Compositions, doses, and methods for treatment of ocular diseases

Abstract

Provided herein are compositions, doses, and methods of using an antibody, or an antigen-binding fragment thereof, for the treatment of ocular disorders and diseases, such as, but not limited to diabetic macular edema, visual impairment that can be associated with DME, or NVAMD.

Description

COMPOSITIONS, DOSES, AND METHODS FOR TREATMENT OF OCULAR DISEASESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The application claims the benefit of priority to U.S. Provisional Application No. 63 / 550,946, filed February 7, 2024, the contents of which are incorporated herein by reference in their entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (26048WOPCT.xml; Size: 29,183 bytes, created on January 23, 2025) are herein incorporated by reference in their entirety.FIELD

[0003] This disclosure relates to compositions, doses, and methods of using an antibody, or an antigenbinding fragment thereof, for the treatment of ocular disorders and diseases.BACKGROUND

[0004] Diabetic macular edema (DME) is the most common cause of vision loss in patients with diabetic retinopathy, with an increasing prevalence tied to the global epidemic of type 2 diabetes mellitus. The pathophysiology of DME starts with decreased retinal oxygen tension or retinal hypoxia that is likely the result of retinal capillary non-perfusion and / or drop out, which in turn leads to the upregulation of vascular endothelial growth factor (VEGF) that causes retinal capillary hyperpermeability. The foundation of treatment for type 2 diabetes is metabolic control of hyperglycemia and blood pressure, which is often insufficient to treat DME. Specific ophthalmic treatments for DME include intravitreal anti- VEGF drug(s) injections, intravitreal corticosteroid injections, focal laser photocoagulation, and vitrectomy, but a substantial fraction of eyes respond incompletely to these modalities, resulting in disordered retinal structure and vasculature and irreversible vision loss (Browning et al, 2018). The Wisconsin Epidemiologic Study of Diabetic Retinopathy found that 20% of patients with type 1 diabetes and 25% of patients with type 2 diabetes will develop DME after 10 years of follow-up, despite treatment for hyperglycemia and blood pressure control (Klein, 1984). In the United Kingdom (UK), it is estimated that 10% of the entire National Health Service budget is spent on the care of people with diabetes, 80% of which is spent on consequences and complications of diabetes, including DME (Al-Husainy et al, 2014), which representsa significant economic burden. Treatment goals for DME include reducing retinal thickness and neurosensory damage and minimizing the risk for potential adverse side effects to preserve vision (Shechtman, 2009).

[0005] Diabetic macular edema describes the accumulation of fluid in and under the central retina (the macula), which arises, in part, secondary to dysfunction of the blood-retinal barrier (BRB). Diabetic macular edema is caused by both extensive capillary leakage and focal leakage from grouped microaneurysms (MAs). Diabetic macular edema can occur in isolation without other signs of microangiopathy in the fundus; therefore, it merits being classified as a separate entity. Diabetic macular edema often results in blurred and distorted central vision, which is reflected in a reduction in measured best-corrected visual acuity (BCVA) (Schmidt-Erfurth, 2017).

[0006] There are a number of treatment options for patients with DME, but all have limited efficacy. As a result, there remains significant unmet need for patients, especially given the predicted increased economic burden associated with increasing diabetes rates.

[0007] Laser photocoagulation was the standard of care for the treatment of DME prior to the advent of the intravitreal injection approach. The efficacy of focal laser treatment has been related to the occlusion of leaking vessels, especially MAs, but the exact mechanism by which focal photocoagulation reduces DME is unknown. Laser photocoagulation is no longer considered the standard of care, as improved efficacy can be obtained with pharmacotherapy (Schmidt-Erfurth, 2017). In addition, laser therapy is not effective in many cases of DME, particularly in cases of diffuse macular edema. Patients also may experience complications following laser treatment, including scotoma and epiretinal membrane formation (Shechtman, 2009).

[0008] Diabetic macular edema stems from a complex pathogenesis, including the presence of a hypoxic environment in the microvasculature of patients with diabetic retinopathy. Hypoxia, in turn, leads to increased levels of VEGF, which is a potent vascular permeability factor and results in a state of hyperpermeability of the retinal vessels. This complex cascade leads to vascular leakage and extracellular fluid accumulation, which eventually manifests as macular edema. Vascular endothelial growth factor has been shown to play a critical role in the development of macular edema, and high levels of VEGF have been found in the aqueous humor of patients with DME (Shechtman, 2009).

[0009] Intravitreal injections with anti-VEGF agents can reduce retinal edema, improve vision, and prevent further visual loss in some patients (Kimoto and Kubota, 2012). These drugs have replaced laser photocoagulation as the standard of care because of their efficacy and safety profiles. Bevacizumab(Avastin®), ranibizumab (Lucentis®), and aflibercept (Eylea®) are all common treatments for DME with proven efficacy (compared with that of laser treatment), improving visual acuity (VA) by 3 or more lines over the course of 1 year in 30-40% of patients (Virgili, 2018). Bevacizumab is a full-length, humanized, monoclonal antibody; ranibizumab is a recombinant humanized Fab fragment of a monoclonal antibody; and aflibercept is a recombinant decoy receptor inhibitor of VEGF (Schmidt- Erfurth, 2017). Ranibizumab and aflibercept are approved for DME, whereas bevacizumab is an approved oncology treatment often used off label in DME. Other anti-VEGF treatments include: brolucizumab (Beovu®), currently approved for DME and neovascular age related macular degeneration; faricimab (Vabysmo®), a bispecific antibody targeting both VEGF and angiopoietin 2, approved in the UK, United States (US), and Japan for DME and NV AMD; and a biosimilar to ranibizumab, ranibizumab-nuna, currently approved for DME in the US.

[0010] Evidence highlights the role of inflammation in the development of DME. The pathological processes include retinal leukostasis, which is defined as the attachment of leukocytes inside the lumen of capillaries and is thought to be a major player and early event in the dysfunction of the BRB. Inflammation leads to the upregulation of intracellular adhesion molecule 1 (ICAM)-l, which mediates attraction of monocytes and neutrophils to the vascular endothelium to further enhance retinal leukostasis, vascular permeability, and breakdown of the BRB in diabetes (Schmidt-Erfurth, 2017).

[0011] Corticosteroids produce an anti-inflammatory effect through various mechanisms, including the decrease in the synthesis of inflammatory mediators, such as interleukin-6 (IL-6), interferon inducible protein 10, monocyte-chemoattractant protein- 1, as well as a decrease in platelet derived growth factor AA and VEGF synthesis. Intravitreal treatment with glucocorticoids has been found to significantly decrease the inflammatory processes by inhibition of ICAM-1 expression and subsequent leukostasis and by improving overall BRB function. Triamcinolone acetonide, dexamethasone, and fluocinolone acetonide have all been used to treat DME (Schmidt-Erfurth, 2017). Long-acting intravitreal corticosteroids can achieve similar results in improving visual acuity to those of anti-VEGF therapies, with longer inter-injection intervals. Because of steroid-induced side effects (progression of cataract, glaucoma), these drugs are regarded as second-line medications (Barth, 2021). Surgery in the form of pars plana vitrectomy also remains an option for patients with associated vitreoretinal traction, but the procedure remains controversial in non-tractional cases.

[0012] Due to limited efficacy and potential side effects of the currently approved treatments for DME, there remains a significant unmet need for additional efficacious and safe treatments. The embodiments provided for herein provide unexpected treatments for DME and other related ocular conditions.SUMMARY

[0013] The present disclosure is directed to a method of treating an ocular disorder in a subject in need thereof, comprising administering to the eye of the subject, a pharmaceutical composition comprising about 0.05 mg to about 1.00 mg of an antibody that binds to LRP5 and FZD4 in a volume of about 100 pL or less. The present disclosure is also directed to a pharmaceutical composition comprising about 0.05 mg to about 1.00 mg of an antibody that binds to LRP5 and FZD4 for use in treating an ocular disorder in a subject in need thereof, wherein the treatment comprises administering about 0.05 mg to about 1.00 mg of said pharmaceutical composition intravitreally to the subject in a volume of about 100 pL or less. The present disclosure is also directed to use of a pharmaceutical composition comprising about 0.05 mg to about 1.00 mg of an antibody that binds to LRP5 and FZD4 in the manufacture of a medicament for treating an ocular disorder in a subject in need thereof, wherein the treatment comprises administering about 0.05 mg to about 1.00 mg of said pharmaceutical composition intravitreally to the subject in a volume of about 100 pL or less. In some aspects, the pharmaceutical composition comprises about 0.05 mg, about 0.15 mg, about 0.40 mg, about 0.48 mg, about 0.50 mg, or about 0.80 mg of the antibody. In some aspects, the pharmaceutical composition comprises about 0.40 mg, about 0.50 mg, or about 0.80 mg of the antibody. In some aspects, the pharmaceutical composition is administered to the subject every two weeks, every 4 weeks, or every 8 weeks. In some aspects, the pharmaceutical composition is administered every 4 weeks (Q4W). In some aspects, the pharmaceutical composition is administered every 8 weeks (Q8W). In some aspects, the pharmaceutical composition is administered in a volume of about 50 pL to about 100 pL. In some aspects, the subject is administered the pharmaceutical composition comprising about 0.40 mg, 0.50 mg, or 0.80 mg of the antibody in a volume of about 50 pL. In some aspects, the subject is administered the pharmaceutical composition comprising about 0.40 mg of the antibody in a volume of about 50 pL. In some aspects, the subject is administered the pharmaceutical composition comprising about 0.50 mg of the antibody in a volume of about 50 pL. In some aspects, the subject is administered the pharmaceutical composition comprising about 0.80 mg of the antibody in a volume of about 50 pL.

