0.2 M ammonium citrate dibasic and 20% (w / v) PEG 3350). Diffraction data were collected from single crystals at the Advanced Photon Source (APS) synchrotron and processed using autPROC (autProc, XDS, POINTLESS, AIMLESS, CCP4, and STARANISO) (see Vonrhein C, Flensburg C, Keller P, Sharff A, Smart O, Paciorek W, Womack T, Bricogne G. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):293-302. [doi:10.1107 / S0907444911007773]).

[0215] Phase solutions were obtained using Phaser (CCP4) (see J. Agre et al. Acta. Cryst. D79, 449-461 (2023) "The CCP4 Suite: integrative software for Macmolecular Crystallography" [doi:10.1107 / S2059798323003595]) using FGF23 and burosumab (PDB numbers 2P32 and 7VEN) as models for molecular replacement. Refinement and model building were performed using RECMAC, BUSTER, and COOT (see J. Agirre et al.; Bricogne G., Blanc E., Brandl M., Flensburg C., Keller P., Paciorek W., Roversi P, Sharff A., Smart OS, Vonrhein C., Womack TO (2017). Cambridge, United Kingdom: Global Phasing Ltd.; and Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 2010;66:486-501. [doi:10.1107 / S0907444910007493]).

[0216] Data collection and refinement statistics for the 3 Å complex structure solved by molecular replacement are shown in FIG.

[0217] Figure 5 shows the crystal structure of hFGF23(R179Q):burosumab Fab, with two antigen:Fab complexes interfaced by an FGF23:FGF23 dimer. It is noteworthy that FGF23 dimerization interface residues are not present in the epitope identified in the crystal structure, although some of them are involved in FGFR1c receptor binding. Furthermore, burosumab binding to FGF23 overlaps with previously reported heparin sulfate binding residues (Goetz, Regina, et al., "Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members," Molecular and Cellular Biology 27.9 (2007):3417-3428). Heparin sulfate is known to be required for dimerization of the FGF23:αKlotho:FGFR1c complex (Chen, G., Liu, Y., Goetz, R. et al., "α-Klotho is a non-enzymatic scaffold for FGF23 hormonal signaling," Nature 553, 461-466 (2018)). The FGF23 dimerization interface residues in this crystal structure correspond to those in FGFR1 binding previously described by Chen et al. (2018). These findings suggest that burosumab binding to FGF23 may further compete with or otherwise interfere with receptor binding and dimerization.

[0218] Figure 6 shows a ribbon model of the FGF23:Fab complex, with FGF23 epitope residues identified as colored purple spheres, and FGF23-dimer / FGFR1c binding residues as colored cyan spheres, burosumab light chain paratope residues as colored green spheres, and burosumab heavy chain paratope residues as colored yellow spheres. Identification of the epitope and paratope residues by crystallography enabled the rational design of burosumab variant molecules, as described in Example 6 herein.

[0219] Overall, the 3 Å resolution crystallographic FGF23:burosumab complex structure provides insight into the antigen:antibody interface, which is distinct from previously reported interfaces in related systems (e.g., FN1 antibody) or predicted interfaces (Kanhasut et al. 2022) (compare epitope residues—purple spheres—in Figure 6 vs. Figure 7). Tables 11 and 12 show amino acid residues in the human FGF23(R179Q) and burosumab long and heavy chain sequences that were found to be involved in the structural interface of the antibody-antigen complex. In Table 11, "Type" refers to the nature of the predicted interaction at the amino acid position(s) of interest, "D" represents a distance-based interaction, "H" represents a hydrogen-bond-based interaction, "I" represents an ionic interaction, "Dist" refers to the distance in Angstroms, "Frequency" refers to the number of interatomic contacts associated with the pair (main chain and side chain), and "Area" is given in squared Angstroms and refers to the difference in surface area when the indicated amino acid is varied.

[0220] [Table 11-1] [Table 11-2] [Table 11-3]

[0221] [Table 12]

[0222] As can be seen from Tables 11 and 12, the amino acid residues found to interact between FGF23 and burosumab fall within the protected region 1 and protected region 3 residues identified by HDX-MS (see Example 2 above), confirming these regions as important mediators of the FGF23-burosumab interaction. However, residues within protected region 2 were not found to be involved in the interaction. With regard to paratope residues, interface residues within the burosumab heavy chain overlap with HDX-MS data describing heavy chain paratope regions 1 and 2, but no interacting residues from heavy chain paratope region 3 were observed (see Example 3 above). Similarly, interface residues within the burosumab light chain overlap with HDX-MS data describing light chain paratope regions 1 and 2, but no interacting residues from light chain paratope region 3 were observed (see Example 3 above).

[0223] Based on the crystal structure, it was concluded that the HDX-MS data in Example 2 correctly identified the protected region of the burosumab-bound FGF23 molecule. However, the crystal structure revealed that protected region 2 does not interact with burosumab. Rather, this region of FGF23 is likely protected by conformational changes in the FGF23 structure upon burosumab binding (i.e., self-protection). Therefore, biased in silico docking attempts on the HDX-MS data (Example 4) led to the selection of molecular poses that did not reflect the actual structure described by crystallography. Nevertheless, the molecular docking study in Example 4 produced models structurally very similar to the final crystal structure (data not shown). However, these models were initially discarded due to the lack of involvement of residues in protected region 2.

[0224] The crystal structure revealed complementary charges and hydrophobic patches involved in the burosumab-FGF23 interaction. Figure 8 shows the FGF23:Fab complex in ribbon (left) and surface (right panel) models, with a rotated view of the FGF23 and burosumab binding interface, overlaid with detailed amino acid positions as a stick model (far-right panel). Positively charged surfaces are shown in blue, negative charges in red, and uncharged surfaces remain white. Positively charged regions of FGF23 (R143 and R140) are seen to contact complementary negatively charged residues of burosumab, and hydrophobic patches on the surface of burosumab are seen to contact complementary uncharged hydrophobic residues of FGF23 (L138, Y51, and H66) (see inset of Figure 8, which details the binding interface).

