Wnt receptor-specific compounds and related methods

Abstract

The present invention provides compounds that specifically bind to one or more Wnt receptors and methods related thereto. In some aspects, compositions and methods are provided for treating eye disorders by regulating the Wnt signaling pathway. In particular, the eye disorder is retinopathy. Also provided are administration methods and pharmaceutical compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to US Ser. No. 62 / 295,805, filed December 31, 2021, US Ser. No. 63 / 298,570, filed January 11, 2022, and US Ser. No. 63 / 398,754, filed August 17, 2022, the contents of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention relates to compounds that specifically bind to one or more Wnt receptors and methods related thereto. Specifically, the present invention provides Wnt signaling modulation for use in treating various ocular diseases, particularly vascular diseases of the eye, including retinopathies.

[0003] Background of the Invention The vertebrate retina is a thin layer of neural tissue at the back of the eye. It is responsible for detecting visual stimuli and is the first station for visual information processing. For proper retinal function, the retinal vasculature is essential as a source of nutrients and oxygen. The retina is metabolically highly active. Due to the high oxygen consumption of photoreceptor cells, the retina exhibits the highest oxygen consumption rate per gram of any organ in the body. To efficiently deliver nutrients and oxygen, the retinal vasculature is arranged in a three-dimensional structure consisting of three planar vascular plexuses on one side and choriocapillaries on the other. Internal angiogenesis begins at the vitreous surface of the retina, giving rise to the primary vascular plexus. After radial superficial expansion of the vascular plexus, blood vessels penetrate vertically into the retina, forming two additional intraretinal capillary plexuses in the inner plexiform layer (IPL) and outer plexiform layer (OPL). Due to the functional and structural relationship between blood vessels and the retina, abnormal development of blood vessels and / or damage to blood vessels is directly linked to retinal dysfunction or disorders, and may cause various types of retinopathies and retinal degenerations.

[0004] Wnt signaling is implicated as a critical pathway in retinal vascular development. Genetic evidence from human and rodent studies is increasingly supporting the importance of Wnt signaling in the retinal vasculature (Wang et al., 2018, Prog Retin Eye Res. 2018 Dec 1. pii: S1350-9462(18)30046-6). Mutations in human genes encoding various receptors (Fzd4, Lrp5, Tspan12) and ligands (norrin) involved in Wnt signaling cause various hereditary vitreoretinopathy diseases. Specific genetic mutations in the mouse orthologues of these genes (Fzd4, Lrp5, Tspan12, and norrin) have also been observed to cause the characteristic vascular abnormalities seen in human retinopathies. These findings have enabled researchers to gain a deeper understanding of retinopathy disease progression and potentially pave the way for treatment of retinopathies through modulation of Wnt signaling.

[0005] Retinopathy is any damage to the retina of the eye that can result in vision loss. Retinopathy encompasses damage to the retina caused by retinal vascular disease and abnormal blood flow. Age-related macular degeneration is considered a type of retinopathy, but is often discussed as a separate disease. Retinopathy, including diabetic retinopathy, can be divided into early and late stages. In the early stage, also known as nonproliferative retinopathy, the small blood vessels of the retina may deteriorate slightly, causing some vessels to swell and leak fluid into the surrounding retinal tissue. In late stages of retinopathy, significant neovascularization and microaneurysms and hemorrhages occur in the retinal region (see, e.g., "Grading Diabetic Retinopathy from Stereoscopic Color Fundus Photographs—An Extension of the Modified Airlie House Classification." (1991) Ophthalmology, 98(5), 786-806). Retinopathy can be an ocular symptom of systemic or metabolic diseases, such as those seen in diabetes and hypertension. Diabetes is a common cause of retinopathy, which is the leading cause of blindness in the working age population and has been designated a priority eye disease by the World Health Organization.

[0006] Familial exudative vitreoretinopathy (FEVR) is a genetic eye disease characterized by hypoplasia of the intraocular vasculature. More than 50% of FEVR patients have mutations in one of the genes encoding Fzd4, Lrp5, Tspan12, or norrin. Norrin, a Wnt signaling ligand, transmits signals to endothelial cells via a receptor complex consisting of Fzd4, Lrp5, and Tspan12, resulting in normal retinal angiogenesis. However, in FEVR patients, mutations in one or more of the norrin, Fzd4, Lrp5, and / or Tspan12 genes result in immature retinal vascular development. This results in the formation of avascular areas, retinal ischemic areas, and primary retinal damage. This ischemic state induces the production of vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2), leading to neovascularization and vascular plexus formation. The newly formed abnormal blood vessels may easily rupture, causing secondary retinal damage due to exudate and bleeding. The disease progression of diabetic retinopathy (DR) is similar to that of FEVR and other genetic vascular malformations or deficiencies. Hyperglycemia induces retinal vascular damage, leading to vascular occlusion, ischemia, neovascularization, and bleeding, ultimately leading to retinopathy.

[0007] Genetic data suggest that Wnt signaling is important in establishing proper vascular structure in the eye, but it is unclear whether activating Wnt signaling after development would lead to improved vascular structure. Some reports even suggest that antagonizing, rather than stimulating, Wnt signaling may be beneficial for treating retinopathy. Therefore, a better understanding of retinopathy, disease progression, and the involvement of Wnt signaling therein would be beneficial, as it may lead to novel treatments. In particular, for effective treatment of retinopathy, it is necessary to regulate Wnt agonist and antagonist signaling at specific stages of the disease. The present invention addresses this need and provides methods for regulating stimulation and antagonism of Wnt signaling at various stages of retinopathy and disease progression.

[0008] Summary of the Invention In one aspect, the present disclosure provides a multispecific antibody or antibody fragment.

[0009] The multispecific antibody or antibody fragment of the present disclosure may comprise at least: (A) a first antigen-binding region that specifically binds frizzled class receptor 4 (Fzd4) and comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); and (B) a second antigen-binding region that specifically binds low-density lipoprotein receptor-related protein 5 and / or 6 (LRP5 and / or LRP6) and comprises at least a second heavy chain variable domain (VH2).

[0010] In some embodiments, the VH1 has (i) (i-1) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 121 or, optionally, the CDRH1 amino acid sequence contained in SEQ ID NO: 181 as defined according to Kabat; and (i-2) the amino acid sequence of SEQ ID NO: 120, where X1 is any amino acid. (i-3) a heavy chain complementarity determining region 1 (CDRH1) having an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 121, 122, 123 or 124 or optionally the CDRH1 amino acid sequence contained in any one of SEQ ID NO: 181 to 188 as defined according to Kabat; and (ii) (ii-1) a heavy chain complementarity determining region 2 (CDRH1) having an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 141 or optionally the CDRH1 amino acid sequence contained in SEQ ID NO: 181 as defined according to Kabat. (ii-1) has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the CDRH2 amino acid sequence of SEQ ID NO: 140 (wherein X1 is any amino acid, optionally E, A, S, or D); or (ii-2) comprises or consists of the amino acid sequence of SEQ ID NO: 140 (wherein X1 is any amino acid, optionally E, A, S, or D); or (ii-3) comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 141, 142, 143, or 144. (iii) a heavy chain complementarity determining region 2 (CDRH2) having an amino acid sequence that comprises or consists of the CDRH2 amino acid sequence contained in any one of SEQ ID NOs: 181 to 188 as defined by Kabat; and (iii) (iii-1) a heavy chain complementarity determining region 2 (CDRH3) having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96% of the amino acid sequence of SEQ ID NO: 161 or, optionally, the CDRH3 amino acid sequence contained in SEQ ID NO: 181 as defined by Kabat;(iii-2) having at least 97%, at least 98%, at least 99%, or 100% identity with the heavy chain complementarity determining region 3 (CDRH3) having an amino acid sequence of SEQ ID NO: 160 (wherein X1 is any amino acid, optionally D or any amino acid that is not S, E, A, R, T, L, H, Y, Q, or K); and (iii-3) a heavy chain complementarity determining region 3 (CDRH3) having an amino acid sequence of SEQ ID NO: 161 or optionally comprising or consisting of a CDRH3 amino acid sequence contained in any one of SEQ ID NOs: 181 to 188 as defined by Kabat.

[0011] In some embodiments, the VL1 (iv) (iv-1) has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 221 or, optionally, the CDRL1 amino acid sequence contained in SEQ ID NO: 281 as defined by Kabat; or (iv-2) comprises or consists of the amino acid sequence of SEQ ID NO: 221 or, optionally, the CDRL1 amino acid sequence contained in SEQ ID NO: 281 as defined according to Kabat. (v) a light chain complementarity determining region 2 (CDRL2) having an amino acid sequence comprising or consisting of (v-1) the amino acid sequence of SEQ ID NO: 241 or (v-2) optionally the CDRL2 amino acid sequence contained in SEQ ID NO: 281 as defined according to Kabat; and (vi) a light chain complementarity determining region 3 (CDRL3) having an amino acid sequence comprising or consisting of (vi-1) the amino acid sequence of SEQ ID NO: 261 or (vi-2) optionally the CDRL3 amino acid sequence contained in SEQ ID NO: 281 as defined according to Kabat.

[0012] In some embodiments, the VH2 comprises: (i) a CDRH1 having an amino acid sequence that comprises or consists of (i-1) the amino acid sequence of SEQ ID NO: 321, or (i-2) optionally the CDRH1 amino acid sequence contained in any one of SEQ ID NOs: 381, 386, and 393-396 as defined according to IMGT or Kabat; and (ii) (ii-1) a CDRH3 amino acid sequence that is at least 90%, at least 90%, of the amino acid sequence of SEQ ID NO: 341, or optionally the CDRH3 amino acid sequence contained in SEQ ID NO: 381 as defined according to IMGT or Kabat. (ii-1) a CDRH2 having an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 341, or (ii-2) a CDRH2 having an amino acid sequence comprising or consisting of the CDRH2 amino acid sequence contained in any one of SEQ ID NOs: 381, 386, and 393 to 396 as defined according to IMGT or Kabat; and (iii) (iii-2) a CDRH2 having an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 341, or (ii-3) a CDRH2 having an amino acid sequence comprising or consisting of the CDRH2 amino acid sequence contained in any one of SEQ ID NOs: 381, 386, and 393 to 396 as defined according to IMGT or Kabat. (iii-1) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 361 or, optionally, the CDRH3 amino acid sequence contained in SEQ ID NO: 381 as defined according to IMGT or Kabat; and (iii-2) the amino acid sequence of SEQ ID NO: 360, wherein X1, X2 and X3 are individually any amino acid, with the proviso that (a) X1 and X2 are each independently selected from the group consisting of: (b) X1 and X3 are D and D, respectively, and / or (c) X2 and X3 are E and D, respectively, optionally with the proviso that X1, X2 and X3 are D, E and D, respectively, D, D and D, respectively, D, E and E, respectively, D, E and S, respectively, D, E and A, or respectively D, E and T; or (iii-3) comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 361, 362, 363, 364, 365 and 366, or optionally,and a CDRH3 having an amino acid sequence comprising or consisting of the CDRH3 amino acid sequence contained in any one of SEQ ID NOs: 381, 382, ​​388, 393, 394, 395, and 396 as defined according to IMGT or Kabat.

[0013] In some cases, a multispecific antibody or antibody fragment may comprise a VH1 as defined above, a VL1 as defined above, and a VH2 as defined above.

[0014] In some instances, the VH2 is, comprises, or is included in a Nanobody.

[0015] In some cases, the multispecific antibody or antibody fragment is a bispecific antibody or antibody fragment.

[0016] In certain embodiments, the VH1 is selected from the group consisting of (i) SEQ ID NOs: 121, 141 and 161, respectively; (ii) SEQ ID NOs: 122, 142 and 161, respectively; (iii) SEQ ID NOs: 121, 142 and 161, respectively; (iv) SEQ ID NOs: 123, 142 and 161, respectively; (v) SEQ ID NOs: 124, 142 and 161, respectively; (vi) SEQ ID NOs: 122, 141 and 161, respectively; (vii) SEQ ID NOs: 122, 143 and 161, respectively; (viii) SEQ ID NOs: 122, 144 and 161, respectively; (ix) SEQ ID NOs: (x) SEQ ID NOs: 121, 143 and 161, respectively; (xii) SEQ ID NOs: 123, 143 and 161, respectively; (xiii) SEQ ID NOs: 123, 144 and 161, respectively; (xiv) SEQ ID NOs: 124, 141 and 161, respectively; (xv) SEQ ID NOs: 124, 143 and 161, respectively; or (xvi) CDRH1, CDRH2 and CDRH3 having the amino acid sequences of SEQ ID NOs: 124, 144 and 161, respectively.

[0017] In certain embodiments, the VL1 comprises CDRL1, CDRL2 and CDRL3 having the amino acid sequences of SEQ ID NOs: 221, 241 and 261, respectively.

[0018] In certain embodiments, the VH2 comprises CDRH1, CDRH2, and CDRH3 having the amino acid sequences of (i) SEQ ID NOs: 321, 341, and 361, respectively; (ii) SEQ ID NOs: 321, 341, and 362, respectively; (iii) SEQ ID NOs: 321, 341, and 363, respectively; (iv) SEQ ID NOs: 321, 341, and 364, respectively; (v) SEQ ID NOs: 321, 341, and 365, respectively; or (vi) SEQ ID NOs: 321, 341, and 366, respectively.

[0019] In some cases, a multispecific antibody or antibody fragment may comprise a VH1 as defined above, a VL1 as defined above, and a VH2 as defined above.

[0020] In a particular embodiment, the VH1 comprises (i) CDRH1, CDRH2 and CDRH3 of VH1 optionally comprising the amino acid sequence of any one of SEQ ID NOs: 181 to 188 according to Kabat, or (ii) CDRH1, CDRH2 and CDRH3 comprising the amino acid sequence of (ii-1) SEQ ID NOs: 121, 141 and 161, respectively; (ii-2) SEQ ID NOs: 122, 142 and 161, respectively; (ii-3) SEQ ID NOs: 121, 142 and 161, respectively; (ii-4) SEQ ID NOs: 123, 142 and 161, respectively; (ii-5) SEQ ID NOs: 124, 142 and 161, respectively; (ii-6) SEQ ID NOs: 122, 141 and 161, respectively; (ii-7) SEQ ID NOs: 122, 143 and 161, respectively; or (ii-8) SEQ ID NOs: 122, 144 and 161, respectively.

[0021] In certain embodiments, the VL1 optionally comprises (i) CDRL1, CDRL2 and CDRL3 of VL1 comprising the amino acid sequence contained in SEQ ID NO: 281 according to Kabat, and (ii) CDRL1, CDRL2 and CDRL3 comprising the amino acid sequences of SEQ ID NOs: 221, 241 and 261, respectively.

[0022] In certain embodiments, the VH2 comprises (i) optionally a CDRH1, CDRH2 and CDRH3 of VH2 comprising the amino acid sequence comprised in any one of SEQ ID NOs: 381, 386, 393, 394, 395 and 396 according to IMGT or Kabat, or (ii) a CDRH1, CDRH2 and CDRH3 comprising the amino acid sequence of (ii-1) SEQ ID NOs: 321, 341 and 361, respectively; (ii-2) SEQ ID NOs: 321, 341 and 362, respectively; (ii-3) SEQ ID NOs: 321, 341 and 363, respectively; (ii-4) SEQ ID NOs: 321, 341 and 364, respectively; (ii-5) SEQ ID NOs: 321, 341 and 365, respectively; (ii-6) SEQ ID NOs: 321, 341 and 366, respectively.

[0023] In some cases, a multispecific antibody or antibody fragment may comprise a VH1 as defined above, a VL1 as defined above, and a VH2 as defined above.

[0024] In a particular embodiment, (A) the VH1 comprises (i) optionally a CDRH1, CDRH2, and CDRH3 of VH1 comprising the amino acid sequence contained in SEQ ID NO: 181 according to Kabat, or (ii) a CDRH1, CDRH2, and CDRH3 of VH1 comprising the amino acid sequence of SEQ ID NOs: 121, 141, and 161, respectively, and the VL1 comprises (i) optionally a CDRL1, CDRL2, and CDRL3 of VL1 comprising the amino acid sequence contained in SEQ ID NO: 281 according to Kabat, or (ii) a CDRL1, CDRL2, and CDRL3 of VL1 comprising the amino acid sequence of SEQ ID NOs: 221, 241, and 261, respectively. and CDRH3, and (B) the VH2 optionally comprises (i) a CDRH1, CDRH2, and CDRH3 of the VH2 comprising the amino acid sequence comprised in any one of SEQ ID NOs: 381, 393, 394, 395, or 396 according to IMGT or Kabat, or (ii) a CDRH1, CDRH2, and CDRH3 comprising the amino acid sequence of SEQ ID NOs: 321, 341, and 361, respectively, SEQ ID NOs: 321, 341, and 363, respectively, SEQ ID NOs: 321, 341, and 364, respectively, SEQ ID NOs: 321, 341, and 365, or SEQ ID NOs: 321, 341, and 366, respectively.

[0025] In certain embodiments, the VH1 (i) (i-1) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 111 or optionally the FRH1 amino acid sequence contained in SEQ ID NO: 181 according to Kabat; (i-2) comprises or consists of the amino acid sequence of SEQ ID NO: 110 (wherein X1 is any amino acid, optionally Q or K); (i-3) comprises or consists of the amino acid sequence of SEQ ID NO: 111 or 112 or optionally comprises or consists of the FRH1 amino acid sequence contained in any one of SEQ ID NOs: 181-188 according to Kabat; and / or (i-4) comprises or consists of the amino acid sequence of human germline IGHV3-23 *04; (ii) (ii-1) a heavy chain framework region 1 (FRH1) having an amino acid sequence consisting of or derived from the FRH1 encoded by SEQ ID NO: 131 or optionally the FRH2 amino acid sequence contained in SEQ ID NO: 181 according to Kabat; (ii-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 130 or 131 or optionally comprising or consisting of the FRH2 amino acid sequence contained in any of SEQ ID NOs: 181 to 188 according to Kabat; and / or (ii-3) a human germline IGHV3-23 * (iii) a heavy chain framework region 2 (FRH2) having an amino acid sequence consisting of or derived from FRH2 encoded by SEQ ID NO: 151 or, optionally, an FRH3 amino acid sequence contained in SEQ ID NO: 181 according to Kabat, which has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 151 or, optionally, the FRH3 amino acid sequence contained in SEQ ID NO: 181 according to Kabat. (iii-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 150 (wherein X1 is any amino acid, optionally N or D, X2 is any amino acid, optionally R or K, and X3 is any amino acid, optionally A or T); (iii-3) comprising or consisting of the amino acid sequence of SEQ ID NO: 151 or 152, or optionally comprising or consisting of the FRH3 amino acid sequence contained in any of SEQ ID NOs: 181 to 188 according to Kabat; and / or (iii-4) human germline IGHV3-23 *A heavy chain framework region 3 (FRH3) consisting of or derived from an amino acid sequence encoded by 04, and / or (iv) (iv-1) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 171 or optionally the FRH4 amino acid sequence contained in SEQ ID NO: 181 according to Kabat, (iv-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 170 or 171 or optionally comprising or consisting of the FRH4 amino acid sequence contained in any of SEQ ID NOs: 181-188 according to Kabat and / or (iv-3) human germ cell line IGHJ4 * Comprising a heavy chain framework region 4 (FRH4) consisting of or derived from an amino acid sequence encoded by 01.

[0026] In certain embodiments, the VL1 comprises: (v) (v-1) a light chain framework region 1 (FRL1) having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 211 or optionally the FRL1 amino acid sequence contained in SEQ ID NO: 281 according to Kabat; or (v-2) a light chain framework region 2 (FRL1) having an amino acid sequence that comprises or consists of the amino acid sequence of SEQ ID NO: 211 or optionally the FRL1 amino acid sequence contained in SEQ ID NO: 281 according to Kabat; (vi) (vi-1) a light chain framework region 3 (FRL2) having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92% identity to the amino acid sequence of SEQ ID NO: 231 or optionally the FRL2 amino acid sequence contained in SEQ ID NO: 281 according to Kabat; (vii) a light chain framework region 2 (FRL2) having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to (vii-1) the amino acid sequence of SEQ ID NO: 251 or optionally the FRL3 amino acid sequence contained in SEQ ID NO: 281 according to Kabat, or (vii-2) comprises or consists of or optionally the amino acid sequence of SEQ ID NO: 251;a light chain framework 3 (FRL3) having an amino acid sequence comprising or consisting of the FRL3 amino acid sequence contained in SEQ ID NO: 281 according to Kabat; and / or (viii) (viii-1) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 271 or optionally the FRL4 amino acid sequence contained in SEQ ID NO: 281 according to Kabat; or (viii-2) a light chain framework 4 (FRL4) having an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 271 or optionally comprising or consisting of the FRL4 amino acid sequence contained in SEQ ID NO: 281 according to Kabat.

[0027] In certain embodiments, the VH2 has (i) (i-1) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 311 or optionally the FRH1 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat; and (i-2) the amino acid sequence of SEQ ID NO: 310, wherein X1 is any amino acid, optionally E or D, and X2 is any X1, X2, and X3 are any amino acid, optionally A or T, and X3 is any amino acid, optionally S, wherein optionally FRH1 does not consist of the amino acid sequence of SEQ ID NO: 310, in which X1, X2, and X3 are E, A, and A, respectively; (i-3) comprising or consisting of the amino acid sequence of SEQ ID NO: 311 or 312, or optionally comprising or consisting of the FRH1 amino acid sequence contained in any one of SEQ ID NOs: 381, 382 to 386, and 393 to 396 according to IMGT or Kabat; and / or (i-4) human germline IGHV3-66 *FRH1 having an amino acid sequence consisting of or derived from FRH1 encoded by 01; (ii) (ii-1) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 331 or optionally the FRH2 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat; and (ii-2) the amino acid sequence of SEQ ID NO: 330, wherein X1 is any amino acid, optionally Y; X2 is any amino acid, optionally Q; X3 is any amino acid, optionally R; X4 is any amino acid, (ii-2) a human germline IGHV3-66 FRH2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 331, 332, or 333, or optionally the FRH2 amino acid sequence contained in any one of SEQ ID NOs: 381, 382 to 386, and 393 to 396 according to IMGT or Kabat; and / or (ii-3) a human germline IGHV3-66 FRH2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 331, 332, or 333, or optionally the FRH2 amino acid sequence contained in any one of SEQ ID NOs: 381, 382 to 386, and 393 to 396 according to IMGT or Kabat; and / or (ii-4) a human germline IGHV3-66 FRH2 amino acid sequence comprising the amino acid sequence of SEQ ID NO: 331, 332, or 333, or optionally the FRH2 amino acid sequence contained in any one of SEQ ID NOs: 381, 382 to 386, and 393 to 396 according to IMGT or Kabat; *(iii) an FRH2 having an amino acid sequence consisting of or derived from an FRH2 encoded by SEQ ID NO: 351, or (iii-1) an FRH3 amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 351 or, optionally, the FRH3 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat. (iii-2) the amino acid sequence of SEQ ID NO: 350 (wherein X1 is any amino acid, optionally D or G, X2 is any amino acid, optionally L, X3 is any amino acid, optionally M, X4 is any amino acid, optionally T, X5 is any amino acid, optionally D or E, X6 is any amino acid, optionally N or S, X7 is any amino acid, optionally S or A, X8 is any amino acid, optionally M, X9 is any amino acid, optionally R or K, and X 10 is any amino acid, optionally A or P, wherein optionally FRH3 is selected from X1, X2, X3, X4, X5, X6, X7, X8, X9 and X 10 (iii-3) comprising or consisting of the amino acid sequence of SEQ ID NO: 351 or 352, or optionally comprising or consisting of the FRH3 amino acid sequence contained in any one of SEQ ID NOs: 381, 382 to 386, and 393 to 396 according to IMGT or Kabat; and / or (iii-4) a human germline IGHV3-66 *01 and / or (iv) (iv-1) an FRH3 having an amino acid sequence consisting of or derived from the FRH3 encoded by SEQ ID NO: 371 or, optionally, an FRH4 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat, which has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 371 or, optionally, the FRH4 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat. (iv-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 370 (wherein X1 is any amino acid, optionally R or W, and X2 is any amino acid, optionally Q or L); (iv-3) comprising or consisting of the amino acid sequence of SEQ ID NO: 351 or 352, or optionally comprising or consisting of the FRH4 amino acid sequence contained in any of SEQ ID NOs: 381, 382 to 386, and 393 to 396 according to IMGT or Kabat; and / or (iv-4) human germline IGHV3-66 * 01 or an FRH4 having an amino acid sequence derived therefrom.

[0028] In some cases, a multispecific antibody or antibody fragment may comprise a VH1 as defined above, a VL1 as defined above, and a VH2 as defined above.

[0029] In certain embodiments, the VH1 has (i) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 181-188, and / or (ii) the amino acid sequence of SEQ ID NO: 180, wherein X1 is any amino acid, optionally Q or K. X2 is any amino acid, optionally S, A, Q or N, X3 is any amino acid, optionally E, A, S or D, X4 is any amino acid, optionally N or D, X5 is any amino acid, optionally R or K, X6 is any amino acid, optionally E or T, and X7 is any amino acid, optionally D or any amino acid that is not S, E, A, R, T, L, H, Y, Q or K).

[0030] In certain embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO:281.

[0031] In certain embodiments, the VH2 has (i) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 381, 386 and 393-396, and / or (ii) the amino acid sequence of SEQ ID NO: 380 (wherein Xi is any amino acid, optionally E or D, X2 is any amino acid, e.g., A or T, X3 is any amino acid, e.g., S, X4 is Q, X5 is any amino acid, e.g., Y, X6 is any amino acid, e.g., Q or G, X7 is any amino acid, e.g., R, X8 is any amino acid, e.g., L or W, X9 is any amino acid, e.g., I, and X 10 is any amino acid, e.g., A, and X 11 is M and X 12 is M and X 13 is any amino acid, e.g., D or G, and X 14 is any amino acid, e.g., L, and X 15 is any amino acid, e.g., M, and X 16 is any amino acid, e.g., T, and X 17 is any amino acid, e.g., D or E, and X 18 is any amino acid, e.g., N or S, and X 19 is any amino acid, e.g., S or A, and X 20 is any amino acid, e.g., M, and X 21 is any amino acid, e.g., R or K, and X 22 is any amino acid, for example, A or P, and (a) X 23 and X 24 are D and E, respectively, and (b) X 23 and X 25 are D and D, respectively, and / or (c) X 24 and X 25 As long as E and D are respectively, X 23 , X 24 and X25 are individually any amino acid, where optionally X 23 , X 24 and X 25 are D, E and D, respectively, D, D and D, respectively, D, E and E, respectively, D, E and S, respectively, D, E and A, or respectively, D, E and T, and X 26 is any amino acid, e.g., R or W, and X 27 is any amino acid, e.g., Q or L).

[0032] In some cases, a multispecific antibody or antibody fragment may comprise a VH1 as defined above, a VL1 as defined above, and a VH2 as defined above.

[0033] In certain embodiments, (A) the VH1 comprises an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 181-188 and / or the VL1 comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 281 and / or (B) the VH2 comprises an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 381, 386 and 393-396.

[0034] In certain embodiments, (A) the VH1 (i) optionally comprises CDRH1, CDRH2, and CDRH3 of VH1 comprising the amino acid sequence contained in SEQ ID NO: 181 according to Kabat, or (ii) CDRH1, CDRH2, and CDRH3 comprising the amino acid sequences of SEQ ID NOs: 121, 141, and 161, respectively; the VL1 (i) optionally comprises CDRL1, CDRL2, and CDRL3 of VL1 comprising the amino acid sequence contained in SEQ ID NO: 281 according to Kabat, or (ii) CDRL1, CDRL2, and CDRL3 comprising the amino acid sequences of SEQ ID NOs: 221, 241, and 261, respectively; and (B) the VH2 (i) optionally comprises CDRH1, CDRH2, and CDRH3 of VH2 comprising the amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat, or (ii) CDRH1, CDRH2, and CDRH3 comprising the amino acid sequences of SEQ ID NOs: 321, 341, and 361, respectively.

[0035] In certain embodiments, the VH1 (i) (i-1) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 111 or optionally the FRH1 amino acid sequence contained in SEQ ID NO: 181 according to Kabat; (i-2) comprises or consists of the amino acid sequence of SEQ ID NO: 111 or optionally comprises or consists of the FRH1 amino acid sequence contained in SEQ ID NO: 181 according to Kabat; and / or (i-3) is a human germline IGHV3-23 *(ii) an FRH1 having an amino acid sequence consisting of or derived from an FRH1 encoded by SEQ ID NO: 04; (ii) (ii-1) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 131 or optionally the FRH2 amino acid sequence contained in SEQ ID NO: 181 according to Kabat; (ii-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 131 or optionally comprising or consisting of the FRH2 amino acid sequence contained in SEQ ID NO: 181 according to Kabat; and / or (ii-3) a human germline IGHV3-23 * 04, (iii) (iii-1) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 151 or optionally the FRH3 amino acid sequence contained in SEQ ID NO: 181 according to Kabat, (iii-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 151 or optionally comprising or consisting of the FRH3 amino acid sequence contained in SEQ ID NO: 181 according to Kabat, and / or (iii-3) a human germline IGHV3-23 *and / or (iv) (iv-1) an FRH3 having an amino acid sequence consisting of or derived from the FRH3 encoded by SEQ ID NO: 171 or optionally the FRH4 amino acid sequence contained in SEQ ID NO: 181 according to Kabat; (iv-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 171 or optionally comprising or consisting of the FRH4 amino acid sequence contained in SEQ ID NO: 181 according to Kabat; and / or (iv-3) a human germline IGHJ4. * 01 or an FRH4 having an amino acid sequence derived therefrom.