[0014] In some aspects, the pharmaceutical composition is administered every 2 weeks (Q2W). In some aspects, the pharmaceutical composition is administered intravitreally. In some aspects, the ocular disorder is visual impairment. In some aspects, the visual impairment is visual impairment secondary to diabetic macular edema. In some aspects, the ocular disorder is diabetic macular edema (DME). In some aspects, the ocular disorder is neovascular age-related macular degeneration (NV AMD). In some aspects, the subject’s Best Corrected Visual Acuity (BCVA) is increased after being treated. In some aspects, the BCVA is increased about 10 to about 15 letters. In some aspects, the subject’s retinal thickness is decreased after administration. In some aspects, the subject’s central subfield thickness (CST) of the retina is reduced after administration. In some aspects, the CST is reduced by about 100pm to about 150pm. In some aspects, the CST is measured by optical coherence tomography. In some aspects, the subject is being treated for vascular leakage, retinal vascular leakage, and / or endothelial cell leakage, and / or disorders characterized by reduced retinal or brain endothelial cell barrier functions, a compromised BBB or BRB, or diabetic retinopathy, retinopathy of prematurity, Coat’s disease, FEVR, Norrie disease, macular degeneration, diabetic macular edema, or pediatric vitreoretinopathy. In some aspects, the ocular disorder is NV AMD, and the subject is administered the antibody in combination with aflibercept. In some aspects, the subject has an increase in BCVA. In some aspects, the BCVA is increased from about 5 to about 10 letters. In some aspects, the subject’s CST thickness is reduced by at least about 50% after administration. In some aspects, the subject has an increase in best-corrected visual acuity (BCVA) using the standardized ETDRS (Early Treatment of Diabetic Retinopathy Study) chart after administration. In some aspects, the subject has an improvement in visual outcomes after administration. In some aspects, the subject has an improvement in at least one symptom after about 1 year of being treated with the antibody. In some aspects, the subject has an improvement in at least one symptom after about 2 years of being treated with the antibody.

[0015] In some aspects, the antibody is a tetravalent antibody. In some aspects, the tetravalent antibody comprises: a first polypeptide comprising: (a) a first heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1, a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3; (b) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10, a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 12; and a second VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 17, a CDR-H2 having the amino acid sequence of SEQ ID NO: 18 and a CDR-H3 havingthe amino acid sequence of SEQ ID NO: 19; a second polypeptide comprising: (a) a first heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 7, a CDR- H2 having the amino acid sequence of SEQ ID NO: 8, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 9; (b) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4, a CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and a CDR- L3 having the amino acid sequence of SEQ ID NO: 6; (c) a second VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 17, a CDR-H2 having the amino acid sequence of SEQ ID NO: 18 and a CDR-H3 having the amino acid sequence of SEQ ID NO: 19; and a third polypeptide comprising a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 20, a CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 22; and a fourth polypeptide comprising a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 20, a CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 22. In some aspects, the first polypeptide further comprises a Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3), and the first polypeptide further comprises a constant heavy chain domain 1 (CHI); the second polypeptide further comprises a Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3), and the second polypeptide further comprises a constant heavy chain domain 1 (CHI); the third polypeptide further comprises a constant light chain domain 1 (CLI); and the fourth polypeptide further comprises a constant light chain domain 1 (CLI).

[0016] In some aspects, the Fc region of the first polypeptide and the second polypeptide are different and the CLI domain of the third polypeptide and the fourth polypeptide are the same. In some aspects, the first VH of the first polypeptide interacts with the VL of the second polypeptide to form a domain that binds LRP5; the first VH of the second polypeptide interacts with the VL of the first polypeptide to form a domain that binds LRP5; the second VH of the first polypeptide interacts with the VL of the third polypeptide to form a domain that binds FZD4; and the second VH of the second polypeptide interacts with the VL of the fourth polypeptide to form a domain that binds FZD4. In some aspects, the first polypeptide and the second polypeptide form a heterodimer. In some aspects, the first polypeptide and the second polypeptide form a heterodimer through the Fc region of the first polypeptide and the second polypeptide. In some aspects, the Fc region of the first polypeptide comprises a serine at position 366, an alanine at position 368 and a valine at position 407; and the Fc region of the second polypeptide comprises a tryptophan at position 366, wherein the positions are according to EU numbering.

[0017] In some aspects, the Fc region of the first polypeptide further comprises an isoleucine at position 354 and a leucine at position 357, and the Fc region of the second polypeptide further comprises a methionine at position 347, a phenylalanine at position 349, an aspartic acid at position 350 and a methionine a position 368, wherein the positions are according to EU numbering. In some aspects, the first polypeptide comprises a cysteine at position 349, and the second polypeptide comprises a cysteine at position 354, wherein the positions are according to EU numbering. In some aspects, the Fc regions of the first polypeptide and / or the second polypeptide lack effector function or have reduced effector function. In some aspects, the Fc regions of the first polypeptide and the second polypeptide comprise a glycine at position 397, an alanine at position 265, or both a glycine at position 397 and an alanine at position 265, wherein the positions according to EU numbering. In some aspects, the Fc regions of the first polypeptide and the second polypeptide comprise an alanine at position 234, an alanine at position 235, or a serine at position 331, or any combination thereof, wherein the positions are according to EU numbering. In some aspects, the Fc regions of the first polypeptide and the second polypeptide comprise an alanine at position 234 and an alanine at position 235, wherein the positions are according to EU numbering. In some aspects, the Fc regions of the first polypeptide and the second polypeptide comprise an alanine at position 234, an alanine at position 235, and a serine a position 331, wherein the positions are according to EU numbering. In some aspects, the first polypeptide, from N-terminus to C-terminus, comprises: i) the first heavy chain variable domain; ii) the light chain variable domain; iii) the Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3); iv) the second heavy chain variable domain; and v) the constant heavy chain domain 1 (CHI), wherein the light chain variable domain of the first polypeptide and the second heavy chain variable domain of the first polypeptide are each, independently, attached to the Fc region of the first polypeptide by a polypeptide linker; the second polypeptide from N-terminus to C-terminus comprises: i) the first heavy chain variable domain; ii) the light chain variable domain; iii) the Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3); iv) the second heavy chain variable domain; and v) the constant heavy chain domain 1 (CHI), wherein the light chain variable domain of the second polypeptide and the second heavy chain variable domain of the second polypeptide are each, independently, attached to the Fc region of the second polypeptide by a polypeptide linker.

[0018] In some aspects, the light chain variable domain of the of the first polypeptide and the light chain variable domain of the second polypeptide are linked to the N-terminus of the Fc region of the first polypeptide and the second polypeptide, respectively, by a polypeptide linker comprising the amino acidsequence of GGGGSGGGGSEPKSSDKTHT (SEQ ID NO: 26). In some aspects, the second heavy chain variable domain of the first polypeptide and the second heavy chain variable domain of the second polypeptide are linked to the C-terminus of the Fc region of the first polypeptide and / or the second polypeptide, respectively, by a polypeptide linker comprising the amino acid sequence of GGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 27). In some aspects, the first VH of the first polypeptide is linked to the VL of the first polypeptide by a polypeptide linker comprising the acid sequence of GGGGS (SEQ ID NO: 25); and the first VH of the second polypeptide is linked to the VL of the second polypeptide by a polypeptide linker comprising the acid sequence of GGGGS (SEQ ID NO: 25). In some aspects, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28, the second polypeptide comprises the amino acid of SEQ ID NO: 29, and the third polypeptide and the fourth polypeptide each comprise the amino acid sequence of SEQ ID NO: 30. In some aspects, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28, the second polypeptide comprises the amino acid of SEQ ID NO: 29, and the third polypeptide and the fourth polypeptide each comprise the amino acid sequence of SEQ ID NO: 30. In some aspects, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28, the second polypeptide comprises the amino acid of SEQ ID NO: 29, and the third polypeptide and the fourth polypeptide each comprise the amino acid sequence of SEQ ID NO: 30. In some aspects, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28, the second polypeptide comprises the amino acid of SEQ ID NO: 29, and the third polypeptide and the fourth polypeptide each comprise the amino acid sequence of SEQ ID NO: 30.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG.l show modalities of tetravalent binding antibody molecules. Illustrated are: a diabody -Fc- diabody format having an FZD-binding monospecific diabody on the N-terminus of the Fc domain and a LPR5 / 6-binding bispecific diabody on the C-terminus of the Fc domain; a Diabody -Fc-scFv format having an N-terminal LPR5 / 6-binding bispecific diabody and two C-terminal FZD binding scFv; an IgG-diabody format having two FZD-binding Fabs forming an N-terminal binding domain and a bispecific LRP5 / 6 binding diabody forming the C-terminal binding domain; an IgG-scFv format having two FZD-binding Fabs forming an N-terminal binding domain and two LRP5 / 6 binding scFvs forming the C-terminal binding domain, and; a diabody-Fc-Fab format having a bispecific LRP5 / 6 binding diabody forming the N-terminal binding domain and two FZD-binding Fabs forming the C-terminalbinding domain, wherein the Fabs are linked to the CH3 of the Fc domain via the Fab variable heavy region.

[0020] FIG. 2 shows a FZD4 agonist having a Diabody-Fc-Fab format having an LRP5-binding bispecific diabody, forming a bivalent bispecific N-terminal LRP5-binding domain and two FZD4- binding Fabs, and an Fc region with attenuated effector functions due to amino acid mutations. The various domains of the tetravalent molecules, VL, VH, CHI, CH2, CH3, CLI and Fc domain, are joined via linkers such as peptide linkers.