[0225] In particular, specific hydrogen-bonding residues that can be targeted to improve antibody binding affinity were identified between burosumab and FGF23. Figure 9 shows the FGF23 surface model interface with burosumab, shown as ribbon and stick models. In the surface model shown, positively charged surfaces are shown in blue, negative charges are shown in red, and uncharged surfaces remain white. Panel A shows the FGF23:Fab complex with the FGF23 moiety rotated 90 degrees to display the binding interface with all burosumab residues in contact with FGF23. Panel B shows the surface model of FGF23 (top) with burosumab hydrogen-bonding residue positions in stick models (top and bottom). Panels C and D provide a more detailed view of the ionic and aromatic stacking interactions at the FGF23:burosumab interface, respectively. In panel B, several selected burosumab residues are marked as hydrogen-bonding interaction sites (black arrows) that could be targets for rational design throughout the interface with FGF23. Panel C highlights the significant ionic interactions with aspartic acid and glutamic acid residues of burosumab, which contact arginine residues (R143 and R140) of FGF23. Panel D highlights the π (pi) stacking interactions at FGF23 residue Y51, which interacts with burosumab valine and tyrosine residues.

[0226] In summary, the combination of HDX-MS (Examples 2 and 3), in silico modeling (Example 4), and crystallography data (Example 7) enabled the generation of an accurate and robustly predictive model of the FGF23:burosumab complex. All three lines of evidence were necessary to confidently identify burosumab residues that could be altered to alter burosumab affinity. Taken together, these findings describe burosumab residues (see Table 12) that likely interface with human FGF23 and may serve as candidate residues for amino acid changes (deletions, additions, substitutions) for the rational design of burosumab variants with improved affinity for FGF23.

[0227] Example 8. Crystal structure-based predictive mutagenesis of burosumab A list of candidate amino acid changes for the variable heavy and variable light chains of burosumab was generated based on the crystal structure and interface-binding contact residues described in Example 7 above. Using molecular mechanics-based tools, single (1X) and double (2X) amino acid changes were generated and optimized, focusing on 19 antigen-contacting residue positions and 10 semi-distal residue positions (i.e., approximately 6-8 Å second shell distance) relative to the antigen residues. Briefly, a crystal structure of the antibody complexed with human FGF23, refined to 3 Å resolution, was used as a basis to generate starting coordinates for molecular mechanics calculations. The structure was assessed for completeness, gaps were filled with corresponding residues, and the termini were capped. The resulting structure was subjected to protonation (pH 7) and energy minimization as implemented in MOE 2022.02 (CCG) to check the structural geometry prior to in silico mutagenesis.

[0228] The antibody-FGF23 interface was analyzed, and relevant contacts were considered based on the distance between the antibody and antigen residues. Residue positions on the antibody were selected and classified into three groups: close contacts (3-4 Å), second shell contacts (up to 4.5 Å), and cavity / VdW interactions.

[0229] We pursued three approaches to improve affinity. First, we designed variants with altered direct interactions. Second, we designed indirect variants to affect already established direct interactions. Third, we designed variants to fill existing cavities. We highlighted hydrogen-bonding interaction sites that could be modified by altering the burosumab residues at the interface to introduce ionic interactions or hydrophobic packing sites into aromatic π-π interactions, thereby increasing affinity. For example, burosumab light chain VL residue S52, which forms a hydrogen-bonding interaction with FGF23 Arg143, could be replaced with negatively charged residues (D, E), bulky hydrogen-bonding residues (Q, T), or stacking residues (F, Y), which could enhance binding / packing interactions. Similarly, heavy chain residue Val101, which stacks with FGF23 Tyr51, could be replaced with bulkier residues (F, I, L, Y) or with hydrogen-bonding properties (T). Additional properties that were considered, but not directly utilized in the selection, were the number of interactions per residue (connectivity), delta (SASA), and interaction delta G. Figures 10 and 11 show the relevant interface positions for the burosumab light and heavy chains, respectively, along with details of each residue's interactions with neighboring residues.

[0230] Initially, each target position was scanned with a 1X mutation screen, with each mutation followed by rotameric optimization and refinement of the surrounding environment (4.5 Å). The resulting set of 420 variants was ranked based on energetic and structural properties, and the top hits (i.e., ΔΔG<0 and dSASA<0) were combined into 2X and 3X variants for further screening and ranking. Based on structural analysis of each solution, a final shortlist of 96 variants (1X and 2X) was generated and used as the basis for creating a second list of expanded mutants to further explore the structural space surrounding the interface of interest.

[0231] An initial 1X scan using predictive mutagenesis yielded 165 engineered FGF23-binding antibody designs, each containing a single amino acid change to either the light chain variable region of burosumab (SEQ ID NO: 4) or the heavy chain variable region of burosumab (SEQ ID NO: 3). All mutational variants were evaluated and screened according to dAffinity (ΔΔG (kcal / mol)); dStability (ΔΔS (kcal / mol)) less than 3.0 kcal / mol; and dSASA (difference in solvent-accessible surface area), as indicators of gain or loss of buried surface area upon mutagenesis. ΔΔG is a metric for predicting how a single point mutation (i.e., a single amino acid change) will affect protein stability. It is a measure of the change in energy between the folded and unfolded states and the change in folding ΔG when the point mutation is present. This metric is used as a predictor of whether a point mutation is favorable for protein stability. The more negative the value, the more stable the protein. A ΔΔG value >0.5 suggests that a mutation is destabilizing; values ​​of 0.5 > ΔΔG > -0.5 suggest that the change should be considered neutral or near-neutral; and ΔΔG < -0.5 suggest that the mutation results in a more stable protein. ΔΔS, on the other hand, is a measure of the difference in vibrational entropy. Positive values ​​indicate increased plasticity, while negative values ​​indicate a more rigid protein. It is used to predict how changing a given amino acid will alter the entropy of a protein. Water-accessible surface area ("VdW SA") was also calculated. Table 13 summarizes the full 1X scan predicted mutagenesis results for the burosumab light chain positions, and Table 14 summarizes the full 1X scan predicted mutagenesis results for the burosumab heavy chain positions.