[0036] In certain embodiments, the VL1 has (v) (v-1) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 211 or optionally the FRL1 amino acid sequence contained in SEQ ID NO: 281 according to Kabat, or (v-2) comprises or consists of the amino acid sequence of SEQ ID NO: 211. (vi) (vi-1) an FRL1 having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 1109%, at least 1111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 1200%, at least 1211%, at least 1221%, at least 1231%, at least 1241%, at least 1251%, at least 1261%, at least 1271%, at least 1282%, at least 1291%, at least 1292%, at least 1293%, at least 1294%, at least 1295%, at least 1296%, at least 1297%, at least 1298%, at least 1299%, at least 1300%, at least 1310%, at least 1311%, at least 1312%, at least 1313%, at least 1314%, at least 1315%, at least 1316%, at least 1317%, at least 1318%, at least 1319%, at least 1320%, at least 13219%, at least 132219, at least 132319, at least 132419 (vii) (vii-1) an FRL2 having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 251 or optionally the FRL2 amino acid sequence contained in SEQ ID NO: 281 according to Kabat; or (vii-2) an FRL3 having an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 251 or optionally the FRL3 amino acid sequence contained in SEQ ID NO: 281 according to Kabat; and / or (viii) (viii-1) the amino acid sequence of SEQ ID NO: 271 or optionally,(viii-2) an FRL4 having an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 271 or optionally comprising or consisting of the FRL4 amino acid sequence comprised in SEQ ID NO: 281 according to Kabat;

[0037] In certain embodiments, the VH2 (i) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to (i-1) the amino acid sequence of SEQ ID NO: 311 or optionally the FRH1 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat; (i-2) comprises or consists of the amino acid sequence of SEQ ID NO: 311 or optionally comprises or consists of the FRH1 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat; and / or (i-3) is a human germline IGHV3-66 *01, (ii) (ii-1) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 331 or optionally the FRH2 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat, (ii-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 331 or optionally comprising or consisting of the FRH2 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat, and / or (ii-3) a human germline IGHV3-66. * 01, (iii) (iii-1) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 351 or optionally the FRH3 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat, (iii-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 351 or optionally comprising or consisting of the FRH3 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat, and / or (iii-3) a human germline IGHV3-66. *01 and / or (iv) (iv-1) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 371 or optionally the FRH4 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat, (iv-2) comprising or consisting of the amino acid sequence of SEQ ID NO: 371 or optionally comprising or consisting of the FRH4 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat, and / or (iv-3) human germline IGHV3-66. * 01 or an FRH4 having an amino acid sequence derived therefrom.

[0038] In some cases, a multispecific antibody or antibody fragment may comprise a VH1 as defined above, a VL1 as defined above, and a VH2 as defined above.

[0039] In certain embodiments, (A) the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NO: 181 and / or the VL1 comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 281. , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 381; and / or (B) the VH2 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 381.

[0040] In certain embodiments, (A) the VH1 comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 181 and / or the VL1 comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 281, and / or (B) the VH2 comprises an amino acid sequence comprising or consisting of any of the amino acid sequences of SEQ ID NO: 381.

[0041] In a specific embodiment, (A) the VH1 comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 181, the VL1 comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 281, and (B) the VH2 comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 381.

[0042] In certain embodiments, a multispecific antibody or antibody fragment of the present disclosure may comprise: (A) a first antigen-binding region that specifically binds to Fzd4 and comprises VH1 and VL1; and (B) a second antigen-binding region that specifically binds to LRP5 and / or LRP6 and comprises at least VH2; wherein (i) CDRH1, CDRH2, and CDRH3 of the VH1 are identical to those of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 181, (ii) CDRL1, CDRL2, and CDRL3 of the VL1 are identical to those of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 281, and (iii) CDRH1, CDRH2, and CDRH3 of the VH2 are identical to those of a VH having the amino acid sequence of SEQ ID NO: 381. In a specific embodiment, (i) the VH1 comprises or consists of the amino acid sequence of SEQ ID NO: 181, (ii) the VL1 comprises or consists of the amino acid sequence of SEQ ID NO: 281, and (iii) the VH2 comprises or consists of the amino acid sequence of SEQ ID NO: 381.

[0043] In some embodiments, (A) the first antigen-binding region is, comprises, or is comprised in an antigen-binding fragment (Fab), a Fab' fragment, F(ab')2, single-chain Fab (scFab), variable fragment (Fv), single-chain Fv (scFv), diabody, or triabody, and / or (B) the second antigen-binding region is (i) is, comprises, or is comprised in a nanobody or Fd fragment, or (ii) further comprises a second light chain variable domain (VL2) and is, comprises, or is comprised in a Fab, Fab' fragment, F(ab')2, scFab, Fv, scFv, diabody, or triabody.

[0044] In some embodiments, the multispecific antibody or antibody fragment comprises one or more of the following: (a) immunoglobulin heavy chain constant domain 1 (CH1) or a variant thereof, (b) immunoglobulin hinge or a variant thereof, (c) immunoglobulin heavy chain constant domain 2 (CH2) or a variant thereof, (d) immunoglobulin heavy chain constant domain 3 (CH3) or a variant thereof, and / or (e) immunoglobulin light chain constant domain (CL) or a variant thereof.

[0045] In certain embodiments, the CH1 or variant thereof (a-1) is that of IgG, IgA, IgE, IgD, IgM, optionally that of IgG1, IgG4, IgG2 or IgG3, further optionally that of a human, and / or (a-2) comprises or consists of the amino acid sequence of SEQ ID NO: 21 or 22 or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0046] In particular embodiments, the hinge or variant thereof is (b-1) that of IgG, IgA, IgE, IgD, IgM, optionally that of IgG1, IgG4, IgG2 or IgG3, further optionally that of a human, and / or (b-2) that comprises or consists of the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0047] In certain embodiments, the CH2 or variant thereof is (c-1) that of IgG, IgA, IgE, IgD, IgM, optionally that of IgG1, IgG4, IgG2, or IgG3, further optionally that of a human, and / or (c-2) that comprises or consists of the amino acid sequence of any of SEQ ID NOs: 41-46, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0048] In certain embodiments, the CH3 or variant thereof (d-1) is that of IgG, IgA, IgE, IgD, IgM, optionally that of IgG1, IgG4, IgG2 or IgG3, further optionally that of a human, and / or (d-2) comprises or consists of the amino acid sequence of any of SEQ ID NOs: 51-54, 61-64 and 66-69 (optionally excluding the C-terminal K), or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0049] In particular embodiments, the CL or variant thereof comprises: (e-1) a kappa light chain constant domain (CLκ), optionally that of a human CLκ, optionally comprising or consisting of the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto; and / or (e-2) a lambda light chain constant domain (CLλ), optionally that of a human CLλ, optionally comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0050] In certain embodiments, the multispecific antibody or antibody fragment comprises the CH2 and CH3 sequences of SEQ ID NOs: 42 and 51, respectively, SEQ ID NOs: 43 and 51, respectively, SEQ ID NOs: 45 and 61, respectively, SEQ ID NOs: 45 and 66, respectively, SEQ ID NOs: 46 and 61, respectively, or SEQ ID NOs: 46 and 66, respectively.

[0051] In some embodiments, the multispecific antibody or antibody fragment comprises a fragment crystallizable (Fc) region.

[0052] In particular embodiments, the Fc region is that of IgG, IgA, IgE, IgD, IgM, optionally IgG1, IgG4, IgG2 or IgG3, further optionally human or a variant thereof.

[0053] In a particular embodiment, the Fc region of the IgG1 or variant thereof comprises any of the following amino acid substitutions according to EU numbering: N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236 deletion, P238A, A327Q, A327G, P329A, P329G, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V305I, P396L, S239D, I3 32E, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y, M428L, N434S, H310A, L328F, M252Y, I253A, S254T, T256E, H435Q, H435A, and / or any combination thereof.

[0054] In certain embodiments, the Fc region of the IgG2 or variant thereof may comprise one or more of the following amino acid substitutions according to EU numbering: P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and / or any combination thereof.

[0055] In a particular embodiment, the Fc region of said IgG3 or variant thereof may comprise the amino acid substitution E235Y according to EU numbering.

[0056] In certain embodiments, the Fc region of the IgG4 or variant thereof may comprise one or more of the following amino acid substitutions according to EU numbering: E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and / or any combination thereof.

[0057] In certain embodiments, the Fc region may comprise one or more amino acid substitutions that reduce Fc effector function.

[0058] In certain embodiments, the one or more amino acid substitutions are at a position selected from the group consisting of 234, 235, 236, 237, 265, 297 and 329 according to EU numbering.

[0059] In certain embodiments, the one or more amino acid substitutions include at least the following amino acid substitutions according to EU numbering: (ii-1) L234A, L235A, and P329G, (ii-2) L234A and L235A, (ii-3) I253A, H310A, and H435Q, (ii-4) I253A, H310A, and H435A, (ii-5) D265A and P329A, (ii-6) M252Y, S254T, and T256E, and / or (ii-7) M428L and N434S. Optionally, such substitutions may be in the human IgG1 Fc region and / or any of SEQ ID NOs: 71-74 (optionally excluding the C-terminal K).

[0060] In certain embodiments, the Fc region may comprise or consist of the amino acid sequence (optionally excluding the C-terminal K) of any one of SEQ ID NOs: 71-74, 81-84, 86-89, 91-94, 96-99, 471-474, 476-479, 481-484, and 486-489, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto.

[0061] In some embodiments, a multispecific antibody or antibody fragment of the present disclosure may comprise a structure shown in any one of Figures 24-35, optionally any of the structures shown in Figure 24.

[0062] In some embodiments, a multispecific antibody or antibody fragment of the present disclosure may comprise (a) a first polypeptide comprising the VH1; and (b) a second polypeptide comprising the VH2 and the VL1, wherein the first and second polypeptides optionally interact with each other via one or more disulfide bonds, thereby enabling the VH1 and VL1 to form the first antigen-binding region.

[0063] In a particular embodiment, (a) the first polypeptide comprises, in order from N-terminus to C-terminus, (a-1) said VH1 and (a-2) a CH1 or variant thereof, optionally wherein said CH1 or variant thereof (i) is of an IgG, optionally of an IgG1 or IgG4, further optionally of a human IgG1 or IgG4, and / or (ii) comprises or consists of, or is at least 80%, at least 85%, at least 90%, at least 91%, at least 100%, at least 120%, at least 140%, at least 160%, at least 18 ... and (a-3) an immunoglobulin hinge or variant thereof, optionally the hinge or variant thereof (i) is that of an IgG, optionally that of an IgG1 or IgG4, further optionally that of a human IgG1 or IgG4, and / or (ii) comprises or consists of or corresponds to the amino acid sequence of SEQ ID NO: 31. (a-4) a CH2 or variant thereof, optionally wherein the CH2 or variant thereof is (i) that of an IgG, optionally that of an IgG1 or IgG4, further optionally that of a human IgG1 or IgG4, and / or (ii) SEQ ID NO: 41 to 46, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto; and (a-5) a CH3 or a variant thereof, optionally the CH3 or variant thereof being (i) that of an IgG, optionally that of an IgG1 or IgG4;and optionally, (ii) an amino acid sequence of human IgG1 or IgG4, and / or (iii) an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 51 to 54, 51 to 64, and 66 to 69 (optionally excluding the C-terminal K), or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto.

[0064] In a particular embodiment, (b) the second polypeptide comprises, in order from N-terminus to C-terminus, (b-1) said VH2; (b-2) optionally a linker, which is optionally a peptide linker and further optionally a flexible linker, wherein optionally the linker (i) comprises one or more amino acids, optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids; (ii) consists of small amino acids consisting of G, S, and / or A; (iii) SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 13 and / or (iv) an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG; and / or (v) an amino acid sequence comprising or consisting of multiple repeats of an amino acid sequence selected from the group consisting of SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG. (b-3) said VL1 and (b-4) a CL or variant thereof, optionally wherein said CL or variant thereof is (i) a CLκ, optionally a human CLκ, and optionally comprises or consists of the amino acid sequence of SEQ ID NO: 11 or is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 1109%, at least 1111%, at least 1120%, at least 1122%, at least 1123%, at least 1124%, at least 1125%, at least 1126%, at least 1127%, at least 1128%, at least 1129%, at least 1130%, at least 1131%, at least 1132, at least 1133, at least 1134, at least 1135, at least 1136, at least 1137, at least 1138, at least 1139, at least 1140, at least 1141, at least 1142, at least 1143, at least 1144, at least 1145, at least 1146, at least 1147, at least 1148, at least 1149, at least 1150, at least 1151, at least 1152, at least 1153, at least 1154, at least 1155, at least 115 or (ii) a CLλ, optionally of human CLλ, optionally comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity thereto.

[0065] In some cases, a multispecific antibody or antibody fragment may comprise both a first polypeptide as described above and a second polypeptide as described above.

[0066] In certain embodiments, the multispecific antibody or antibody fragment comprises two of said first polypeptides and two of said second polypeptides that optionally form a dimer with one another via one or more disulfide bonds, wherein one of the second polypeptides optionally interacts with one of the first polypeptides via a disulfide bond and another of the second polypeptides optionally interacts with another of the first polypeptides via a disulfide bond.

[0067] In certain embodiments, the multispecific antibody or antibody fragment comprises an Fc region, wherein the Fc region is (I) that of an IgG, optionally that of IgG1 or IgG4, and further optionally that of a human or variant thereof; and (II) optionally that of a human IgG1. The Fc region and / or any of SEQ ID NOS: 71 to 74 (optionally excluding the C-terminal K) includes at least the following amino acid substitutions according to EU numbering: (ii-1) L234A, L235A, and P329G, (ii-2) L234A and L235A, (ii-3) I253A, H310A, and H435Q, (ii-4) I253A, H310A, and H435A, (ii-5) D265A and P329A, (ii-6) M252Y, S254T, and T256E, and / or (ii-7) M428L and N434S, and / or (III) SEQ ID NOS: 71 to 74. or an amino acid sequence which comprises or consists of the amino acid sequence (optionally excluding the C-terminal K) of any one of 4, 81-84, 86-89, 91-94, 96-99, 471-474, 476-479, 481-484 and 486-489, or which comprises an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0068] In certain embodiments, (a) the first polypeptide comprises (i) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 2, 7, 8, 9, 450, 451, 452, and 453 (optionally excluding the C-terminal K); and (ii) the amino acid sequence of any one of SEQ ID NOs: 402, 407, 408, 409, 460, 461, 462, and 463 (optionally excluding the C-terminal K), wherein X1 is any amino acid, optionally Q or X2 is any amino acid, optionally S, A, Q or N, X3 is any amino acid, optionally E, A, S or D, X4 is any amino acid, optionally N or D, X5 is any amino acid, optionally R or K, X6 is any amino acid, optionally E or T, X7 is any amino acid, optionally D or any amino acid that is not S, E, A, R, T, L, H, Y, Q or K; and / or (iii) an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 2, 7, 8, 9, 450, 451, 452 and 453 (optionally excluding the C-terminal K).

[0069] In certain embodiments, (b) the second polypeptide comprises (i) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1 and / or (ii) the amino acid sequence of SEQ ID NO:401, wherein X1 is any amino acid, optionally E or D, X2 is any amino acid, for example, A or T, X3 is any amino acid, for example, S, X4 is Q, X5 is any amino acid, for example, Y, X6 is any amino acid, for example, Q or G, X7 is any amino acid, for example, R, X8 is any amino acid, for example, L or W, X9 is any amino acid, for example, I, and X 10 is any amino acid, e.g., A, and X 11 is M and X 12 is M and X 13 is any amino acid, e.g., D or G, and X 14 is any amino acid, e.g., L, and X 15 is any amino acid, e.g., M, and X 16 is any amino acid, e.g., T, and X 17 is any amino acid, e.g., D or E, and X 18 is any amino acid, e.g., N or S, and X 19 is any amino acid, e.g., S or A, and X 20 is any amino acid, e.g., M, and X 21 is any amino acid, e.g., R or K, and X 22 is any amino acid, for example, A or P, and (a) X 23 and X 24 are D and E, respectively, and (b) X 23 and X 25 are D and D, respectively, and / or (c) X 24 and X 25 As long as E and D are respectively, X 23 , X 24 and X25 are individually any amino acid, where optionally X 23 , X 24 and X 25 are D, E and D, respectively, D, D and D, respectively, D, E and E, respectively, D, E and S, respectively, D, E and A, or respectively, D, E and T; X 26 is any amino acid, e.g., R or W, and X 27 is any amino acid, e.g., Q or L), and / or (iii) comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:1.

[0070] In certain embodiments, (a) the first polypeptide comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:2, 7, 450 and 451 (optionally excluding the C-terminal K), and (b) the second polypeptide comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:1.

[0071] In a further particular embodiment, the multispecific antibody or antibody fragment comprises two of said first polypeptides and two of said second polypeptides that form a dimer with each other via one or more disulfide bonds, wherein one of the second polypeptides interacts with one of the first polypeptides via a disulfide bond and another of the second polypeptides interacts with another of the first polypeptides via a disulfide bond.

[0072] In further embodiments, the multispecific antibody or antibody fragment of the present disclosure may comprise (A) two or more Fzd4-binding regions, wherein at least one of the Fzd4-binding regions is, comprises, or is contained within the first antigen-binding region, and / or (B) two or more LRP5 and / or LRP6-binding regions, wherein at least one of the LRP5 and / or LRP6-binding regions is, comprises, or is contained within the second antigen-binding region.

[0073] In some cases, the ratio of the number of the one or more Fzd4-binding regions to the number of the one or more LRP5 and / or LRP6-binding regions contained in the multispecific antibody or antibody fragment may be selected from the group consisting of 2:1, 1:2, 2:2, 3:1, 1:3, 3:2, 2:3, 3:3, 4:1, 1:4, 4:2, 2:4, 4:3, 3:4 and 4:4.

[0074] In some cases, the number of the one or more Fzd4 binding regions and the number of the one or more LRP5 and / or LRP6 binding regions contained in the multispecific antibody or antibody fragment may be 2 and 1, respectively, 1 and 2, respectively, 2 and 2, respectively, 3 and 1, respectively, 1 and 3, respectively, 3 and 2, respectively, 2 and 3, respectively, 3 and 3, respectively, 4 and 1, respectively, 1 and 4, respectively, 4 and 2, respectively, 2 and 4, respectively, 4 and 3, respectively, 3 and 4, or respectively, 4 and 4.

[0075] In certain cases, a multispecific antibody or antibody fragment of the disclosure may comprise two identical Fzd4-binding regions, each of which is, comprises, or is contained within the first antigen-binding region, and one LRP5 and / or LRP6-binding region, each of which is, comprises, or is contained within the second antigen-binding region.

[0076] In certain cases, a multispecific antibody or antibody fragment of the present disclosure may comprise two Fzd4-binding regions that are different from each other, (i) at least one of which is, comprises, or is comprised in the first antigen-binding region, or (ii) each of which is, comprises, or is comprised in the first antigen-binding region, and one LRP5 and / or LRP6-binding region that is, comprises, or is comprised in the second antigen-binding region.

[0077] In certain cases, a multispecific antibody or antibody fragment of the present disclosure may comprise one Fzd4-binding region that is, comprises, or is contained within the first antigen-binding region and two identical LRP5 and / or LRP6-binding regions, each of which is, comprises, or is contained within the second antigen-binding region.

[0078] In certain cases, a multispecific antibody or antibody fragment of the present disclosure may comprise one Fzd4-binding region that is, comprises, or is contained within the first antigen-binding region and two LRP5- and / or LRP6-binding regions that are different from each other and (i) at least one of which is, comprises, or is contained within the second antigen-binding region, or (ii) each of which is, comprises, or is contained within the second antigen-binding region.

[0079] In certain cases, a multispecific antibody or antibody fragment of the disclosure may comprise two identical Fzd4-binding regions, each of which is, comprises, or is contained within the first antigen-binding region, and two identical LRP5 and / or LRP6-binding regions, each of which is, comprises, or is contained within the second antigen-binding region.

[0080] In certain cases, a multispecific antibody or antibody fragment of the present disclosure may comprise two Fzd4-binding regions that are different from each other and (i) at least one of which is, comprises, or is included in the first antigen-binding region, or (ii) each of which is, comprises, or is included in the first antigen-binding region, and two identical LRP5 and / or LRP6-binding regions, each of which is, comprises, or is included in the second antigen-binding region.

[0081] In certain cases, a multispecific antibody or antibody fragment of the present disclosure may comprise two identical Fzd4-binding regions, each of which is, comprises, or is contained within the first antigen-binding region, and two LRP5- and / or LRP6-binding regions that are different from each other and (i) at least one of which is, comprises, or is contained within the second antigen-binding region, or (ii) each of which is, comprises, or is contained within the second antigen-binding region.

[0082] In certain cases, a multispecific antibody or antibody fragment of the present disclosure may comprise two Fzd4-binding regions that are different from each other and (i) at least one of which is, comprises, or is included in the first antigen-binding region, or (ii) each is, comprises, or is included in the first antigen-binding region, and two LRP5- and / or LRP6-binding regions that are different from each other and (i) at least one of which is, comprises, or is included in the second antigen-binding region, or (ii) each is, comprises, or is included in the second antigen-binding region.

[0083] In another aspect, the present disclosure provides nucleic acids, where optionally, a nucleic acid or combination of nucleic acids of the disclosure can encode any of the multispecific antibodies and antibody fragments described herein.

[0084] In some embodiments, the nucleic acid may be DNA, cDNA, RNA, mRNA, modified mRNA, or a DNA / RNA hybrid.

[0085] In some embodiments, a nucleic acid or combination of nucleic acids may comprise (a) a first nucleic acid that encodes any of the first polypeptides described herein, and (b) a second nucleic acid that encodes any of the second polypeptides described herein.

[0086] In another aspect, the disclosure provides vectors comprising these nucleic acids.

[0087] In some embodiments, a vector or combination of vectors of the present disclosure may comprise any of the nucleic acids or any of the combinations of nucleic acids described herein.

[0088] In some embodiments, the vector comprises one or more promoters operably linked to the nucleic acid.

[0089] In some embodiments, the vector is an expression vector.

[0090] In some embodiments, the vector comprises a plasmid, a viral vector, optionally an adeno-associated virus, adenovirus, lentivirus or retrovirus, a lipid-based vector, a self-replicating RNA vector, a virus-like particle, a polymer-based vector and / or a nanoparticle, optionally a lipid-based nanoparticle.

[0091] In some embodiments, a vector combination may include (a) a first vector comprising a first nucleic acid encoding a first polypeptide, and (b) a second vector comprising a second nucleic acid encoding a second polypeptide.

[0092] In another aspect, the disclosure provides host cells and cell populations comprising such nucleic acids or vectors.

[0093] A host cell of the disclosure may comprise (A) any of the multispecific antibodies or antibody fragments described herein, (B) any of the nucleic acids or any combination of nucleic acids described herein, and / or (C) any of the vectors or any combination of vectors described herein.

[0094] In some embodiments, the host cell is mammalian, optionally a human, non-human primate, monkey, rabbit, rodent, hamster, rat, or mouse.

[0095] In some embodiments, the host cell is non-mammalian, optionally a plant, bacterium, fungus, yeast, protozoan, or insect.

[0096] In certain embodiments, the host cell is (i) a human embryonic kidney (HEK) cell, optionally an HEK293 cell or a variant thereof, and further optionally an Expi293F™ cell, (ii) a CHO (Chinese Hamster Ovary) cell, (iii) an immune cell, or (iv) a hybridoma.

[0097] In some embodiments, the cell populations of the present disclosure may comprise any of two or more of the host cells described herein.

[0098] In another aspect, the present disclosure provides pharmaceutical compositions containing any of the foregoing. The pharmaceutical compositions of the present disclosure may comprise (A) any of the multispecific antibodies and antibody fragments described herein, (B) any of the nucleic acids or any combination of nucleic acids described herein, (C) any of the vectors or any combination of vectors described herein, and / or (D) any of the host cells or cell populations described herein, and (II) a pharmaceutically acceptable carrier and / or excipient.

[0099] In another aspect, the disclosure provides in vivo methods, e.g., methods of treating a subject, treating or preventing a disease, disorder, or condition in a subject, and methods of inducing, promoting, stimulating, enhancing, and / or supporting Wnt signaling in target cells in a subject.

[0100] In some aspects, methods of treating a subject in need of treatment of the present disclosure can include administering to the subject an effective amount of (A) any of the multispecific antibodies or antibody fragments described herein, (B) any of the nucleic acids or combinations of nucleic acids described herein, (C) any of the vectors or combinations of vectors described herein, (D) any of the host cells or cell populations described herein, and / or (E) any of the pharmaceutical compositions described herein.

[0101] In some embodiments, the subject comprises or has a risk of developing a disease, disorder, or condition, for example, due to genetics or an underlying or co-morbid condition.

[0102] In some embodiments, the present invention provides methods of treating or preventing a disease, disorder, or condition in a subject in need thereof, which may comprise administering an effective amount of (A) any of the multispecific antibodies or antibody fragments described herein, (B) any of the nucleic acids or combinations of nucleic acids described herein, (C) any of the vectors or combinations of vectors described herein, (D) any of the host cells or cell populations described herein, and / or (E) any of the pharmaceutical compositions described herein.

[0103] In some aspects, a method of inducing, promoting, stimulating, enhancing and / or supporting Wnt signaling in a target cell of a subject of the present disclosure may comprise administering to the subject an effective amount of (A) any of the multispecific antibodies or antibody fragments described herein, (B) any of the nucleic acids or combinations of nucleic acids described herein, (C) any of the vectors or combinations of vectors described herein, (D) any of the host cells or cell populations described herein, and / or (E) any of the pharmaceutical compositions described herein.

[0104] In some embodiments, the Wnt signaling is or comprises Wnt / β-catenin signaling.

[0105] In some embodiments, the methods promote, stimulate, enhance and / or assist the recruitment of multiple receptor complexes, each of which comprises (i) Fzd4 and (ii) LRP5 or LRP6.

[0106] In any of the in vivo methods disclosed herein, the subject is typically a mammal, optionally a human, non-human primate, monkey, horse, cow, sheep, goat, pig, dog, cat, rabbit, rodent, hamster, rat or mouse.

[0107] In any of the in vivo methods disclosed herein, the subject may include a non-mammalian vertebrate, optionally a bird, fish, amphibian, or reptile.

[0108] In any of the in vivo methods disclosed herein, in some cases, the subject includes or is at risk of developing the disease, disorder, or condition due to, for example, genetics or another underlying or co-morbid condition.

[0109] In any of the in vivo methods disclosed herein, the method may further comprise administering to the subject an additional agent, optionally an adjuvant or therapeutic agent.

[0110] In any of the in vivo methods disclosed herein, the disease, disorder, or condition may comprise a retinopathy, optionally a retinal vascular disease (optionally caused by inhibited vascular development or excessive angiogenesis), and / or optionally a retinopathy selected from the group consisting of exudative vitreoretinopathy, familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie's disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease.

[0111] In any of the in vivo methods disclosed herein, the disease, disorder or condition may include a vascular disorder, optionally a vascular malformation or vascular insufficiency, and optionally a vascular disorder associated with ischemia-induced neovascularization.

[0112] In any of the in vivo methods disclosed herein, the disease, disorder, or condition may comprise a bone disease or injury optionally selected from the group consisting of osteoporosis (including osteoporotic pseudoglioma (OPPG) syndrome and juvenile primary osteoporosis), bone mineral density alterations, and cancer-related osteolysis.

[0113] In any of the in vivo methods disclosed herein, the disease, disorder or condition may comprise a muscle wasting disease optionally selected from the group consisting of sarcopenia, cachexia and muscular dystrophy.

[0114] In any of the in vivo methods disclosed herein, the disease, disorder or condition may comprise a neurological or neurodegenerative disease optionally selected from the group consisting of stroke, traumatic brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease and Creutzfeldt-Jakob disease.

[0115] In any of the in vivo methods disclosed herein, administration of the active agent can be to one or more eyes of the subject, optionally intravitreally or by eye drops.

[0116] In any of the in vivo methods disclosed herein, administration of the active agent can be topical, optionally to the eye, ear, nose (optionally intranasally), skin (optionally transdermally or epicutaneously), mucosa, skin or vagina, or by inhalation.

[0117] In any of the in vivo methods disclosed herein, administration of the active agent can be parenteral, optionally by injection (optionally intravenous, intramuscular, subcutaneous, intradermal, intrathecal, intraarterial, intraarticular, intraosseous or intraperitoneal) or by inhalation.

[0118] In any of the in vivo methods disclosed herein, administration of the active agent can be by enteral administration, optionally orally, sublingually, bucally, or rectally.

[0119] In any of the in vivo methods disclosed herein, administration of an active agent may involve administering any of the multispecific antibodies and antibody fragments described herein to one or more eyes of the subject at about 0.1 to 100,000 ng per eye, about 1 to 100,000 ng per eye, about 1 to 10,000 ng per eye, about 10 to 10,000 ng per eye, about 1 to 100 ng per eye, about 200 to 300 ng per eye, about 300 to 400 ng per eye, about 400 to 500 ng per eye, about 500 to 600 ng per eye, about 600 to 700 ng per eye, about 700 to 800 ng per eye, about 800 to 900 ng per eye, about 900 to 1,000 ng per eye, about 1,000 to 2,000 ng per eye, about 2,000 to 3,000 ng per eye, about 1,000 to 2,000 ng per eye, about 1,000 to 3,000 ng per eye, about 1,000 to 3,000 ng per eye, about 1,000 to 4,000 ng per eye, about 1,000 to 5 ... Approximately 3000-4000ng per eye, approximately 4000-5000ng per eye, approximately 5000-6000ng per eye, approximately 6000-7000ng per eye, approximately 7000-8000ng per eye, approximately 8000-9000ng per eye, approximately 9000-10000ng per eye, approximately 10000-20000ng per eye, approximately 20000-3000ng per eye This may include administering at about 0 ng per eye, about 30,000-40,000 ng per eye, about 40,000-50,000 ng per eye, about 50,000-60,000 ng per eye, about 60,000-70,000 ng per eye, about 70,000-80,000 ng per eye, about 80,000-90,000 ng per eye, or about 90,000-100,000 ng per eye.

[0120] In another aspect, the present disclosure provides methods for inducing, promoting, stimulating, enhancing and / or supporting Wnt signaling in target cells.

[0121] In some aspects, such methods of inducing, promoting, stimulating, enhancing and / or supporting Wnt signaling in a target cell may comprise contacting the target cell with an effective amount of (A) any of the multispecific antibodies or antibody fragments described herein, (B) any of the nucleic acids or combinations of nucleic acids described herein, (C) any of the vectors or combinations of vectors described herein, (D) any of the host cells or cell populations described herein, and / or (E) any of the pharmaceutical compositions described herein.

[0122] In some embodiments, the contacting occurs in vitro.

[0123] In some embodiments, the contacting occurs ex vivo.

[0124] In some embodiments, the contacting occurs in vivo.

[0125] In some embodiments, the methods include (i) preparing cells and / or tissues for transplantation, (ii) differentiating stem cells, and / or (iii) preparing organoids.

[0126] In some embodiments, the method induces or promotes osteogenesis, angiogenesis and / or neurogenesis and / or differentiation.

[0127] In a further aspect, the present disclosure provides methods of producing any of the multispecific antibodies and antibody fragments described herein.

[0128] In some aspects, methods of producing any of the multispecific antibodies and antibody fragments described herein may comprise: (a) culturing cells comprising any nucleic acid or combination of nucleic acids encoding the multispecific antibody or antibody fragment under conditions that allow for expression of said multispecific antibody or antibody fragment; and (b) collecting and purifying the multispecific antibody or antibody fragment obtained from the cell culture from (a).

[0129] In a further aspect, the present disclosure provides methods of producing any of the host cells or any of the cell populations described herein.

[0130] In some aspects, methods of producing any of the host cells or populations of such cells described herein may include introducing any of the nucleic acids or combinations of nucleic acids described herein and / or any of the vectors or combinations of vectors described herein into one or more cells.