[0021] FIG. 3 shows two formats, ANT39 and inverted format ANT39i.

[0022] FIG. 4 shows the FZD4 agonist ANT39 with a Diabody-Fc-Fab format with the Fc region having attenuated effector functions due amino acid mutations to N297G and D265A (DANG) variants or L234A, L235A, P331S (LALAPS) variants.

[0023] FIG. 5 shows the FZD4 agonist ANT39 having a Diabody-Fc-Fab with the Fc region having attenuated effector functions due amino acid mutations to L234A, L235A, P331S (LALAPS) variants, and with the Fc region further comprising knob-in-hole heterodimerization variants Merrimack, Merchant, or Merchant S:S as described herein.

[0024] FIG. 6 shows the FZD4 agonist ANT39i having an IgG-Fc-Diabody format and an Fc region with attenuated effector functions due to LALAPS variants.

[0025] FIG. 7 shows the FZD4 agonist ANT39i having an IgG-Fc-Diabody format and an Fc region with attenuated effector functions due to LALAPS variants, and Merrimack, Merchant, or Merchant S:S heterodimerization variants as described herein.

[0026] FIG. 8 shows the FZD4 agonist ANT42 having a Diabody-Fc-Fab format with the Fc region having attenuated effector functions due amino acid mutations to N297G and D265A (DANG) variants or L234A, L235A, P331 S (LALAPS) variants, and with the Fc region further comprising knob-in-hole heterodimerization variants Merrimack, Merchant or Merchant S:S as described herein.

[0027] FIG. 9 shows the FZD4 agonist ANT42 having a Diabody-Fc-Fab format with the Fc region having attenuated effector functions due amino acid mutations to N297G and D265A (DANG) variants or L234A, L235A, P331 S (LALAPS) variants, and with the Fc region further comprising knob-in-hole heterodimerization variants Merrimack, Merchant or Merchant S:S as described herein.

[0028] FIG. 10 shows the FZD4 agonist ANT42 having a Diabody-Fc-Fab format with the Fc region having attenuated effector functions due amino acid mutations to N297G and D265A (DANG) variantsor L234A, L235A, P331 S (LALAPS) variants, and with the Fc region further comprising knob-in-hole Merchant heterodimerization variant as described herein.

[0029] FIG. 11 shows the FZD4 agonist ANT42 having a Diabody -Fc-Fab format with the Fc region having attenuated effector functions due amino acid mutations to N297G and D265A (DANG) variants or L234A, L235A, P331 S (LALAPS) variants, and with the Fc region further comprising knob-in-hole Merchant S:S heterodimerization variant as described herein.

[0030] FIGs. 12A-12I show various additional antibody formats.DETAILED DESCRIPTION

[0031] Provided herein are multispecific / multivalent antibodies that bind to LRP5 and FZD4 that can be used to treat ocular disorders and diseases, such as those provided for herein. Also provided herein are pharmaceutical compositions of such antibodies. Methods of using such antibodies and pharmaceutical compositions are also provided.

[0032] It is to be understood that the embodiments described herein are not limited to particular formulations, compositions and experimental conditions disclosed, as such formulations, compositions, and experimental conditions may vary. It is also to be understood that the terminology used herein is only for the purpose of describing particular embodiments, and it is not intended to be limiting.

[0033] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.

[0034] Those of ordinary skill in the art will understand that given the amino acid sequence of a variable region of the binding molecules (e.g., antibodies) set forth herein can determine the CDR sequences therein by these or any other conventions used to define CDR, such as, for example, IMGT, Kabat, Chothia, or Contact using web-based tools are available for determining the CDRs in such variable regions based on any known convention. Such tools include those found at www. aby si s . org / aby si s / sequence_input / key_annotati on / key_annotati on . cgi and at www.novoprolabs.com / tools / cdr. Accordingly, the disclosure herein of a set of three CDRs in a heavy or light chain variable region based upon one CDR convention is considered the equivalent of that same set of CDRs as determined by any other convention, which can be referred to as “convention equivalents” or “convention equivalent CDRs”.

[0035] Furthermore, the formulations, compositions, and experimental conditions described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunologicaltechniques known within the skill of the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition), by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2ndedition).

[0036] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0037] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions are performed according to manufacturer’ s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0038] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0039] As used herein, the terms “a” or “and” means that “at least one” or “one or more” unless the context clearly indicates otherwise.

[0040] As used herein, the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments. Additionally, where a phrase recites “about x to y”, the term “about” modifies both x and y and can be used interchangeably with the phrase “about x to about y” unless context dictates differently.

[0041] As used herein, the terms “comprising” (and any form of comprising, such as “comprise”, “comprises” and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any step or composition that uses the transitional phrase of “comprise” or “comprising” can also be said to describe the same with the transitional phase of “consisting of’ or “consists of.” Further, the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. For example, reference to “comprising a therapeutic agent” includes one or a plurality of such therapeutic agents. The term “or” refers to a single element of stated alternative elements, unless the context clearly indicates otherwise. For example, the phrase “A or B” refers to A alone or B alone. The phrase “A, B, or a combination thereof’ refers to A alone, B alone, or a combination of A and B. Similarly, “one or more of A and B” refers to A, B, or a combination of both A and B. The phrase “A and B” refers to a combination of A and B. Furthermore, the various elements, features and steps discussed herein, as well as other known equivalents for each such element, feature, or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in particular examples.

[0042] A “disease” in an animal is a state of health wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0043] “Parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intraci sternal injection, intrathecal, intravitreal, or infusion techniques.

[0044] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0045] Throughout this disclosure, various aspects of the embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to havespecifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3, to 6 etc., as well as the individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Unless otherwise explicitly stated to the contrary, a range that is disclosed also includes the endpoints of the range.

[0046] The term “composition” as used herein means a product which results from the mixing or combining of more than one element or ingredient.

[0047] As used herein, the term “pharmaceutical composition” refers to a medicinal or pharmaceutical formulation that contains an active ingredient as well as one or more excipients and diluents to enable the active ingredient suitable for the method of administration.

[0048] The term “carrier” as used herein encompasses carriers, excipients, and diluents, meaning a material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material involved in carrying or transporting a pharmaceutical, cosmetic or other agent across a tissue layer.

[0049] The phrase “pharmaceutically acceptable” is employed herein to refer to those agents of interest / compounds, salts, compositions, pharmaceutical dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and / or other mammals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments, pharmaceutical acceptable means approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for the use in animals (e.g., mammals), and more particularly, in humans.

[0050] As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. The nature of the carrier differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is generally used.

[0051] As used herein, “stable” a “stable composition” or a “stable pharmaceutical composition” refers to a composition or pharmaceutical composition that maintains one or more of the characteristics of the composition within a defined margin when subject to various stressors such as, but not limited to, heat, agitation, light, temperature, humidity, repeated freeze thaw cycles, and extended storage. Such characteristics include, but are not limited to, pH, presence of aggregates, functional titer, or concentrationof protein excipient. In some embodiments, the defined margins for pH, presence of aggregates, functional titer, or concentration of protein excipient are as provided for herein.

[0052] As used herein, the term “antibody” refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized, fully human antibodies, chimeric antibodies, and camelid single domain antibodies. An antibody can comprise four polypeptide chains; two heavy chains and two light chains that can also be connected by disulfide bonds. Each chain is a series of domains: Light chains consist of one variable domain (VL) and one constant domain (CL), while heavy chains contain one variable domain (VH) and three to four constant domains (CHI, CH2, CH3, CH4). Multispecific antibodies may comprise additional polypeptide domains as required to construct additional variable domain pairs (VH and VL) capable of binding a target.

[0053] “Parental antibodies” are antibodies obtained by exposure of an immune system to an antigen prior to modification of the antibodies for an intended use, such as before humanization of an antibody raised in a species other than human, for use as a humanized therapeutic antibody.

[0054] As used herein, unless otherwise indicated, “antibody fragment” or “antigen binding fragment” refers to antigen binding fragments of antibodies, i.e., antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions, or VH and VL pairs. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., scFv; nanobodies and multispecific antibodies formed from antibody fragments.

[0055] A “Fab fragment” is comprised of one light chain and the CHI and variable regions of one heavy chain.

[0056] An “Fc domain” contains two “Fc regions”, which are heavy chain fragments, or a subset of residues located in a complete heavy chain, comprising the CH2 and CH3 domains of an antibody. The Fc domain is held together by disulfide bonds and / or by hydrophobic interactions of the CH3 domains of each Fc region. The Fc region of either heavy chain fragment can also comprise mutations to create homodimers or heterodimers. Non-limiting examples of such mutations are provided for herein.

[0057] As used herein, the term “fused” or “linked” when used in reference to a protein having different domains or heterologous sequences means that the protein domains are part of the same peptide chain that are connected to one another with either peptide bonds or other covalent bonding. The domains or sectioncan be linked or fused directly to one another, or another domain or peptide sequence can be between the two domains or sequences and such sequences would still be considered to be fused or linked to one another. In some embodiments, the various domains or proteins provided for herein are linked or fused directly to one another or via a linker sequence, such as a glycine / serine, glycine / alanine, or other type of peptide linker. Two peptide sequences are linked directly if they are directly connected to one another or indirectly if there is a linker or other structure that links the two regions. A linker can be directly linked to two different peptide sequences or domains.

[0058] A “Fab1fragment” contains one light chain and a portion or fragment of one heavy chain that contains the VH domain and the CHI domain and also the hinge region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab') 2 molecule.

[0059] A “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, the hinge domain, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab') 2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[0060] The “Fv region” comprises the variable regions from both the heavy and light chains but lacks the constant regions.