[0232] [Table 13-1] [Table 13-2] [Table 13-3]

[0233] [Table 14-1] [Table 14-2] [Table 14-3]

[0234] A 2X scan using predictive mutagenesis of double amino acid changes to the heavy and light chain variable regions of burosumab resulted in 20 additional engineered FGF23-binding antibody designs, each containing two amino acid changes to the light chain variable region of burosumab (SEQ ID NO: 4) and / or the heavy chain variable region of burosumab (SEQ ID NO: 3). Table 15 shows a summary of the predictive mutagenesis results of the 2X scan.

[0235] [Table 15]

[0236] Example 9. Design Panel 2 of Engineered FGF23 Binding Antibodies Based on Crystal Structure

[0237] Table 16 shows 76 engineered FGF23-binding antibody designs from a 1X scan according to Examples 7 and 8. Each variant is labeled with a consecutive engineered FGF23-binding antibody structure ID numbered 101 to 176 and has one amino acid change compared to the parent burosumab heavy chain variable region amino acid sequence (SEQ ID NO: 3) or the parent burosumab light chain variable region amino acid sequence (SEQ ID NO: 4). For clarity, each antibody represented by antibody structure ID numbers UGX101 to UGX176 has the parent burosumab heavy chain sequence (SEQ ID NO: 1) and light chain sequence (SEQ ID NO: 2), excluding the indicated amino acid change compared to these sequences.

[0238] Table 17 shows 20 engineered FGF23-binding antibody designs incorporating two amino acid changes (2X scan) according to Examples 7 and 8. Each variant is labeled with a consecutive engineered FGF23-binding antibody structure ID numbered 177-196 and has two amino acid changes compared to the parent burosumab heavy chain variable region amino acid sequence (SEQ ID NO: 3) and / or the parent burosumab light chain variable region amino acid sequence (SEQ ID NO: 4). For clarity, each antibody represented by antibody structure ID numbers UGX177-UGX196 has the parent burosumab heavy chain sequence (SEQ ID NO: 1) and light chain sequence (SEQ ID NO: 2), excluding the indicated amino acid changes compared to these sequences.

[0239] [Table 16-1] [Table 16-2]

[0240] [Table 17]

[0241] Example 10. In vitro testing of selected variants for increased affinity for FGF23 FGF23-binding antibody designs from the 1X and 2X antibody design panels shown in Tables 16 and 17, respectively, as well as additional antibody variants with one or two amino acid changes compared to burosumab, were evaluated for affinity by surface plasmon resonance (SPR).

[0242] Ninety-six antibody variants were expressed in small-scale (3 ml) and screened by SPR analysis against human FGF23(R179Q) at a single concentration (100 nM). Experimental configuration for SPR analysis: SPR device - 8K+; chip preparation - CM5 hIgG chip; channels - 1-8; immobilization concentration - 1.5 μg / ml; immobilization method - capture; flow rate - 5 μl / min; contact time - 60 s; running buffer - 1x HBS-EP + pH 7.4; temperature - 20 °C; flow rate - 50 μl / min; association time - 270 s; dissociation time - 600 s; antigen - rhFGF23(R179Q) maximum concentration - 0.1 μM.

[0243] All antibody variants were analyzed using the off-rate k d or the equilibrium dissociation constant K D The top hits with the greatest affinity improvement compared to unmodified burosumab were confirmed by concentration-response curves (CRC). Experimental configuration for SPR analysis of CRC: SPR device—8K+; chip preparation—CM5 hIgG chip; channels—1–8; immobilization concentration—1.5 μg / ml; immobilization method—capture; flow rate—5 μl / min; contact time—60 s; running buffer—1x HBS-EP+ pH 7.4; temperature—20°C; flow rate—50 μl / min; association time—120 s; dissociation time—1800 s; antigen—rhFGF23(R179Q) highest concentration—0.1 μM; 9-pt3x single-cycle kinetics (single-cycle binding kinetics analysis).

[0244] The results of the SPR screening and CRC evaluation are shown in Table 18 and indicate the K D and the final reaction rate parameter k a and k dThe determination of surface activity, as well as surface activity, are shown in Table 18. In Table 18, column headings are abbreviated as follows: "AS ID" refers to "Antibody Structure ID," which is a unique identifier for each antibody as defined herein; "AA change(s)" refers to an amino acid residue change relative to either the parent burosumab heavy chain ("HC") variable region amino acid sequence (SEQ ID NO: 3) or the parent burosumab light chain ("LC") variable region amino acid sequence (SEQ ID NO: 4), or both; "CL" refers to capture level (RU); "K D " refers to the equilibrium dissociation constant (M); "k a " refers to the association rate or constant (1 / Ms); "k d " refers to the dissociation rate or constant (1 / s); "R max " is the theoretical R max or refers to the calculated maximum observable signal (relative units); "SA" refers to surface activity (%); "Ratio" refers to the ratio of the variant antibody K D / Burosumab K D This refers to the calculated ratio of

[0245] [Table 18-1] [Table 18-2]

[0246] These data demonstrate that the improved affinity of the engineered burosumab variants is due to the increased dissociation rate (off-rate, or k d ), but the association rate (on-rate, or k a ) remained very similar across all variants. These data also show that K > 2 DAt least 13 different engineered burosumab variants with a ratio of 0.01 to 0.02 are shown, meaning that these antibody variants have at least a 2-fold improvement in affinity (up to a 5-fold improvement) compared to burosumab. Antibodies that showed slower or similar off-rates compared to burosumab were selected for further analysis using concentration-response curves (CRCs) to confirm kinetic parameters.