[0131] In some embodiments, the introducing occurs in vitro.

[0132] In some embodiments, the introducing occurs ex vivo.

[0133] In some embodiments, the introducing occurs in vivo.

[0134] Any of the multispecific antibodies or antibody fragments described herein, any of the nucleic acids or combinations of nucleic acids described herein, any of the vectors or combinations of vectors described herein, any of the host cells or cell populations described herein and / or any of the pharmaceutical compositions described herein may be for use in medicine.

[0135] Any of the multispecific antibodies or antibody fragments described herein, any of the nucleic acids or combinations of nucleic acids described herein, any of the vectors or combinations of vectors described herein, any of the host cells or cell populations described herein and / or any of the pharmaceutical compositions described herein may be for use in treating a disease, disorder or condition, such as one in which Wnt signaling is implicated.

[0136] In some embodiments, the disease, disorder, or condition includes any one or more of the diseases described herein.

[0137] The present disclosure further encompasses the use of any of the multispecific antibodies or antibody fragments described herein, any of the nucleic acids or combinations of nucleic acids described herein, any of the vectors or combinations of vectors described herein, any of the host cells or cell populations described herein and / or any of the pharmaceutical compositions described herein for the manufacture of a medicament for the treatment of a disease, disorder or condition.

[0138] In some embodiments, the disease, disorder, or condition includes any one or more of the diseases described herein.

[0139] The present invention is based, in part, on the use of Wnt signaling agonists and antagonists to control abnormal angiogenesis in retinopathy indications.

[0140] In some embodiments, the present invention provides a method of treating a subject suffering from retinopathy, comprising administering to the subject a modified Wnt signaling agonist. In further embodiments, the modified Wnt agonist comprises a binding composition that binds to one or more Fzd receptors and a binding composition that binds to one or more LRP receptors.

[0141] In one aspect, the disclosure provides a modified Wnt signaling agonist, wherein the Wnt signaling agonist comprises two light chains (each comprising a polypeptide sequence having at least 95% identity to SEQ ID NO: 1) and two heavy chains (each comprising a polypeptide sequence having at least 95% identity to SEQ ID NO: 2), wherein the two heavy chains are linked to each other and the two light chains are each linked to a different heavy chain of the Wnt signaling agonist. In certain embodiments, the two light chains each comprise a VHH domain comprising one or more of the following amino acid residue modifications: hVHH3-H4 DDD (amino acids at positions 101-103) substituted with N29Q and / or DED. In certain embodiments, the two heavy chains each comprise one or more of the following amino acid residues: CDR1 S31, CDR2 E62, and / or CDR3 D108.

[0142] In another aspect, the present disclosure provides a nucleic acid comprising a sequence encoding one or more light chains and / or one or more heavy chains of a modified Wnt agonist disclosed herein, e.g., hp4SD1-03, or a fragment or variant thereof. In certain embodiments, the nucleic acid is an mRNA, e.g., a modified mRNA.

[0143] In a related aspect, the present disclosure provides a vector comprising a nucleic acid disclosed herein, e.g., a nucleic acid comprising a sequence encoding one or more light chains and / or one or more heavy chains of hp4SD1-03, or a fragment or variant thereof. In certain embodiments, the vector is an expression vector, such as a viral vector, comprising a promoter operably linked to the nucleic acid.

[0144] In related embodiments, the present disclosure provides host cells comprising a vector or nucleic acid disclosed herein, e.g., a vector or nucleic acid comprising a sequence encoding one or more light chains and / or one or more heavy chains of hp4SD1-03 or a fragment or variant thereof. In certain embodiments, the host cell is a recombinant cell. In certain embodiments, the host cell is a mammalian cell or an insect cell.

[0145] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable diluent, excipient, or carrier; a) an engineered Wnt signaling agonist as disclosed herein; b) a nucleic acid disclosed herein and / or c) the vectors disclosed herein; The present invention provides a pharmaceutical composition comprising:

[0146] In certain embodiments, the pharmaceutical composition comprises hp4SD1-03 or a fragment or variant thereof, or the nucleic acid encodes one or more light chains and / or one or more heavy chains of hp4SD1-03 or a fragment or variant thereof, or the vector comprises a nucleic acid comprising a sequence encoding one or more light chains and / or one or more heavy chains of hp4SD1-03 or a fragment or variant thereof.

[0147] In a further aspect, the present disclosure provides a method of treating retinopathy in a subject, comprising: To the subject, a) a modified Wnt signaling agonist as disclosed herein; b) the nucleic acids disclosed herein c) the vectors disclosed herein and / or d) a pharmaceutical composition disclosed herein The method includes administering

[0148] In certain embodiments, the modified Wnt signaling agonist is hp4SD1-03 or a fragment or variant thereof, or the pharmaceutical composition comprises hp4SD1-03 or a fragment or variant thereof, or the nucleic acid encodes one or more light chains and / or one or more heavy chains of hp4SD1-03 or a fragment or variant thereof, or the vector comprises a nucleic acid comprising a sequence encoding one or more light chains and / or one or more heavy chains of hp4SD1-03 or a fragment or variant thereof.

[0149] In certain embodiments of the disclosed methods of treatment, the retinopathy being treated is a retinal vascular disease. In certain embodiments, the retinal vascular disease is caused by inhibition of vascular development. In certain embodiments, the retinopathy is caused by excessive neovascularization. In certain embodiments, the retinopathy is early or late retinopathy. In some embodiments, the retinal vascular disease is selected from the group consisting of familial exudative vitreoretinopathy (FEVR), exudative vitreoretinopathy, Norrie's disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease. The modified Wnt signaling agonist can be administered systemically or locally via a variety of routes of administration, including, but not limited to, intravenous, subcutaneous, oral, topical, nasal, intravitreal, suprachoroidal, or subretinal. In certain embodiments, the engineered Wnt signaling agonist is administered to one or more eyes of the subject, for example, intravitreally. [Brief explanation of the drawings]

[0150] [Figure 1]1 provides the amino acid sequence of 4SD-03_LALAPG, a tetravalent, bispecific Wnt signaling agonist, and its related IgG-like antibody structure, which comprises two heavy chains and two light chains of the anti-Fzd4 antibody 4SD1, where an anti-LRP5 / 6 VHH domain is fused to the N-terminus of each anti-Fzd4 antibody light chain via the linker sequence GGSGS (SEQ ID NO: 102), where the two heavy chains are linked via disulfide bonds and each light chain is linked to a heavy chain via a disulfide bond. The VHH domain is shown in italics, the linker sequence is GGSGS (SEQ ID NO: 102), the CDRs are underlined and in bold, the LALAPG mutation in the Fc domain of the heavy chain is shown in grey, the Fc domain is highlighted in grey, and the anti-Fzd4 VL and VH domains each contain three CDRs, which are also highlighted (anti-Fzd4 CDRs are defined according to Kabat numbering). [Figure 2A] Figure 2A shows the various amino acid modifications made to the VHH3 domain of 4SD1-03_LALAPG to humanize the VHH camelid sequence. VHH represents the parent camelid sequence, 3-66*01 represents the closest human sequence identified in the human genome database, and hVHH3-H1 to hVHH3-H4 contain the indicated amino acid substitutions. The amino acid residue numbers, based on the actual residue number ("seq") within the VH and based on Martin numbering ("AbM"), are indicated above the sequence. [Figure 2B] Figure 2B shows the SEC (size exclusion chromatography) profiles of various humanized forms of VHH3 shown in Figure 2A after Protein A purification. The x-axis shows elution volume (ml) and the y-axis shows milliabsorbance units (mAU). hVHH3-H4 showed the best expression and highest purity, as indicated by a single peak and high mAU value. The arrow indicates the expected retention volume. [Figure 3]Figure 3 shows the SEC profile of VHHs associated with modification of NS, a potential deamidation motif in hVHH3-H4. The x-axis shows elution volume (ml) and the y-axis shows milliabsorbance units (mAU). The graph shows that N29 in hVHH3-H4 can be replaced by, for example, Q, without affecting purity. [Figure 4] Figure 4 shows the SEC profile of VHHs associated with modifications of the potential isomerization motif DDD in hVHH3-H4. The x-axis shows elution volume (ml) and the y-axis shows milliabsorbance units (mAU). The chromatograms show the effect of various amino acid substitutions in the DDD motif on purity after Protein A elution. [Figure 5] Figure 5 shows the modification of potential oxidation-sensitive residues M51 and M57 in VHH3. The table on the left shows various amino acid substitutions made to the M51 or M57 residues, demonstrating that substitution of M51 or M57 with any of the amino acids tested was not tolerated and resulted in very poor yields. The chromatograms on the right show examples of the purity resulting from different M51 and M57 mutations. The x-axis shows elution volume (ml) and the y-axis shows milli-absorbance units (mAU). [Figure 6] Figure 6 provides a diagram of the modifications to the VHH3 domain of 4SD1-03_LALAPG (VHH3 was humanized and incorporated N29Q and D102E substitutions) and the modifications to the VH domain of 4SD1-03_LALAPG made to derive hp4SD1-03 (N31S and DF62E substitutions were incorporated and the framework region sequence was altered to further simulate the human germline coding sequence). [Figure 7]Figure 7 shows various amino acid substitutions made to the 4SD1-03_LALAPG heavy chain to modify potentially unstable amino acids. N31 in CDR1 was modified to Q, S, or A, D62 in CDR2 was modified to E, S, or A, and D108 in CDR3 was modified to E, S, or A. The graph shows that mutations in CDR1 N31 and CDR2 D62 have similar or even greater Wnt signaling activity compared to the parent molecule. The N31S mutation increased potency by 5-fold. However, CDR3 D108 could not be replaced without losing potency. Further mutations were tested, but they reduced Wnt signaling activity, as shown in the graph on the right. Combinations of N31S, D62E, and D108 were tested by mass spectrometry and showed no isomerization even under accelerated stress conditions. [Figure 8] Figure 8 shows the alignment between the VH region sequence of 4SD1 and its closest human germline IGHV3-23*04a counterpart. Based on this alignment, framework mutations of 4SD1-03_LALAPG containing changes in the FR sequence (shown in the table in the upper right) were constructed to simulate the human germline coding sequence. The framework mutation hp4SD1-03 had a good expression and purification profile, as shown by the SEC (size exclusion chromatography) profile. The framework mutation hp4SD1-03 exhibited five-fold higher potency than the parent molecule 4SD1-03_LALAPG in a cell-based STF assay. Furthermore, hp4SD1-03 showed no tendency for isomerization when analyzed by mass spectrometry after two weeks of heat stress at pH 5.5 and 42°C. [Figure 9]Figure 9 provides the amino acid sequence of hp4SD1-03, a tetravalent, bispecific Wnt signaling agonist, resulting from the parent sequence 4SD1-03_LALAPG, including the modifications described above. This construct has the same general structure as the parent construct. The VHH domain is shown in italics, the linker sequence is GGGGS (SEQ ID NO: 101), the CDRs are underlined and in bold, the LALAPG mutation in the Fc domain of the heavy chain is shown in gray, the Fc domain is highlighted in gray, and the anti-Fzd4 VL and VH domains each contain three CDRs, also highlighted (anti-Fzd4 CDRs are defined according to Kabat numbering). [Figure 10A] Figure 10A is a graph showing the Wnt signaling activity of the modified construct hp4SD1-03 compared to the parent construct 4SD1-03_LALAPG. Both constructs contain the LALAPG Fc variant. The EC50 values ​​shown in the table indicate that hp4SD1-03 has approximately 10-fold greater Wnt signaling activity. [Figure 10B] Figure 10B shows exemplary results of the Axin2 expression-based Wnt / β-catenin signaling activity assessment described in Example 5, comparing hp4SD1-03 and 4SD1-03_LALAPG (both containing the LALAPG Fc variant) with Norrin as a control. As shown in the table, hp4SD1-03 showed an approximately 11.5-fold decrease in EC50. [Figure 11]Figure 11 shows the effects of treatment with the modified construct hp4SD1-03 compared to the parent construct 4SD1-03_LALAPG in a mouse retinal injury model. The timeline at the top outlines the induction of retinal injury with 75% oxygen, followed by intravitreal injection of either construct and examination of retinal tissue by staining for avascular (AV) and vascular plexus areas. The retinal flat-mount images below, stained with isolectin B4 (IB4), show uninjured retinas, injured retinas (negative control), and retinas from animals treated with Eylea® (positive control), the modified construct hp4SD1-03, or the parent construct 4SD1-03_LALAPG at the doses indicated in the table on the right. The graphs on the right show the percentage of avascular tissue area or vascular plexus area relative to the total area obtained with each treatment. This demonstrates the superior results achieved with hp4SD1-03 compared to 4SD1-03_LALAPG. From left to right in each graph, the bars correspond to no treatment, Eylea®, 4SD1-03_LALAPG, and hp4SD1-03. In the ANOVA test, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. [Figure 12] Figure 12 shows the effect of treatment with the indicated amounts of the modified construct hp4SD1-03 in a rabbit retinal injury model of VEGF-induced retinal vascular leakage. Doses are shown in the table, and VEGF-induced retinal leakage scores are shown in the accompanying graph. Treatment with hp4SD1-03 at 10 μg / eye or 2 μg / eye reduced leakage by approximately 75%, and treatment with hp4SD1-03 at 0.4 μg / eye or 0.08 μg / eye also significantly reduced vascular leakage. In the ANOVA test, * indicates p<0.05, and ** indicates p<0.01. Comparison of retinal vascular leakage between vehicle and hp4SD1-03 treatment on day 3. [Figure 13] Figure 13 provides fluorescein angiography images of rabbit retinal tissue treated as indicated. Efficacy was observed in all test animals. [Figure 14]Figure 14 shows the activity of various mutants with amino acid substitutions within the hVHH3-H4 sequence to replace D at position 103 or DED at positions 101-103. The top graph and bottom table show the STF activity of the various mutants compared to the activity of hp4SD1-03, which contains DED at positions 101-103. The chromatograms show the purity after Protein A purification following modification of DED in hVHH3-H4. The x-axis shows elution volume (ml) and the y-axis shows milliabsorbance units (mAU). These data demonstrate that both D101 and D103 of hp4SD1-03 can be replaced without reducing the activity of the molecule. [Figure 15A-C] Figures 15A-15C show the effect of treatment with high concentrations of hp4SD1-03 in a mouse OIR model. Figure 15A shows the OIR study design. Figure 15B shows staining of the retinal avascular (AV) and neovascular (NV) regions. Retinal flat-mount images stained with isolectin B4 (IB4) show untreated and treated with 40 nM, 400 nM, and 4000 nM hp4SD1-03. Figure 15C provides a quantitative graph of the imaging results in Figure 15B. In the ANOVA test, * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. [Figure 16A-B] Figures 16A-B show the increased expression of selected genes and proteins upon treatment with parental 4SD1-03_LALAPG: Figure 16A shows the mRNA expression of LEF1 and MFSD2A, and Figure 16B shows the protein expression of two tight junction proteins, ZO-1 and CLDN5. [Figure 16C] Figure 16C provides exemplary Western blot analysis results from Example 7. Treatment of human retinal microvascular endothelial cells (HRMECs) with hp4SD1-03 increased the phosphorylation of DVL3, a key cytoplasmic molecule in Wnt signaling (increased the ratio of phosphorylated to unphosphorylated DVL3). [Figures 17A-C]Figures 17A-17C provide exemplary results obtained from the binding kinetics assessment described in Example 8. Monovalent binding of human Fzd4 CRD to 4SD1-03_LALAPG or hp4SD1-03 was measured by BLI. Figure 17A provides an exemplary binding sensorgram of human Fzd4 CRD to 4SD1-03_LALAPG. The red line represents a global fit generated by fitting the real-time binding sensorgram (blue line) using a 1:1 Langmuir binding model. Figure 17B provides an exemplary binding sensorgram of human Fzd4 CRD to hp4SD1-03. The red line represents a global fit generated by fitting the real-time binding sensorgram (green line) using a 1:1 Langmuir binding model. FIG. 17C provides a table showing the KD, k on and k dis (also called k off ) values ​​observed for binding of human Fzd4 CRD to 4SD1-03_LALAPG or hp4SD1-03 at the indicated human Fzd4 CRD concentrations. [Figures 18A-C] Figures 18A-18C provide exemplary results from the assessment of binding kinetics as described in Example 8. Binding of 4SD1-03_LALAPG or hp4SD1-03 to human Fzd4 CRD was measured by SPR. Figure 18A provides an exemplary binding sensorgram of human Fzd4 CRD to 4SD1-03_LALAPG. Figure 18B provides an exemplary binding sensorgram of human Fzd4 CRD to hp4SD1-03. Figure 18C provides a table showing the KD, ka (also called k) and kd (also called k) values ​​observed for the binding of 4SD1-03_LALAPG or hp4SD1-03 to human Fzd4 CRD. Averages of the values ​​observed at four capture concentrations are shown. [Figure 19A-B] Figures 19A-19B provide the amino acid sequences of the heavy and light chains of hp4SD1-03 AAQ and hp4SD1-03 AAA. Additional amino acid substitutions introduced relative to hp4SD1-03 are highlighted. [Figure 20]Figure 20 provides exemplary SEC chromatograms obtained in Example 9 after Protein A affinity resin purification of hp4SD1-03, hp4SD1-03 AAQ, and hp4SD1-03 AAA produced in transiently transfected Expi293F™ cells. Highlighted fractions were analyzed on an SDS-PAGE gel. [Figure 21] Figure 21 provides exemplary results from the melting temperature evaluation described in Example 9. The top three graphs are exemplary melting curves for hp4SD1-03, hp4SD1-03 AAQ, and hp4SD1-03 AAA, each analyzed in duplicate. The table (top right) provides the average Tm1 values ​​from each sample performed in duplicate. The bottom graph shows an overlay of Tagg values ​​from each sample analyzed in duplicate. [Figure 22] Figure 22 provides exemplary results obtained from the Wnt signaling assay in Example 9. The graph (left) shows exemplary dose-response curves for hp4SD1-03, hp4SD1-03 AAQ, and hp4SD1-03 AAA obtained using a luciferase-reporter system in bEnd.3 cells that natively express Fzd4 and LRP5. The table (right) shows EC50 values ​​from the fitted curves. [Figure 23] Figure 23 provides exemplary results from the assessment of binding to the fetal Fc receptor FcRn in Example 9. The graph shows exemplary sensorgrams for hp4SD1-03, hp4SD1-03-AAQ, and hp4SD1-03-AAA. hp4SD1-03 showed binding to FcRn as expected; in contrast, no binding was observed with the hp4SD1-03-AAQ or hp4SD1-03-AAA mutants. [Figure 24-34]Figures 24 to 34 provide exemplary, non-limiting embodiments of various multispecific antibody structures that may include any of the variable domain sequences of the anti-Fzd4 and anti-LRP5 / 6 antibodies disclosed herein. In Figures 24 to 34, the following applies unless otherwise noted: (1) Each domain is represented as a rectangle with text indicating the domain name (e.g., CH3, VH1, etc.) inside it. (2) A set of multiple domains linked together represents a polypeptide (e.g., a heavy chain polypeptide, a light chain polypeptide, etc.). (3) The orientation of domains within a polypeptide is from the N-terminus to the C-terminus, following the direction of the text indicating the domain name. (4) Linkers or hinges can be used between domains as needed, and disulfide bonds may exist between polypeptides (and / or within domains) to ensure proper formation of antigen-binding sites, even if linkers, hinges, or disulfide bonds are not explicitly shown in the figures. (5) A line extending from VH2 to another domain indicates that a linker may or may not exist between VH2 and the other domain. (6) The CH1, CH2, CH3, and / or CL domains shown in the diagrams may be omitted if possible and replaced with hinges or linkers where appropriate. (7) The CH1, CH2, and CH3 domains may individually be of any (heavy chain) isotype. (8) If two or more CH1 domains are present in the structure, the CH1 domain sequences may or may not be identical and may or may not be of the same isotype. If two or more CH2 domains are present in the structure, the CH2 domain sequences may or may not be identical and may or may not be of the same isotype. If two or more CH3 domains are present in the structure, the CH3 domain sequences may or may not be identical and may or may not be of the same isotype. (9) The light chain constant (CL) domain may be a kappa CL domain or a lambda CL domain.(10) When two or more CL domains are present in a structure, all of the CL domains may be kappa CL or all of the CL domains may be lambda CL, or alternatively, one CL may be a kappa CL and another CL may be a lambda CL domain, and the CL domain sequences may or may not be identical. (11) When both kappa and lambda CL domains are present, the CH1 domains paired with the CL domains may in some cases be variant CH1 domains, one of which may be a variant CH1 that preferentially binds kappa CL and another CH1 domain may be a variant CH1 that preferentially binds lambda CL (having kappa and lambda CL and kappa-preferring CH1s and lambda-preferring CH1s within a molecule may enable efficient manufacturing). (12) Generally, VH1 and VL1 (if VL1 is present) form an antigen-binding region for a first antigen (e.g., Fzd4), and VH2 forms an antigen-binding region for a second antigen (e.g., LRP5 and / or LRP6). (13) In a VH1-VL1 pair, even if VL1 is shown in the figure, VL1 can be omitted if VH1 alone confers sufficient specificity for the first antigen (i.e., nanobody). (14) Even if VL2 is not explicitly shown in the figure, an antigen-binding region comprising VH2 may further include VL2 as long as the VH2-VL2 pair has binding ability for a second antigen. [Figure 24]Figure 24 provides exemplary, non-limiting embodiments of some of the various multispecific antibody structures in which any of the variable domain sequences of the anti-Fzd4 and anti-LRP5 / 6 antibodies disclosed herein may be used. The structure in the upper left (boxed) is an exemplary structure possessed by some of the specific bispecific antibodies disclosed herein (e.g., hp4SD1-03, hp4SD1-03 AAQ, and hp4SD1-03 AAA). The boxed structure comprises two first polypeptides (sometimes referred to herein as heavy chains), each comprising, from N- to C-terminus, a VH1, a CH1, a CH2, and a CH3, and two second polypeptides (sometimes referred to herein as light chains), each comprising, from N- to C-terminus, a VH2, an optional linker, a VL1, and a CL. Each pair of VH1 and VL1 forms an antigen-binding region specific for a first antigen, and each VH2 forms an antigen-binding region specific for a second antigen. Although hinges or disulfide bonds are not explicitly shown in the framed structures, the framed structures may include, for example, a hinge between CH1 and CH2 within a single polypeptide, and disulfide bonds (shown in dashed lines) may exist between the hinge and between the CH1 and CL domains, as shown in the structure at the bottom left. Alternatively, the framed antibody may include, for example, a hinge between CH1 and CH2 within a single polypeptide, and disulfide bonds (shown in dashed lines) may exist between the hinge and between the CL and hinge, as shown in the structure at the bottom right. Hinges and disulfide bonds, such as those shown in the structures at the bottom left and bottom right, may be present in any structure shown in the figures and described herein, even if not explicitly shown. [Figure 25]Figure 25 provides exemplary variations of the antibody structures shown in Figure 24. In some variations of the boxed antibodies, VH2 may be rearranged to a different position, e.g., N-terminal to VH1 in the first polypeptide (top right), C-terminal to CL in the second polypeptide (bottom left), or C-terminal to CH3 in the first polypeptide (bottom right). Variations equivalent to those shown in Figure 25 (VH2 rearrangements) may also be applied to any of the structures shown in Figures 24-34 or variations thereof, as appropriate, even if not explicitly or otherwise described. [Figure 26] Figure 26 shows further exemplary variations of the antibody structures shown in Figure 24. In some variations of the boxed antibodies, a VH2 may further be included, for example, N-terminal to another VH2 in a second polypeptide (top left), N-terminal to a VH1 in a first polypeptide (top right), C-terminal to a CL in a second polypeptide (bottom left), or C-terminal to a CH3 in a first polypeptide (bottom right). The equivalent variations shown in Figure 26 (adding a VH2 to the N- or C-terminus of a first and / or second polypeptide) may also be applied to any of the structures shown in Figures 24-34 or variations thereof, as appropriate, even if not explicitly or otherwise described. [Figure 27] Figure 27 provides exemplary variations of some of the antibody structures shown in Figure 25. Specifically, the positions of VH1 and VL1 are swapped relative to the antibody structures shown in Figure 25. The equivalent variations shown in Figure 27 (swaps of VH1 and VL1) may also be applied to any of the structures shown in Figures 24-34 or variations thereof, as appropriate, even if not explicitly stated. [Figure 28]Figure 28 provides exemplary variations of some of the antibody structures shown in Figure 25. Specifically, the positions of CH1 and CL1 are swapped relative to the antibody structure shown in Figure 25. The equivalent variations shown in Figure 28 (swaps of CH1 and CL) may also be applied to any of the structures shown in Figures 24-34 or variations thereof, as appropriate, even if not explicitly stated. [Figure 29] Figure 29 provides exemplary variations of some of the antibody structures shown in Figure 25. Specifically, the CH3 domain is absent relative to the antibody structures shown in Figure 25. The equivalent variation shown in Figure 29 (removal of CH3) may also be applied to any of the structures shown in Figures 24-34 or variations thereof, as appropriate, even if not explicitly stated. [Figure 30] Figure 30 provides exemplary variations of some of the antibody structures shown in Figure 25. Specifically, the CH2 domain is absent relative to the antibody structures shown in Figure 25. The equivalent variation shown in Figure 30 (removal of CH2) may also be applied to any of the structures shown in Figures 24-34 or variations thereof, as appropriate, even if not explicitly stated. [Figure 31] Figure 31 provides exemplary variations of some of the antibody structures shown in Figure 25. Specifically, the CH2 and CH3 domains are absent relative to the antibody structures shown in Figure 25. In some cases, as shown, hinge and disulfide bonds (disulfide bonds shown as dashed lines) may be present, allowing dimerization of two first polypeptides. The equivalent variation shown in Figure 31 (removal of CH2 and CH3) may also be applied to any of the structures shown in Figures 24-34 or variations thereof, as appropriate, even if not explicitly stated. [Figure 32A]Figure 32A provides exemplary variations of some of the antibody structures shown in Figure 25. Specifically, the structures shown are half of the antibody structures shown in Figure 25, i.e., they comprise one first polypeptide and one second polypeptide, and contain one first antigen-binding region and one second antigen-binding region. The equivalent variations (half structures or half antibodies) shown in Figure 32A may also apply to any of the structures or variations thereof shown in Figures 24-34, as appropriate, even if not explicitly stated. [Figure 32B] Figure 32B provides a further exemplary variation of the half antibody structure shown in Figure 32A. This structure does not include CH2 and CH3, but does include CH1. This structure may include a hinge or partial hinge C-terminal to CH1 (top four structures), and VH2 may be located N-terminal to VL1 (top left), N-terminal to VH1 (top second from left), C-terminal to CL (top second from right), or C-terminal to the hinge or partial hinge (top right). Alternatively, this structure may not include a hinge (bottom four structures), and VH2 may be located N-terminal to VL1 (bottom left), N-terminal to VH1 (bottom second from left), C-terminal to CL (bottom second from right), or C-terminal to CH1 (bottom right). [Figure 33]Figure 33 provides further exemplary, non-limiting embodiments of various multispecific antibody structures that may include any of the variable domain sequences of the anti-Fzd4 and anti-LRP5 / 6 antibodies disclosed herein. In the structures shown above, VH1 and VL1 form an scFv, which is further fused to VH2. The scFv may include VH1, an optional linker, and VL1, or VL1, an optional linker, and VH1, in an N- to C-terminal orientation, with VH2 positioned N-terminal to the scFv (top left and second from the left at the top) or C-terminal to the scFv (top right and second from the right at the top). Such structures can be further linked or dimerized. For example, as shown in the lower structure, the boxed structure may be fused to another boxed structure (bottom left) or to each of the three other variants above (bottom left, bottom right, bottom second from the right at the bottom). [Figure 34] Figure 34 provides further exemplary variations of the boxed structure in Figure 24. Specifically, VH1 and VL1 form an scFv, which is further fused to VH2. Thus, the antibody structure may comprise two first polypeptides, each comprising, from N- to C-terminus, VH2, VL1, an optional linker, VH1, CH1, CH2, and CH3 (top left, boxed). In further variations of the boxed structure, the CH3 domain may be absent (middle left), the CH2 domain may be absent (middle center), or the CH2 and CH3 domains may be absent (middle right). Hinge and disulfide bonds may be present (disulfide bonds shown with dashed lines), for example, as shown in the lower structure.

[0151] Detailed Description As used in this specification, including the appended claims, the singular forms of words, e.g., "a," "an," and "the," include references to their corresponding plural forms unless specifically stated otherwise.

[0152] All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent was specifically and individually indicated to be incorporated by reference. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0153] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0154] It is to be understood that the terminology used in this description is for the purpose of describing particular versions or embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0155] As used herein, the term "about," when used in connection with a particular stated numerical value, means that the value may vary by 10% or less from the stated value. For example, as used herein, the expression "about 100" includes 90 and 110 and all values ​​therebetween (e.g., 91, 92, 93, 99, 99.1, 99.2, 99.3, 99.4, 100, 100.8, 100.9, 101, 106, 107, 108, 109, etc.).

[0156] The "activity" of a molecule may describe or refer to the binding of the molecule to a ligand or receptor, catalytic activity, the ability to induce, promote, enhance, and / or support multimerization (e.g., dimerization, trimerization, tetramerization, etc.) of one or more target molecules (e.g., antigens), the ability to induce, promote, enhance, and / or support signal transduction in cells expressing one or more target molecules (e.g., antigens), the ability to alter (e.g., stimulate or inhibit) gene expression, antigenic activity, the activity of another molecule, e.g., modulation of the activity of a ligand specific for one or more target molecules (e.g., antigens), etc. The "activity" of a molecule may also refer to the activity of modulating or maintaining cell-cell interactions, e.g., adhesion, or maintaining cellular structure, e.g., cell membrane or cytoskeleton, or a therapeutic effect. "Activity" may also refer to specific activity, e.g., [catalytic activity] / [mg protein] or [immune activity] / [mg protein], etc.

[0157] As used herein, the terms "administering" or "introducing" refer to delivering a compound or composition of the invention to a cell, a cell, tissue, and / or organ of a subject, or to a subject. Such administration or introduction can occur in vivo, in vitro, or ex vivo.

[0158] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that comprises the variable region sequences necessary to specifically bind to an antigen epitope. Accordingly, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binding to a specific target antigen. Thus, antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including (but not limited to) scFv, Fab, and F(ab')2, so long as they exhibit the desired biological activity.