[0061] The term “single-chain Fv” or “scFv” antibody refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also, International Patent Application Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946, 778 and 5,260,203.

[0062] A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

[0063] A “bivalent antibody” comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. Such bivalent antibodies may thus be biparatopic, such as provided for herein.

[0064] A “single-domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody.

[0065] In some embodiments, as provided for herein, antibody molecules can be monospecific (e.g., monovalent or bivalent), bispecific (e.g., bivalent, trivalent, tetravalent, pentavalent, or hexavalent), trispecific (e.g., trivalent, tetravalent, pentavalent, hexavalent), or with higher orders of specificity (e.g., tetraspecific) and / or higher orders of valency beyond hexavalency. An antibody molecule can comprise a functional fragment of a light chain variable region and a functional fragment of a heavy chain variable region, or heavy and light chains may be fused together into a single polypeptide.

[0066] In certain embodiments, monoclonal antibodies herein also include camelid single domain antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci. 26:230; Reichmann etal. (1999) J. Immunol. Methods 231 :25; WO 94 / 04678; WO 94 / 25591; U.S. Pat. No. 6,005,079). In one embodiment, the invention provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.

[0067] As used herein, the term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat.Biotechnol. 23: 1126-1136.

[0068] Typically, a variant antibody or antigen binding fragment of the antibodies provided herein retain at least 10% of its binding activity (when compared to a parental antibody that is modified) when that activity is expressed on a molar basis. In some embodiments, a variant antibody (or antigen fragment thereof), or antigen binding fragment of an antibody provided herein, retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity as the parental antibody against the antigen. As described herein, it is also intended that an antibody or antigen binding fragment thereof can include conservative or non-conservative amino acid substitutions, which can also be referred to as “conservativevariants” or “function conserved variants” of the antibody, that do not substantially alter its biologic activity.

[0069] “Isolated antibody” refers to the purification status of a binding compound and in such context means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to an absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with experimental or therapeutic use of the binding compound as described herein.

[0070] The term “monoclonal antibody”, as used herein, refers to population of substantially homogeneous antibodies, z.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations and / or post-translational modifications that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs, that are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by the hybridoma method first described by Kohler etal. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624- 628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0071] As used herein, a “chimeric antibody” is an antibody having the variable domain from a first antibody and constant domain from a second antibody, where the first and second antibodies are from different species. (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81 : 6851-6855). Typically, the variable domains are obtained from an antibody from an experimental animal (the “parental antibody”), such as a rodent, and the constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human subject than the parental (e.g. rodent) antibody.

[0072] As used herein, the term “humanized antibody” refers to forms of antibodies that contain sequences from both human and non-human (e.g., murine, rat) antibodies. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.

[0073] The term “fully human antibody” refers to an antibody that comprises human immunoglobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” refers to an antibody that comprises mouse immunoglobulin sequences only. Alternatively, a fully human antibody may contain rat carbohydrate chains if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell. Similarly, “rat antibody” refers to an antibody that comprises rat immunoglobulin sequences only.

[0074] In some embodiments, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy -terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W ., ed., 2nd ed. Raven Press, N.Y. (1989).

[0075] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. However, in bifunctional or bispecific antibodies, the two binding sites are, in general, not the same. The heavy and light chain pair can be on different polypeptides that interact with one another to form the binding site.

[0076] Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminus to C-terminus, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domainis, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al , National Institutes of Health, Bethesda, Md.; 5thed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0077] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” (i.e. residues 24-34 (CDRL1), 50-56 (CDRL2) and 89- 97 (CDRL3) in the light chain variable domain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95- 102 (CDRH3) in the heavy chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) and / or those residues from a “hypervariable loop” (i.e. residues 26-32 (CDRL1), 50-52 (CDRL2) and 91- 96 (CDRL3) in the light chain variable domain and 26-32 (CDRH1), 53-55 (CDRH2) and 96-101 (CDRH3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917). The CDRs can also be referenced according to the IMGT system for the identification of CDRs, which is described in Lefranc MP. Unique database numbering system for immunogenetic analysis. Immunol Today (1997) 18:509. As used herein, the term “framework” or “FR” residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. CDRs of interest can be derived from donor antibody variable heavy and light chain sequences, and include analogs of the naturally occurring CDRs, which analogs also share or retain the same antigen binding specificity and / or neutralizing ability as the donor antibody from which they were derived.

[0078] As used herein, “specific binding” or “immunospecific binding” or “binds immunospecifically” refer to antibody binding to a predetermined antigen at a much higher affinity than for another antigen(s) (e.g. selectively binds FZD4 or LRP5 as compared to other antigens). In some embodiments, the antibody binds the predetermined antigen with a dissociation constant (KD) of 10'7M or less, and such Kois at least two-fold less than its Ko for binding to a non-specific antigen (e.g., BSA, casein, or another non-specific polypeptide).

[0079] The phrases “an antibody recognizing” an antigen and “an antibody specific for” an antigen are used interchangeably herein with the term “an antibody which binds immunospecifically to” the antigen.

[0080] Methods for determining mAb specificity and affinity by competitive inhibition can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589 601 (1983), which references are entirely incorporated herein by reference.

[0081] In some embodiments, the sequences provided herein can be varied to create homologs or variants of the antibodies. The term “homolog” or variant means protein sequences having between 40% and 100% sequence identity to a reference sequence. Percent identity between two peptide chains can be determined by pair wise alignment using the default settings of various software, such as BLAST. In some embodiments, the antibody, or antigen binding fragment thereof has, at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology or identity to a sequence described herein. In some embodiments, the antibody has conservative substitutions as compared to a sequence described herein. Exemplary conservative substitutions are illustrated in Table 1 and are encompassed within the scope of the disclosed subject matter. The conservative substitution may reside in the framework regions, or in antigen-binding sites, as long they do not adversely affect the properties of the antibody. Substitutions may be made to improve antibody properties, for example stability or affinity. Conservative substitutions will produce molecules having functional and chemical characteristics similar to those molecules into which such modifications are made. Exemplary amino acid substitutions are shown in the table below.Table 1: Exemplary Conservative Substitutions

[0082] In some embodiments, variants of the proteins and peptides provided herein are provided. In some embodiments, a variant comprises a substitution, deletions, or insertion. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) substitutions. As described herein, the substitutions can be conservative substitutions. In some embodiments, the substitution is non-conservative. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) deletions. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) insertions. In some embodiments, the substitutions, deletions, or insertions are present in the CDRs provided for herein. In some embodiments, the substitutions, deletions, or insertions are not present in the CDRs provided for herein.

[0083] The term “in combination with” as used herein means that the described agents can be administered to an animal or subject together in a mixture, concurrently as single agents, or sequentially as single agents in any order.

[0084] The term “epitope” is meant to refer to that portion of any molecule capable of being recognized by and bound by an antibody at one or more of the Ab’s antigen binding regions. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0085] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0086] In some embodiments, an antibody, or antigen binding fragment thereof is provided, wherein the antibody or antibody fragment comprises the following polypeptides:EVQL VESGGGL VQPGGSLRLSC AASGFDF S S S SIHW VRQ APGKGLEW VASIS S S YGYT YYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSWAMDYWGQGTLVTV SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSAS DLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYAGAGLITFGQGTKVEIKGGGGSGGGGSEPKS SDKTHTCPPCPAPEAAGGP S VFLFPPKPKDTLMISRTPEVTC VVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNKALPASIEKTISKAKGQPREPQVYTLPPIRELMTSNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGFTL SSYSMHWVRQAPGKGLEWVAYISSYDSITDYADSVKGRFTISADTSKNTAYLQMNSL RAEDTAVYYCARPAVGHMAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 28);EVQLVESGGGLVQPGGSLRLSCAASGFDFTAYAMHWVRQAPGKGLEWVASIYPSGGYTAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRSYYFALDYWGQGT LVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLL IYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYWAYYSPITFGQGTKVE IKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYKCKVSNKALPASIEKTISKAKGQPREPMVFDLPPSREEMTKNQVSLWCMVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSC AASGFTLSSYSMHWVRQAPGKGLEWVAYISSYDSITDYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCARPAVGHMAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 29); andDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQWYNAPITFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 30)

[0087] The polypeptides can be expressed together or separately to form a multispecific antibody that binds to FZD4 and LRP5. In some aspects, one copy of SEQ ID NO: 28, one copy of SEQ ID NO:29, andtwo copies of SEQ ID NO: 30 form a trispecific, tetravalent antibody that binds to FZD4 and LRP5, and may also be referred to EYB-500 herein. The antibody can bind to different epitopes on the antigen as provided for herein. The configuration of numerous alternate antibodies are illustrated in FIG. 2. The Fc portions of the heavy chains associated with one another may contain specific mutations described herein creating an electrostatic protrusion on one chain and an electrostatic crevice on the second chain to further encourage heterodimerization of the heavy chains, and is commonly referred to as Knobs in Holes (KiH).

[0088] Methods for dimerizing peptides via a knob-in-hole configuration are described in WO20 18 / 026942, inventors Van Dyk et al., Carter P. (2001) J. Immunol. Methods 248, 7-15; Ridgway et al. (1996) Protein Eng. 9, 617-621; Merchant, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35. The Fc regions may be Merrimack (knob chain: Q347M, Y349F, T350D, T366W and L368M; hole chain: S354I, E357L, T366S, L368A and Y407V), Merchant (knob chain: T366W; hole chain: T336S, L368A and Y407V) or Merchant S:S (Merchant mutations with additional S354C variant in the knob chain and Y349C in the hole chain). The Fc regions may also contain mutations that alter their effector function, e.g., the Fc region may have attenuated effector functions due to amino acid mutations, e.g., DANG variants and LALAPS variants.