[0247] The results are shown in Table 19, ranked according to off-rate. In Table 19, column headings are abbreviated as follows: "AS ID" refers to "Antibody Structure ID," which is a unique identifier for each antibody as defined herein; "AA change(s)" refers to an amino acid residue change relative to either the parent burosumab heavy chain ("HC") variable region amino acid sequence (SEQ ID NO: 3) or the parent burosumab light chain ("LC") variable region amino acid sequence (SEQ ID NO: 4), or both; "K" refers to an amino acid residue change relative to either the parent burosumab heavy chain ("HC") variable region amino acid sequence (SEQ ID NO: 4), or both; D " refers to the equilibrium dissociation constant (M); "k a " refers to the association rate or constant (1 / Ms); "k d " refers to the dissociation rate or constant (1 / s); "R max " is the theoretical R max or refers to the calculated maximum observable signal (relative units); "SA" refers to surface activity (%); "Ratio" refers to the ratio of the variant antibody K D / Burosumab K D For clarity, each antibody represented in Table 19 by antibody structure ID numbers UGX205, UGX201, UGX144, UGX157, UGX202, UGX175, UGX206, UGX182, UGX156, UGX203, UGX126, UGX145, and UGX204 has the parent burosumab heavy chain sequence (SEQ ID NO: 1) and light chain sequence (SEQ ID NO: 2), excluding the indicated amino acid changes compared to these sequences.

[0248] [Table 19]

[0249] SPR data suggest that multiple paratope residues act through various mechanisms to support burosumab:FGF23 binding. In particular, amino acid substitutions S52D / E, N92F / Y, and Y94D / E in the parent burosumab light chain sequence and I54F / Y, S59D / E, V101I, and A103S in the parent burosumab heavy chain sequence primarily affect the off-rate kappa (kappa) of burosumab. d Reducing the amount of FGF23 significantly affects the affinity of the antibody for FGF23.

[0250] Without wishing to be bound by theory, the inventors believe that the light chain Y94D / E substitution may form an ionic interaction with the epitope residue Arg48; the heavy chain S59E and light chain Y94D / E substitutions may function in concert to stabilize the sandwiching interaction with the epitope residue Arg48; the light chain N92F / Y substitution, particularly the heavy chain S59E substitution, appears to enhance hydrophobic packing interactions with epitope residues such as Pro153; and the heavy chain A103S substitution appears to enhance hydrophobic packing interactions with epitope residues such as Pro153. The heavy chain S59E and light chain S52D / E substitutions likely introduce hydrogen bonds with nearby epitope residues, including rg140; the heavy chain V101I substitution likely promotes stronger hydrophobic packing interactions with epitope residues Tyr51, L138, and Tyr154; and the heavy chain I54F / Y substitutions may promote stacking interactions with epitope residue His66 (Figure 12).

[0251] Example 11. Characterization of selected variant antibodies against cynoFGF23 The binding kinetics of the 13 antibody variants shown in Table 19 were characterized against cynomolgus monkey (Macaca fascicularis) FGF23 (R179Q) (i.e., "cynoFGF23") (SEQ ID NO: 372). The antibody variants were expressed at small scale (3 ml) and screened by SPR analysis at a single concentration (100 nM). Experimental configuration for SPR analysis of CRC: SPR device—8K+, chip preparation—CM5 hIgG chip; channels—1 to 8; immobilization concentration—1.5 μg / ml; immobilization method—capture; flow rate—5 μl / min; contact time—60 sec; running buffer—1xHBS-EP+pH 7.4; temperature—20°C; flow rate—50 μl / min; association time—120 sec; dissociation time—1800 sec; antigen—cynoFGF23 (R179Q); highest concentration—0.1 μM, 9-pt3x single-cycle kinetics. The results are shown in Table 20, which shows the variant antibody K for each antibody variant against human FGF23 and cynoFGF23. D / Burosumab K D K D and calculated ratios are shown.

[0252] [Table 20]

[0253] Binding of selected antibody variants to cynoFGF23 was comparable to that observed with human FGF23.

[0254] Example 12. Confirmation of HDX-MS epitope / paratope mapping by SPR analysis The HDX-MS epitope and paratope mapping data described in Examples 2 and 3, respectively, were reevaluated in light of the SPR data described in Example 10 to determine the impact of changing key residues within the HDX-MS protected regions. Epitope mapping identified three putative epitopes (protected regions 1-3 in Table 3), and paratope mapping identified six regions potentially involved in antigen binding (see Table 4).

[0255] SPR data confirmed that burosumab light chain residue S52 in light chain paratope region 2 (see Table 4) and I54 and S59 in heavy chain paratope region 2 (see Table 4) are critical residues within the HDX-MS protected region of burosumab, suggesting that these residues are important mediators of burosumab-FGF23 binding.

[0256] SPR data also identified burosumab light chain residues S52, N92, and Y94 and burosumab heavy chain residues S59 and V101 as critical residues for interfacial binding with FGF23 protected regions 1 to 3 (Table 3).

[0257] Thus, of the seven key burosumab residues identified by SPR as potentially substitutable to improve burosumab affinity (light chain S52, N92, Y94 and heavy chain I54, S59, V101, and A103), six of them were also identified by HDX-MS epitope-paratope mapping, demonstrating strong correlation and agreement between the results of the initial HDX-MS studies and subsequent crystal structure-based modeling and antibody variant design.