[0159] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, Fab'-SH, and Fv fragments, diabodies, linear antibodies (e.g., Zapata et al., Protein Eng. 8(10):1057-1062 (1995)), single-chain antibody molecules (e.g., scFv), half antibodies, nanobodies, or multispecific antibodies formed from only the VH or only the VL and antibody fragments. The term "half molecule" or "half antibody" (when referring to IgG, IgE, or IgD, may also be referred to as "half-IgG," "half-IgE," or "half-IgD," respectively) refers to one heavy chain and one light chain set of the referenced antibody. Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0160] Terms such as "intact antibody," "full length antibody," and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody. In some examples, antibodies comprise a heavy (H) chain and a light (L) chain inter-connected by disulfide bonds. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. For example, an intact IgG (or IgD or IgE) antibody comprises two heavy immunoglobulin chains and two light immunoglobulin chains. Thus, in some examples, antibodies of the present disclosure may comprise two pairs of heavy and light chains or antigen-binding fragments thereof inter-connected by disulfide bonds. Some intact antibodies comprise multiple units, each comprising two pairs of heavy and light chains inter-connected by disulfide bonds. For example, intact IgA comprises two units, and intact IgM comprises five units. Thus, in other examples, antibodies of the present disclosure may instead comprise multiple (e.g., 2, 3, 4, 5, etc.) units, each comprising two pairs of heavy and light chains or antigen-binding fragments thereof inter-connected by disulfide bonds.

[0161] In an intact antibody, each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant domain (CH), and the CH is typically composed of domains CH1, CH2, and CH3. Each light chain is composed of a light chain variable domain (VL) and a light chain constant domain (CL). Typically (with some exceptions, such as nanobodies, camelid heavy chain antibodies, and IgNARs), one VH and one VL can form an antigen-binding region. The VH and VL can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are separated by highly conserved regions called framework regions (FRs). Each VH and VL polypeptide is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs in the heavy chain are designated "CDRH1," "CDRH2," and "CDRH3," respectively, and the CDRs in the light chain are designated "CDRL1," "CDRL2," and "CDRL3." The FRs in the heavy chain are designated "FRH1," "FRH2," "FRH3," and "FRH4," respectively, and the FRs in the light chain are designated "FRL1," "FRL2," "FRL3," and "FRL4." In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline coding sequences (e.g., heavy chain FR sequences encoded by the VH1-03 germline and / or light chain FR sequences encoded by the VK4-01 germline), or may be naturally occurring or artificially modified. An amino acid consensus sequence can be defined based on side-by-side analysis of two or more CDRs.

[0162] The term "heavy chain" or "HC" is used in its broadest sense and includes polypeptides comprising at least one immunoglobulin heavy chain domain or portion (i.e., at least one of VH, CH1, hinge, CH2, and CH3). Similarly, the term "light chain" or "LC" is used in its broadest sense and includes polypeptides comprising at least one immunoglobulin light chain domain (i.e., at least one of VL and CL).

[0163] The numbering of amino acid residues in the variable and / or constant domains of an antibody can be performed according to any suitable numbering scheme, method, and definition. For example, the CDR sequences can be determined according to Kabat or IMGT numbering, or alternatively, Chothia, Martin (also known as AbM numbering), Gelfand, Honneger (also known as AHo numbering), or Chemical Computing Group (CCG) numbering for the variable region, or can be determined structurally (see, for example, IgBlast (https: / / www.ncbi.nlm.nih.gov / igblast / ); Dondelinger et al, Front Immunol. 2018 Oct 16;9:2278; http: / / www.bioinf.org.uk / abs / info.html#cdrid; http: / / opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / ). For constant domains, for example, EU numbering, IMGT numbering or Kabat numbering can be used. Unless otherwise specified, EU numbering is used for constant domains and regions.

[0164] According to IMGT (the international ImMunoGeneTics information system for immunoglobulins or antibodies, T-cell receptors, MH, immunoglobulin superfamilies IgSF and MhSF), the CH1 domain, hinge region, CH2 domain, and CH3 domain correspond to amino acids 118-215, 216-230, 231-340, and 341-446, respectively (EU numbering). The terms "CH1 domain," "hinge," "CH2 domain," and "CH3" are used broadly herein to encompass allotypes of any naturally occurring corresponding heavy chain constant domain and / or region and variants thereof, which may contain fewer or more amino acids (e.g., the CH1 domain may include part of the hinge region) and / or amino acid modifications.

[0165] An exemplary CH1 domain of human IgG1 may comprise the amino acid sequence of SEQ ID NO: 21 or 22, an exemplary hinge of human IgG1 may comprise the amino acid sequence of SEQ ID NO: 31, and a CH2 domain of human IgG1 may comprise the amino acid sequence of SEQ ID NO: 41. An exemplary CH3 domain of human IgG1 may comprise the amino acid sequence of SEQ ID NO: 51, 52, 53, or 54, where the C-terminal K may be removed from such CH3 sequences. Any variant of such exemplary sequences may be used in combination with the CDR and / or variable domain sequences described herein.

[0166] An "Fc region" is a C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region, including native sequence Fc regions and variant Fc regions. The Fc region of a human IgG heavy chain may extend from Asp221 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0167] The phrase "effector function" of an antibody refers to a biological activity attributable to the Fc region of the antibody and varies depending on the antibody isotype. Exemplary effector functions include complement (e.g., C1q) binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0168] There are two major light chain isotypes, kappa (κ) and lambda (λ), and the corresponding light chain constant domains are called kappa CL domains (CLκ) and lambda CL domains (CLλ), respectively.

[0169] According to IMGT, the CLκ domain is amino acids 108-214 (EU numbering). An exemplary CLκ domain of human IgG may comprise the amino acid sequence of SEQ ID NO: 11. According to IMGT, the CLλ domain is amino acids 107-215 (EU numbering). An exemplary CLλ domain of human IgG may comprise the amino acid sequence of SEQ ID NO: 12.

[0170] The terms "CLκ" and "CLλ" are used broadly herein to encompass allotypes of any naturally occurring corresponding light chain constant domains and / or regions and variations thereof, which may contain fewer or more amino acids and / or amino acid modifications.

[0171] Various standard sequences (corresponding to different allotypes) of the constant domains of human IgG1, IgG2, IgG3, and IgG4 are known in the art and can be found, for example, in Vidarsson et al., Front Immunol. 2014 Oct 20;5:520 and US Pat. No. 9,150,663, the disclosures of which are incorporated herein by reference in their entireties. Again, these reference sequences are intended to be exemplary, as the applicant intends by human IgG1, IgG2, IgG3, and IgG4 sequences to include any naturally occurring human IgG1, IgG2, IgG3, and IgG4 allotypes.

[0172] "Antigen-binding region" refers to the portion of an antibody or antigen-binding fragment that has specificity for an antigen. In some cases, the antigen-binding region may comprise a VH and a VL. In some cases, the antigen-binding region may comprise a VH but not a VL, for example, in the case of nanobodies.

[0173] The term "antigen" refers to a molecule or portion of a molecule that is capable of binding to a selective binding agent, e.g., an antibody, and that can be used in an animal to generate antibodies capable of binding to an epitope of that antigen. In certain embodiments, a binding agent (e.g., a Wnt surrogate molecule or binding region thereof, or a Wnt antagonist) is said to specifically bind to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0174] The phrase "different antigens" can refer to different and / or distinct proteins, polypeptides, or molecules, as well as to different and / or distinct epitopes, which may be contained in one protein, one polypeptide, or one molecule. An "antigen-binding fragment" or "antigen-binding antibody fragment" refers to a portion of an intact antibody or a combination of portions derived from one or more intact antibodies that bind to the antigen to which the intact antibody binds. As used herein, the term "antigen-binding fragment" can refer to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and / or light chain, or a VHH / sdAb (single domain antibody) or Nanobody® (Nab), and that binds to an antigen of interest, in particular one or more Fzd receptors or LRP5 and / or LRP6. In this regard, antigen-binding fragments of the antibodies described herein can comprise one, two, three, four, five, or all six CDRs of the VH and VL from an antibody that binds to one or more Fzd receptors or LRP5 and / or LRP6. Antigen-binding fragments of antibodies include any natural, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0175] As used herein, the terms "biological activity" and "biologically active" refer to activity resulting from a specific biological element in a cell. For example, the "biological activity" of a Wnt agonist or a fragment or variant thereof refers to its ability to mimic or enhance Wnt signaling. As another example, the biological activity of a polypeptide or a functional fragment or variant thereof refers to the ability of the polypeptide or a functional fragment or variant thereof to perform its native function, such as binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element, such as a promoter, enhancer, Kozak sequence, etc., refers to the ability of the regulatory element or a functional fragment or variant thereof to control the expression of a gene to which it is operably linked, i.e., to promote, enhance, or activate translation, respectively.

[0176] As used herein, the term "bifunctional antibody" refers to an antibody that contains a first arm with specificity for one antigenic site and a second arm with specificity for a different antigenic site. That is, a bifunctional antibody has dual specificities.

[0177] With respect to multispecific antibodies (e.g., bispecific, trispecific, tetraspecific, etc.), such antibodies comprise at least two different antigen-binding regions that recognize and specifically bind to at least two different antigens or epitopes. The different epitopes may or may not be within the same antigen. A "bispecific antibody" is a type of multispecific antibody that comprises two different antigen-binding regions that recognize and specifically bind to two different antigens or two epitopes. Bispecific antibodies may, for example, target two different surface receptors on the same or different cells. As used herein, "bispecific antibodies" is used to encompass full-length antibodies generated by quadroma technology (see Milstein et al., Nature, 305(5934): 537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012): 628-631 (1985)), or by knob-into-hole or similar approaches to introduce mutations into the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14): 6444-6448 (1993)), resulting in multiple different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds to one antigen (or epitope) with one of its two binding arms (one HC / LC pair) and to a different antigen (or epitope) with its second arm (a different HC / LC pair). By this definition, a bispecific antibody has two distinct antigen-binding arms (both in specificity and CDR sequences).

[0178] "Comprising" means that the recited element is essential, e.g., in a composition, method, kit, etc., but that other elements may be included to form, e.g., a composition, method, kit, etc., within the scope of the claim. For example, an expression cassette "comprising" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, e.g., polyadenylation sequences, enhancer elements, other genes, linker domains, etc.

[0179] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution will not substantially alter the functional properties of a protein. When two or more amino acid sequences differ by conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331). Exemplary groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, in some embodiments, a conservative replacement includes any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45. In some embodiments, a "moderately conservative" replacement includes any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0180] "Consisting essentially of" refers to limitations in the scope of, e.g., a composition, method, kit, etc., described to specified materials or steps that do not substantially affect the basic and novel characteristics of, e.g., the composition, method, kit, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and polyadenylation sequence may include additional sequences, e.g., linker sequences, so long as they do not substantially affect transcription or translation of the gene. As another example, a variant or mutant polypeptide fragment "consisting essentially of" a recited sequence has the amino acid sequence of the recited sequence plus or minus about 10 amino acid residues at the sequence boundaries, based on the full-length native polypeptide from which it is derived, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than the recited linking amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the recited linking amino acid residues.

[0181] "Consisting of" means excluding from a composition, method, or kit any element, step, or ingredient not specified in the claim. For example, a polypeptide or polypeptide domain "consisting of" a recited sequence contains only the recited sequence.

[0182] A "control element" or "control sequence" is a nucleotide sequence involved in a molecular interaction that contributes to the functional control of a polynucleotide, including its replication, duplication, transcription, splicing, translation, or degradation. This control may affect the frequency, rate, or specificity of the process and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a DNA region capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region typically located downstream (3') of the promoter.

[0183] An "effective amount" of an antibody or composition (e.g., a pharmaceutical composition) disclosed herein is at least the minimum amount necessary to achieve a desired therapeutic or prophylactic result, e.g., measurable improvement or prevention of a particular disorder, e.g., retinopathy, preferably with minimal or no toxic or adverse effects. The effective amount may vary depending, among other things, on the disease state, the age, sex, and weight of the patient, the ability of the antibody (or antigen-binding fragment thereof) to elicit a desired response in the individual, and in some cases, by co-administration of one or more additional therapeutic agents.

[0184] An "expression vector" is a vector discussed herein or known in the art, e.g., a plasmid, minicircle, viral vector, liposome, etc., that contains a region encoding a gene product of interest and is used to effect expression of the gene product in an intended target cell. An expression vector also contains regulatory elements, e.g., a promoter, enhancer, UTR, miRNA targeting sequence, etc., operably linked to the coding region to promote expression of the gene product in the target. The combination of regulatory elements and one or more genes to which they are operably linked for expression may be referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

[0185] As used herein, the term "FR set" refers to the four adjacent amino acid sequences that form the CDRs of a CDR set of a heavy or light chain V region. While some FR residues may contact a bound antigen, FRs, particularly those directly adjacent to the CDRs, are primarily responsible for folding the V region into the antigen-binding site. Certain amino acid residues and structural features within FRs are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form the antigen-binding surface. It is generally recognized that FRs contain conserved structural regions that influence the folded shape of the CDR loops within a particular "canonical" structure, regardless of the exact amino acid sequence of the CDR. Furthermore, certain FR residues are known to participate in noncovalent interdomain contacts that stabilize the interaction between the heavy and light chains of an antibody.

[0186] The frizzled family is a family of frizzled-class receptors, which are G protein-coupled receptors that function as receptors in Wingless / Integrated (Wnt) and other signaling pathways, often with co-receptors such as LRP5, LRP6, or ROR1 / 2. Members of the frizzled family include frizzled-class receptors 1 to 10 (Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10).

[0187] Frizzled class receptor 4 (Fzd4) is a member of the frizzled family, which may also be referred to as CD344, EVR1, FEVR, frizzled4, frizzled family receptor 4, frizzled homolog 4, Fz4, Fz-4, Fzd4S, FzE4, MGC34390, or Wnt receptor frizzled-4. In humans, Fzd4 is encoded by the Fzd4 gene on chromosome 11, located at 11q14.2 (NCBI, Gene ID: 8322). In some embodiments, human Fzd4 may have the amino acid sequence AAR23924.1 or SEQ ID NO: 4 provided in GenBank, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, etc.

[0188] Several diseases and / or phenotypes are caused by one or more alterations in the Fzd4 gene or altered expression of the Fzd4 gene. According to the OMIM® database (https: / / www.omim.org / ), genetic disorders of the Fzd4 gene include exudative vitreoretinopathy 1 (phenotype MIM number 133780, autosomal dominant inheritance) and retinopathy of prematurity (phenotype MIM number 133780, autosomal dominant inheritance). According to MedlinePlus (NIH, https: / / medlineplus.gov / genetics / gene / ), diseases associated with genetic alterations in the Fzd4 gene include familial exudative vitreoretinopathy.

[0189] The term "host cell" refers to a cell into which an exogenous nucleic acid sequence has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, including the primary transformed cell and progeny derived therefrom without regard to the number of transfers.

[0190] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, human and non-human primates (including apes and humans), mammalian sport animals (e.g., horses), mammalian farm animals (e.g., sheep, goats, etc.), mammalian pets (dogs, cats, etc.), and rodents (e.g., mice, rats, etc.).

[0191] Low-density lipoprotein receptors (LDLRs) are involved in various cellular functions, including endocytosis of low-density lipoproteins (LDLs), the primary carrier of cholesterol in the blood. LDLR family members include the LDL receptor, low-density lipoprotein receptor-related proteins 1, 1b, 2, 3, 4, 5, 6, and 8 (LRP1, LRP1b, LRP2, LRP3, LRP4, LRP5, LRP6, and LRP8), LDLR11, and the very-low-density lipoprotein (VLDL) receptor (Ren et al., Front Cell Dev Biol. 2021 May 6;9:670960).

[0192] Among the LDLRs, LRP5 and LRP6 are unique in their structure and function: for example, both LRP5 and LRP6 can individually function as co-receptors for Wnt and other ligands and mediate signal transduction by forming receptor complexes with other receptors, such as other Wnt receptors (e.g., Fzd receptors).

[0193] LRP5 may also be referred to as BMND1, EVR1, EVR4, HBM, LR3, low-density lipoprotein receptor-related protein 7 (LRP7), OPPG, OPS, OPTA1, or VBCH2. In humans, LRP5 is encoded by the LRP5 gene on chromosome 11, with the gene location being 11q13.2 (NCBI, Gene ID: 4041). In some embodiments, human LRP5 may have the amino acid sequence provided as NCBI Reference Sequence: NP_002326.2 or SEQ ID NO: 5, or equivalent residues from non-human species, such as mouse, rodent, monkey, ape, etc.

[0194] Several diseases and / or phenotypes are caused, mediated, and / or exacerbated by one or more alterations in the LRP5 gene or alterations in its expression. Loss-of-function mutations in LRP5 have been shown to cause osteoporotic pseudoglioma (OPPG), a rare syndrome associated with prematurity, a systemic osteoporosis leading to fractures. Familial exudative vitreoretinopathy (FEVR) has also been associated with mutations in LRP5. Upregulation of LRP5 has been associated with calcific aortic valve disease (e.g., expression of bone matrix proteins in the aortic valve and vasculature in the presence of hypercholesterolemia is dependent on LRP5, and increased tissue staining and total tissue protein levels of LRP5 have been observed in degenerative human mitral valve and calcific aortic valve specimens). LRP5 gene expression has been found to be upregulated in hip and knee joint tissues from human patients with osteoarthritis (OA), and truncated forms of LRP5 generated by alternative splicing have been associated with parathyroid tumors and breast cancer (Joiner et al., Trends Endocrinol Metab. 2013 Jan;24(1):31-9). In colorectal cancer (CRC), tissue LRP5 expression is upregulated and correlates with clinical stage of CRC, and LRP5 has been shown to promote tumor stem-like traits (Nie et al., J Cell Mol Med. 2022 Feb;26(4):1095-1112). Meanwhile, LRP5 expression in osteosarcoma cells has also been associated with disease progression (Hoang et al., Int J Cancer. 2004 Mar;109(1):106-11.), while overexpression of LRP5 in bone cells has been shown to enhance the anti-breast cancer activity of bone cells in bone (Liu et al., Bone Res. 2021 Jul 6;9(1):32.).

[0195] According to the OMIM® database (https: / / www.omim.org / ), inherited disorders of the LRP5 gene include exudative vitreoretinopathy type 4 (phenotype MIM number 601813, autosomal dominant and autosomal recessive), endosteal hyperplasia of bone (phenotype MIM number 144750, autosomal dominant), osteopetrosis autosomal dominant type 1 (phenotype MIM number 607634, autosomal dominant), osteoporosis-pseudoglioma syndrome (phenotype MIM number 259770, autosomal recessive), osteosclerosis (phenotype MIM number 144750, autosomal dominant), polycystic liver disease type 4 with or without renal cysts (phenotype MIM number 617875, autosomal dominant), van der Waals syndrome (phenotype MIM number 617875, autosomal dominant), and glaucoma. These include Buchem disease type 2 (phenotype MIM number 607636), bone mineral density variation type 1 (phenotype MIM number 601884, autosomal dominant), and osteoporosis (phenotype MIM number 166710, autosomal dominant). According to MedlinePlus (NIH, https: / / medlineplus.gov / genetics / gene / ), diseases associated with genetic alterations in the LRP5 gene include familial exudative vitreoretinopathy, juvenile primary osteoporosis, and osteoporosis-pseudoglioma syndrome.

[0196] LRP6 may also be referred to as ADCAD2 or STHAG7. In humans, LRP6 is encoded by the LRP6 gene on chromosome 12, with the gene location being 12p13.2 (NCBI, Gene ID: 4040). In some embodiments, human LRP6 may have the amino acid sequence provided as NCBI Reference Sequence: NP_002327.2 or SEQ ID NO: 6, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, etc.

[0197] Several diseases and / or phenotypes are caused, mediated, and / or exacerbated by one or more changes in the LRP6 gene or changes in its expression. For example, a single nucleotide polymorphism (SNP) in exon 18 of LRP6 is associated with Alzheimer's disease. A causal relationship between a mutation in LRP6 (R611C) and premature coronary artery disease has been observed. LRP6 gene expression has been found to be upregulated in hip and knee joint tissues from human patients with osteoarthritis (OA) (Joiner et al., Trends Endocrinol Metab. 2013 Jan;24(1):31-9.). Furthermore, LRP6 has been reported to be upregulated and promote proliferation in various cancers, including breast cancer (e.g., triple-negative breast cancer (TNBC)), primary chronic lymphocytic leukemia (CLL), non-small cell lung cancer (NSCL), lung squamous cell carcinoma (LSCC), and hepatocellular carcinoma (HCC) (Li et al., Oncogene. 2004 Dec 2;23(56):9129-35.; Roslan et al., J Oncol. 2019 Mar 26;2019:4536302.) In this context, several antagonistic anti-LRP6 antibodies have been reported to inhibit Wnt-driven tumors in vivo (Joiner et al., Trends Endocrinol Metab. 2013 Jan;24(1):31-9.).

[0198] According to the OMIM® database (https: / / www.omim.org / ), inherited disorders of the LRP6 gene include selective odontopathy type 7 (phenotype MIM number 616724, autosomal dominant) and coronary artery disease autosomal dominant type 2 (phenotype MIM number 610947, autosomal dominant).

[0199] As used herein, the term "native" or "wild-type" refers to a nucleotide sequence, e.g., a gene or gene product, e.g., RNA or protein, present in a wild-type cell, tissue, organ, or organism. As used herein, the term "variant" refers to a variant of a reference polynucleotide or polypeptide sequence, e.g., a native polynucleotide or polypeptide sequence, i.e., one having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence. Stated differently, a variant contains at least one amino acid difference (e.g., an amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polynucleotide sequence, e.g., a native polynucleotide or polypeptide sequence. For example, a variant can be a polynucleotide having 50% or more, 60% or more, or 70% or more sequence identity with a full-length native polynucleotide sequence, e.g., 75% or 80% or more, e.g., 85%, 90%, or 95% or more, e.g., 98% or 99% identity with a full-length native polynucleotide sequence. As another example, a variant can be a polypeptide having 70% or more sequence identity to a full-length native polypeptide sequence, e.g., 75% or 80% or more identity to a full-length native polypeptide sequence, e.g., 85%, 90%, or 95% or more, e.g., 98% or 99% identity. A variant can also include a variant fragment of a reference sequence, e.g., a native sequence, that shares 70% or more sequence identity with a reference sequence, e.g., a fragment of a native sequence, e.g., 75% or 80% or more identity to the native sequence, e.g., 85%, 90%, or 95% or more, e.g., 98% or 99% identity.

[0200] "Norrin" is a secreted protein with a cystine-knot motif that activates the Wnt / β-catenin signaling pathway. In humans, Norrin is encoded by the NDP gene on the X chromosome, located at Xp11.3 (NCBI, Gene ID: 4693). Norrin may also be referred to as Norry disease protein, NDP, ND, X-linked exudative vitreoretinopathy type 2 protein, or EVR2. In some embodiments, human Norrin may have the amino acid sequence provided as NCBI Reference Sequence: NP_000257.1 or SEQ ID NO: 3, or equivalent residues from non-human species, such as mouse, rodent, monkey, ape, etc.

[0201] Several diseases and / or phenotypes are caused, mediated, and / or exacerbated by one or more alterations in the NDP gene or alterations in its expression. According to the OMIM® database (https: / / www.omim.org / ), genetic disorders of the NDP gene include X-linked exudative vitreoretinopathy type 2 (phenotype MIM number 305390, X-linked dominant or X-linked recessive) and Norrie disease (phenotype MIM number 310600, X-linked recessive). According to MedlinePlus (NIH, https: / / medlineplus.gov / genetics / gene / ), diseases associated with genetic alterations in the NDP gene include familial exudative vitreoretinopathy, Norrie disease, and retinal dystrophies.

[0202] Norrin binds to one or more Wnt receptors, such as Fzd4, LRP5, or LRP6 (often referred to as LRP5 / 6) and TSPAN12, and induces the canonical Wnt signaling pathway, the downstream effects of which include, for example, angiogenesis via activation of the transcription factor Sox17 (Junge et al., Cell. 2009 Oct 16;139(2):299-311. and Ye et al., Cell. 2009 Oct 16;139(2):285-98.). Mutations in Fz4, Lrp5, Norrin, or TSPAN12 are further known to cause reduced retinal neovascularization. Blocking Norrin-induced Fzd4-mediated Wnt signaling has also been reported to cause defects in central nervous system (CNS) angiogenesis and the blood-CNS barrier (Zhang et al., Nat Commun. 2017 Jul 4;8:16050).

[0203] "Operably linked" or "operably linked" refers to the juxtaposition of genetic elements wherein the elements are in a relationship permitting them to function in their expected manner. For example, a promoter is operably linked to a coding region if the promoter serves to initiate transcription of the coding sequence. There can be intervening residues between the promoter and the coding region so long as this functional relationship is maintained.

[0204] A "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical formulation or composition, which is largely non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some embodiments, a pharmaceutical formulation comprises any of the antibodies provided herein and at least one additional therapeutic agent.

[0205] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. The terms also encompass amino acid polymers that have been modified to include, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety.

[0206] The terms "polynucleotide" or "nucleic acid" refer to a polymeric form of nucleotides of any length, comprising deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0207] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide. This means that when two sequences are compared and aligned, that percentage of bases or amino acids are the same. The terms "identical" or "identity," when used in the context of two or more nucleic acid or polypeptide sequences, refer to the number or percentage of residues that are the same in a sequence of interest and a reference sequence. This percentage can be calculated by optimally aligning the sequence of interest to the reference sequence, comparing the two sequences across the entire length of the reference sequence, determining the number of positions where identical amino acid residues or nucleic acid bases occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence adjusted by adding the number of gap positions introduced into the reference sequence when generating the alignment, and multiplying the result by 100 to obtain the percentage of sequence identity. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Sequence identity can be determined by using a standalone executable BLAST engine program (bl2seq) to blast two sequences. This can be searched from the National Center for Biotechnology Information (NCBI) ftp site or on the World Wide Web at ncbi.nlm.nih.gov / BLAST / using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250, which is incorporated herein by reference in its entirety).

[0208] As used herein, a "promoter" includes a DNA sequence that directs the binding of RNA polymerase, thereby facilitating RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression can be ubiquitous, meaning they are strongly active in a wide range of cells, tissues, and species, or they can be cell-type-, tissue-, or species-specific. Promoters can be "constitutive," meaning they are continuously active, or "inducible," meaning they can be activated or inactivated by the presence or absence of biotic or abiotic factors. A nucleic acid construct or vector of the invention also includes enhancer sequences, which may or may not be contiguous with the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5' or 3' regions of the native gene.

[0209] "Recombinant," when applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction, or ligation steps and other procedures that result in a construct that is distinct from polynucleotides found in nature.

[0210] As used herein, the terms "treatment," "treating," and the like are generally used to mean achieving a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially inhibits or prevents a disease or its symptoms, e.g., reduces the likelihood of a disease or its symptoms occurring in a subject, and / or may be therapeutic, in that it partially or completely cures the disease and / or side effects resulting from the disease. As used herein, "treatment" covers any treatment of a disease in a mammal, including (a) inhibiting the disease, i.e., preventing its onset, or (b) alleviating the disease, i.e., causing the disease to regress. Therapeutic agents can be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease, which stabilizes or alleviates undesirable clinical symptoms in a patient, is of particular interest. Such treatment is desirably administered before complete loss of function of the affected tissue, e.g., the eye. The subject treatment will desirably be administered during the symptomatic stage of the disease, and in some cases, after the symptomatic stage of the disease.

[0211] As used herein, the phrase "retinal vascular disease" refers to a disease of the eye, particularly the retina, caused by abnormal vasculature formation. In some aspects, the abnormal vasculature is caused by inhibition of vasculature development, while in other aspects, the abnormal vasculature is caused by excessive neovascularization.

[0212] A "vector" is a compound or composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including (but not limited to) linear polynucleotides, polynucleotides associated with ionic or amphipathic compounds, plasmids, viruses, and virus-like particles (VLPs). Thus, the term "vector" includes autonomously replicating plasmids, self-replicating RNA, or viral particles. The term "vector" therefore encompasses expression vectors. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0213] The "Wnt signaling pathway" includes a group of signaling pathways that can be induced by the binding of a Wnt ligand (also referred to as Wnt) or other ligand to one or more Wnt receptors. Wnt ligands are a diverse family of secreted glycoproteins that are cysteine-rich, highly hydrophobic, and typically 350-400 amino acids in length. In humans, Wnt ligands include WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16. Wnt receptors are receptors to which Wnt ligands can bind, and include frizzled class receptors and co-receptors such as tyrosine kinase-related (Ryk) LRP5, LRP6 and receptor tyrosine kinase-like orphan receptors (ROR).

[0214] The Wnt signaling pathway controls complex normal cellular processes, such as cell differentiation, development, tissue homeostasis, and wound healing. Abnormal regulation of Wnt signaling can be associated with developmental disorders, cancer, and other diseases. Major Wnt signaling pathways include the canonical Wnt signaling pathway (also known as the Wnt / β-catenin signaling pathway), the Wnt / planar cell polarity (PCP) pathway, and the Wnt / Ca2+ pathway (Sharma and Pruitt. Int J Mol Sci. 2020 Oct 28;21(21):8018.). These pathways are described below.

[0215] "Canonical Wnt signaling pathway": In the absence of a Wnt ligand, cytoplasmic β-catenin typically forms a complex (often referred to as the "degradation complex") with axin and adenomatous polyposis coli (APC) and is phosphorylated by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). The phosphorylated β-catenin is recognized by the E3 ubiquitin ligase β-Trcp, which targets β-catenin for proteosomal degradation, preventing it from entering the nucleus. In the presence of a Wnt ligand, Fzd and LRP (e.g., LRP5 or LRP6) form a receptor complex upon binding to the Wnt ligand, and this complex is phosphorylated by CK1 and GSK3. This allows binding of axin to the LRP cytoplasmic tail and discheveled (DVL) to the Fzd cytoplasmic tail, disrupting the degradation complex and allowing cytoplasmic β-catenin to enter the nucleus. Within the nucleus, β-catenin mediates direct and indirect transcriptional activation, for example, by binding to TCF / LEF family transcription factors as coactivators (Wiese et al., Development. 2018 Jun 26;145(12):dev165902; Sharma and Pruitt. Int J Mol Sci. 2020 Oct 28;21(21):8018; Ren et al., Front Cell Dev Biol. 2021 May 6;9:670960). The canonical Wnt signaling pathway can also be initiated by the binding of a non-Wnt ligand to one or more Wnt receptors, for example, by the binding of Norrin (NDP) to Fzd4, LRP5 or LRP6 (often referred to as LRP5 / 6), and TSPAN12 (Junge et al., Cell. 2009 Oct 16;139(2):299-311. and Ye et al., Cell. 2009 Oct 16;139(2):285-98.).