[0089] Other mutations can also be utilized to create this heteromultimer polypeptide and, thus, the Fc regions that are incorporated into these polypeptides can be modified.

[0090] Other examples of antibodies that could be substituted for this antibody are provided for in WO2022130342, which is hereby incorporated by reference in its entirety.

[0091] Accordingly, in some embodiments, the antibody comprises the polypeptides having an amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.

[0092] In some embodiments, the antibody comprises a polypeptide that binds to LRP5, wherein the antibody comprises a HCDR1 comprising the amino acid sequence of FSSSSI (SEQ ID NO: 1), a HCDR2 of SISSSYGYTY (SEQ ID NO: 2); and a HCDR3 of SWAM (SEQ ID NO: 3), a LCDR1 of SVSSA (SEQ ID NO: 10); a LCDR2 of SASDLYS (SEQ ID NO: 11) and AGAGLI (SEQ ID NO: 12).

[0093] In some embodiments, the antibody comprises a polypeptide that binds to LRP5 comprising a HCDR1 of FTAYAM (SEQ ID NO: 7), a HCDR2 of SIYPSGGYTA (SEQ ID NO: 8), and a HCDR3 of RSYYFAL (SEQ ID NO: 9) and a LCDR1 of SVSSA (SEQ ID NO: 4); a LCDR2 of SASSLYS (SEQ ID NO: 5) and a LCDR3 of YWAYYSPI (SEQ ID NO: 6).

[0094] In some embodiments, the antibody comprises a polypeptide that binds to FZD4 comprising a HCDR1 of LSSYSM (SEQ ID NO: 17), a HCDR2 of YISSYDSITD (SEQ ID NO: 18) and a HCDR3 of PAVGHMAF (SEQ ID NO: 19).

[0095] In some embodiments, the polypeptide comprises a VH domain that forms a binding domain with a VL domain to bind LRP5 and a VH domain that can form a binding domain with another VL domain that binds to FZD4. The VL domain can be on a separate polypeptide. In some embodiments, the polypeptide comprises a HCDR1 comprising the amino acid sequence of FSSSSI (SEQ ID NO: 1), a HCDR2 of SISSSYGYTY (SEQ ID NO: 2); and a HCDR3 of SWAM (SEQ ID NO: 3), a LCDR1 of SVSSA (SEQ ID NO: 10); a LCDR2 of SASDLYS (SEQ ID NO: 11) and AGAGLI (SEQ ID NO: 12) that form with another VH and VL domain, to bind to LRP5 and a polypeptide that forms a binding site to bind FZD4 comprising a HCDR1 of LSSYSM (SEQ ID NO: 17), a HCDR2 of YISSYDSITD (SEQ ID NO: 18) and a HCDR3 of PAVGHMAF (SEQ ID NO: 19).

[0096] In some embodiments, the polypeptide comprises a VH domain that forms a binding domain with a VL domain to bind LRP5 and a VH domain that can form a binding domain that binds to FZD4. The VL domain can be on a separate polypeptide. In some embodiments, the polypeptide comprises a HCDR1 of FTAYAM (SEQ ID NO: 7), a HCDR2 of SIYPSGGYTA (SEQ ID NO: 8), and a HCDR3 of RSYYFAL (SEQ ID NO: 9) and a LCDR1 of SVSSA (SEQ ID NO: 4); a LCDR2 of SASSLYS (SEQ ID NO: 5) and a LCDR3 of YWAYYSPI (SEQ ID NO: 6) that form with another VH and VL domain, to bind LRP5 and a polypeptide that forms a binding site to bind FZD4 comprising a HCDR1 of LSSYSM (SEQ ID NO: 17), a HCDR2 of YISSYDSITD (SEQ ID NO: 18) and a HCDR3 of PAVGHMAF (SEQ ID NO: 19).

[0097] In some embodiments, a third polypeptide is provided that provides a FZD4 VL that binds FZD4 in combination with the polypeptide that comprises the VH of FZD4, such as those provided for herein. In some embodiments, the FZD4 VL comprises a LCDR1 of SVSSA (SEQ ID NO: 20), a LCDR2 of SASSLYS (SEQ ID NO: 21); and a LCDR3 of WYNAPI (SEQ ID NO: 22).

[0098] The CDR sequences and antibody sequences provided herein are based on those provided in PCT publication WO2022130342, which is hereby incorporated by reference. These sequences are also provided for in the tables appended hereto, and therefore, the antibody can be according to any of those provided for herein that bind to LRP5 and FZD4.

[0099] In some embodiments, the antibody comprises CDR sets as provided in the tables provided for herein.

[0100] In some embodiments, the antibody comprises the polypeptides of the clones as provided for in the tables provided for herein.

[0101] In some embodiments, the antibody comprises polypeptides comprising a first VH and VL that binds to first LRP5 epitope, a second VH and VL that binds to a second LRP5 epitope, and a VH and VL that binds to FZD4. The VH and VL can be on different polypeptides that interact with one another to form the antigen binding domain. In some embodiments, the VH and VL regions are as provided for in the tables. In some embodiments, the antibody comprises three polypeptides to form such a molecule. In some embodiments, the VH and VL domains that form an antigen binding domain that binds to one or both epitopes of LPR5 can be replaced with other VH and VL domains that binds to another LRP protein.

[0102] The format of the molecule can be as illustrated in the drawings provided for herein and in Example 1.

[0103] In some embodiments, the antibody comprises a light chain variable region (VL) polypeptide as provided in WO2022130342, which is hereby incorporated by reference in its entirety. In some embodiments, the antibody comprises a heavy chain variable region (VH) polypeptide as provided in WO2022130342, which is hereby incorporated by reference in its entirety.

[0104] Accordingly, the antibody, which can be referred to as a tetravalent antibody, comprises a first polypeptide comprising:(a) a first heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1, a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and a CORED having the amino acid sequence of SEQ ID NO: 3;(b) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10, a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 12; and(c) a second VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 17, a CDR-H2 having the amino acid sequence of SEQ ID NO: 18 and a CDR-H3 having the amino acid sequence of SEQ ID NO: 19; a second polypeptide comprising:(a) a first heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 7, a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and a CORED having the amino acid sequence of SEQ ID NO: 9;(b) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4, a CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6;(c) a second VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 17, a CDR-H2 having the amino acid sequence of SEQ ID NO: 18 and a CDR-H3 having the amino acid sequence of SEQ ID NO: 19; and a third polypeptide comprising a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 20, a CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 22; and a fourth polypeptide comprising a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 20, a CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 22.

[0105] In the context of the antibodies for herein, a tetravalent antibody refers to an antibody that comprises 4 antigen binding domains. The tetravalent antibody can bind to 4 different epitopes or fewer. For example, a tetravalent antibody can comprise two antigen binding domains that bind to the same antigen and the same or different epitopes on the antigen. Thus, in some embodiments, the tetravalent antibody comprises two antigen binding domains that bind to LRP5 and two antigen binding domains that binds to FZD4. In some embodiments, the two antigen binding domains that bind to LRP5 bind to different epitopes. In some embodiments the two antigen binding domains that bind to LRP5 bind to a Wntl binding site on LRP5 and a Wnt3 binding site on LRP5. For example, a domain that binds to LRP5 may for example bind to a Wnt 1 binding site on LRP5 or a Wnt 3 binding site on LRP5.In some embodiments, the two antigen binding domains that bind to LRP5 bind to the same epitopes. In some embodiments, the two antigen binding domains that bind to FZD4 are the same, that is comprise the same VH and VL and CDRs contained therein. In some embodiments, the two antigen binding domains that bind to FZD4 have the same CDRs but different VH and VL framework regions. In some embodiments, the two antigen binding domains that bind to FZD4 have different CDRs and / or bind to different epitopes.

[0106] In some embodiments, the antibody, the first polypeptide further comprises a Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3), and the first polypeptide further comprises a constant heavy chain domain 1 (CHI); the second polypeptide further comprises a Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavychain domain 3 (CH3), and the second polypeptide further comprises a constant heavy chain domain 1 (CHI); the third polypeptide further comprises a constant light chain domain 1 (CLI); and the fourth polypeptide further comprises a constant light chain domain 1 (CLI).

[0107] In some embodiments, the Fc region of the first polypeptide and the second polypeptide are different and the CLi domain of the third polypeptide and the fourth polypeptide are the same.

[0108] In some embodiments, the first VH of the first polypeptide interacts with the VL of the second polypeptide to form a domain that binds LRP5; the first VH of the second polypeptide interacts with the VL of the first polypeptide to form a domain that binds LRP5; the second VH of the first polypeptide interacts with the VL of the third polypeptide to form a domain that binds FZD4; and the second VH of the second polypeptide interacts with the VL of the fourth polypeptide to form a domain that binds FZD4.

[0109] In some embodiments, the first polypeptide and the second polypeptide form a heterodimer. In some embodiments, the first polypeptide and the second polypeptide form a heterodimer through the Fc region of the first polypeptide and the second polypeptide. In some embodiments, the Fc region of the first polypeptide comprises a serine at position 366, an alanine at position 368 and a valine at position 407; and the Fc region of the second polypeptide comprises a tryptophan at position 366, wherein the positions are according to EU numbering. In some embodiments, the Fc region of the first polypeptide further comprises an isoleucine at position 354 and a leucine at position 357, and the Fc region of the second polypeptide further comprises a methionine at position 347, a phenylalanine at position 349, an aspartic acid at position 350 and a methionine a position 368, wherein the positions are according to EU numbering. In some embodiments, the first polypeptide comprises a cysteine at position 349, and the second polypeptide comprises a cysteine at position 354, wherein the positions are according to EU numbering.