[0258] Example 13. In silico modeling of additional antibody variants based on SPR analysis Examples 10-12 identify and confirm at least seven burosumab residues (light chain S52, N92, Y94, and heavy chain I54, S59, V101, and A103) that can be substituted to improve the antibody's affinity for FGF23. These key burosumab residues were further evaluated by in silico modeling to identify additional antibody variants with comparable or improved affinity compared to the parent burosumab and / or the variants identified in Tables 18 and 19.

[0259] Briefly, all seven key burosumab residues were probed using in silico modeling predictive mutagenesis based on the burosumab Fab:FGF23 crystal structure and SPR data. Specifically, all residues with a K > 2 were probed. D Ratio (variant antibody KD / Burosumab K D Antibody variants in Tables 18 and 19 with a calculated ratio of β-amyloid to β-amyloid (i.e., 3 or greater; UGX205, UGX201, UGX202, and UGX206) were analyzed using the Molecular Operating Environment "MOE" (2022.02) to define structural determinants of binding (i.e., which single residues or pairs of residues may act synergistically to improve affinity). All possible combinations of amino acid changes to seven key burosumab residues in these antibodies were computationally generated. Specific amino acid changes (per amino acid position) consistent with the initial empirical SPR data were then selected for further analysis. All amino acid variant combinations predicted to improve affinity compared to parent burosumab from a structural / computational perspective are shown in Table 21, where "AS ID" refers to the "Antibody Structure ID" (a unique identifier for each antibody as defined herein); "AA change(s)" refers to the amino acid residue change to either the parent burosumab heavy chain ("HC") variable region amino acid sequence (SEQ ID NO: 3) or the parent burosumab light chain ("LC") variable region amino acid sequence (SEQ ID NO: 4), or both; "ΔΔG" refers to the calculated dAffinity (kcal / mol); "ΔΔS" refers to the calculated dStability (kcal / mol); and "Rationale" refers to the main considerations noted during the MOE evaluation.

[0260] [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4] [Table 21-5]

[0261] In summary, mutation solutions derived from lead antibody variants (e.g., Table 19) were analyzed using structural computational methods to generate a series of novel variants with designs predicted to have similar or superior affinity. Additional designs included several burosumab antibody variants predicted to have improved affinity compared to the parent burosumab.

[0262] Example 14. Data Summary The work described herein sought to rationally design burosumab antibody variants with improved affinity for FGF23 by introducing one or more amino acid change(s) into either the heavy chain amino acid sequence, the light chain amino acid sequence, or both the heavy chain amino acid sequence and the light chain amino acid sequence.

[0263] Amino acid residue changes were modeled based on input from epitope and paratope mapping studies (Examples 2 and 3), molecular docking and predictive mutagenesis studies (Example 4), structural analysis by crystallography (Example 7), and predictive mutagenesis based on solved crystal structures (Example 8), refinement of antibody variant design to identify the most promising antibody structural variants with at least a two-fold improvement in affinity for human FGF23, and in vitro testing of selected variants by SPR analysis (Examples 9 and 10).

[0264] In the studies reported herein, certain lead antibody variants were further characterized for affinity to cynoFGF23 (Example 11), and additional antibody variants were identified by computationally exploring alternative amino acid substitutions at key residues identified as important for modulating burosumab-FGF23 binding (Example 13).

[0265] Table 22 lists the unique burosumab antibody variants designed according to the studies described herein. In Table 22, each variant antibody is assigned a unique "AS ID" number. Furthermore, each antibody variant has one or more light chain amino acid changes ("LC AA changes") compared to the parent burosumab light chain amino acid sequence of SEQ ID NO: 2, or one or more light chain variable region amino acid changes ("VL AA changes") compared to the parent burosumab light chain amino acid sequence of SEQ ID NO: 4, and / or one or more heavy chain amino acid changes ("HC AA changes") compared to the parent burosumab heavy chain amino acid sequence of SEQ ID NO: 1, or one or more heavy chain variable region amino acid changes ("VH AA changes") compared to the parent burosumab heavy chain variable region amino acid sequence of SEQ ID NO: 3. In Table 22, the "AKA" column refers to the alternative identifiers assigned to specific AS IDs provided herein. Sequence identification numbers ("SID#") are provided for the amino acid sequences of each heavy chain (HC), light chain (LC), heavy chain variable region (VH), and light chain variable region (VL) disclosed herein.

[0266] [Table 22-1] [Table 22-2] [Table 22-3] [Table 22-4] [Table 22-5] [Table 22-6] Table 22-7 Table 22-8 Table 22-9

Claims

1. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 3, but containing one or more amino acid changes relative to SEQ ID NO: 3, wherein said one or more amino acid changes are T30E, T30F, T30Q, T30S, T30Y, N31D, N31E, N31F, N31Q, N31R, N31Y, H32E, H32F, H32Q, H32S, H32T, I50D, I50E, I50F, I50S, I50T, I50Y, N52E, N52F, N52Q, N52Y, I54E, I54F, I54L, I54Q, I54 S, I54T, I54V, I54Y, S55E, S55F, S55Q, S55T, S55Y, S59D, S59E, S59F, S59I, S59L, S59 Q, S59T, S59V, S59Y, D99E, D99Q, D99Y, I100D, I100E, I100F, I100L, I100Q, I100V, I10 0Y, V101F, V101I, V101L, V101T, V101Y, D102E, D102F, D102I, D102L, D102Q, D102Y, A103D, A103E, A103F, A103Q, A103S, A103T, and A103Y.

2. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, comprising a light chain variable region (VL) amino acid sequence according to SEQ ID NO:4, but containing one or more amino acid changes relative to SEQ ID NO:4, wherein the one or more amino acid changes are A25L, A25V, I29F, I29L, I29V, S30D, S30E, S30F, S30I, S30L, S30Q, S30T, S30V, S30Y, S31D, S31E, S31F, S31I, S31L, S31Q, S31T, S31V, S31Y, A32F, A32I, A32L, A32T, A32V, A32Y, V34F, V34I, V34L, V34Y, D50E, D50 F, D50I, D50L, D50Q, D50R, D50S, D50T, D50V, A51F, A51I, A51L, A51S, A51T, A51V, S52D, S52 E, S52F, S52Q, S52T, S52Y, S53D, S53E, S53F, S53I, S53L, S53Q, S53R, S53T, S53V, S53Y, Q90F , Q90Y, F91K, F91R, F91Y, N92D, N92E, N92F, N92Q, N92S, N92T, N92Y, D93E, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94D, Y94E, Y94F, Y94Q, Y94S, and Y94T.

3. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, comprising a VH amino acid sequence according to SEQ ID NO:3 and a VL amino acid sequence according to SEQ ID NO:4, wherein the VH amino acid sequence is selected from the group consisting of T30E, T30F, T30Q, T30S, T30Y, N31D, N31E, N31F, N31Q, N31R, N31Y, H32E, H32F, H32Q, H32S, H32T, I50D, I50E, I50F, I50S, I50T, I50Y, N52E, N52F, N52Q, N52Y, I54E, I55 4F, I54L, I54Q, I54S, I54T, I54V, I54Y, S55E, S55F, S55Q, S55T, S55Y, S59D , S59E, S59F, S59I, S59L, S59Q, S59T, S59V, S59Y, D99E, D99Q, D99Y, I100D, I100E, I100F, I100L, I100Q, I100V, I100Y, V101F, V101I, V101L, V101T, V1 01Y, D102E, D102F, D102I, D102L, D102Q, D102Y, A103D, A103E, A103F, A103Q , A103S, A103T, and A103Y, wherein the VL amino acid sequence comprises one or more amino acid changes selected from A25L, A25V, I29F, I29L, I29V, S30D, S30E, S30F, S30I, S30L, S30Q, S30T, S30V, S30Y, S31D, S31E, S31F, S31I, S31L, S31Q, S31T, S31V, S31Y, A32F, A32I, A32L, A32T, A32V, A32Y, V34F, V34I, V34L, V34Y, D50E, D50F, D50I, D50L, D50Q, D 50R, D50S, D50T, D50V, A51F, A51I, A51L, A51S, A51T, A51V, S52D, S52E, S52 F, S52Q, S52T, S52Y, S53D, S53E, S53F, S53I, S53L, S53Q, S53R, S53T, S53V, S53Y, Q90F, Q90Y, F91K, F91R, F91Y, N92D, N92E, N92F, N92Q, N92S, N92T, N9 2Y, D93E, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94D, Y94E, Y94F, Y94Q, Y94S,and Y94T.

4. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to an amino acid sequence selected from SEQ ID NOs: 22-32.

5. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain complementarity determining region 2 (HCDR2) according to an amino acid sequence selected from SEQ ID NOs: 33-65.

6. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain complementarity-determining region 3 (HCDR3) according to an amino acid sequence selected from SEQ ID NOs: 66-93.

7. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain complementarity determining region 1 (LCDR1) according to an amino acid sequence selected from SEQ ID NOs: 94-126.

8. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain complementarity-determining region 2 (LCDR2) according to an amino acid sequence selected from SEQ ID NOs: 127-158.

9. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a light chain complementarity-determining region 3 (LCDR3) according to an amino acid sequence selected from SEQ ID NOs: 159-185.

10. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, the antibody or fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; the HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NOs: 22-32; the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 12 and SEQ ID NOs: 33-65; the HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 13 and SEQ ID NOs: 66-93; the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 14 and SEQ ID NOs: 94-126; the LCDR2 comprises an amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NOs: 127-158; The isolated antibody or fragment thereof, wherein the LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 16 and SEQ ID NOs: 159-185.

11. 11. The isolated antibody or fragment thereof according to any one of claims 1 to 10, comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186-270; or The isolated antibody or fragment thereof, wherein the VL comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

12. 12. The isolated antibody or fragment thereof according to any one of claims 1 to 11, the antibody or fragment thereof comprises a VH comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186-270; or The isolated antibody or fragment thereof, wherein the antibody or fragment thereof comprises a VL comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

13. 13. The isolated antibody or fragment thereof of any one of claims 1 to 12, the antibody or fragment thereof comprises a VH comprising an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NOs: 186-270; or The isolated antibody or fragment thereof, wherein the antibody or fragment thereof comprises a VL comprising an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

14. 14. The isolated antibody or fragment thereof according to any one of claims 1 to 13, comprising a VH and a VL, the VH comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO:3 and SEQ ID NOs:186-270; and The isolated antibody or fragment thereof, wherein the VL comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

15. 15. The isolated antibody or fragment thereof of any one of claims 1 to 14, comprising a VH and a VL, the VH comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:3 and SEQ ID NOs:186-270; and The isolated antibody or fragment thereof, wherein the VL comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

16. 16. The isolated antibody or fragment thereof of any one of claims 1 to 15, comprising a VH and a VL, the VH comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO:3 and SEQ ID NOs:186-270; and The isolated antibody or fragment thereof, wherein the VL comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271 to 368.

17. 17. The isolated antibody or fragment thereof of any one of claims 1 to 16, comprising a VH and a VL, the VH comprises an amino acid sequence selected from SEQ ID NO:3 and SEQ ID NOs:186-270; or The isolated antibody or fragment thereof, wherein the VL comprises an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271-368.

18. 17. The isolated antibody or fragment thereof of any one of claims 1 to 16, comprising a VH and a VL, the VH comprises an amino acid sequence selected from SEQ ID NO:3 and SEQ ID NOs:186-270; and The isolated antibody or fragment thereof, wherein the VL comprises an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NOs: 271-368.