[0216] "Wnt / planar cell polarity (PCP) pathway": Upon binding to a Wnt ligand, Fzd and a coreceptor (e.g., ROR1 or ROR2) form a receptor complex and recruit DVL to the cell membrane. DVL interacts with small GTPases, such as RHO and RAC, which further activate ROCK and / or JNK. This activates cytoskeletal rearrangements through a transcriptional response that activates transcription factors, such as JUN and ATF2 (Sharma and Pruitt. Int J Mol Sci. 2020 Oct 28;21(21):8018.).

[0217] "Wnt / Ca2+ pathway": When Fzd and a coreceptor (e.g., ROR1 or ROR2) bind to a Wnt ligand, they form a receptor complex, activate phospholipase C (PLC), and induce intracellular Ca2+ influx. This in turn activates CDC42, which then induces Ca2+-dependent cell motility and polarity via various transcription factors, such as NFA (Sharma and Pruitt. Int J Mol Sci. 2020 Oct 28;21(21):8018).

[0218] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, microbiology, biochemistry and immunology, which are within the skill of those in the art. Such techniques are described in the scientific literature, e.g., "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (MJ Gait, ed., 1984), "Animal Cell Culture" (RI Freshney, ed., 1987), "Methods in Enzymology" (Academic Press, Inc.), "Handbook of Experimental Immunology" (DM Weir & CC Blackwell, eds.), "Gene Transfer Vectors for Mammalian Cells" (JM Miller & MP Calos, eds., 1987), "Current Protocols in Molecular Biology" (FM Ausubel et al., eds., 1987), "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994) and "Current Protocols in Immunology" (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0219] Several aspects of the present invention will now be described with reference to illustrative examples. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the present invention. However, one skilled in the art will readily recognize that the present invention can be practiced without one or more of the specific details or in other ways. The present invention is not limited by the illustrated order of acts or events, as some acts may occur in a different order and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement the method of the present invention.

[0220] Where the terms "including," "including," "having," "having," "with," or variations thereof, are used in either the detailed description and / or claims, such terms are intended to be inclusive in a similar manner to the term "comprising."

[0221] All publications mentioned herein are incorporated by reference as if disclosed and described in connection with the methods and / or materials for which the publications are cited, and the present disclosure will be understood to supersede the disclosure of the incorporated publications in case of conflict.

[0222] It is further noted that the claims may be drafted to exclude any element, and as such, this statement is intended to serve as a predicate for using such exclusive terminology, e.g., "solely," "only," etc., or a "negative" limitation in connection with the recitation of claim elements.

[0223] Unless otherwise specified, all terms used herein have the same meaning as they would to one of ordinary skill in the art, and the practice of the present invention will employ conventional techniques in microbiology and recombinant DNA technology that are within the knowledge of those skilled in the art.

[0224] II. General The present disclosure provides Wnt signal modulation and methods of use thereof, for example, to treat diseases, disorders, and conditions involving aberrant Wnt signaling, such as, but not limited to, various ocular disorders, including methods of modulating Wnt signaling to treat retinopathies, including, but not limited to, FEVR and other genetic disorders, DR, and AMD. In certain embodiments, the present invention provides Wnt / β-catenin agonists for inhibiting aberrant neovascularization in the progression of retinopathies.

[0225] Wnt ("Wingless-related integration site" or "Wingless and Int-1" or "Wingless-Int") ligands and their signals play important roles in regulating the development, homeostasis, and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste buds, ovary, cochlea, lung, and many other tissues (reviewed, e.g., in Clevers, Loh, and Nusse, Science 2014; 346:1248012). Modulation of the Wnt signaling pathway has potential for the treatment of degenerative diseases and tissue injury.

[0226] One of the challenges in therapeutically modulating Wnt signaling is the existence of multiple Wnt ligands and Wnt receptors, Frizzled 1-10 (Fzd1-10), and the overlapping expression of multiple Fzds in many tissues. Furthermore, in addition to Fzds, canonical Wnt signaling also involves the co-receptors low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and low-density lipoprotein (LDL) receptor-related protein 6 (LRP6), which are widely expressed in various tissues.

[0227] R-spondins 1-4 are a family of ligands that amplify Wnt signaling. Each R-spondin functions through a receptor complex containing zinc-and-ring finger 3 (ZNRF3) or ring finger protein 43 (RNF43) on one side and leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-6) on the other side (reviewed, e.g., in Knight and Hankenson 2014, Matrix Biology; 37: 157-161). R-spondins may also function through additional mechanisms of action. ZNRF3 and RNF43 are two membrane-bound E3 ligases that specifically target Wnt receptors (Fzd1-10 and LRP5 or LRP6) for degradation. Binding of R-spondins to ZNRF3 / RNF43 and LGR4-6 results in clearance or blockage of the trimolecular complex. This removes the E3 ligase from the Wnt receptor, stabilizing the Wnt receptor and resulting in enhanced Wnt signaling. Each R-spondin contains two furin domains (1 and 2), with furin domain 1 binding to ZNRF3 / RNF43 and furin domain 2 binding to LGR4-6. A fragment of R-spondin containing furin domains 1 and 2 is sufficient to amplify Wnt signaling. Although the effect of R-spondin depends on Wnt signaling, the effect of R-spondin is not tissue-specific because both LGR4-6 and ZNRF3 / RNF43 are widely expressed in various tissues.

[0228] In certain embodiments, the Wnt / β-catenin agonist (or Wnt signaling agonist) comprises an antibody, antibody fragment, and / or antibody domain that binds to one or more Fzds and an antibody domain that binds to LRP5 and / or Lrp6. In certain embodiments, the antibody domain preferentially binds to Fzd4 compared to other Fzds. In certain embodiments, the Fzd-binding domain has a K D At least 10 times lower K DIn certain embodiments, the Fzd-binding domain has a K D At least 100 times lower than D In certain embodiments, the Fzd-binding domain has a K D At least 1000 times lower than D and preferentially binds to one or more Fzds. In certain embodiments, the antibody domain comprises the corresponding CDRs present in a Wnt signaling agonist disclosed herein, e.g., hp4SD1-03, or a fragment or variant of hp4SD1-03, e.g., the variable heavy chain region or the variable light chain region. Non-limiting exemplary sequences can be found in the attached Tables A-J.

[0229] In certain embodiments, the Wnt / β-catenin agonist comprises an IgG antibody-like structure comprising two antibody light chains and two antibody heavy chains, further comprising two VHH domains fused to either of the antibody chains (see, e.g., Figures 24-25). In certain embodiments, a VHH domain is fused to the N-terminus of each antibody light chain (see, e.g., Figure 24). In certain embodiments, the antibody heavy and light chains bind to one or more Fzds or preferentially bind to Fzd4, and the VHH domains bind to LRP5 and / or LRP6. In certain embodiments, the VHH domains preferentially bind to LRP5 compared to LRP6. In certain embodiments, the LRP5 / 6 binding domains have a K D At least 100 times lower than D In certain embodiments, the LRP5 / 6 binding domain has a K D At least 1000 times lower than D and preferentially binds to LRP5 or LRP6.

[0230] In related embodiments, Wnt / β-catenin agonists can employ different bispecific binding structures, including (but not limited to) those disclosed herein (including but not limited to, the structures shown in Figures 24-34). As is well known in the art, antibodies are immunoglobulin molecules capable of specifically binding to a target, e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one epitope binding domain located on the variable region of the immunoglobulin molecule. As used herein, this term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof containing the epitope-binding domain (e.g., dAb, Fab, Fab', (F(ab')2, Fv, single-chain (scFv), VHH or single-domain antibody (sdAb), DVD-Ig, synthetic variants thereof, natural variants, fusion proteins containing the epitope-binding domain, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding site or fragment (epitope recognition site) of the required specificity). "Diabodies," which are multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993), are also a specific form of antibody contemplated herein. Minibodies containing scFv linked to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996).See, for example, Ward, ES et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883, (1988); PCT / US No. 92 / 09965; WO No. 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0231] The proteolytic enzyme papain preferentially cleaves IgG molecules to generate several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites. Fv fragments for use in accordance with certain embodiments of the present disclosure can be produced by preferential proteolytic cleavage of IgM and, in rare cases, IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments contain a noncovalently linked VH:VL heterodimer containing an antigen-binding site, which retains much of the antigen recognition and binding capabilities of native antibody molecules. Inbar et al. (1972) Proc. Natl. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710 and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0232] In certain embodiments, single-chain Fv or scFv antibodies are contemplated. For example, kappabodies (Ill et al., Prot. Eng. 10: 949-57 (1997)), minibodies (Martin et al., EMBO J 13: 5305-9 (1994)), diabodies (Holliger et al., PNAS 90: 6444-8 (1993)), or Janusins ​​(Traunecker et al., EMBO J 10: 3655-59 (1991) and Traunecker et al., Int. J. Cancer Suppl. 7: 51-52 (1992)) can be prepared using standard molecular biology techniques, following the teachings of the present application for selection of antibodies with the desired specificity. In yet other embodiments, bispecific or chimeric antibodies can be prepared that incorporate the ligands of the present disclosure. For example, chimeric antibodies may contain CDRs and framework regions from different antibodies, while bispecific antibodies may be generated that specifically bind to one or more Fzd receptors through one binding domain and to a second molecule through a second binding domain. These antibodies may be produced by recombinant molecular biology techniques or may be physically conjugated to each other. Single-chain Fv (scFv) polypeptides are covalently linked VH:VL heterodimers expressed from a gene fusion containing a VH-encoding gene and a VL-encoding gene linked by a peptide-encoding linker. Exemplary linkers that can be used to link VH and VL in scFvs may include flexible linkers. In some embodiments, the linker may comprise one or more amino acids, optionally 1 to 50 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the linker may consist of small amino acids consisting of G, S and / or A.In some embodiments, a linker may comprise an amino acid sequence that comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG. In some embodiments, a linker may comprise an amino acid sequence that comprises or consists of multiple repeats (e.g., 2, 3, 4, or 5 repeats) of an amino acid sequence selected from the group consisting of SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG. In certain embodiments, a linker may comprise three or four repeats of SEQ ID NO:101, which may sometimes be referred to as a (G4S)3 linker and a (G4S)4 linker, respectively. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Numerous methods have been described for discerning chemical structures for converting naturally aggregated, but chemically separated, light and heavy chain polypeptides from antibody V regions into scFv molecules that will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, for example, US Pat. Nos. 5,091,513 and 5,132,405 (Huston et al.) and 4,946,778 (Ladner et al.).

[0233] In certain embodiments, the antibodies described herein are in the form of diabodies, which are multimers of polypeptides, each polypeptide comprising a first domain comprising the binding region of an immunoglobulin light chain and a second domain comprising the binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site, which is formed by the association of the first domain of one polypeptide in the multimer with the second domain of another polypeptide in the multimer (WO 94 / 13804).

[0234] A dAb fragment of an antibody consists of the VH domain (Ward, ES et al., Nature 341, 544-546 (1989)).

[0235] When bispecific antibodies are used, they may include conventional bispecific antibodies, which can be produced by various known methods, for example, as described in Holliger, P. and Winter G., Current Opinion Biotechnol. 4, 446-449 (1993), or may be prepared chemically or from hybrid hybridomas, or they may include any of the bispecific antibody fragments mentioned above. Diabodies and scFvs can be constructed using only variable domains, without the Fc region, which may reduce the effects of anti-idiotypic reaction.

[0236] Bispecific diabodies, as opposed to whole bispecific antibodies, can be particularly useful, in part because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides, e.g., antibody fragments) of appropriate binding specificities can be readily selected from libraries using phage display (see, e.g., WO 94 / 13804). If one arm of the diabody is held constant, e.g., specificity for antigen X, libraries can be prepared in which the other arm is varied, and antibodies of appropriate specificity can be selected. Whole bispecific antibodies can be prepared by knobs-into-holes engineering (JBB Ridgeway et al., Protein Eng., 9, 616-621 (1996)).

[0237] In certain embodiments, the antibodies described herein can be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US 20090226421). This proprietary antibody technology generates a stable, small antibody format, which is expected to have a longer therapeutic window than current small antibody formats. IgG4 antibodies are thought to be inert and therefore do not interact with the immune system. Fully human IgG4 antibodies can be modified by removing the hinge region of the antibody to yield half-fragments with distinct stability relative to the corresponding intact IgG4 (GenMab, Utrecht). By halving the IgG4 molecule, the UniBody® is left with only one region capable of binding to its cognate antigen (e.g., disease target); therefore, the UniBody® binds monovalently to only one site on the target cell.

[0238] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy and light chain CDRs, respectively, inserted between a set of heavy and light chain framework regions (FRs), which provide support for the CDRs and define the spatial relationship between the CDRs. As used herein, the term "CDR set" refers to the three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3," respectively. Thus, an antigen-binding site comprises six CDRs, comprising the CDR sets from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of many antigen-antibody complexes has demonstrated that amino acid residues in the CDRs form extensive contacts with bound antigens. Here, the most extensive antigen contacts are with the heavy chain CDR3. Thus, the specificity of an antigen-binding site is primarily mediated by the molecular recognition unit.

[0239] As used herein, the term "FR set" refers to the four adjacent amino acid sequences that form the CDRs of a CDR set of a heavy or light chain V region. While some FR residues may contact a bound antigen, FRs, particularly those immediately adjacent to the CDRs, are primarily responsible for folding the V region into the antigen-binding site. Certain amino acid residues and structural features within FRs are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form the antigen-binding surface. It is generally recognized that FRs contain conserved structural regions that influence the folded shape of the CDR loops within a particular "canonical" structure, regardless of the exact amino acid sequence of the CDR. Furthermore, certain FR residues are known to participate in noncovalent interdomain contacts that stabilize the interaction between the heavy and light chains of an antibody.

[0240] "Monoclonal antibody" refers to a homogeneous antibody population in which the individual antibodies comprising the population are identical and bind to the same epitope, except for possible variant antibodies (e.g., containing natural mutations or arising during production of a monoclonal antibody preparation) (such variants are generally present in small amounts), where the monoclonal antibody is composed of amino acids (both natural and non-natural) that are involved in selective binding of the epitope. Monoclonal antibodies are highly specific for a single epitope. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv), Nanobodies®, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule (including the Wnt surrogate molecules disclosed herein) containing an antigen-binding fragment (epitope recognition site) with the required specificity and ability to bind to the epitope. It is not intended to be limited with respect to the source of the antibody or the manner of preparation (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term encompasses whole immunoglobulins and fragments, etc., as defined above in the definition of "antibody."

[0241] In certain embodiments, the antibodies of the present disclosure can take the form of Nanobodies®. Nanobody® technology was initially developed following the discovery and identification that Camelidae (e.g., camels and llamas) possess fully functional antibodies consisting only of heavy chains and therefore devoid of light chains. These heavy-chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3). Cloned and isolated single variable domains possess full antigen-binding capacity and are highly stable. These single variable domains possess unique structural and functional properties and are the basis for "Nanobodies®." Nanobodies® are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as E. coli (see, e.g., U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see, e.g., U.S. Pat. No. 6,838,254)). The production process is scalable, and multi-kilogram quantities of Nanobodies® have been produced. Nanobodies® can be formulated as ready-to-use solutions with long shelf lives. The Nanoclone® method (see, e.g., WO 06 / 079372) is a patented method for generating Nanobodies® against desired targets based on automated, high-throughput selection of B cells. Nanobodies® are single-domain antigen-binding fragments of camelid-specific heavy-chain-only antibodies. Nanobodies®, also called VHH antibodies, typically have a small size of around 15 kDa.

[0242] Another contemplated antibody fragment is the dual variable domain immunoglobulin (DVD-Ig), an engineered protein that combines the functions and specificities of two monoclonal antibodies in a single molecule. DVD-Igs are designed as IgG-like molecules, except that instead of the single variable domain found in IgGs, the light and heavy chains each contain two variable domains in tandem connected via a short peptide bond. The orientation of the fusion of the two variable domains and the selection of the linker sequence are important for the functional activity and efficient expression of the molecule. DVD-Igs can be produced as a single species using conventional mammalian expression systems for production and purification. DVD-Igs retain the specificity of the parent antibody, are stable in vivo, and exhibit IgG-like physicochemical and pharmacokinetic properties. DVD-Igs and methods for producing them are described in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007). The DVD-Ig structure can be applied when at least one of the antigen binding regions comprises a VHH domain (see, for example, Figure 26 (e.g., top right)).

[0243] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are humanized. This generally refers to chimeric molecules, prepared using recombinant techniques, having an antigen-binding site (i.e., a complete set of CDRs) derived from an immunoglobulin from a non-human species, with the remainder of the immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. In some cases, for example, when the FR sequences derived from the non-human species match or closely match the corresponding human FR sequences, the humanized antibody or antigen-binding antibody fragment may comprise a complete variable domain derived from the non-human species fused onto a constant domain. In some cases, for example, when the FR sequences derived from the non-human species are different or significantly different from the corresponding human FR sequences, the humanized antibody or antigen-binding antibody fragment may comprise CDRs derived from the non-human species grafted onto appropriate framework regions in a variable domain fused onto a constant domain. In some cases, appropriate residues in the FRs are substituted with corresponding human residues or similar residues (e.g., conservative amino acids). In certain cases, some residues in the CDRs may also be substituted with corresponding human residues or residues similar thereto (e.g., conservative amino acids). The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but leaves open the possibility of an immune response against the foreign variable region (LoBuglio, AF et al., (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327). Exemplary methods for humanizing the anti-Fzd or LRP antibodies disclosed herein include those described in US Pat. No. 7,462,697 and other well-known humanization methods.

[0244] Such further modifications to the CDRs allow the CDRs to be reshaped to resemble human forms as closely as possible. It is known that the variable regions of both heavy and light chains are relatively conserved in a given species and contain three complementarity-determining regions (CDRs) flanked by four framework regions (FRs) that are presumed to provide a scaffold for the CDRs. The CDRs vary depending on the epitope and determine binding ability. When a non-human antibody is prepared for a specific epitope, the variable region can be "reshaped" or "humanized" by grafting the CDRs derived from the non-human antibody onto the FRs present in the modified human antibody. Application of this approach to various antibodies has been reported in Sato, K., et al., (1993) Cancer Res 53:851-856; Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al., (1988) Science 239:1534-1536; Kettleborough, CA, et al., (1991) Protein Engineering 4:773-3783; Maeda, H., et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman, SD, et al., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, PR, et al., (1991) Bio / Technology 9:266-271; Co, MS, Carter, P., et al., (1992) Proc Natl Acad Sci USA 89:4285-4289, and Co, MS et al., (1992) J Immunol 148:1149-1154. In some embodiments, the humanized antibody contains all CDR sequences (e.g., a humanized mouse antibody contains all six CDRs from the mouse antibody).In other embodiments, a humanized antibody has one or more CDRs (1, 2, 3, 4, 5, 6) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0245] In certain embodiments, antibodies of the present disclosure may be chimeric antibodies. In this regard, a chimeric antibody is composed of an antigen-binding fragment of an antibody operably linked or otherwise fused to a heterologous constant or Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is human. In other embodiments, the heterologous Fc domain may be derived from a different Ig class than the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be composed of CH2 and CH3 domains from one or more of the different Ig classes. As noted above with respect to humanized antibodies, the antigen-binding fragment of a chimeric antibody may include only one or more CDRs of an antibody described herein (e.g., one, two, three, four, five, or six CDRs of an antibody described herein) or may include the entire variable domain (VL, VH, or both).

[0246] In some embodiments, an antibody or antibody fragment of the present disclosure comprises an Fc region.

[0247] Specific amino acid modifications in the Fc region are known to modulate Ab effector functions and properties, including but not limited to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and half-life (Wang X. et al., Protein Cell. 2018 Jan; 9(1): 63-73; Dall'Acqua WF et al., J Biol Chem. 2006 Aug 18; 281(33): 23514-24. Epub 2006 Jun 21; Monnet C. et al., Front Immunol. 2015 Feb 4; 6:39. doi: 10.3389 / fimmu.2015.00039. eCollection 2015). Mutations can be symmetric or asymmetric. In certain cases, antibodies with asymmetrically mutated Fc regions (i.e., the two Fc regions are not identical) may provide better function, e.g., ADCC (Liu Z. et al. J Biol Chem. 2014 Feb 7; 289(6): 3571-3590).

[0248] In some embodiments, antibodies or antibody fragments, e.g., multispecific antibodies or antibody fragments, of the present disclosure may comprise an Fc region comprising one or more amino acid substitutions that reduce or alleviate effector function compared to the corresponding wild-type Fc region (also referred to as "Fc-silencing mutations or substitutions"). In certain embodiments, the Fc region may comprise one or more amino acid substitutions at positions selected from the group consisting of 234, 235, 236, 237, 265, 297, and 329 according to EU numbering. In certain embodiments, the Fc region may comprise L234A, L235A, and P329G according to EU numbering (also referred to herein as "LALAPG"). In certain embodiments, the Fc region may comprise L234A and L235A according to EU numbering (also referred to herein as "LALA"). In certain embodiments, the Fc region may comprise D265A and P329A (also referred to herein as "DAPA") according to EU numbering. In certain embodiments, the Fc region may comprise N297A according to EU numbering.

[0249] In some embodiments, antibodies or antibody fragments, e.g., multispecific antibodies or antibody fragments, of the present disclosure may comprise an Fc region comprising one or more amino acid substitutions that reduce the half-life of the antibody or antibody fragment compared to the corresponding wild-type Fc region. In certain embodiments, the Fc region may comprise I253A, H310A, and H435Q according to EU numbering (also referred to herein as "IAHAHQ" or "AAQ"). In certain embodiments, the Fc region may comprise I253A, H310A, and H435A according to EU numbering (also referred to herein as "IAHAHA" or "AAA").

[0250] In some embodiments, antibodies or antibody fragments, e.g., multispecific antibodies or antibody fragments, of the present disclosure may comprise an Fc region comprising one or more amino acid substitutions that improve FcRn binding and / or extend the half-life of the antibody or antibody fragment compared to the corresponding wild-type Fc region. In certain embodiments, the Fc region may comprise M252Y, S254T, and T256E according to EU numbering (also referred to herein as "YTE"). In certain embodiments, the Fc region may comprise M428L and N434S according to EU numbering (also referred to herein as "LS").

[0251] In some embodiments, when an antibody comprises an IgG1 Fc region or an Fc region derived from an IgG1, the Fc region may comprise one or more amino acid substitutions, such as, for example, N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236 deletion, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V305I, P396L, S239D, I332E, S298A, E333A, K334A, L23 4Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y, M428L, N434S, L328F, M252Y, S254T, T256E and / or any combination thereof (residue numbering according to EU numbering) (Dall'Acqua (WF et al., J. Biol. Chem. 2006 Aug. 18;281(33):23514-24. Epub 2006 Jun. 21; Wang X. et al., Protein Cell. 2018 Jan.;9(1):63-73). The Fc region may further comprise one or more additional amino acid substitutions. The substitutions may be, for example, but not limited to, A330L, L234F, L235E, P331S, and / or any combination thereof (residue numbering according to EU numbering).

[0252] In some embodiments, when the antibody comprises an IgG2 Fc region or an Fc region derived from IgG2, the Fc region may comprise one or more amino acid substitutions, such as, but not limited to, P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and / or any combination thereof (residue numbering according to EU numbering). The Fc region may further comprise one or more additional amino acid substitutions, such as, but not limited to, M252Y, S254T, T256E, and / or any combination thereof (residue numbering according to EU numbering).

[0253] In some embodiments, when the antibody comprises an IgG3 Fc region or an Fc region derived from an IgG3, the Fc region may comprise one or more amino acid substitutions, such as, but not limited to, E235Y (residue numbering according to EU numbering).

[0254] In some embodiments, when an antibody comprises an IgG4 Fc region or an Fc region derived from IgG4, the Fc region may comprise one or more amino acid substitutions, such as, but not limited to, E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and / or any combination thereof (residue numbering according to EU numbering). Such a substitution may be, for example, S228P (residue numbering according to EU numbering).

[0255] In some embodiments, the glycosylation of the human-like Fc region may be altered to modify effector function (see, e.g., Li T. et al., Proc Natl Acad Sci US A. 2017 Mar 28;114(13):3485-3490. doi: 10.1073 / pnas.1702173114. Epub 2017 Mar 13).

[0256] In some embodiments, the structure and location of immunoglobulin CDRs and variable domains can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. U.S. Department of Health and Human Services. 1987 and updates thereto (now available on the internet at immuno.bme.nwu.edu).

[0257] In certain embodiments, the antagonist or agonist binding agent binds one or more Fzd and / or LRP5 / 6 with a dissociation constant (K) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, or about 10 nM or less. D For example, in certain embodiments, an Fzd-binding domain or antibody described herein that binds to one or more Fzds binds to those Fzds with a K of about 100 nM or less, about 20 nM or less, or about 10 nM or less. D In certain embodiments, the binding domain binds to one or more of its target antigens with an EC50 of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM or less.

[0258] Antibodies or other agents of the invention can be assayed for specific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as biolayer interferometry (BLI) analysis, surface plasmon resonance (SPR), FACS analysis, immunofluorescence, immunocytochemistry, Western blot, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, and protein A immunoassay. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety).

[0259] For example, the specific binding of an antibody to a target antigen can be determined using ELISA. An ELISA assay involves preparing the antigen, coating the antigen onto the wells of a 96-well microtiter plate, adding an antibody or other binding agent conjugated to a detectable compound, such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase), to the wells, incubating for a period of time, and detecting the presence of the antigen. In some embodiments, the antibody or agent is not conjugated to a detectable compound, but instead, a second conjugated antibody that recognizes the first antibody or agent is added to the well. In some embodiments, instead of coating the wells with the antigen, the antibody or agent can be coated to the well, and a second antibody conjugated to a detectable compound can be added after the antigen has been added to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and other variations of ELISAs known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).

[0260] The binding affinity of an antibody or other drug to a target antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay, which involves incubating a labeled antigen (e.g., Fzd, LRP) or a fragment or variant thereof with the antibody of interest in the presence of increasing amounts of unlabeled antigen, followed by detecting antibody binding to the labeled antigen. Antibody affinity and binding off-rates can be determined from the data by scatterplot analysis. In some embodiments, BLI analysis is used to determine the binding on-rate and off-rate of an antibody or drug. BLI kinetic analysis involves analyzing the binding and dissociation of an antibody from a sensor with an antigen immobilized on its surface.

[0261] In certain embodiments, the present disclosure provides Wnt signaling agonists, including (but not limited to) hp4SD1-03, hp4SD1-03 AAQ, hp4SD1-03 AAA, and fragments and variants thereof. In certain embodiments, the Wnt signaling agonist has the structure shown in Figure 1 or the structure boxed in Figure 24 and / or comprises one or both of the sequences disclosed in Figure 9 or Figure 19A or Figure 19B, or any functional fragment or variant thereof, such as an antigen-binding fragment thereof, e.g., a VHH domain, a VL domain, a VH domain, or an Fc domain, or a variant of any of these functional domains, such as a variant having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any functional fragment of the antibody sequences disclosed herein. In some embodiments, the Wnt signaling agonist comprises a variant sequence having at least 90% identity (e.g., 95%, 98% or 100% identity) to the sequence disclosed in Figure 9 or Figure 19A or Figure 19B or set forth in SEQ ID NO: 1 and / or 2, SEQ ID NO: 1 and / or 7, SEQ ID NO: 1 and / or 450, SEQ ID NO: 1 and / or 451, SEQ ID NO: 1 and / or 452, SEQ ID NO: 1 and / or 453, or any functional fragment thereof, e.g., an antigen-binding fragment thereof. In some embodiments, the Wnt signaling agonist comprises a wild-type or variant sequence having at least 90% identity (e.g., 95%, 98% or 100% identity) to the light chain sequence disclosed in Figure 9 or Figure 19A or Figure 19B or set forth in SEQ ID NO: 1, and a wild-type or variant sequence having at least 90% identity (e.g., 95%, 98% or 100% identity) to the heavy chain sequence disclosed in Figure 9 or Figure 19A or Figure 19B or set forth in SEQ ID NO: 2, 7, 450, 451, 452 or 453.In some embodiments, the Wnt signaling agonist comprises two wild-type or variant sequences, each having at least 90% identity (e.g., 95%, 98% or 100% identity) to the light chain sequence disclosed in Figure 9 or Figure 19A or Figure 19B or set forth in SEQ ID NO: 1, and two wild-type or variant sequences, each having at least 90% identity (e.g., 95%, 98% or 100% identity) to the heavy chain sequence disclosed in Figure 9 or Figure 19A or Figure 19B or set forth in SEQ ID NO: 2, 7, 450, 451, 452 or 453, wherein the two heavy chains are linked to each other and each light chain is linked to a different heavy chain, e.g., by a disulfide bond. In certain embodiments, one or more amino acid residues are introduced into the parent molecule 4SD1-03_LALAPG to generate hp4SD1-03, including, but not limited to, hVHH3-H4 (Figure 2), hVHH3-H4 N29Q (Figure 3), hVHH3-H4 DDD substituted with DED (Figure 4), 4SD1 HC CDR1 N31S and CDR2 D62E (Figure 7), and / or the humanized 4SD1 framework depicted in Figure 8. In certain embodiments, VHH3 CDR3 D108 is maintained. In certain embodiments, VHH3 M51 is maintained. In certain embodiments, the present disclosure provides variants of hp4SD1-03 (or fragments or variants of fragments or chains thereof) comprising one or more amino acid substitutions. The variants may optionally comprise an amino acid substitution within the DDDY (SEQ ID NO: 418) motif of hVHH3-H4 at positions 101-104 (corresponding to DEDY (SEQ ID NO: 410) of the VHH domain of hp4SD1-03 at positions 101-104), where optionally the D at position 102 is replaced by another amino acid, e.g., E, S, A, or T, or where optionally DEDY (SEQ ID NO: 410) is modified to DEEY (SEQ ID NO: 411), DESY (SEQ ID NO: 412), DEAY (SEQ ID NO: 413), DETY (SEQ ID NO: 414), ESEY (SEQ ID NO: 415), ESTY (SEQ ID NO: 416), or ESSY (SEQ ID NO: 417) (Figure 14).The sequence of hp4SD1-03 is shown below.

[0262] Light chain [ka]

[0263] heavy chain [ka]

[0264] The VHH domain of hp4SD1-03 has the following sequence: [ka] and the CDRs are underlined and in bold.

[0265] GGGGS (SEQ ID NO: 101) is the linker sequence between the VHH and VL domains.

[0266] The VL domain of hp4SD1-03 has the following sequence: [ka] and the CDRs are underlined and in bold.

[0267] The VH domain of hp4SD1-03 has the following sequence: [ka] and the CDRs are underlined and in bold.

[0268] The Fc domain of hp4SD1-03 has the following sequence: [ka] It has.

[0269] In particular, some Fc variants of hp4SD1-03 are further encompassed, which contain the same light chain as hp4SD1-03 (i.e., SEQ ID NO: 1) and a heavy chain that differs in Fc region sequence from that of hp4SD1-03 (hp4SD1 HC contains an Fc region with a LALAPG substitution (SEQ ID NO: 81)).