[0110] In some embodiments, the Fc regions of each of the first polypeptide and the second polypeptide lack effector function or have reduced effector function. In some embodiments, the Fc regions of the first polypeptide and the second polypeptide comprise a glycine at position 397, an alanine at position 265, or both a glycine at position 397 and an alanine at position 265, wherein the positions according to EU numbering. In some embodiments, the Fc regions of the first polypeptide and the second polypeptide comprise an alanine at position 234, an alanine at position 235, a serine at position 331, or any combination thereof, wherein the positions are according to EU numbering. In some embodiments, the Fc regions of the first polypeptide and the second polypeptide comprise an alanine at position 234 andan alanine at position 235, wherein the positions are according to EU numbering. In some embodiments, the Fc regions of the first polypeptide and the second polypeptide comprise an alanine at position 234, an alanine at position 235, and a serine a position 331, wherein the positions are according to EU numbering.[OHl] In some embodiments, the antibody comprising the first polypeptide and the second polypeptide, wherein: the first polypeptide from N-terminus to C-terminus comprises: i) the first heavy chain variable domain; ii) the light chain variable domain; iii) a Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3); iv) the second heavy chain variable domain; and v) the constant heavy chain domain 1 (CHI), wherein the light chain variable domain of the first polypeptide and the second heavy chain variable domain of the first polypeptide are each, independently, attached to the first Fc region of the first polypeptide by a polypeptide linker; the second polypeptide from N-terminus to C-terminus comprises: i) the first heavy chain variable domain; ii) the light chain variable domain; iii) a Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3); iv) the second heavy chain variable domain; and v) the constant heavy chain domain 1 (CHI), wherein the light chain variable domain of the second polypeptide and the second heavy chain variable domain of the second polypeptide are each, independently, attached to the first Fc region of the second polypeptide by a polypeptide linker.

[0112] In some embodiments, the light chain variable domain of the of the first polypeptide and the light chain variable domain of the second polypeptide are linked to the N-terminus of the Fc region of the first polypeptide and / or the second polypeptide, respectively, by a polypeptide linker comprising the amino acid sequence of GGGGSGGGGSEPKSSDKTHT (SEQ ID NO: 26).

[0113] In some embodiments, the second heavy chain variable domain of the first polypeptide and the second heavy chain variable domain of the second polypeptide are linked to the C-terminus of the Fc region of the first polypeptide and the second polypeptide, respectively, by a polypeptide linker comprising the amino acid sequence of GGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 27).

[0114] In some embodiments, the first VH of the first polypeptide is linked to the VL of the first polypeptide by a polypeptide linker comprising the acid sequence of GGGGS (SEQ ID NO: 25); and the first VH of the second polypeptide is linked to the VL of the second polypeptide by a polypeptide linker comprising the acid sequence of GGGGS (SEQ ID NO: 25). In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 29, and the third polypeptide and the fourth polypeptide each comprise the sequence of SEQ ID NO: 30.

[0115] Methods for making such antibodies are provided for in WO2022 / 130342, which is hereby incorporated by reference in its entirety.COMPOSITIONS, DOSES, AND METHODS OF ADMINISTRATION

[0116] In some embodiments, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises an antibody or antigen-binding fragment thereof as provided for herein.

[0117] In some embodiments, to prepare pharmaceutical or sterile compositions of the antibodies or other proteins provided herein, the antibody, or antigen binding fragment thereof, or other proteins provided herein are admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0118] Formulations of therapeutic or the antibodies provided herein may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman ’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al.(eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, el al.(eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner andKotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In some embodiments, the antibodies are diluted to an appropriate concentration in a sodium acetate solution pH 5-6, and NaCl or sucrose is added for tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.

[0119] Toxicity and therapeutic efficacy of the antibody compositions, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LDso / EDso). In particular aspects, antibodies exhibiting high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

[0120] In some embodiments, a composition is administered to a subject in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).

[0121] The mode of administration can vary. Suitable routes of administration include intravitreal, oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial. In some embodiments, the composition is an injectable pharmaceutical composition. In some embodiments, the composition is formulated for intravenous or subcutaneous injection. In some embodiments, the composition is formulated for intravenous injection. In some embodiments, the composition is formulated for subcutaneous injection.

[0122] In some embodiments, the antibody, or antigen binding fragment thereof, can be administered by an invasive route such as by injection. In some embodiments, the antibodies or antigen binding fragment thereof, or pharmaceutical composition thereof, is administered intravitreally, intraocularly, intravenously, subcutaneously, intramuscularly, or intraarterially. In some embodiments, the composition is administered intravitreally.

[0123] In some embodiments, the antibodies provided for herein, or antigen binding fragment thereof, is administered in combination with at least one additional therapeutic agent, such as, but not limited toany therapeutic used to treat the disorders provided for herein. In some embodiments, the antibody is administered in combination with another treatment for the disorders provided for herein.

[0124] Compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector.

[0125] The pharmaceutical compositions may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

[0126] In some embodiments, the pharmaceutical composition can also be administered in the form of eye drops.

[0127] The pharmaceutical compositions may also be administered by infusion. Examples of well- known implants and modules form administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0128] Alternately, one may administer the antibody in a local rather than systemic manner, for example, via injection of the antibody directly into the eye or via eye drops into the eye.

[0129] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies is available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY ; Baert, el al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341 : 1966-1973;Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602).

[0130] Determination of the appropriate dose can be made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. In general, it is desirable that a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent. In the case of human subjects, for example, chimeric, humanized and fully human antibodies may be desirable.

[0131] Polypeptides, antibodies, or antigen binding fragments thereof provided for herein may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, biweekly, every three weeks, monthly, bimonthly, or quarterly. The dose can be administered to each eye, such as at the doses provided for herein.

[0132] Accordingly, in some embodiments, provided are methods of administering a polypeptide, an antibody, or an antigen-binding fragment thereof, such as those provided herein.

[0133] In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose from about 0.05 mg to about 1 mg. In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.05 mg. In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.15 mg. In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.40 mg. In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.48 mg. In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.50 mg. In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.75 mg. In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.80 mg. In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose from about 0.50 mg to about 0.90 mg. In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose from about 0.40 mg to about 0.80 mg.

[0134] In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.05 mg every 4 weeks (Q4W). In some embodiments, the antibody, or antigen binding fragmentthereof, is administered at a dose of about 0.15 mg every 4 weeks (Q4W). In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.40 mg every 4 weeks (Q4W). In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.48 mg every 4 weeks (Q4W). In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.50 mg every 4 weeks (Q4W). In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.75 mg every 4 weeks (Q4W). In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose of about 0.80 mg every 4 weeks (Q4W). In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose from about 0.50 mg to about 0.90 mg every 4 weeks (Q4W). In some embodiments, the antibody, or antigen binding fragment thereof, is administered at a dose from about 0.40 mg to about 0.80 mg every 4 weeks (Q4W).

[0135] In some embodiments, the antibody, or antigen binding fragment thereof, is administered in a volume of about 25 to about 100 pL to an eye, such as by intravitreal administration. In some embodiments, the antibody, or antigen binding fragment thereof, is administered in a volume of about 40 to about 60 pL. In some embodiments, the antibody, or antigen binding fragment thereof, is administered in a volume of about 50 to about 100 pL. In some embodiments, the antibody, or antigen binding fragment thereof, is administered in a volume of about 45 to about 55 pL. In some embodiments, the antibody, or antigen binding fragment thereof, is administered in a volume of about 50 pL.

[0136] In some embodiments, the concentration of the antibody in the volume being administered is about 0.5 mg / mL to about 16 mg / mL, about 4 mg / mL to about 16 mg / mL. In some embodiments, the concentration is about 8 mg / mL. In some embodiments, the concentration is about 16 mg / mL. In some embodiments, the concentration is about 10 mg / mL.

[0137] In some embodiments, a dosage form in a container is provided. In some embodiments, the dosage form comprises a pharmaceutical composition such as those provided herein. In some embodiments, the container is a plastic vial or a glass vial.

[0138] In some embodiments, a polypeptide, an antibody, or an antigen-binding fragment thereof, such as those provided for herein, is administered as part of a pharmaceutical composition comprising the polypeptide, the antibody, or the antigen-binding fragment thereof, such as those provided for herein, and at least one pharmaceutically acceptable excipient.

[0139] Polypeptides, antibodies, antigen-binding fragments thereof, and pharmaceutical compositions may be administered with medical devices known in the art. For example, a pharmaceutical compositionof the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector.

[0140] In some embodiments, a kit, comprising a pharmaceutical composition, such as those provided herein, is provided.

[0141] In some embodiments, to prepare pharmaceutical or sterile compositions of the antibodies or other proteins provided herein, the antibody or antigen binding fragment thereof or other proteins provided herein are admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0142] Polypeptides, antibodies, antigen-binding fragments thereof, and pharmaceutical compositions may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman ’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis,, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In some embodiments, the formulation be an aqueous formulation.

[0143] In some embodiments, the polypeptide, the antibody, the antigen-binding fragment thereof, or the pharmaceutical composition can be administered by an invasive route such as by injection. In some embodiments, the polypeptide, the antibody, the antigen-binding fragment thereof, or the pharmaceutical composition is administered intravitreally, intravenously, subcutaneously, intramuscularly, intraarterially, intra-articularly (e.g., in arthritis joints), or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present embodiments.