19. 19. The isolated antibody or fragment thereof according to any one of claims 1 to 18, comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 3 and a light chain variable region (VL) according to SEQ ID NO: 4, wherein the VH and VL sequences comprise at least two amino acid changes, wherein the at least two amino acid changes are VL:S31E / VL:D50F, VH:N31F / VH:S59F, VL:D50F / VL:S53E, VL:S31F / VL:S52E, VL:D50F / VH:V101I, VH:S59D / VH:V101I, VL:S31F / VL:S53E, VL:N92E / VL:D93F, VL:S31E / VL:S53E, VL:S31F / VL :D50F, VH:N31E / VH:S59E, VH:S57E / VH:S59D, VH:N31E / VH:I54F, VH:S59T / VH:V101L, VH: I54Y / VH:V101F, VH:N52F / VH:V101I, VL:N92D / VL:D93F, VH:N52F / VH:V101L, VL:S31F / VL :D50E, VL:Y94D / VH:S59D, VL:Y94D / VH:S59E, VL:N92F / VH:S59E, VL:N92Y / VH:S59E, VL:Y94E / VH:S59E, VL:S52D / VH:S59E, and VL:S52E / VH:S59E.

20. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, comprising a VH amino acid sequence according to SEQ ID NO: 3 and a VL amino acid sequence according to SEQ ID NO: 4, wherein the VH amino acid sequence contains one or more amino acid changes selected from I54F, I54Y, S59D, S59E, V101I, and A103S compared to SEQ ID NO:

3.

21. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, comprising a VH amino acid sequence according to SEQ ID NO: 3 and a VL amino acid sequence according to SEQ ID NO: 4, wherein the VL amino acid sequence contains one or more amino acid changes selected from S52D, S52E, N92F, N92Y, Y94D, and Y94E compared to SEQ ID NO:

4.

22. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-001 and a light chain variable region (VL) according to VL-027 (UGX126).

23. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-001 and a light chain variable region (VL) according to VL-066 (UGX144).

24. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-001 and a light chain variable region (VL) according to VL-067 (UGX145).

25. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-028 and a light chain variable region (VL) according to VL-001 (UGX156).

26. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-029 and a light chain variable region (VL) according to VL-001 (UGX157).

27. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-061 and a light chain variable region (VL) according to VL-001 (UGX175).

28. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-055 and a light chain variable region (VL) according to VL-001 (UGX182).

29. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-050 (UGX201).

30. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-054 (UGX202).

31. An isolated antibody that specifically binds to human fibroblast growth factor 23 (FGF23) polypeptide.

1. An isolated antibody or fragment thereof, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-027 (UGX203).

32. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-030 (UGX204).

33. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-066 (UGX205).

34. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) according to VH-031 and a light chain variable region (VL) according to VL-067 (UGX206).

35. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, comprising a heavy chain (HC) amino acid sequence according to SEQ ID NO: 1 and a light chain (LC) amino acid sequence according to SEQ ID NO: 2, wherein the antibody or fragment thereof comprises one or more HC amino acid changes relative to SEQ ID NO: 1 and / or one or more LC amino acid changes relative to SEQ ID NO: 2, wherein the one or more HC amino acid changes are T28Y, T30F, T30K, N31D, N31E, N31F, N31Q, N31R, H32K, H3 2Y, Y33D, Y33L, Y33R, N52E, N52F, N52Q, N52R, N52Y, I54A, I54D, I54F, I54G , I54H, I54K, I54L, I54M, I54N, I54P, I54R, I54V, I54W, I54Y, S55E, S55F, S5 5I, S55Q, S55R, S55Y, S57E, S57F, S57R, S57Y, T58F, T58R, S59A, S59D, S59E , S59F, S59G, S59H, S59I, S59K, S59L, S59M, S59N, S59P, S59Q, S59R, S59T, S5 9V, S59W, S59Y, N60E, N60R, N60Y, A61F, A61R, A61Y, Q62L, Q62R, Q62Y, Q65E , Q65R, Q65Y, D99F, D99R, D99Y, I100D, I100E, I100F, I100Y, V101A, V101D, V 101E, V101F, V101G, V101H, V101I, V101K, V101L, V101M, V101N, V101P, V10 1Q, V101R, V101S, V101W, D102E, D102F, D102K, D102R, D102Y, A103D, A103F, and the one or more LC amino acid changes are selected from A103G, A103H, A103I, A103K, A103L, A103M, A103N, A103P, A103R, A103S, A103T, A103V, and A103W, and the one or more LC amino acid changes are selected from Q27F, Q27R, G28D, G28Q, G28R, G28Y, I29F, I29L, S30D, S30E, S30F, S30L, S30Q, S30R, S30Y, S31D, S31E, S31F, S31Q, S31Y, A32F, A32V, V34F, V34I, D50E, D50F, D50L, D50Q, D50R,D50S, D50T, D50V, D50Y, A51V, S52A, S52D, S52E, S52F, S52G, S52H, S52I, S52K, S52L, S52M, S52N, S52P, S52Q, S52R, S52V, S52W, S5 2Y, S53D, S53E, S53F, S53R, S53Y, L54E, L54Y, E55R, E55Y, S67E, S67R, S67Y, T69R, T69Y, F91E, F91R, F91Y, N92A, N92D, N92E, N92F, The isolated antibody or fragment thereof is selected from N92G, N92H, N92I, N92K, N92L, N92M, N92P, N92Q, N92R, N92S, N92T, N92V, N92W, N92Y, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94A, Y94D, Y94E, Y94F, Y94G, Y94H, Y94I, Y94K, Y94L, Y94M, Y94N, Y94P, Y94Q, Y94R, Y94S, Y94V, and Y94W.

36. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide, said antibody or fragment thereof comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO:3 and a light chain variable region (VL) amino acid sequence according to SEQ ID NO:4, wherein said antibody or fragment thereof comprises one or more VH amino acid changes relative to SEQ ID NO:3 and / or one or more VL amino acid changes relative to SEQ ID NO:4, wherein the one or more VH amino acid changes are T28Y, T30F, T30K, N31D, N3 1E, N31F, N31Q, N31R, H32K, H32Y, Y33D, Y33L, Y33R, N52E, N52F, N52Q, N52R , N52Y, I54A, I54D, I54F, I54G, I54H, I54K, I54L, I54M, I54N, I54P, I54R, I5 4V, I54W, I54Y, S55E, S55F, S55I, S55Q, S55R, S55Y, S57E, S57F, S57R, S57Y , T58F, T58R, S59A, S59D, S59E, S59F, S59G, S59H, S59I, S59K, S59L, S59M, S5 9N, S59P, S59Q, S59R, S59T, S59V, S59W, S59Y, N60E, N60R, N60Y, A61F, A61R , A61Y, Q62L, Q62R, Q62Y, Q65E, Q65R, Q65Y, D99F, D99R, D99Y, I100D, I100E, I100F, I100Y, V101A, V101D, V101E, V101F, V101G, V101H, V101I, V101K, V1 01L, V101M, V101N, V101P, V101Q, V101R, V101S, V101W, D102E, D102F, D102K , D102R, D102Y, A103D, A103F, A103G, A103H, A103I, A103K, A103L, A103M, A103N, A103P, A103R, A103S, A103T, A103V, and A103W, and the one or more VL amino acid changes are selected from Q27F, Q27R, G28D, G28Q, G28R, G28Y, I29F, I29L, S30D, S30E, S30F, S30L, S30Q, S30R, S30Y, S31D, S31E, S31F, S31Q, S31Y, A32F, A32V, V34F, V34I,D50E, D50F, D50L, D50Q, D50R, D50S, D50T, D50V, D50Y, A51V, S52A, S52D, S52E, S52F, S52G, S52H, S52I, S52K, S52L, S52M, S52N, S52P, S52 Q, S52R, S52V, S52W, S52Y, S53D, S53E, S53F, S53R, S53Y, L54E, L54Y, E55R, E55Y, S67E, S67R, S67Y, T69R, T69Y, F91E, F91R, F91Y, N92A, N9 2D, N92E, N92F, N92G, N92H, N92I, N92K, N92L, N92M, N92P, N92Q, N92R, N92S, N92T, N92V, N92W, N92Y, D93F, D93Q, D93R, D93S, D93T, D93Y, Y94A, Y94D, Y94E, Y94F, Y94G, Y94H, Y94I, Y94K, Y94L, Y94M, Y94N, Y94P, Y94Q, Y94R, Y94S, Y94V, and Y94W.

37. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide with greater binding affinity than burosumab, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) amino acid sequence and a light chain variable region (VL) amino acid sequence according to Table 22.

38. 1. An isolated antibody or fragment thereof that specifically binds to a human fibroblast growth factor 23 (FGF23) polypeptide with greater binding affinity than burosumab, wherein the antibody or fragment thereof comprises a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence according to Table 22.

39. 39. The isolated antibody or fragment thereof of any one of claims 1 to 38, wherein the antibody or fragment thereof has increased affinity for human FGF23 compared to burosumab.

40. 40. The isolated antibody or fragment thereof of any one of claims 1 to 39, wherein the antibody or fragment thereof has about a 2-fold to about a 10-fold increased binding affinity for human FGF23 compared to burosumab.

41. The binding affinity (K Ｄ ) is about 1 × 10 －１２ M ~ approx. 7 x 10 －１２ 41. The isolated antibody or fragment thereof according to any one of claims 1 to 40, wherein said antibody or fragment is M.

42. 42. The isolated antibody or fragment thereof of any one of claims 1 to 41 for use in a method for lowering serum FGF23 in a subject in need thereof.

43. 42. The isolated antibody or fragment thereof of any one of claims 1 to 41 for use in a method for increasing the renal maximum threshold value (TmP) of phosphorus reabsorption in a subject in need thereof.

44. 42. The isolated antibody or fragment thereof of any one of claims 1 to 41 for use in a method for increasing serum inorganic phosphorus (Pi) in a subject in need thereof.

45. 42. The isolated antibody or fragment thereof of any one of claims 1 to 41 for use in a method for increasing serum 1,25 dihydroxyvitamin D (1,25[OH]2D) levels in a subject in need thereof.

46. 42. The isolated antibody or fragment thereof of any one of claims 1 to 41 for use in a method for treating a hypophosphatemic condition in a subject in need thereof.

47. 47. The isolated antibody or fragment thereof for use according to any one of claims 42 to 46, wherein the subject has been diagnosed with X-linked hypophosphatemia (XLH) or tumor-induced osteomalacia (TIO).

48. 42. A method in a subject in need of lowering serum FGF23, the method comprising administering to the subject an isolated antibody or fragment thereof according to any one of claims 1 to 41.

49. 42. A method in a subject in need of increasing the renal maximal threshold for phosphorus reabsorption (TmP), comprising administering to the subject an isolated antibody or fragment thereof according to any one of claims 1 to 41.

50. 42. A method in a subject in need of increasing serum inorganic phosphorus (Pi), comprising administering to said subject an isolated antibody or fragment thereof according to any one of claims 1 to 41.

51. 42. A method for increasing serum 1,25 dihydroxyvitamin D (1,25[OH]2D) levels in a subject in need thereof, comprising administering to the subject an isolated antibody or fragment thereof described in any one of claims 1 to 41.

52. 42. A method of treating a hypophosphatemic condition in a subject in need thereof, said method comprising administering to said subject the isolated antibody or fragment thereof of any one of claims 1-41.

53. 53. The method of any one of claims 48 to 52, wherein the subject has been diagnosed with X-linked hypophosphatemia (XLH) or tumor-induced osteomalacia (TIO).

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