[0270] For example, a LALAPG substitution can be introduced into another Fc region reference sequence to give SEQ ID NOs:82-84.

[0271] In another example, "hp4SD1-03 AAQ" comprises a light chain of SEQ ID NO: 1 and a heavy chain that is similar to the heavy chain of hp4SD1-03 but that comprises an Fc region (e.g., an Fc region of SEQ ID NOs: 471-474) that contains an IAHAHQ substitution (also referred to herein as an AAQ substitution) in addition to the LALAPG substitution. Such a heavy chain may comprise, for example, the amino acid sequence of SEQ ID NO: 450.

[0272] In another example, "hp4SD1-03 AAA" comprises a light chain of SEQ ID NO: 1 and a heavy chain similar to the heavy chain of hp4SD1-03, but comprising an Fc region (e.g., an Fc region of SEQ ID NOs: 476-479) that contains an IAHAHA substitution (also referred to herein as an AAA substitution) in addition to the LALAPG substitution. Such a heavy chain may comprise, for example, the amino acid sequence of SEQ ID NO: 451.

[0273] Other non-limiting examples of Fc variants of hp4SD1-03 include: (I) a bispecific antibody comprising a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 7 comprising an Fc region comprising a LALA substitution (such an Fc region sequence may be, for example, SEQ ID NOs: 86-89); (II) a bispecific antibody comprising a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 8 comprising an Fc region comprising an IAHAHQ substitution but not a LALAPG substitution (such an Fc region sequence may be, for example, SEQ ID NOs: 91-94); (III) a bispecific antibody comprising a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 8 comprising an Fc region comprising an IAHAHA substitution but not a LALAPG substitution (such an Fc region sequence may be, for example, SEQ ID NOs: 91-94); Such Fc region sequences include (IV) bispecific antibodies comprising a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 452 comprising an Fc region that, in addition to the LALA substitution, includes an IAHAHQ substitution (such Fc region sequences may be, for example, SEQ ID NOs: 481 to 484); and (V) bispecific antibodies comprising a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 453 comprising an Fc region that, in addition to the LALA substitution, includes an IAHAHA substitution (such Fc region sequences may be, for example, SEQ ID NOs: 486 to 489).

[0274] Further non-limiting Fc variants of hp4SD1-03 include: (I) hp4SD1-03 variants in which the Fc region has been replaced with an Fc region containing one or more amino acid substitutions at positions selected from 234, 235, 236, 237, 265, 297, and 329 according to EU numbering; (II) hp4SD1-03 variants in which the Fc region has been replaced with an Fc region containing D265A and P329A substitutions (also referred to herein as "DAPA") according to EU numbering; and (III) hp4SD1-03 variants in which the Fc region has been replaced with an Fc region containing E (IV) hp4SD1-03 variants in which the Fc region has been replaced with an Fc region comprising an N297A substitution according to EU numbering, (IV) hp4SD1-03 variants in which the Fc region has been replaced with an Fc region comprising M252Y, S254T, and T256E (also referred to herein as "YTE") according to EU numbering, and (V) hp4SD1-03 variants in which the Fc region has been replaced with an Fc region comprising M428L and N434S (also referred to herein as "LS") according to EU numbering. Such substitutions may be made in any Fc region sequence, including, but not limited to, the Fc region reference sequences of SEQ ID NOs: 71-74.

[0275] Also specifically encompassed are variants of hp4SD1-03. In the variants, one or more of the anti-Fzd4 VH and / or VL and / or anti-LRP5 / 6 VH sequences are replaced by any of the corresponding variants contained in the attached Tables B, C and E; one or more of the anti-Fzd4 VH CDR and / or VL CDR and / or anti-LRP5 / 6 VH CDR sequences are replaced by any of the corresponding variants contained in the attached Tables A, C and D or any of the corresponding variants contained in the anti-Fzd4 VH and / or VL and / or anti-LRP5 / 6 VH sequences contained in the attached Tables B, C and E; one or more of the anti-Fzd4 VH FR and / or VL FR and / or anti-LRP5 / 6 VH FR sequences are replaced by any of the corresponding variants contained in the attached Tables A, C and D or any of the corresponding variants contained in the anti-Fzd4 VH and / or VL and / or anti-LRP5 / 6 VH sequences contained in the attached Tables B, C and E; and a linker (anti-LRP5 / 6 wherein the linker between the VH and anti-Fzd4 VL) sequence is replaced by any of the corresponding variants contained in the attached Table F; one or more of the CH1, hinge, CH2, CH3 and / or CL sequences are replaced by any of the corresponding variants contained in the attached Table G; one or more of the Fc region sequences are replaced by any of the corresponding variants contained in the attached Table H; and / or one or more of the heavy chain (HC) and / or light chain (LC) sequences are replaced by any of the corresponding variants contained in the attached Tables I-J. In further embodiments, the present disclosure provides nucleic acid sequences encoding a Wnt agonist molecule or polypeptide component thereof described herein, e.g., encoding the polypeptide of SEQ ID NO: 1 and / or the polypeptide of SEQ ID NO: 2, 7, 8, 9, 450, 451, 452, or 453, or a variant or fragment of any polypeptide. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., a modified mRNA. In certain embodiments, the polynucleotide is a modified mRNA that further comprises a 5' cap sequence and / or a 3' tailing sequence, eg, a polyA tail.

[0276] In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to the coding sequence. In further embodiments, the polynucleotide is present in an expression vector, e.g., a viral vector, comprising a polynucleotide comprising a nucleic acid sequence encoding a Wnt agonist polypeptide sequence described herein, or a variant or fragment thereof.

[0277] The present disclosure further contemplates a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a Wnt agonist polypeptide sequence or a variant or fragment thereof. In certain embodiments, the cell comprises an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid sequence encoding a Wnt agonist polypeptide. In certain embodiments, the host cell is a mammalian cell, an insect cell, or a yeast cell. Various cells that can be used to express and produce mammalian polypeptides are known in the art. In certain embodiments, the cell is a xenogeneic cell, an allogeneic cell, or an autologous cell obtained from the subject to be treated. In certain embodiments, the cell is a stem cell, such as an adipose-derived stem cell or a hematopoietic stem cell.

[0278] The present disclosure also provides methods for producing the Wnt agonists or polypeptide sequences thereof disclosed herein. The methods include culturing cells (e.g., host cells) containing one or more expression vectors encoding Wnt agonist polypeptides under conditions suitable for expressing the Wnt agonist polypeptides. In certain embodiments, the polynucleotide encoding the heavy chain polypeptide is present in a first expression vector and the polynucleotide encoding the light chain polypeptide is present in a second expression vector. In certain embodiments, the polynucleotide encoding the heavy chain polypeptide and the polynucleotide encoding the light chain polypeptide are present in the same expression vector. In certain embodiments, the Wnt agonist or polypeptide sequence thereof is secreted and / or purified from the host cell after expression.

[0279] III. Pharmaceutical Compositions Also disclosed are pharmaceutical compositions comprising a Wnt agonist molecule described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients.

[0280] In further embodiments, pharmaceutical compositions are also disclosed, comprising a polynucleotide comprising a nucleic acid sequence encoding a Wnt agonist molecule described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., a modified mRNA. In certain embodiments, the polynucleotide is a modified mRNA further comprising a 5' cap sequence and / or a 3' tailing sequence, e.g., a polyA tail. In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to a coding sequence.

[0281] In further embodiments, pharmaceutical compositions are also disclosed that include an expression vector, e.g., a viral vector, comprising a polynucleotide that includes a nucleic acid sequence encoding a Wnt agonist molecule described herein, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the nucleic acid sequence encoding the Wnt antagonist molecule and the nucleic acid sequence encoding the Wnt agonist are in the same polynucleotide, e.g., an expression cassette.

[0282] The present disclosure further contemplates a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a Wnt antagonist / agonist molecule, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid sequence encoding a Wnt agonist. In certain embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In certain embodiments, the cell is a stem cell, for example, an adipose-derived stem cell or a hematopoietic stem cell.

[0283] The subject molecules, alone or in combination, can be combined with generally safe, non-toxic, and pharmaceutically acceptable carriers, diluents, excipients, and reagents useful for preparing desired formulations, including excipients acceptable for use in mammals, e.g., humans or primates. Such excipients can be solid, liquid, semisolid, or, in the case of aerosol compositions, gaseous. Examples of such carriers, diluents, and excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulation. The solutions or suspensions used in the formulation may contain sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents, antibacterial compounds such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating compounds such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetates, citrates, and phosphates, surfactants to prevent aggregation and / or absorption of the substance, such as Tween 20®, and compounds for adjusting osmotic pressure, such as sodium chloride or glucose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical compositions are sterile.

[0284] Pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). In some cases, the composition should be sterile and fluid so that it can be drawn into a syringe and delivered to a subject from a syringe. In certain embodiments, the composition is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal composition can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0285] Sterile solutions can be prepared by incorporating the Wnt antagonist / agonist antibody or its antigen-binding fragment (or encoding polynucleotide or cells containing it) in the required amount in an appropriate solvent containing one or a combination of the ingredients listed above, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0286] In one embodiment, the pharmaceutical compositions, including controlled-release formulations, including implants and microencapsulated delivery systems, are prepared using carriers that will protect the antibody or antigen-binding fragment thereof from rapid elimination from the body. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0287] For ease of administration and uniformity of dosage, it may be advantageous to formulate the pharmaceutical composition in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unitary doses for the subject to be treated, each unit containing a predetermined amount of active antibody or antigen-binding fragment thereof calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The specifications for the dosage unit form can be determined by, or will depend directly on, the inherent characteristics of the antibody or antigen-binding fragment thereof and the particular therapeutic effect to be achieved, as well as the limitations inherent in the technical field of formulating such active antibody or antigen-binding fragment thereof to treat individuals.

[0288] The pharmaceutical compositions can be included in a container, pack, or dispenser, eg, a syringe, eg, a pre-filled syringe, together with instructions for administration.

[0289] The pharmaceutical compositions of the present disclosure include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compounds that, upon administration to a mammal, are capable of providing (directly or indirectly) a biologically active antibody or antigen-binding fragment thereof.

[0290] The present disclosure includes pharmaceutically acceptable salts of the Wnt agonist molecules described herein. The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound of the present disclosure, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects to the parent compound. Various pharmaceutically acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its more recent revisions), "Encyclopedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66:2 (1977). Also, for a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.

[0291] Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base-addition salts of the acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms may differ somewhat from the respective salt forms in certain physical properties, such as solubility in polar solvents, but otherwise, for purposes of this disclosure, the salts are equivalent to the respective free acids.

[0292] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a Wnt agonist molecule or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable carrier, diluent, and / or excipient, such as saline, phosphate-buffered saline, phosphate, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, and sugars include octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, and polyethylene glycol. Preferably, the formulation is stable at 4°C for at least 6 months.

[0293] In some embodiments, the pharmaceutical compositions provided herein comprise a buffer, such as phosphate-buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those of skill in the art, such as those described in Good et al. (1966) Biochemistry 5:467. The pH of the buffer can range from 6.5 to 7.75, preferably from 7 to 7.5, and most preferably from 7.2 to 7.4.

[0294] IV.How to use The present disclosure also provides methods for using Wnt agonist molecules, e.g., to modulate the Wnt signaling pathway, e.g., to increase Wnt signaling, and the administration of Wnt agonist molecules in various therapeutic settings. Provided herein are methods of treatment using Wnt agonist molecules. In one embodiment, a Wnt agonist molecule is provided to a subject with a disease involving inappropriate or dysregulated Wnt signaling.

[0295] In certain embodiments, Wnt agonist molecules can be used to enhance the Wnt signaling pathway in tissues or cells. Stimulating the Wnt signaling pathway can include, for example, increasing Wnt signaling or enhancing Wnt signaling in tissues or cells. Thus, in some aspects, the present disclosure provides a method for stimulating the Wnt signaling pathway in cells, comprising contacting the tissue or cells with an effective amount of a Wnt agonist molecule disclosed herein or a pharmaceutically acceptable salt thereof, wherein the Wnt agonist molecule is a Wnt signaling pathway agonist. In some embodiments, the contacting occurs in vitro, ex vivo, or in vivo. In certain embodiments, the cells are cultured cells, and the contacting occurs in vitro. In certain embodiments, such methods can be useful for inducing differentiation of cells and / or tissues of interest. Thus, such methods are for preparing cells and / or tissues for transplantation (e.g., neural cells or tissues, which may be useful, for example, in treating neurological or neurodegenerative diseases), for differentiating stem cells (e.g., differentiating stem cells into neural progenitor cells or neural cells), and / or for preparing organoids (e.g., neural organoids).

[0296] Wnt agonist molecules can be used to treat various diseases, disorders, and conditions, such as retinopathies. In particular, activation of Wnt signaling is necessary for retinal angiogenesis during ocular vascular development. Genetic deficiency of norrin, Fzd4, Lrp5, or Tspan12 significantly retards not only vascular development in the superficial retina but also vascular invasion into the deeper retina. Furthermore, avascular areas resulting from premature angiogenesis lead to ischemia-induced neovascularization. Therefore, timely and controlled administration of Wnt agonists and / or antagonists will not only reverse the progression of retinopathy disease, but also lead to disease improvement. In certain embodiments, the Wnt agonist will be administered at either the early or late stage of the progression of retinopathy disease in a subject.

[0297] Retinal vascular diseases that can be treated include, but are not limited to, familial exudative vitreoretinopathy (FEVR), exudative vitreoretinopathy, Norrie's disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease.

[0298] In certain embodiments, retinal diseases or retinopathies are treated with Wnt signaling agonists including (but not limited to) hp4SD1-03, hp4SD1-03 AAQ, or hp4SD1-03 AAA, or Wnt signaling agonists comprising a fragment or variant of hp4SD1-03 or a polypeptide component thereof. In certain embodiments, the fragment or variant is a functional fragment or variant that retains Fzd and / or LRP5 / 6 binding activity and the ability to induce the Wnt signaling pathway in the context of a Wnt signaling agonist, e.g., in certain embodiments, at least 100%, at least 90%, at least 80%, at least 70%, or at least 60% of the Fzd and / or LRP5 / 6 binding activity and / or at least 100%, at least 90%, at least 80%, at least 70%, or at least 60% of the ability to induce the Wnt signaling pathway compared to the parent molecule. These may be, for example, hp4SD1-03, hp4SD1-03 AAQ or hp4SD1-03 AAA

[0299] In certain embodiments, the Wnt signaling agonist has the structural format depicted in Figure 1 and / or comprises one or both of the sequences disclosed in Figure 9 or Figure 19A or Figure 19B or any functional fragment or variant thereof, such as an antigen-binding fragment thereof, such as a VHH domain, a VL domain or a VH domain or an Fc domain or a variant of any of these functional domains, such as a variant having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any functional fragment of the antibody sequences disclosed herein. In some embodiments, the Wnt signaling agonist comprises a variant sequence having at least 90% identity (e.g., 95%, 98% or 100% identity) to the sequence disclosed in Figure 9 or Figure 19A or Figure 19B or set forth in SEQ ID NO:1 and / or 2, SEQ ID NO:1 and / or 7, SEQ ID NO:1 and / or 8, SEQ ID NO:1 and / or 9, SEQ ID NO:1 and / or 450, SEQ ID NO:1 and / or 451, SEQ ID NO:1 and / or 452 and / or SEQ ID NO:1 and / or 453 or any functional fragment thereof, e.g., an antigen-binding fragment thereof. In some embodiments, the Wnt signaling agonist comprises a wild-type or variant sequence having at least 90% identity (e.g., 95%, 98% or 100% identity) to the light chain sequence disclosed in Figure 9 or set forth in SEQ ID NO: 1, and a wild-type or variant sequence having at least 90% identity (e.g., 95%, 98% or 100% identity) to the heavy chain sequence disclosed in Figure 9 or Figure 19A or Figure 19B, or set forth in any one of SEQ ID NOs: 2, 7-9 and 450-453.In some embodiments, the Wnt signaling agonist comprises two wild-type or variant sequences, each having at least 90% identity (e.g., 95%, 98%, or 100% identity) to the light chain sequence disclosed in Figure 9 or Figure 19A or Figure 19B or set forth in SEQ ID NO: 1, and two wild-type or variant sequences, each having at least 90% identity (e.g., 95%, 98%, or 100% identity) to the heavy chain sequence disclosed in Figure 9 or Figure 19A or Figure 19B or set forth in any one of SEQ ID NOs: 2, 7-9, and 450-453, wherein the two heavy chains are linked to each other and each light chain is linked to a different heavy chain, e.g., by a disulfide bond. In certain embodiments, amino acid modifications can be introduced into the parent molecule 4SD1-03_LALPG to generate hp4SD1-03 or variants thereof. Such modifications include, for example, those of hVHH3-H4 (Figure 2), hVHH3-H4 N29Q, hVHH3-H4 DDD substituted with DED (Figure 4), hVHH3-H4 DDDY (SEQ ID NO: 418) (amino acids at positions 101-104) substituted with DEEY (SEQ ID NO: 411), DESY (SEQ ID NO: 412), DEAY (SEQ ID NO: 413), DETY (SEQ ID NO: 414), ESEY (SEQ ID NO: 415), ESTY (SEQ ID NO: 416), or ESSY (SEQ ID NO: 417) (Figure 14), 4SD1 HC CDR1 N31S and 4SD1 HC CDR2 D62E (Figure 7), and / or those of the humanized 4DS1 framework shown in Figure 8. In certain embodiments, CDR3 D108 is maintained. In certain embodiments, VHH3 M51 is maintained.

[0300] In certain embodiments, the methods of treatment disclosed herein result in one or more of a reduction in retinal vascular leakage, a reduction in retinal ischemia, or a decrease in the size of retinal neovascular lesions. In certain embodiments, the methods of treatment disclosed herein result in an improvement in one or more symptoms or pathology of any of the diseases or disorders disclosed herein.

[0301] The present invention also provides combination treatments with known treatments for FEVR and / or DR. For example, a Wnt agonist can be administered in combination with another treatment for retinopathy, including (but not limited to) an anti-VEGF agent, a VEGF inhibitor, or a VEGF antagonist, such as an anti-VEGF antibody. In some embodiments, an anti-Ang2 antibody will also be administered to a subject in combination with a Wnt agonist. Hypoxia-induced VEGF and Ang2 expression is an important clue to pathological neovascularization, and indeed, antagonistic Ang2 antibodies have been investigated for the treatment of patients with retinopathy (Gadkar et al., Invest Ophthalmol Vis Sci. 2015 Aug;56(9):5390-400). An anti-VEGF antibody or anti-Ang2 antibody can be administered sequentially or simultaneously with the molecule of the present invention. VEGF antagonists can include, but are not limited to, bevacizumab, ranibizumab, aflibercept, ramucirumab, and tanibirumab. Ang2 antagonists can also include, but are not limited to, nesbacumab, AMG780, and MEDI3617.

[0302] In further embodiments, the agonistic molecule may also incorporate a tissue-targeting moiety, such as an antibody or fragment thereof, that recognizes a retinal tissue-specific receptor or cell surface molecule.

[0303] Therapeutic agents (e.g., Wnt agonists) can be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease, which stabilizes or alleviates undesirable clinical symptoms in patients, is of particular interest. Such treatment is desirably administered before complete loss of function in affected tissues. The subject treatments will desirably be administered during the symptomatic phase of disease, and in some cases, after the symptomatic phase of disease. In some embodiments, the subject methods provide a therapeutic benefit, e.g., prevention of the onset of a disorder, arresting the progression of a disorder, reversing the progression of a disorder, etc. In some embodiments, the subject methods include detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measurements of therapeutic effect will be applicable to the particular disease being modified and will be aware of appropriate detection methods to use to measure the therapeutic effect.

[0304] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced in this specification and / or listed in the Application Data Sheets are incorporated herein by reference in their entirety.

[0305] Thus, although specific embodiments of the present disclosure have been described herein for purposes of illustration, it will be understood that various modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited except as by the appended claims.

[0306] The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific embodiments.

[0307] Example Standard methods in molecular biology are described in Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods are also provided in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc., New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycocompound and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0308] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described in Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation are also described. See, for example, Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; and Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391, which describe the production, purification, and fragmentation of polyclonal and monoclonal antibodies. Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra. Standard techniques are available for characterizing ligand / receptor interactions.See, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York.

[0309] Methods for flow cytometry, including fluorescence-activated cell sorting detection systems (FACS®), are available. See, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2 nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies for use, for example, as diagnostic reagents, are available. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0310] Standard methods for immune system histology are described, see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY.

[0311] For example, software packages and databases are available for determining antigenic fragments, leader sequences, protein folds, functional domains and glycosylation sites. For example, GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp., Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16: 741-742; Menne et al. (2000) Bioinformatics See Applications Note 16:741-742;Wren et al. (2002) Comput. Methods Programs Biomed. 68:177-181;von Heijne (1983) Eur. J. Biochem. 133:17-21;von Heijne (1986) Nucleic Acids Res. 14:4683-4690.

[0312] Example 1 Production and characterization of novel Wnt signaling agonists Humanized and optimized Wnt signaling agonists exhibiting enhanced activity were developed and characterized as described.

[0313] Protein production All recombinant proteins were produced in Expi293F™ cells (Thermo Fisher Scientific) by transient transfection. Proteins were first purified using a MabSelect SuRe column (often referred to herein as Protein A) and then further refined by size-exclusion chromatography (SEC) on a Superdex 200 Increase 10 / 300 GL (GE Healthcare Life Sciences) using 1x HBS buffer (20 mM HEPES pH 7.4, 150 mM NaCl) or a HiLoad 16 / 600 Superdex 200 using 2x HBS buffer. Proteins were then examined by SDS-polyacrylamide gel electrophoresis, and purity was estimated to be greater than 90%.

[0314] Super Top Flash (STF) Assay (Wnt Signaling Assay) Wnt signaling activity was measured using bEND3 cells containing a luciferase gene controlled by a Wnt-responsive promoter (Super Top Flash reporter assay, STF) as previously reported (Chen et al., STAR Protoc. 2020 Jun 3;1(1):100043). Briefly, cells were seeded at a density of 10,000 cells per well in 96-well plates 24 hours before treatment with 3 μM IWP2 to inhibit endogenous Wnt ligand production. Various concentrations of recombinant protein were then added to the cells overnight. Cells were lysed with Luciferase Cell Culture Lysis Reagent (Promega), and luciferase activity was measured using the Luciferase Assay System (Promega) using the vendor's recommended procedure.

[0315] Example 2 Oxygen-induced retinopathy (OIR) mouse study The oxygen-induced retinopathy (OIR) mouse model was performed as previously described (Smith et al., Oxygen-induced retinopathy in the mouse, Investigative Ophthalmology & Visual Science, January 1994, Vol. 35, 101-111) with some experimental modifications. Briefly, postnatal day 7 (P7) neonatal mice were exposed to a hyperoxic environment with an inhaled oxygen concentration of 75% for 5 days. As a result, between P7 and P12, newly formed capillaries in the central retina underwent vascular occlusion, forming an avascular (AV) retina. After the pups were transferred to a room at P12, the avascular central retina became hypoxic, and between P12 and P17, an intravitreal neovascular (NV) plexus formed at the junction between the vascular and avascular retina. For treatment, P12 pups were intravitreally injected with one of the following test articles: 60 μg / eye Eylea® (aflibercept, decoy VEGF receptor), 68 ng / eye 4SD1-03_LALAPG, 13.6 ng / eye hp4SD1-03, or vehicle control. To evaluate the efficacy of the test articles in reducing AV and NV areas, retinal whole-tissue preparations from P17 pups were stained with isolectin B4 and imaged. AV area, NV area, and total retinal area were manually scored and expressed as a percentage of either AV or NV area relative to total retinal area. To assess the dose-efficacy relationship, a second OIR study was performed as described above, injecting hp4SD1-03 at doses of 13.6 ng, 136 ng, or 1360 ng per eye or vehicle control (Figures 15A-C). To evaluate the effectiveness of test articles in reducing AV and NV areas, retinal whole-tissue specimens from pups at P17 were stained with isolectin B4 and imaged. AV area, NV area, and total retinal area were manually scored and expressed as the percentage of either AV or NV area relative to total retinal area. Statistical significance between each treatment group and the vehicle control group was determined using one-way ANOVA followed by Tukey's multiple comparison test.hp4SD1-03 was effective in reducing the NV vascular plexus area and AV area in the OIR model at concentrations of 40 nM to 4000 nM.

[0316] All tested doses of hp4SD1-03 showed significant reductions in both pathological NV area and retinal AV area, which were greater than those in the vehicle-treated group (Figures 15B and 15C). These results suggest that hp4SD1-03 not only potently inhibits pathological NV vascular plexus formation under retinal ischemic conditions, but also potentially promotes revascularization suitable for reperfusion of the injured retina.

[0317] Example 3 VEGF-induced retinal vascular leakage rabbit study A rabbit model of VEGF-induced retinal vascular leakage was performed with some experimental modifications as previously described (Arana, LA, et al. Fluorescein angiography, optical coherence tomography, and histopathologic findings in a VEGF-induced retinal vascular leakage rabbit model). An animal model of retinal angiogenesis was performed similarly to that described in Graefes Arch Clin Exp Ophthalmol 250, 1421-1428 (2012). On day 0, rabbit eyes were intravitreally administered with the test product, followed by 1.0 μg of recombinant human VEGF165 (R&D Systems) for retinal leakage. Fluorescein angiography (FA) was performed on both eyes on day 0, before VEGF administration, and on days 3 and 5. Initial retinal photography was performed 1-2 minutes after intravenous injection of sodium fluorescein (12 mg kg-1). Animals were photographed using a RetCam3 (Natus) imaging system. The test article and collected images were evaluated (masked) and retinal leakage was scored as follows: Score retinal leakage 0: major vessels appear very straight with limited tortuosity of small vessels; 1: increased tortuosity and / or dilated vessels are observed in major vessels; 2: leakage is observed between major vessels and no leakage is observed between small vessels; 3: leakage is observed between major and small vessels and 4: leakage is observed between major and small vessels with no small vessels visible.

[0318] Example 4 VHH3 humanization To humanize camelid antibody (VHH3) sequences, a human germline IgG heavy chain (HC) sequence database was searched. * 01 was found to be the closest match. Four variants (H1 to H4) were generated and purified. hVHH3-H4, which showed the best production yield, was selected as the humanized VHH3 (Figure 3).

[0319] Example 5 VHH3 refinement To eliminate the risk of potentially unstable tendencies toward deamidation, isomerization, and oxidation in the CDR regions of VHH3, VHH3 mutants were generated and tested as follows (Figures 3 to 5).

[0320] N29Q mutation in CDR1 (Figure 3) Three N29 mutants (N29Q, N29A, and N29S) of hVHH3-H4 were generated and purified. For example, N29Q VHH3 showed good yields for high-purity expression. The asparagine in CDR1 was replaced with glutamine to mitigate its potentially unstable tendency to deamidation.

[0321] D102E mutation in CDR3 (Figure 4) To eliminate the risk of "DD" motif isomerization in VHH3, five mutants (DED, SDE, EDE, ESD, SSD) in hVH3-H4 CDR3 were generated and purified. All mutants showed good STF response and expression. The DED mutant, which may have minimal structural changes, was selected. The aspartic acid in CDR3 was replaced with glutamic acid to reduce the potentially unstable tendency of isomerization.

[0322] D103 mutation in CDR3 (Figure 14) hp4SD1-03 contained various amino acid substitutions within amino acids 101 to 104 of the CDR3 of the VHH domain. These substitutions included the following: DEEY (SEQ ID NO: 411), DESY (SEQ ID NO: 412), DEAY (SEQ ID NO: 413), DETY (SEQ ID NO: 414), ESEY (SEQ ID NO: 415), ESTY (SEQ ID NO: 416), and ESSY (SEQ ID NO: 417). These mutants demonstrated good expression and stability. The magnitude of the STF response (Emax) was increased for all mutants tested. Some mutants (e.g., ESSY (SEQ ID NO: 417)) had decreased EC50 values.

[0323] M51 and M57 in VHH3 CDR2 remain unchanged (Figure 5) Three mutants at each methionine residue in VHH3 CDR2 (M51K, M51L, M51I, M57K, M57L, M57I) were generated as VHHs and purified to eliminate the risk of potentially unstable oxidation. All mutants showed a significant decrease in expression and were determined to maintain both methionine residues. Oxidation was tested by mass spectrometry and no oxidation was observed (data not shown).

[0324] Refinement of 4SD1 IgG LALAPG To eliminate the risk of potentially unstable tendencies towards deamidation, isomerization, and oxidation in the 4SD1 variable domain, HC mutants were generated and tested as follows: There were no potentially prone amino acids in the light chain CDR regions.

[0325] N31S mutation in 4SD1 HC_CDR1 (Figure 7) We generated and purified bispecific antibodies containing 4SD1 LC and one of three N31 mutants of 4SD1 HC (N31Q, N31A, and N31S). All mutants showed no reduction in activity in the STF assay compared to their parent forms. The N31S and N31A mutants showed improved activity in the STF assay, and N31S was selected as the optimal molecule.

[0326] D62E mutation in 4SD1 HC_CDR2 (Figure 7) Bispecific antibodies containing one of three D62 mutants (D62E, D62A, and D62S) of 4SD1 LC and 4SD1 HC were generated and purified. All mutants exhibited STF activity (e.g., increased magnitude of response) comparable to or improved over their parental forms. D62E was selected as the optimal molecule due to the amino acid similarity between D and E.

[0327] Do not change D108 in 4SD1 HC_CDR3 (Figure 7) We generated and purified bispecific antibodies containing 4SD1 LC and one of ten D108 mutants of 4SD1 HC (D108E, D108A, D108S, D108R, D108T, D108L, D108H, D108Y, D108Q, and D108K). All mutants showed significantly reduced potency of STF activity compared to the parent form. We determined that D108 should not be altered, and its isomerization tendency was assessed by mass spectrometry.

[0328] Humanization of the 4SD1 framework (Figure 8) The sequence of the 4SD1 antibody was derived from a synthetic human antibody library containing several mutations that mismatched the human germline sequence. Mutations in the framework were modified based on the human IgG germline sequence shown in Figure 8. hp4SD1-03, which incorporates these sequence modifications and is shown in Figure 9, showed improved STF activity relative to the parent 4SD1-03_LALAPG, i.e., a lower EC50 and an increased Emax.

[0329] Example 6 Biological activity of hp4SD1-03 Wnt signaling activity The final humanized and refined 4SD1-03_LALAPG, hp4SD1-03 (FIG. 9), exhibited approximately 10-fold increased potency (assessed based on a decrease in EC50) over its parent form, 4SD1-03_LALAPG, in the STF assay performed in Example 1 (one exemplary side-by-side dose-response comparison shown in FIG. 10A). More specifically, the mean EC50±SEM for hp4SD1-03 from seven independent experiments was 0.055±0.014 nM, and the mean EC50±SEM for 4SD1-03_LALAPG from ten independent experiments was 0.477±0.069 nM.