[0144] The polypeptide, the antibody, the antigen-binding fragment thereof, or the pharmaceutical composition may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

[0145] The polypeptide, the antibody, the antigen-binding fragment thereof, or the pharmaceutical composition may also be administered by infusion. Examples of well-known implants and modules form administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0146] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection to the tissue of interest, such as the eye or retina.

[0147] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies is available (see, e.g., Wawrzynczak 1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies andPeptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, etal. (2003) New Engl. J. Med. 348:601- 608; Milgrom et al. (1999) New Engl. J. Med. 341 : 1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602).

[0148] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. In general, it is desirable that a biologic that will be used is derived from the same species as the animal targeted fortreatment, thereby minimizing any immune response to the reagent. In the case of human subjects, for example, chimeric, humanized and fully human antibodies may be desirable.

[0149] The antibody, the antigen-binding fragment thereof, or the pharmaceutical composition provided herein can be administered once a week, once every two weeks, once every three weeks, once every four weeks, monthly, bimonthly (every two months), or quarterly. In some embodiments, the antibody is administered every four weeks, which can be represented by the term “Q4W”.

[0150] As used herein, “inhibit” or “treat” or “treatment” includes a postponement of development of the symptoms associated with a disorder and / or a reduction in the severity of the symptoms of such disorder. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject with a disorder, disease, or symptom, or with the potential to develop such a disorder, disease, or symptom.

[0151] As used herein, the terms “therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to an amount of the antibody, or antigen binding fragment thereof, that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition. A therapeutically effective dose further refers to that amount of the binding compound sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention, or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity. In some embodiments, an amount is a therapeutically effective amount if it is an amount that can be used to treat or ameliorate a condition as provided for herein.

[0152] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective toachieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that when administered to a mammal, causes a detectable level of immune cell activation compared to the immune cell activation detected in the absence of the composition. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0153] As used herein, the term “individual” or “subject” or “patient” used interchangeably, means any organism, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans. In one embodiment, the subject is a human. A subject can also be referred to as a patient. In some embodiments, the subject is a subject in need thereof. A subject that is “in need thereof’ refers to a subject that has been identified as requiring treatment for the condition that is to be treated and is treated with the specific intent of treating such condition. The conditions can be, for example, any of the conditions described herein.

[0154] Accordingly, in some embodiments, methods of treating a subject with an ocular disorder are provided. In some embodiments. Pharmaceutical compositions comprising the antibody that binds to LRP5 and FZD4 for use in treating an ocular disorder in a subject in need thereof, are provided. In some embodiments, the treatment comprises administering about 0.05 mg to about 1.00 mg of said pharmaceutical composition (e.g., intravitreally) to the subject in a volume of about 100 pL or less. In some embodiments, uses of a pharmaceutical composition comprising the antibody that binds to LRP5 and FZD4 in the manufacture of a medicament for treating an ocular disorder in a subject in need thereof are provided. In some embodiments, the treatment comprises administering about 0.05 mg to about 1.00 mg of said pharmaceutical composition to the subject in a volume of about 100 pL or less.

[0155] In some embodiments, the method comprises administering a pharmaceutical composition comprising an antibody, or antigen binding fragment thereof, as provided herein. As provided for herein, the antibodies, or antigen binding fragments thereof, can be administered with other therapeutics. These can be administered simultaneously or sequentially. In some embodiments, the antibody is administered with aflibercept. In some embodiments, the amount of aflibercept is at least 2, 4, 6, or 8 mg. In some embodiments, the additional therapeutic is an anti-VEGF therapy, such as, but not limited to, an antibodythat targets the VEGF pathway. Non-limiting examples of such antibodies include bevacizumab, ranibizumab, and the like.

[0156] In some embodiments, the subject is a subject who has previously been treated with a different antibody than those provided herein.

[0157] Kits are also provided which are useful for carrying out embodiments described herein. The present kits comprise a first container containing or packaged in association with the above-described antibodies. The kit may also comprise another container containing or packaged in association solutions necessary or convenient for carrying out the embodiments. The containers can be made of glass, plastic or foil and can be a vial, bottle, pouch, tube, bag, etc. The kit may also contain written information, such as procedures for carrying out the embodiments or analytical information, such as the amount of reagent contained in the first container means. The container may be in another container apparatus, e.g., a box or a bag, along with the written information.

[0158] In some embodiments, the antibody comprises a sequence as provided for herein or antigen binding fragment thereof. In some embodiments, the antibody comprises a heavy chain CDR or an antigen binding fragment thereof described herein. The heavy chain may be one or more of the heavy chains described herein. In some embodiments, the antibody comprises a light chain, or an antigen binding fragment thereof as described herein.METHODS OF USE

[0159] The present antibodies and compositions provided for herein can be used to treat ocular disorders. Examples of such ocular disorders include, but are not limited to, disorders characterized by vascular leakage, particularly retinal vascular leakage, and / or endothelial cell leakage, and disorders characterized by reduced retinal or brain endothelial cell barrier functions or a compromised BBB or BRB, e.g., diabetic retinopathy, retinopathy of prematurity, Coat’s disease, FEVR, Norrie disease, macular degeneration, diabetic macular edema, and pediatric vitreoretinopathies. Other disorders include visual impairment. In some embodiments the visual impairment can be associated with or due to diabetic macular edema. In some embodiments, the disorder is NV AMD. Other disorders also include, but are not limited to, retinal vein occlusion and other causes of choroidal neovascularization (CNV) besides age-related macular degeneration (e.g., myopic macular degeneration, histoplasmosis).

[0160] In some embodiments, the visual impairment is visual impairment secondary to diabetic macular edema. In some embodiments, the ocular disorder is diabetic macular edema (DME). In some embodiments, the ocular disorder is neovascular age-related macular degeneration (NV AMD).

[0161] In some embodiments, the subject that is treated has an increase in Best Corrected Visual Acuity (BCVA) after being treated. In some embodiments, the BCVA is increased about 1 to about 15 letters, about 5 to about 15 letters, or about 10 to about 15 letters. In some embodiments, the BCVA is measured using the standardized ETDRS (Early Treatment of Diabetic Retinopathy Study) chart. In some embodiments, the BCVA is improved at, or after, about 1 week, 2 weeks, 1 month, 2 months, 3 months, about 6 months, about 9 months, about 48 weeks to about 52 weeks, about 1 year, about 48 weeks, or 2 years.

[0162] BCVA measurement can be, for example, performed according to the standard ETDRS refraction and visual acuity testing protocol. For example, the test can be performed at 4 meters first from the eye chart. The participant can be asked to read the letters slowly, approximately one letter per second. The participant can be told that only one chance is given to read each letter but may change their mind before moving to the next letter. If the participant is unsure about the identity of the letter, then the participant can be encouraged to guess. The participant should begin by reading the top line of the chart and continue reading every letter on each smaller line, from left to right on each line. The participant should be encouraged to continue reading even if making mistakes. Each letter read is counted. The examiner circles every correct letter read and totals each line and the whole column (0 if no letters are correct) on the data collection form. An X is put through letters read incorrectly. Letters, for which no guess was attempted, are not marked. When a participant reaches a level where he / she cannot guess, the examiner may stop the test provided that the participant has made errors on previous guesses, which is a clear indication that the best visual acuity has been obtained. When a participant cannot read at least 20 letters on the chart at 4.0 meters, the participant is tested at 1.0 meter. The distance from the participant to the chart should be measured again using the rigid one-meter stick. The distance is measured from the outer canthus to the center of the fourth letter (right eye) or the second letter (left eye) of the third line of the chart. The spherical correction in the trial frame should be changed by adding +0.75 to correct for the closer test distance. The participant may fixate eccentrically or turn or shake his / her head to improve visual acuity. Particular care should be taken to make sure the participant does not move forward when testing at 1 meter. The participant should be reminded to blink. When 20 or more letters are read at 4 meters the visual acuity score for that eye is recorded as the number of letters correct at 4 meters plus 30. The participantgets credit for the 30 IM letters even though they did not have to read them. Otherwise, the visual acuity score is the number of letters read correctly at 1.0 meter plus the number, if any, read at 4 meters. If no letters are read correctly at either 4.0 meters or 1 meter, then the visual acuity score is recorded as 0.

[0163] In some embodiments, the retinal thickness is decreased in the subject. In some embodiments, the central subfield thickness (CST) of the retina is reduced. In some embodiments, the CST is reduced by about 100 pm to about 150 pm. In some embodiments, the CST is measured by optical coherence tomography.

[0164] In some embodiments, the patient or subject is not one that is pregnant or breastfeeding.

[0165] In some embodiments, the patient or subject is not one that, if being treated for DME, have a history of any of the following treatments within the noted time windows: have had an IVT anti-VEGF treatment (aflibercept, ranibizumab, bevacizumab, brolucizumab, pegaptanib sodium) in the study eye within 12 weeks (84 days) of being treated; had prior IVT investigational agents in either eye at any time; had treatment with ocriplasmin (JETREA®) in the study eye at any time; had previous use of intraocular or periocular corticosteroids in the study eye within 16 weeks (112 days) of being treated.

[0166] In some embodiments, the patient or subject is not one that has had previous use ILUVIEN® or OZURDEX® IVT implants at any time.

[0167] In some embodiments the subject has not previously been treated with a treatment for the ocular disorder, such as DME or visual impairment, including visual impairment associated with DME.