[0330] Similarly, when we performed a separate assay to measure Wnt / β-catenin signaling (methods detailed below), similar to the STF assay but using bEND3 cells that do not contain an exogenous reporter gene controlled by a Wnt-responsive promoter (i.e., using a less artificial setup), we observed an 11.5-fold decrease in EC50 for hp4SD1-03 relative to 4SD1-03_LALAPG without loss in response magnitude (Emax) (Figure 10B).

[0331] Materials and Methods Wnt / β-catenin signaling activity was measured in mouse brain microvascular endothelial cells (bEnd.3 cells) by quantifying the expression level of Axin2, a gene induced by active Wnt signaling and acting in a negative feedback loop (Yan, PNAS, 98(26):14973-14978, (2001); Lustig, Mol. Cell. Biol. 22(4): 1184-1193 (2002); Jho, Mol. Cell. Biol. 22(4): 1172-1183 (2002)).

[0332] Cells were seeded at a density of 500,000 cells per well in 6-well plates and starved in DMEM containing 0.5% BSA for 24 hours before treatment. 4SD1-03_LALAPG or hp4SD1-03 protein was then added to the cells for 24 hours. Starvation was performed to exclude the effects of nonspecific FCS signaling. Recombinant human Norrin (R&D Systems, Lot NSO0717041, 3014-NR-025) was used as a reaction control. Cells were lysed, and RNA was extracted with 350 μl of RLT buffer (Qiagen RNeasy 96 Kit, Catalog No. 74181, Lot 172036621) according to the manufacturer's protocol. cDNA was synthesized, and Axin2 cDNA was amplified according to the manufacturer's procedures (Applied Biosystems High Capacity cDNA Reverse Transcription Kit, catalog number 4368813, lot 01216654; Applied Biosystems TaqMan Universal PCR Master Mix, catalog number 4304437, lot 2208187; Axin2 primer: Mm00443610_m1 Applied Biosystems).

[0333] Dose-response curves (DRCs) were generated using GraphPad Prism 9.3.1. For normalization, the maximum response of recombinant Norrin was set to 100%.

[0334] Mouse retinal injury model As shown in Figure 11, treatment with the modified construct hp4SD1-03 compared to the parent construct 4SD1-03_LALAPG in a mouse model of retinal injury demonstrated better results with hp4SD1-03 compared to 4SD1-03_LALAPG.

[0335] Rabbit retinal vascular leakage model Treatment with the modified construct, hp4SD1-03, reduced vascular leakage in a VEGF-induced retinal vascular leakage rabbit model of retinal injury, as shown in Figure 12. Treatment with 2 μg / eye hp4SD1-03 reduced leakage by 44%, and treatment with 0.4 μg / eye hp4SD1-03 also significantly reduced vascular leakage.

[0336] Example 7 Western blot analysis - 4SD1-03_LALAPG Western blot analysis was performed as previously reported with minor modifications. Briefly, human retinal microvascular endothelial cells (HRMECs) were treated with 10 nM 4SD1-03_LALAPG and harvested for cell lysis 24 hours later. Primary antibodies used were anti-ZO-1 (Abcam; ab216880), anti-CLDN5 (Abcam; ab131259), and anti-β-actin (Sigma-Aldrich; A5441), followed by horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies for chemiluminescence signaling.

[0337] Accumulation of lymphocyte enhancer-binding factor 1 (Lef1) and Mfsd2a has been observed in blood-brain barrier (BBB) ​​vessels, which contain specialized endothelial cell (EC) tight junctions under the control of Wnt / β-catenin signaling (see, e.g., Wang Z et al. (2020) Sci Adv 2020;6:eaba7457; Wang Y et al. (2019) Elife 8; and Ben-Zvi A, et al. (2014) Nature 509:507-511). Expression of LEF1 and MFSD2A mRNA was significantly upregulated by 4SD1-03_LALAPG treatment in HRMECs (Figure 16A). Furthermore, expression of EC tight junction proteins CLDN5 and ZO-1 was upregulated in retinal vascular ECs after 4SD1-03_LALAPG treatment (Figure 16B). These results suggest that biologically engineered tetravalent antibodies targeting Fzd4 and LRP5 are active in vascular ECs and may enhance vascular integrity through upregulation of functional barrier proteins.

[0338] Western blot analysis - hp4SD1-03 Similarly, treatment of HRMECs with 10 nM hp4SD1-03 (detailed in Materials and Methods below) induced Wnt / β-catenin signaling, as measured by increased phosphorylation of Disheveled Segment Polarity Protein 3 (DVL3), a key component of Wnt signaling that is phosphorylated upon activation of the Wnt signaling pathway (Gonzalez-Sancho et al., Mol Cell Biol. 2004 Jun;24(11):4757-68.) (Figure 16C).

[0339] Materials and Methods HRMEC cells were seeded in 6-well plates at a density of 500,000 cells per well and starved in DMEM containing 0.5% BSA 24 hours before treatment. Cells were incubated with recombinant Norrin, R2M3_null control, or hp4SD1-03 protein for 1 hour. Recombinant human Norrin (R&D Systems, lot NSO0717041, 3014-NR-025) was used as a response control. Cells were solubilized in 300 μl of cell lysis buffer (Cell Signaling 9803S) containing Complete (Roche 11873580001) or Pefabloc SC (Roche 505648900) for 30 min at 4°C. The extract was centrifuged at 14,000 g for 15 min at 4°C, and the supernatant was digested with 3 μl of DNAse I (Qiagen 79254). Lysates were heated in SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and subjected to Western blot analysis using the indicated antibodies (NuPage 7% Tris Acetate Gel, Invitrogen EA03585BOX, iBlot® NC Transfer Stacks Invitrogen IB 23001, rabbit anti-Dvl3 antibody Cell Signaling 3218, and anti-rabbit IgG (H+L), HRP-conjugated Promega W4011, according to the manufacturer's protocol). Protein amounts were quantified by measuring band intensity using a Fuji LumiImager LAS 300 and AIDA software (Advanced Data Image Analyzer Version 4.15.025). The intensity ratio between the upper band (corresponding to phosphorylated DVL3) and the lower band (corresponding to unphosphorylated DVL3) was calculated.

[0340] Example 8 Binding kinetics by BLI We observed an increased binding affinity of hp4SD1-03 to the CRD of human Fzd4 relative to 4SD1-03_LALAPG, as measured by biolayer interferometry (BLI) (detailed in Materials and Methods below) (Figures 17A-17C). At all concentrations tested, the sensorgrams of human Fzd4 CRD using hp4SD1-03 showed slower dissociation than using 4SD1-03_LALAPG (Figure 17C). Kinetic analysis in the two graphs (Figures 17A-17B) also revealed that k off (k dis ) decrease in K of hp4SD1-03 D It is clear that this has had a significant effect on the reduction of the k off Decrease in K D This decrease may at least partially explain the decreased EC50 values ​​observed in the assays measuring STF and Wnt / β-catenin signaling described above.

[0341] Materials and Methods The kinetics of monovalent binding of 4SD1-03_LALAPG or hp4SD1-03 to the cysteine-rich domain (CRD) of human Fzd4 was measured by BLI using an OCTET® Red 96 (PALL ForteBio, Fremont, CA) instrument equipped with an AHC biosensor (Sartorius) at 30°C and 1000 rpm. 4SD1-03_LALAPG or hp4SD1-03 was diluted to 50 nM in running buffer (PBS, 0.05% Tween-20®, 0.5% BSA, pH 7.2) and captured by the AHC biosensor. Subsequently, wells containing the CRD of human Fzd4 at different concentrations (1000, 333.3, 111.1, 37, 12.4, 4.12, and 1.37 nM) in running buffer or wells containing only running buffer as a reference channel were immersed. Dissociation of the interaction was followed in running buffer. Monovalent K for each binder D was calculated by the OCTET® system software based on fitting to a 1:1 binding model.

[0342] Binding kinetics by SPR Relative to 4SD1-03_LALAPG, improved binding affinity was observed for hp4SD1-03 to the CRD of human Fzd4 as measured by surface plasmon resonance (SPR) (detailed in Materials and Methods below) (Figures 18A-18C). As shown in the sensorgrams (Figures 18A-18B) and kinetics values ​​(Figure 18C), hp4SD1-03 exhibited slower dissociation and improved affinity (decreased K) than 4SD1-03_LALAPG. D Again, without wishing to be bound by theory, k off Decrease in K D This decrease may at least partially explain the decreased EC50 values ​​observed in the assays measuring STF and Wnt / β-catenin signaling described above.

[0343] Materials and Methods Binding of hp4SD1-03 or 4SD1-03_LALAPG to human Fzd4 CRD (TPP-48723, PPB-51327) was measured by SPR using a Biacore® 8K instrument with HBS-EP+ (Biacore #BR-100668) as the running buffer. CM5 biosensor chips (Biacore #BR100530) were activated with a 50:50 solution of EDC / NHS (Amine Coupling Kit, Biacore #BR-100633) according to the manufacturer's recommendations. 10 μg / ml Protein AG in 10 mM acetic acid, pH 4.5, was immobilized on the surface, followed by inactivation with 1 M ethanolamine HCl.

[0344] hp4SD1-03 or 4SD1-03_LALAPG was captured at four concentrations (4, 2, 1, and 0.5 μg / ml in HBS-EP+) for 30 seconds at a flow rate of 10 μl / min. Serial dilutions of human Fzd CRD were prepared in HBS-EP+ (2.5 μM to 10.29 nM) and injected at 30 μl / min for 90 seconds to allow association, followed by 200 seconds to allow dissociation. The surface was regenerated by injecting 0.85% phosphoric acid for 30 seconds at a flow rate of 30 μl / min.

[0345] The interaction of the analyte with the sensor surface (flow cell 1) and the blank (HBS-EP+ or 0 nM analyte) was subtracted from the raw data. The sensorgrams were then fitted using a 1:1 binding model to obtain the rate constant (k a and k d ) and affinity (K D ) values ​​were provided. Results from different capture concentrations were averaged and the standard deviation was calculated in Excel to obtain the values ​​reported here. The highest concentration of analyte used was 2.5 μM, thus a K > 250 nM. D The value is the apparent K D is.

[0346] Example 9 Design of Fc mutants of hp4SD1-03 In an effort to generate variants of hp4SD1-03 that may have shorter in vivo circulation times, the Fc region of hp4SD1-03 (which already has L234A, L235A, and P329G (also referred to herein as LALAPG)) was modified to further contain the following amino acid substitutions: I253A, H310A, and H435Q (also referred to herein as IAHAHQ or AAQ) or I253A, H310A, and H435A (also referred to herein as IAHAHA or AAA). The hp4SD1-03 variant containing the additional IAHAHQ was designated hp4SD1-03 AAQ, and the hp4SD1-03 variant containing the additional IAHAHA was designated hp4SD1-03 AAA. The light chain of hp4SD1-03 was not altered. The amino acid sequences of the heavy and light chains of hp4SD1-03 AAQ and hp4SD1-03 AAA are shown in Figures 19A and 19B, respectively. Without wishing to be bound by theory, the in vivo circulation time (half-life, T 1 / 2 ) may provide reduced toxicity.

[0347] Production of hp4SD1-03 AAQ and hp4SD1-03 AAA Both hp4SD1-03 AAQ and hp4SD1-03 AAA were successfully purified using Protein A affinity resin (FIG. 20).

[0348] Materials and Methods Recombinant protein was produced in Expi293F™ cells (Thermo Fisher Scientific) by transient transfection. Protein was captured by protein A affinity chromatography, eluted under acidic conditions, and then refined and buffer-exchanged by Superdex 200 Increase 10 / 300 GL (Cytiva) size-exclusion chromatography (SEC) using HBS buffer. The purified protein was examined by SDS-polyacrylamide electrophoresis, based on which the protein was estimated to have a purity of >90%. The SEC chromatogram is shown in Figure 20.

[0349] Protein stability of hp4SD1-03 AAQ and hp4SD1-03 AAA For hp4SD1-03 AAQ and hp4SD1-03 AAA, no significant change in the first melting temperature (Tm1) relative to hp4SD1-03 was observed (FIG. 21).

[0350] Materials and Methods Melting curves were determined and melting temperatures (Tm1 and Tm2) were measured using an UNcle Instrument (UNchained Labs) that monitors intrinsic fluorescence. The protein concentration was 0.5 mg / mL in HBS, pH 7.4. The temperature was increased from 15°C to 95°C at a rate of 0.6°C / min. In the same experiment, the onset of aggregation (Tagg) was measured by static light scattering with increasing particle size. Each sample was analyzed in duplicate.

[0351] STF activity of hp4SD1-03 AAQ and hp4SD1-03 AAA Wnt signaling activity was measured by the STF assay described in Example 1. Exemplary results are shown in Figure 22. hp4SD1-03 AAQ showed a slightly reduced EC50, and hp4SD1-03 AAA showed an increased magnitude of response (Emax) relative to hp4SD1-03.

[0352] Binding of hp4SD1-03 AAQ and hp4SD1-03 AAA to FcRn Neither hp4SD1-03 AAQ nor hp4SD1-03 AAA showed any binding to FcRn (see Figure 23).

[0353] Materials and Methods Binding assays were performed by BLI on an Octet® Red 96 (PALL ForteBio, Fremont, CA) instrument equipped with an NTA biosensor at 25°C and 1000 rpm. His-tagged FcRn (Acro Biosystems) was diluted to 100 nM in running buffer (PBS, 0.05% Tween-20, 0.5% BSA, pH 7.2) and captured on an NTA biosensor (Sartorius) and subsequently immersed in wells containing 100 nM hp4SD1-03, hp4SD1-03 AAQ, and hp4SD1-03 AAA in running buffer or running buffer alone as a reference channel. Samples were assayed in duplicate.

[0354] Example 10 Pharmacokinetics (PK) Various pharmacokinetic parameters, including half-life, for hp4SD1-03, hp4SD1-03 AAQ, and hp4SD1-03 AAA will be determined.

[0355] hp4SD1-03, hp4SD1-03 AAQ, and hp4SD1-03 AAA will be suitably produced and purified, for example, as described above. Prior to administration, model animals (e.g., rabbits, e.g., New Zealand White rabbits or chinchilla rabbits, monkeys, e.g., cynomolgus monkeys, etc.) will be randomly assigned to different experimental groups designed based on the desired dose (e.g., 100 ng, 1 μg, 10 μg, 100 μg, 1 mg, and 10 mg, etc.), the desired route (e.g., intravitreal, topical via eye drops, intravenous, etc.), the type of sample to be collected, and the time point of sample collection. Blood samples will be collected for baseline measurements.

[0356] On the day of administration, hp4SD1-03, hp4SD1-03 AAQ, and hp4SD1-03 AAA will be reconstituted to the desired concentration (e.g., 300 nM, 3 μM, 30 μM, 300 μM, 3 mM, and 30 mM, etc.) and administered to the model animals at the desired dose by the desired route.

[0357] For analysis after intravenous administration, suitable volumes of blood (e.g., 0.2 mL in small animals, e.g., rabbits, and 3 mL in large animals, e.g., monkeys) should be collected serially, e.g., via the central ear artery, at various time points, e.g., 5 and 30 minutes and 1, 2, 4, 8, 24, 32, 48, 72, and 96 hours after administration. For analysis after intravitreal or topical administration, suitable volumes of blood should also be collected serially, and suitable tissues, e.g., ocular tissues (e.g., vitreous humor, aqueous humor, vitreous humor, retinal tissues, including e.g., disc, macula, and periretina), should also be collected at various preselected endpoints.

[0358] All blood samples should be collected into anticoagulant-free serum separator tubes, allowed to clot at ambient temperature, e.g., for at least 20 minutes, and centrifuged at 2-8°C. Serum should be collected within approximately 20 minutes of centrifugation and stored at -60°C to -80°C until PK analysis. All collected tissues should be weighed and stored at -80°C until analysis.

[0359] The hp4SD1-03, hp4SD1-03 AAQ, or hp4SD1-03 AAA concentration in serum samples will be analyzed, for example, by subjecting the serum samples to ELISA. The hp4SD1-03, hp4SD1-03 AAQ, or hp4SD1-03 AAA concentration in tissue samples will be analyzed, for example, by homogenizing the tissue samples and subjecting the homogenates to ELISA. Concentration will be plotted against time to visualize systemic and local pharmacokinetics. Systemic and local half-lives will be calculated. PK analysis can involve fitting to any suitable PK model (see, e.g., Gadkar et al., Invest Ophthalmol Vis Sci. 2015 Aug;56(9):5390-400; Le et al., J Pharmacol Exp Ther. 2015 Nov;355(2):288-96; Li et al., Exp Eye Res. 2012 Apr;97(1):154-9; and Park et al., Invest Ophthalmol Vis Sci. 2016 May 1;57(6):2612-7).

[0360] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced in this specification and / or listed in the Application Data Sheets are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, as appropriate, to utilize concepts from the various patents, applications, and publications to provide further embodiments. These and other changes can be made to the embodiments in light of the above detailed description.

[0361] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

[0362] Exemplary Implementations Some exemplary embodiments of the present disclosure are described herein below.

[0363] Embodiment P1. 1. A modified WNT signaling agonist comprising: The WNT signaling agonist comprises: a) two light chains, each comprising a polypeptide sequence having at least 95% identity to SEQ ID NO:1; and b) two heavy chains, each comprising a polypeptide sequence having at least 95% identity to SEQ ID NO:2; wherein the two heavy chains are bound to each other and the two light chains are each bound to a different heavy chain of a WNT signaling agonist; Engineered WNT signaling agonists.

[0364] Embodiment P2. The modified WNT signaling agonist of embodiment P1, wherein each of the two light chains comprises a VHH domain comprising one or more of the following amino acid residues: VHH3-H4 (or hVHH3-H4) DDD substituted with N29Q and / or DED.

[0365] Embodiment P3. The modified WNT signaling agonist of embodiment P1, wherein each of the two heavy chains comprises one or more of the following amino acid residues: CDR1 S31, CDR2 E62 and / or CDR3 D108.

[0366] Embodiment P4. comprising a sequence encoding the light chain and / or the heavy chain of the modified WNT signaling agonist according to any one of embodiments P1 to P3. Nucleic acid.

[0367] Embodiment P5. The nucleic acid of embodiment P4, wherein the nucleic acid is an mRNA, e.g., a modified mRNA.

[0368] Embodiment P6. comprising a nucleic acid according to embodiment P4 or embodiment P5, vector.

[0369] Embodiment P7. The vector of embodiment P6, wherein the vector is an expression vector comprising a promoter operably linked to the nucleic acid of embodiment P4 or embodiment P5.

[0370] Embodiment P8. comprising a nucleic acid according to embodiment P4 or embodiment P5 or a vector according to embodiment P6 or embodiment P7, host cell.

[0371] Embodiment P9. a pharmaceutically acceptable diluent, excipient or carrier, and a) a modified WNT signaling agonist according to any one of embodiments P1 to P3, b) a nucleic acid according to embodiment P4 or P5, and / or c) a vector according to embodiment P6 or P7, Pharmaceutical compositions.

[0372] Embodiment P10. 1. A method of treating retinopathy in a subject, comprising: administering to the subject a) a modified WNT signaling agonist according to any one of embodiments P1 to P3, b) a nucleic acid according to embodiment P4 or P5, c) a vector according to embodiment P6 or P7, and / or d) a pharmaceutical composition according to embodiment P9. method.

[0373] Embodiment P11. The method of embodiment P10, wherein the retinopathy is a retinal vascular disease.

[0374] Embodiment P12. The method of embodiment P11, wherein the retinal vascular disease is caused by inhibition of vascular development.

[0375] Embodiment P13. The method of embodiment P10, wherein the retinopathy is caused by excessive neovascularization.

[0376] Embodiment P14. The method of any one of embodiments P10 to P13, wherein the retinal vascular disease is optionally selected from the group consisting of familial exudative vitreoretinopathy (FEVR), exudative vitreoretinopathy, Norrie's disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease.

[0377] Embodiment P15. The method of any one of embodiments P10 to P14, wherein the modified WNT signaling agonist is administered to one or more eyes of the subject.

[0378] Embodiment P16. The method of embodiment P15, wherein the modified WNT signaling agonist is administered intravitreally.

[0379] Embodiment A1. (A) (i) (i-1) optionally a CDRH1 amino acid sequence included in any one of SEQ ID NOs: 181 to 188 according to Kabat, (i-2) an amino acid sequence included in any one of SEQ ID NOs: 121 to 124, and / or (i-3) an amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 120 (wherein X1 is any amino acid, optionally S, A, Q, or N); (ii) (ii-1) optionally a CDRH2 amino acid sequence included in any one of SEQ ID NOs: 181 to 188 according to Kabat, (ii-2) an amino acid sequence included in any one of SEQ ID NOs: 141 to 144, and / or (ii-3) an amino acid sequence of SEQ ID NO: and (iii) a VH comprising a CDRH2 comprising an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 140 (wherein X1 is any amino acid, optionally E, A, S, or D); and (iii) a VH comprising a CDRH3 comprising (iii-1) optionally an amino acid sequence of SEQ ID NO: 181 to 188 according to Kabat, (iii-2) the amino acid sequence of SEQ ID NO: 161, and / or (iii-3) an amino acid sequence of SEQ ID NO: 160 (wherein X1 is any amino acid, optionally D or any amino acid that is not S, E, A, R, T, L, H, Y, Q, or K); (B) (i) (i-1) optionally a CDRL1 comprising an amino acid sequence comprising or consisting of the CDRL1 amino acid sequence contained in SEQ ID NO: 281 according to Kabat and / or (i-2) an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 221; (ii) (ii-1) optionally a CDRL2 comprising an amino acid sequence comprising or consisting of the CDRL2 amino acid sequence contained in SEQ ID NO: 281 according to Kabat and / or (ii-2) an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 241; and (iii) (iii-1) optionally a CDRL3 comprising an amino acid sequence comprising or consisting of the CDRL3 amino acid sequence contained in SEQ ID NO: 281 according to Kabat and / or (iii-2) an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 261. Including, Anti-Fzd4 antibodies or antibody fragments.

[0380] Embodiment A2. (A) The VH comprises (i) (i-1) optionally an FRH1 amino acid sequence comprising or consisting of an FRH1 amino acid sequence included in any one of SEQ ID NOS: 181 to 188 according to Kabat, (i-2) an amino acid sequence of SEQ ID NOS: 111 or 112, and / or (i-3) an amino acid sequence of SEQ ID NOS: 110 (wherein X1 is any amino acid, optionally Q or K); (ii) (ii-1) optionally an FRH2 amino acid sequence comprising or consisting of an FRH2 amino acid sequence included in any one of SEQ ID NOS: 181 to 188 according to Kabat, and / or (ii-2) an FRH2 amino acid sequence comprising or consisting of an amino acid sequence of SEQ ID NOS: 130 or 131; (iii) (iii-1) optionally an FRH2 amino acid sequence comprising or consisting of an FRH2 amino acid sequence included in any one of SEQ ID NOS: 181 to 188 according to Kabat; (iii-2) the amino acid sequence of SEQ ID NO: 151 or 152 and / or (iii-3) an FRH3 comprising an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 150 (wherein X1 is any amino acid, optionally N or D, X2 is any amino acid, optionally R or K, and X3 is any amino acid, optionally E or T); and / or (iv) (iv-1) optionally an FRH4 amino acid sequence comprising any one of SEQ ID NOs: 181 to 188 according to Kabat and / or (iv-2) an FRH4 comprising an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 170 or 171; (B) The anti-Fzd4 antibody or antibody fragment of embodiment A1, wherein the VL comprises: (i) (i-1) optionally an FRL1 comprising the FRL1 amino acid sequence contained in SEQ ID NO: 281 according to Kabat and / or (i-2) an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 221; (ii) (ii-1) optionally an FRL2 amino acid sequence contained in SEQ ID NO: 281 according to Kabat and / or (ii-2) an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 231; (iii) (iii-1) optionally an FRL3 amino acid sequence contained in SEQ ID NO: 281 according to Kabat and / or (iii-2) an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 251; (iv) (iv-1) optionally an FRL4 amino acid sequence contained in SEQ ID NO: 281 according to Kabat and / or (iv-2) an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 271.

[0381] Embodiment A3. (A) In the VH, (I) (I-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 181 according to Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 121, 141, and 161; and (I-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 181; (II) (II-1) CDRH1, CDRH2, and CDRH3 (i) optionally comprise the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 182 according to Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 122, 142, and 162; and (II-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 182; (III) (III-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 183 according to Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 124, 142, and 162, and (III-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 183; (IV) (IV-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 184 according to Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 121, 142, and 162; and (IV-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 184; (V) (V-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 185 according to Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 123, 142, and 162; and (V-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 185; (VI) (VI-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 186 according to Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 122, 144, and 162; and (VI-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 186; (VII) (VII-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 187 according to Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 122, 141, and 162; and (VII-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 187; (VIII) (VIII-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 188 according to Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 122, 143, and 162; and (VIII-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 188; (B) An anti-Fzd4 antibody or antibody fragment according to embodiment A1 or A2, wherein in the VL, (1) CDRL1, CDRL2 and CDRL3 (i) optionally comprise the CDRL1 amino acid sequence, CDRL2 amino acid sequence and CDRL3 amino acid sequence contained in SEQ ID NO: 281 according to Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 221, 241 and 261, and (2) optionally, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 281.

[0382] Embodiment A4. An anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A3, wherein (A) the VH comprises an amino acid sequence comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 181 to 188 and / or (B) the VL comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 281.

[0383] Embodiment A5. An anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A4, wherein (A) the VH comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 181, and (B) the VL comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 281.

[0384] Embodiment A6. (i) an IgG, optionally an IgG1, IgG2, IgG3 or IgG4, IgA, IgE, IgD or IgM, comprising said VH and said VL; (ii) a multispecific antibody or antibody fragment (optionally comprising any one of Figures 24 to 35, and further optionally comprising the structure shown in Figure 24) comprising a first antigen-binding region comprising said VH and said VL and a second antigen-binding site specific for a second antigen (wherein optionally the second antigen is LRP5 and / or LRP6); and / or (iii) an antigen-binding fragment (Fab), a Fab' fragment, a F(ab')2, a single-chain Fab (scFab), a variable fragment (Fv), a single-chain Fv (scFv), a diabody, or a triabody comprising the VH and the VL; The anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A5, which is, comprises or is comprised in:

[0385] Embodiment A7. the below described: (a) an immunoglobulin heavy chain constant domain 1 (CH1) or a variant thereof, optionally wherein the CH1 or variant thereof is (a-1) that of IgG, IgA, IgE, IgD, IgM, optionally that of IgG1, IgG4, IgG2, or IgG3, further optionally that of a human; and / or (a-2) that comprises or consists of the amino acid sequence of SEQ ID NO: 21 or 22, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto; (b) an immunoglobulin hinge or variant thereof, optionally wherein the hinge or variant thereof is (b-1) that of IgG, IgA, IgE, IgD, IgM, optionally that of IgG1, IgG4, IgG2 or IgG3, further optionally that of a human; and / or (b-2) that comprises or consists of the amino acid sequence of SEQ ID NO: 31 or is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto; (c) an immunoglobulin heavy chain constant domain 2 (CH2) or a variant thereof, optionally wherein the CH2 or variant thereof is (c-1) that of IgG, IgA, IgE, IgD, or IgM, optionally that of IgG1, IgG4, IgG2, or IgG3, and further optionally that of a human; and / or (c-2) that comprises or consists of the amino acid sequence of any of SEQ ID NOs: 41 to 46, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto; (d) an immunoglobulin heavy chain constant domain 3 (CH3) or a variant thereof, optionally wherein the CH3 or variant thereof is (d-1) that of IgG, IgA, IgE, IgD, or IgM, optionally that of IgG1, IgG4, IgG2, or IgG3, and further optionally that of a human; and / or (d-2) that comprises or consists of the amino acid sequence of any of SEQ ID NOs: 51 to 54, 61 to 64, and 66 to 69 (optionally excluding the C-terminal K), or that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto; and / or (e) an immunoglobulin light chain constant domain (CL) or variant thereof, optionally wherein the CL or variant thereof is (e-1) a kappa light chain constant domain (CLκ), optionally of a human CLκ, optionally comprising or consisting of the amino acid sequence of SEQ ID NO: 11, or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least or (e-2) a lambda light chain constant domain (CLλ), optionally of human CLλ, optionally comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. An anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A6, comprising one or more of the following: wherein optionally the anti-Fzd4 antibody or antibody fragment comprises the CH2 and CH3 sequences of SEQ ID NOs: 42 and 51, respectively, SEQ ID NOs: 43 and 51, respectively, SEQ ID NOs: 45 and 61, respectively, SEQ ID NOs: 45 and 66, respectively, SEQ ID NOs: 46 and 61, respectively, or SEQ ID NOs: 46 and 66, respectively.

[0386] Embodiment A8. and a fragment crystallizable (Fc) region, wherein optionally the Fc region comprises: (I) IgG, IgA, IgE, IgD, IgM, optionally IgG1, IgG4, IgG2, or IgG3, and optionally human or a variant thereof; (II) (i) IgG1 or a variant thereof, optionally containing any of the following amino acid substitutions according to EU numbering: N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236 deletion, P238A, A327Q, A327G, P329A, P329G, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V3 05I, P396L, S239D, I332E, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y, M428L, N434S, H310A, L328F, M252Y, I253A, S254T, T256E, H435Q, H435A (ii) IgG2 or a variant thereof, optionally comprising one or more of the following amino acid substitutions according to EU numbering: P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and / or any combination thereof; (iii) IgG3 or a variant thereof, optionally comprising one or more of the following amino acid substitutions according to EU numbering: P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and / or any combination thereof. (iv) IgG4 or a variant thereof, optionally comprising the amino acid substitution E235Y according to EU numbering, or (iv) IgG4 or a variant thereof, optionally comprising one or more of the following amino acid substitutions according to EU numbering: E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q and / or any combination thereof; (III) one or more amino acid substitutions that reduce an Fc effector function, optionally wherein the one or more amino acid substitutions are at a position selected from the group consisting of 234, 235, 236, 237, 265, 297, and 329 according to EU numbering; (IV) at least the following amino acid substitutions according to EU numbering: (ii-1) L234A, L235A, and P329G, (ii-2) L234A and L235A, (ii-3) I253A, H310A, and H435Q, (ii-4) I253A, H310A, and H435A, (ii-5) D265A and P329A, (ii-6) M252Y, S254T, and T256E, and / or (ii-7) M428L and N434S, optionally with respect to a human IgG1 Fc region and / or any of SEQ ID NOs: 71 to 74 (optionally excluding the C-terminal K); and / or (V) An anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A7, comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 71 to 74, 81 to 84, 86 to 89, 91 to 94, 96 to 99, 471 to 474, 476 to 479, 481 to 484 and 486 to 489 (optionally excluding the C-terminal K), or comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0387] Embodiment A9. A nucleic acid or a combination of nucleic acids encoding the anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A8, wherein optionally the nucleic acid is DNA, cDNA, RNA, mRNA, modified mRNA or a DNA / RNA hybrid. A nucleic acid or a combination of nucleic acids.

[0388] Embodiment A10. A nucleic acid or combination of nucleic acids according to embodiment A9, comprising (a) a first nucleic acid encoding a first polypeptide comprising said VH, and (b) a second nucleic acid encoding a second polypeptide comprising said VL.

[0389] Embodiment A11. A vector or a combination of vectors comprising a nucleic acid or a combination of nucleic acids according to embodiment A9 or A10, wherein optionally (i) the vector comprises one or more promoters operably linked to the nucleic acid; (ii) the vector is an expression vector; and / or (iii) the vector comprises a plasmid, a viral vector, optionally an adeno-associated virus, an adenovirus, a lentivirus, or a retrovirus, a lipid-based vector, a self-replicating RNA vector, a virus-like particle, a polymer-based vector, and / or a nanoparticle, optionally a lipid-based nanoparticle. A vector or combination of vectors.

[0390] Embodiment A12. A vector or combination of vectors described in embodiment A11, encoding an anti-Fzd4 antibody or antibody fragment described in any one of embodiments A1 to A8, comprising (a) a first vector comprising a first nucleic acid encoding a first polypeptide comprising the VH, and (b) a second vector comprising a second nucleic acid encoding a second polypeptide comprising the VL.

[0391] Embodiment A13. A host cell comprising (A) an anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A8, (B) a nucleic acid or combination of nucleic acids according to embodiment A9 or A10, and / or (C) a vector or combination of vectors according to embodiment A11 or A12, wherein optionally the host cell is (i) a mammal, optionally a human, a non-human primate, a monkey, a rabbit, a rodent, a hamster, a rat or a mouse, or (ii) a non-mammal, optionally a plant, a bacterium, a fungus, a yeast, a protozoan or an insect, wherein optionally the host cell is (i) a human embryonic kidney (HEK) cell, optionally an HEK293 cell or a variant thereof, further optionally an Expi293F™ cell, (ii) a CHO (Chinese Hamster Ovary) cell, (iii) an immune cell or (iv) a hybridoma. host cell.

[0392] Embodiment A14. comprising two or more host cells of embodiment A13, Cell population.

[0393] Embodiment A15. (I) A method for producing a cell population comprising: (A) an anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A8; (B) a nucleic acid or a combination of nucleic acids according to embodiment A9 or A10; (C) a vector or a combination of vectors according to embodiment A11 or A12; and / or (D) a host cell according to embodiment A13 or a cell population according to embodiment A14; and (II) a pharmaceutically acceptable carrier and / or excipient. Pharmaceutical compositions.

[0394] Embodiment A16. 1. A method of treating a subject in need thereof, comprising: administering to the subject an effective amount of (A) an anti-Fzd4 antibody or antibody fragment according to any one of Embodiments A1 to A8, (B) a nucleic acid or combination of nucleic acids according to Embodiment A9 or A10, (C) a vector or combination of vectors according to Embodiment A11 or A12, and / or (D) a host cell according to Embodiment A13 or a cell population according to Embodiment A14, and / or (E) a pharmaceutical composition according to Embodiment A15, wherein optionally (a) the subject is (i) a mammal, optionally a human, a non-human primate, monkey, horse, cow, sheep, goat, pig, dog, cat, rabbit, rodent, hamster, rat or mouse, or (ii) a vertebrate other than a mammal, optionally a bird, fish, amphibian or reptile, (b) the subject comprises or has a risk of developing a disease, disorder or condition, and / or (c) the method further comprises administering to the subject an additional agent, optionally an adjuvant or therapeutic agent. method.

[0395] Embodiment A17. 1. A method of treating or preventing a disease, disorder, or condition in a subject in need thereof, comprising: administering an effective amount of (A) an anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A8, (B) a nucleic acid or combination of nucleic acids according to embodiment A9 or A10, (C) a vector or combination of vectors according to embodiment A11 or A12, and / or (D) a host cell according to embodiment A13 or a cell population according to embodiment A14, and / or (E) a pharmaceutical composition according to embodiment A15, wherein optionally (a) the subject is (i) a mammal, optionally a human, a non-human primate, monkey, horse, cow, sheep, goat, pig, dog, cat, rabbit, rodent, hamster, rat or mouse, or (ii) a vertebrate other than a mammal, optionally a bird, fish, amphibian or reptile, and / or (b) the method further comprises administering to the subject an additional agent, optionally an adjuvant or therapeutic agent, method.

[0396] Embodiment A18. (a) culturing cells comprising the nucleic acid or combination of nucleic acids of embodiment A9 or A10 under conditions that allow expression of the anti-Fzd4 antibody or antibody fragment; and (b) collecting and purifying the anti-Fzd4 antibody or antibody fragment from the cell culture from (a). A method for producing an anti-Fzd4 antibody or antibody fragment according to any one of embodiments A1 to A8.

[0397] Embodiment A19. introducing a nucleic acid or a combination of nucleic acids according to embodiment A9 or A10 and / or a vector or a combination of vectors according to embodiment A11 or A12 into one or more cells, wherein optionally said introduction occurs in vitro, ex vivo or in vivo, A method of producing a host cell according to embodiment A13 or a population of such cells.

[0398] Embodiment B1. (i) CDRH1 optionally comprising (i-1) a CDRH1 amino acid sequence included in any one of SEQ ID NOS: 381 to 399 according to IMGT or Kabat, (i-2) an amino acid sequence included in any one of SEQ ID NOS: 321 to 323, and / or (i-3) an amino acid sequence of SEQ ID NO: 320, wherein X1 is any amino acid, optionally an amino acid sequence comprising or consisting of any amino acid that is Q, N, or S, or is not A; (ii) (ii-1) optionally, a CDRH2 comprising an amino acid sequence of SEQ ID NOs: 381 to 399 according to IMGT or Kabat, (ii-2) the amino acid sequence of SEQ ID NO: 341, and / or (ii-3) the amino acid sequence of SEQ ID NO: 340, wherein X1 is any amino acid and X2 comprises or consists of any amino acid, and wherein the CDRH2 does not comprise the amino acid sequence of SEQ ID NO: 340 (X1 and X2 are K and M, L and M, I and M, M and K, M and L, or M and I). (iii) (iii-1) optionally, a CDRH3 comprising an amino acid sequence comprising any one of SEQ ID NOS: 381 to 399 according to IMGT or Kabat, (iii-2) any one of SEQ ID NOS: 361 to 369 and 300 to 303, and / or (iii-3) an amino acid sequence of SEQ ID NO: 360 (X1 is any amino acid, optionally D or E; X2 is any amino acid, optionally E, D, or S; and X3 is any amino acid, optionally D, E, S, A, T, or D). wherein optionally the VH is, comprises or is comprised in a Nanobody; Anti-LRP5 / 6 antibodies or antibody fragments.

[0399] Embodiment B2. The VH is (i) (i-1) optionally, an FRH1 comprising an amino acid sequence of FRH1 included in any one of SEQ ID NOS: 381 to 399 according to IMGT or Kabat, (i-2) the amino acid sequence of SEQ ID NOS: 311 or 312, and / or (i-3) the amino acid sequence of SEQ ID NOS: 310 (X1 is any amino acid, optionally E or D, X2 is any amino acid, optionally A or T, and X3 is any amino acid, optionally S), or an amino acid sequence consisting of these, and optionally, the FRH1 does not comprise the amino acid sequence consisting of SEQ ID NOS: 310 (X1, X2, and X3 are E, A, and A, respectively), (ii) (ii-1) optionally, an FRH2 comprising an amino acid sequence comprising or consisting of an FRH2 amino acid sequence included in any one of SEQ ID NOS: 381 to 399 according to IMGT or Kabat, (ii-2) any one of SEQ ID NOS: 331 to 333, and / or (ii-3) the amino acid sequence of SEQ ID NO: 330 (X1 is any amino acid, optionally Y; X2 is any amino acid, optionally Q or G; X3 is any amino acid, optionally R; X4 is any amino acid, optionally L or W; X5 is any amino acid, optionally I; and X6 is any amino acid, optionally A), wherein optionally, the FRH2 does not comprise an amino acid sequence consisting of SEQ ID NO: 330 (X1, X2, X3, X4, X5, and X6 are V, G, L, W, V, and S, respectively), (iii) (iii-1) optionally an FRH3 amino acid sequence included in any one of SEQ ID NOS: 381 to 399 according to IMGT or Kabat, (iii-2) the amino acid sequence of SEQ ID NOS: 351 or 352, and / or (iii-3) the amino acid sequence of SEQ ID NOS: 350 (X1 is any amino acid, optionally D or G, X2 is any amino acid, optionally L, X3 is any amino acid, optionally M, X4 is any amino acid, optionally T, X5 is any amino acid, optionally D or E, X6 is any amino acid, optionally N or S, X7 is any amino acid, optionally S or A, X8 is any amino acid, optionally M, X9 is any amino acid, optionally R or K, and X 10 is any amino acid, optionally A or P), and optionally the FRH3 comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 350 (X1, X2, X3, X4, X5, X6, X7, X8, X9 and X 10 are D, V, I, R, D, N, S, L, R and E respectively) and / or (iv) (iv-1) optionally, an FRH4 comprising an amino acid sequence comprising or consisting of an FRH4 amino acid sequence included in any one of SEQ ID NOS: 381 to 399 according to IMGT or Kabat, (iv-2) an amino acid sequence of SEQ ID NOS: 371 or 372, and / or (iv-3) an amino acid sequence of SEQ ID NOS: 370 (X1 is any amino acid, optionally R or W, and X2 is any amino acid, optionally Q or L). The anti-LRP5 / 6 antibody or antibody fragment of embodiment B1, comprising:

[0400] Embodiment B3. In the VH, (I) (I-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 381 according to IMGT or Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 321, 341, and 361; and (I-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 381; (II) (II-1) CDRH1, CDRH2, and CDRH3 (i) optionally comprise the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence, respectively, contained in any one of SEQ ID NOs: 382 to 385 according to IMGT or Kabat, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 322, 341, and 362, and (II-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 382 to 385, (III) (III-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 386 according to IMGT or Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 321, 341, and 362, and (III-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 386; (IV) (IV-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 387 according to IMGT or Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 323, 341, and 362, and (IV-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 387; (V) (V-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 388 according to IMGT or Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 322, 341, and 361; and (V-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 388; (VI) (VI-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence contained in SEQ ID NO: 389 according to IMGT or Kabat, respectively, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 322, 341, and 300, and (VI-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 389; (VII) (VII-1) CDRH1, CDRH2, and CDRH3 optionally comprise (i) the CDRH1 amino acid sequence, CDRH2 amino acid sequence, and CDRH3 amino acid sequence, respectively, contained in SEQ ID NO: 390 according to IMGT or Kabat, and / or (ii) an amino acid sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 322, 341, and 301; and (VII-2) optionally, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 390; (VIII) (VIII-1) CDRH1, CDRH2, and ...

Claims

1. at least: (A) a first antigen-binding region that specifically binds to frizzled class receptor 4 (Fzd4) and includes a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); (B) a second antigen-binding region that specifically binds to low-density lipoprotein receptor-related protein 5 and / or 6 (LRP5 and / or LRP6) and includes at least a second heavy chain variable domain (VH2); A multispecific antibody comprising: (A) the VH1 is (i) a CDRH1 consisting of the amino acid sequence of SEQ ID NO: 121 or the heavy chain complementarity-determining region 1 (CDRH1) amino acid sequence contained in SEQ ID NO: 181 as defined according to Kabat; (ii) a CDRH2 consisting of the amino acid sequence of SEQ ID NO: 141 or the heavy chain complementarity-determining region 2 (CDRH2) amino acid sequence contained in SEQ ID NO: 181 as defined according to Kabat; and (iii) a CDRH3 consisting of the amino acid sequence of SEQ ID NO: 161 or the heavy chain complementarity-determining region 3 (CDRH3) amino acid sequence contained in SEQ ID NO: 181 as defined according to Kabat Including; and, The VL1 is (iv) a light chain complementarity-determining region 1 (CDRL1) consisting of the amino acid sequence of SEQ ID NO: 221 or the light chain complementarity-determining region 1 (CDRL1) amino acid sequence contained in SEQ ID NO: 281 as defined according to Kabat; (v) a CDRL2 consisting of the amino acid sequence of SEQ ID NO: 241 or the light chain complementarity-determining region 2 (CDRL2) amino acid sequence contained in SEQ ID NO: 281 as defined according to Kabat; and (vi) a light chain complementarity-determining region 3 (CDRL3) consisting of the amino acid sequence of SEQ ID NO: 261 or the light chain complementarity-determining region 3 (CDRL3) amino acid sequence contained in SEQ ID NO: 281 as defined according to Kabat Including; and (B) the VH2 is (i) a CDRH1 consisting of the amino acid sequence of SEQ ID NO: 321 or the CDRH1 amino acid sequence contained in SEQ ID NO: 381 as defined according to IMGT or Kabat; (ii) a CDRH2 consisting of the amino acid sequence of SEQ ID NO: 341 or the CDRH2 amino acid sequence contained in SEQ ID NO: 381 as defined according to IMGT or Kabat; and (iii) a CDRH3 consisting of the amino acid sequence of SEQ ID NO: 361 or the CDRH3 amino acid sequence contained in SEQ ID NO: 381 as defined according to IMGT or Kabat A multispecific antibody comprising:

2. The multispecific antibody of claim 1 , wherein the VH2 is, comprises or is comprised in a nanobody.

3. The multispecific antibody of claim 1, which is a bispecific antibody.

4. (A) (i) the VH1 comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 181; and / or (ii) the VL1 comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 281; and / or (B) The multispecific antibody of claim 1, wherein VH2 comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

381.

5. (A) the VH1 comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 181; and / or the VL1 comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 281; and / or (B) The multispecific antibody of claim 1, wherein VH2 comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:

381.

6. (A) the VH1 comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 181; the VL1 comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 281; (B) The multispecific antibody of claim 1, wherein VH2 comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:

381.

7. (A) a first antigen-binding region that specifically binds to Fzd4 and includes VH1 and VL1; (B) a second antigen-binding region that specifically binds to LRP5 and / or LRP6 and includes at least VH2; (i) the VH1 comprises or consists of the amino acid sequence of SEQ ID NO: 181; (ii) the VL1 comprises or consists of the amino acid sequence of SEQ ID NO: 281; (iii) The multispecific antibody of claim 1, wherein the VH2 comprises or consists of the amino acid sequence of SEQ ID NO:

381.

8. a fragment crystallizable (Fc) region, wherein the Fc region comprises: (I) The multispecific antibody according to claim 1, which is an IgG, IgA, IgE, IgD, or IgM antibody.

9. The Fc region is (I) The multispecific antibody according to claim 8, which is IgG1, IgG4, IgG2 or IgG3.

10. The Fc region is (I) is human or a variant thereof; (II) one or more amino acid substitutions that reduce an Fc effector function, wherein the one or more amino acid substitutions are at a position selected from the group consisting of 234, 235, 236, 237, 265, 297, and 329, according to EU numbering, of any of a human IgG1, human IgG4, human IgG2, or human IgG3 Fc region; (III) the following amino acid substitutions according to EU numbering in the human IgG1 Fc region and / or any of SEQ ID NOs: 71 to 74: (ii-1) L234A, L235A and P329G, (ii-2) L234A and L235A, (ii-3) I253A, H310A and H435Q, and / or (ii-4) I253A, H310A and H435A and / or (IV) The multispecific antibody according to claim 8, comprising an amino acid sequence comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 71-74, 81-84, 86-89, 91-94, 96-99, 471-474, 476-479, 481-484, and 486-489, or an amino acid sequence that is at least 90% identical thereto.

11. (a) a first polypeptide comprising said VH1; and (b) the following, in order from N-terminus to C-terminus: (b-1) said VH2; (b-2) a linker, wherein: (i) comprising one or more amino acids; (ii) consists of small amino acids consisting of G, S and / or A; (iii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS and SSG; and / or (iv) a linker comprising, or consisting of, multiple repeats of an amino acid or peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG; and (b-3) the VL1 and a second polypeptide comprising The multispecific antibody of claim 1 , comprising:

12. (a) the first polypeptide is (i) comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2, 7, 8, 9, 450, 451, 452 and 453; and / or (ii) comprising an amino acid sequence comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 2, 7, 8, 9, 450, 451, 452, and 453; and (b) the second polypeptide is (i) comprises an amino acid sequence having at least 90% identity to SEQ ID NO:1; and / or (ii) comprising an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 1; The multispecific antibody of claim 11.

13. (a) the first polypeptide comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 2, 7, 450, or 451; and (b) the second polypeptide comprises an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 1; The multispecific antibody of claim 11.

14. two of the first polypeptides and two of the second polypeptides that form a dimer with each other via one or more disulfide bonds; 12. The multispecific antibody of claim 11 , wherein one of the second polypeptides interacts with one of the first polypeptides via a disulfide bond, and another of the second polypeptides interacts with another of the first polypeptides via a disulfide bond.

15. (A) two or more Fzd4-binding regions, wherein at least one of the Fzd4-binding regions is, comprises, or is included in the first antigen-binding region; and / or (B) two or more LRP5 and / or LRP6 binding regions, wherein at least one of the LRP5 and / or LRP6 binding regions is the second antigen-binding region, comprises the second antigen-binding region, or is included in the second antigen-binding region; The multispecific antibody of claim 1 , comprising an antigen-binding region.

16. (I) The ratio of the number of the one or more Fzd4 binding regions to the number of the one or more LRP5 and / or LRP6 binding regions contained in the multispecific antibody is selected from the group consisting of 2: 1, 1: 2, 2: 2, 3: 1, 1: 3, 3: 2, 2: 3, 3: 3, 4: 1, 1: 4, 4: 2, 2: 4, 4: 3, 3: 4 and 4: 4; (II) The number of the one or more Fzd4 binding regions and the number of the one or more LRP5 and / or LRP6 binding regions contained in the multispecific antibody are 2 and 1, 1 and 2, 2 and 2, 3 and 1, 1 and 3, 3 and 2, 2 and 3, 3 and 3, 4 and 1, 1 and 4, 4 and 2, 2 and 4, 4 and 3, 3 and 4, or 4 and 4; and / or (III) The multispecific antibody comprises: (a) two identical Fzd4 binding regions, each of which is, comprises, or is included in the first antigen-binding region, and one LRP5 and / or LRP6 binding region, each of which is, comprises, or is included in the second antigen-binding region; (b) two Fzd4 binding regions, which are different from each other, (i) at least one of which is the first antigen-binding region, comprises the first antigen-binding region, or is included in the first antigen-binding region, or (ii) each of which is the first antigen-binding region, comprises the first antigen-binding region, or is included in the first antigen-binding region, and one LRP5 and / or LRP6 binding region, which is the second antigen-binding region, comprises the second antigen-binding region, or is included in the second antigen-binding region; (c) one Fzd4 binding region that is the first antigen-binding region, that includes the first antigen-binding region, or that is included in the first antigen-binding region, and two identical LRP5 and / or LRP6 binding regions that are the second antigen-binding region, that include the second antigen-binding region, or that are included in the second antigen-binding region; (d) one Fzd4 binding region that is the first antigen-binding region, that includes the first antigen-binding region, or that is included in the first antigen-binding region, and two LRP5 and / or LRP6 binding regions that are different from each other, and (i) at least one of which is the second antigen-binding region, that includes the second antigen-binding region, or that is included in the second antigen-binding region, or (ii) each of which is the second antigen-binding region, that includes the second antigen-binding region, or that is included in the second antigen-binding region. (e) two identical Fzd4 binding regions, each of which is the first antigen-binding region, comprises the first antigen-binding region, or is included in the first antigen-binding region, and two identical LRP5 and / or LRP6 binding regions, each of which is the second antigen-binding region, comprises the second antigen-binding region, or is included in the second antigen-binding region; (f) two Fzd4 binding regions, which are different from each other, (i) at least one of which is the first antigen-binding region, comprises the first antigen-binding region, or is included in the first antigen-binding region, or (ii) each is the first antigen-binding region, comprises the first antigen-binding region, or is included in the first antigen-binding region; and two identical LRP5 and / or LRP6 binding regions, each of which is the second antigen-binding region, comprises the second antigen-binding region, or is included in the second antigen-binding region. (g) two identical Fzd4 binding regions, each of which is, comprises, or is comprised in the first antigen-binding region, and two LRP5 and / or LRP6 binding regions that are different from each other, and (i) at least one of which is, comprises, or is comprised in the second antigen-binding region, or (ii) each of which is, comprises, or is comprised in the second antigen-binding region, and / or (h) two Fzd4 binding regions, which are different from each other, (i) at least one of which is the first antigen-binding region, comprises the first antigen-binding region, or is included in the first antigen-binding region, or (ii) each of which is the first antigen-binding region, comprises the first antigen-binding region, or is included in the first antigen-binding region; and two LRP5 and / or LRP6 binding regions, which are different from each other, (i) at least one of which is the second antigen-binding region, comprises the second antigen-binding region, or is included in the second antigen-binding region, or (ii) each of which is the second antigen-binding region, comprises the second antigen-binding region, or is included in the second antigen-binding region. The multispecific antibody of claim 15.

17. A nucleic acid or a combination of nucleic acids encoding the multispecific antibody of claim 1, A nucleic acid or a combination of nucleic acids, wherein the nucleic acid is DNA, cDNA, RNA, mRNA, modified mRNA or a DNA / RNA hybrid.

18. encoding the multispecific antibody according to claim 11 instead of the multispecific antibody according to claim 1, and (a) a first nucleic acid encoding the first polypeptide; (b) a second nucleic acid encoding the second polypeptide; and 18. The nucleic acid or nucleic acid combination of claim 17, comprising:

19. 18. A vector or a combination of vectors comprising a nucleic acid or a combination of nucleic acids according to claim 17, where: (i) the vector comprises one or more promoters operably linked to the nucleic acid; (ii) the vector is an expression vector; and / or (iii) A vector or combination of vectors, wherein the vector comprises a plasmid, a viral vector, a lipid-based vector, a self-replicating RNA vector, a virus-like particle, a polymer-based vector and / or a nanoparticle.

20. encoding the multispecific antibody according to claim 11 instead of the multispecific antibody according to claim 1, and (a) a first vector comprising a first nucleic acid encoding the first polypeptide; (b) a second vector comprising a second nucleic acid encoding the second polypeptide; and 20. The vector or vector combination of claim 19, comprising:

21. (A) the multispecific antibody of any one of claims 1 to 16; (B) a nucleic acid or a combination of nucleic acids according to claim 17 or 18; and / or (C) A vector or a combination of vectors according to claim 19 or 20. A host cell comprising: The host cell (i) a mammal, or (ii) is non-mammalian; and / or The host cell (i) human embryonic kidney (HEK) cells; (ii) CHO (Chinese Hamster Ovary) cells, or (iii) A host cell that is an immune cell.

22. A cell population comprising two or more host cells of claim 21.

23. (I) (A) the multispecific antibody of any one of claims 1 to 16; (B) a nucleic acid or a combination of nucleic acids according to claim 17 or 18; and / or (C) a vector or a combination of vectors according to claim 19 or 20; (II) a pharmaceutically acceptable carrier and / or excipient; A pharmaceutical composition comprising:

24. (a) The subject is: (i) a mammal, or (ii) is a vertebrate other than a mammal; (b) the subject comprises or has a risk of developing a disease, disorder, or condition; and / or (c) the use of the pharmaceutical composition further comprises administering to the subject an additional agent; 24. The pharmaceutical composition of claim 23.

25. 1. A composition for inducing, promoting, stimulating, enhancing and / or supporting Wnt signaling in a target cell of a subject, comprising: (A) the multispecific antibody of any one of claims 1 to 16; (B) a nucleic acid or a combination of nucleic acids according to claim 17 or 18; and / or (C) a vector or a combination of vectors according to claim 19 or 20, where: (a) the subject is (i) a mammal, or (ii) is a vertebrate other than a mammal; (b) the subject comprises or has a risk of developing a disease, disorder, or condition; and / or (c) using the composition further comprises administering to the subject an additional agent; where: (i) the Wnt signaling is or includes Wnt / β-catenin signaling; and / or (ii) the composition promotes, stimulates, enhances and / or supports the recruitment of multiple receptor complexes, each of which comprises (ii-1) Fzd4 and (ii-2) LRP5 or LRP6.

26. The disease, disorder or condition of interest is one of the following: (a) a retinopathy which is a retinal vascular disease and / or is selected from the group consisting of exudative vitreoretinopathy, familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie's disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease; (b) vascular disorders that are vascular malformations or vascular insufficiencies or that involve ischemia-induced neovascularization; (c) a bone disease or injury selected from the group consisting of osteoporosis (including osteoporotic pseudoglioma (OPPG) syndrome and juvenile primary osteoporosis), bone mineral density abnormalities, and cancer-related osteolysis; (d) a muscle wasting disorder selected from the group consisting of sarcopenia, cachexia, and muscular dystrophy; and / or (e) a neurological or neurodegenerative disease selected from the group consisting of stroke, traumatic brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, and Creutzfeldt-Jakob disease.

24. The pharmaceutical composition of claim 23, comprising one or more of:

27. (I) administering the pharmaceutical composition (i) administration to one or more eyes of a subject; (ii) topical administration, or (iii) parenteral administration, or (iv) enteral administration; and / or (II) The administration comprises administering the multispecific antibody of claim 1 to one or more eyes of the subject at about 0.1 to 100,000 ng per eye, about 1 to 100,000 ng per eye, about 1 to 10,000 ng per eye, about 10 to 10,000 ng per eye, about 1 to 100 ng per eye, about 200 to 300 ng per eye, about 300 to 400 ng per eye, about 400 to 500 ng per eye, about 500 to 600 ng per eye, about 600 to 700 ng per eye, about 700 to 800 ng per eye, about 800 to 900 ng per eye, about 900 to 1,000 ng per eye, about 1,000 to 2,000 ng per eye, about 2,000 to 3,000 ng per eye, about 3,000 to 4,000 ng per eye, about 4 000-5000 ng per eye, about 5000-6000 ng per eye, about 6000-7000 ng per eye, about 7000-8000 ng per eye, about 8000-9000 ng per eye, about 9000-10000 ng per eye, about 10000-20000 ng per eye, about 20000-30000 ng per eye, about 30000-4000 ng per eye 24. The pharmaceutical composition of claim 23, comprising administering about 0,000 ng per eye, about 40,000-50,000 ng per eye, about 50,000-60,000 ng per eye, about 60,000-70,000 ng per eye, about 70,000-80,000 ng per eye, about 80,000-90,000 ng per eye, or about 90,000-100,000 ng per eye.

28. 1. A composition for inducing, promoting, stimulating, enhancing and / or supporting Wnt signaling in a target cell, comprising: (A) the multispecific antibody of any one of claims 1 to 16; (B) a nucleic acid or a combination of nucleic acids according to claim 17 or 18; and / or (C) a vector or a combination of vectors according to claim 19 or 20, wherein using the composition comprises contacting a target cell with an effective amount of the composition; where: (I) the contacting occurs in vitro, ex vivo, or in vivo; (II) The use is for (i) the preparation of cells and / or tissues for transplantation, (ii) the differentiation of stem cells, and / or (iii) the preparation of organoids; (III) The composition, wherein the use is for osteogenesis, angiogenesis, and / or neurogenesis and / or differentiation.

29. (a) culturing a cell comprising a nucleic acid or a combination of nucleic acids according to claim 17 or 18 under conditions allowing expression of said multispecific antibody; (b) harvesting and purifying the multispecific antibodies from the cell culture from (a); A method for producing a multispecific antibody according to any one of claims 1 to 16, comprising:

30. A nucleic acid or combination of nucleic acids according to claim 17 for producing a host cell according to claim 21, said producing comprising introducing said nucleic acid or combination of nucleic acids into one or more cells; wherein the introduction occurs in vitro, ex vivo, or in vivo; A nucleic acid or a combination of nucleic acids.

31. A multispecific antibody according to any one of claims 1 to 16, a nucleic acid or a combination of nucleic acids according to claim 17 or 18, and / or a vector or a combination of vectors according to claim 19 or 20 for use in medicine.

32. for use in treating a disease, disorder or condition, wherein the disease, disorder or condition is one of the following: (a) a retinopathy which is a retinal vascular disease and / or is selected from the group consisting of exudative vitreoretinopathy, familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie's disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease; (b) vascular disorders that are vascular malformations or vascular insufficiencies or that involve ischemia-induced neovascularization; (c) a bone disease or injury selected from the group consisting of osteoporosis (including osteoporotic pseudoglioma (OPPG) syndrome and juvenile primary osteoporosis), bone mineral density abnormalities, and cancer-related osteolysis; (d) a muscle wasting disorder selected from the group consisting of sarcopenia, cachexia, and muscular dystrophy; and / or (e) a neurological or neurodegenerative disease selected from the group consisting of stroke, traumatic brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, and Creutzfeldt-Jakob disease.

21. The multispecific antibody of any one of claims 1 to 16, the nucleic acid or combination of nucleic acids of claim 17 or 18, and / or the vector or combination of vectors of claim 19 or 20, comprising one or more of:

33. for the manufacture of a medicament for the treatment of a disease, disorder or condition, wherein the disease, disorder or condition is one of the following: (a) a retinopathy which is a retinal vascular disease and / or is selected from the group consisting of exudative vitreoretinopathy, familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie's disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease; (b) vascular disorders that are vascular malformations or vascular insufficiencies or that involve ischemia-induced neovascularization; (c) a bone disease or injury selected from the group consisting of osteoporosis (including osteoporotic pseudoglioma (OPPG) syndrome and juvenile primary osteoporosis), bone mineral density abnormalities, and cancer-related osteolysis; (d) a muscle wasting disorder selected from the group consisting of sarcopenia, cachexia, and muscular dystrophy; and / or (e) a neurological or neurodegenerative disease selected from the group consisting of stroke, traumatic brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, and Creutzfeldt-Jakob disease.

21. The multispecific antibody of any one of claims 1 to 16, the nucleic acid or combination of nucleic acids of claim 17 or 18, and / or the vector or combination of vectors of claim 19 or 20, comprising one or more of:

34. (I) a host cell according to claim 21; (II) a pharmaceutically acceptable carrier and / or excipient; A pharmaceutical composition comprising:

35. (I) a cell population according to claim 22; (II) a pharmaceutically acceptable carrier and / or excipient; A pharmaceutical composition comprising:

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