[0168] Throughout the present disclosure, all expressions of percentage, ratio, and the like are “by weight” unless otherwise indicated. As used herein, “by weight” is synonymous with the term “by mass,” and indicates that a ratio or percentage defined herein is done according to weight rather than volume, thickness, or some other measure.Table 2: Sequences

[0169] The subject matter is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the claims should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.EXAMPLESExample 1

[0170] Tetravalent Antibody for the treatment of Diabetic Macular Edema and Neovascular Age- related Macular Degeneration: Results from a phase lb / 2a clinical trial. EYB-500, which is a tetravalent, tri-specific antibody, was tested in a Phase lb / 2a clinical trial for the treatment of diabetic macular edema (DME) and neovascular age-related macular degeneration (NV AMD). The antibody contains the polypeptides of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30 to form the tetraval ent antibody. The antibody can be visualized as shown in FIG. 2.

[0171] 4 cohorts were tested in a multiple ascending dose (MAD) trial. The doses were 0.05 mg, 0.15 mg, 0.48 mg, and 0.80 mg. Patients were treated for diabetic macular edema or NV AMD. The patients were previously untreated. The antibody was administered in an aqueous solution. Those treated for NV AMD were treated with a combination of the antibody and aflibercept (2 mg). The treatments were administered intravitreally. Patients were treated on Day 1, Week 4 or Week 8 and followed through Week 12.

[0172] The key inclusion / exclusion criteria for NV AMD were as follows: Diagnosed with NV AMD; treatment naive; BCVA range 65 to 35 letters (20 / 50 to 20 / 200); CST > 350 pm; and those that were excluded were those subject that had large PED or other confounding ocular pathology.

[0173] The key inclusion / exclusion criteria for DMA were as follows: diagnosed with DME; treatment naive; BCVA range 65 to 35 letters (20 / 50 to 20 / 200); CST > 350 pm and subjects were excluded PDR or confounding ocular pathology.

[0174] For the Phase 2a part of the trial, only subjects with DME were treated.

[0175] The primary endpoints were to assess the safety and tolerability of 3 repeat doses of intravitreal EYB-500 given every 4 weeks in an ascending dose escalation fashion. The secondary endpoints were to assess preliminary efficacy of intravitreal antibody administration in patients with diabetic macular edema (DME) or neovascular age-related macular degeneration (NV AMD).

[0176] The patient demographics and baseline characteristics were as follows.

[0177] 31 of the 33 patients completed the study. No rescue therapy was given to any participant. The patients that completed the study were as follows: DME=26 (monotherapy); NVAMD= 5 (in combination with aflibercept 2mg). No drug related SAE or AE were observed. There was also no evidence of intraocular inflammation or vasculitis.

[0178] Patients being treated for DME were found to have an increase of BCVA with a mean change in BCVA at week 12 of 11.2 letters (range of -4 to 24). The mean was found not to be driven by outliers. No need for rescue in any patient. The mean and median change is illustrated in the following Table.Gain in ETDRS BCVA (Baseline 59.6 Liters)N= 26 at all timepoints, per protocol population shown

[0179] The subject’s CST was also measured and found to have a mean change of 143 pm. The mean was not driven by any outliers. The data is represented in the following table.Absolute Reduction in OCT CST (um)N= 26 at all timepoints, per protocol population shown

[0180] At 12 weeks the data can be summarized as follows. There was Mean visual acuity change: +11.2 letters; mean reduction in excess OCT CST thickness: 80%; absolute reduction in OCT CST thickness: - 143 um (-27%); continued improvement with sequential doses; data are mean of 0.15, 0.48, 0.80 mg dose groups; all dose levels were effective; and Statistical outliers did not drive mean changes.

[0181] The data over the period of 12 weeks for % change in excess OCT thickness and absolute OCT thickness are shown in the following table:

[0182] For patients being treated for NV AMD with the combination of EYB-500 and aflibercept, the following results were obtained. The patient (n=5) had a mean VA change: +6.8 letters; mean reduction in excess OCT CST thickness: 100%; absolute reduction in OCT CST thickness: -268um (51%); and data are mean of 0.05, 0.48, 0.80 mg dose groups. All dose levels were effective and statistical outliers did not drive mean changes. The data are shown in the following tables.12 Week NVAMD BCVA: Gain in ETDRS BCVA (Baseline 47.6 Liters)% Change in Excess OCT Thickness (>300 pM)

[0183] The conclusions of the study found that antibody was well tolerated without observing any safety signals. The structural and functional improvements are encouraging and teach that the antibody is a valuable approach to treated exudative retinal diseases, which can be performed independent of VEGF inhibitors.Example 2

[0184] A randomized, double-masked, multi-center, 3-arm phase 2 / 3 study to evaluate the efficacy and safety of intravitreal tetravalent antibody compared with intravitreal ranibizumab (0.5mg) in participants with visual impairment secondary to diabetic macular edema.

[0185] The trial is designed with the following primary and secondary endpoints.

[0186] Primary Endpoint: To demonstrate that EYB-500 (0.5mg or 0.8mg per eye) is non-inferior (NI) to ranibizumab 0.5mg, as measured through the mean change in best-corrected visual acuity (BCVA) using the standardized ETDRS (Early Treatment of Diabetic Retinopathy Study) chart from Day 1 to Year 1 (average of Week 48 and Week 52).

[0187] Secondary Endpoint Efficacy Objectives:a. To evaluate the efficacy of EYB-500 (0.5mg or 0.8mg) compared to ranibizumab 0.5 mg over the study duration by assessing changes in visual outcomes from Day 1 to Year 1 (average of Week 48 and Week 52); b. To evaluate the efficacy of EYB-500 (0.5mg or 0.8mg) compared to ranibizumab 0.5 mg by assessing changes in structural outcomes from Day 1 to Week 52; c. To evaluate the mean change in BCVA using the standardized ETDRS chart from Day 1 to Year 2 (average of Week 100 and Week 104); d. To evaluate the efficacy of EYB-500 (0.5mg or 0.8mg) compared to ranibizumab 0.5 mg over the study duration by assessing changes in visual outcomes from Day 1 to Year 2 (average of Week 100 and Week 104); e. To evaluate the efficacy of EYB-500 (0.5mg and 0.8mg) compared to ranibizumab 0.5 mg by assessing changes in structural outcomes from Day 1 to Week 104

[0188] The secondary efficacy endpoints can also be summarized as follows: a. From Day 1 to Year 1 (average of Week 48 and Week 52): the proportion of participants who gain > 5, >10 and >15 ETDRS letters and the proportion of participants who lose > 5, >10 and >15 ETDRS letters; b. From Day 1 to Week 52: the mean change in optical coherence tomography (OCT) central subfield thickness (CST) and the ability to lead to an absence of intraretinal and / or subretinal fluid over time.

[0189] Additionally, the systemic PKs (exposure) of EYB-500 and immunogenicity of EYB-500 will be measured. From the time of randomization of treatment, each participant will be in the study for up to 104 weeks, which includes a 100-week Treatment Period and a Safety Follow-Up visit approximately 4 weeks after the last injection. The additional Screening Period can last up to 28 days. EYB-500 (0.5 mg or 0.8 mg, depending on treatment assignment) will be administered by intravitreal (IVT) injection with a volume of 50 pL. The IVT injection schedule is Q4W.

[0190] The present embodiments are not to be limited in scope by the specific embodiments described herein. Indeed, various modifications in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the embodiments and any appended claims.

Claims

WHAT IS CLAIMED:

1. A method of treating an ocular disorder in a subject in need thereof, comprising administering to the eye of the subject, a pharmaceutical composition comprising about 0.05 mg to about 1.00 mg of an antibody that binds to LRP5 and FZD4 in a volume of about 100 pL or less.2 A pharmaceutical composition comprising about 0.05 mg to about 1.00 mg of an antibody that binds to LRP5 and FZD4 for use in treating an ocular disorder in a subject in need thereof, wherein the treatment comprises administering about 0.05 mg to about 1.00 mg of said pharmaceutical composition intravitreally to the subject in a volume of about 100 pL or less.3 Use of a pharmaceutical composition comprising about 0.05 mg to about 1.00 mg of an antibody that binds to LRP5 and FZD4 in the manufacture of a medicament for treating an ocular disorder in a subject in need thereof, wherein the treatment comprises administering about 0.05 mg to about 1.00 mg of said pharmaceutical composition intravitreally to the subject in a volume of about 100 pL or less.4 The method, pharmaceutical composition, or use of any one of claims 1-3, wherein the pharmaceutical composition comprises about 0.05 mg, about 0.15 mg, about 0.40 mg, about 0.48 mg, about 0.50 mg, or about 0.80 mg of the antibody.5 The method, pharmaceutical composition, or use of any one of claims 1-4, wherein the pharmaceutical composition comprises about 0.40 mg, about 0.50 mg, or about 0.80 mg of the antibody.6 The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the pharmaceutical composition is administered to the subject every two weeks, every 4 weeks, or every 8 weeks.7 The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the pharmaceutical composition is administered every 4 weeks (Q4W).

8. The method, pharmaceutical composition, or use of any one of the preceding claims wherein the pharmaceutical composition is administered in a volume of about 50 pL to about 100 pL.

9. The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the subject is administered the pharmaceutical composition comprising about 0.40 mg, 0.50 mg, or 0.80 mg of the antibody in a volume of about 50 pL.

10. The method, pharmaceutical composition, or use of any one of claims 1-6, wherein the pharmaceutical composition is administered every 8 weeks (Q8W).

11. The method, pharmaceutical composition, or use of any one of claims 1-6, wherein the subject is administered a pharmaceutical composition comprising about 0.80 mg of the antibody in a volume of about 50 pL.

12. The method, pharmaceutical composition, or use any one of claims 1-6, wherein the pharmaceutical composition is administered every 2 weeks (Q2W).

13. The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the pharmaceutical composition is administered intravitreally.

14. The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the ocular disorder is visual impairment.

15. The method, pharmaceutical composition, or use of claim 14, wherein the visual impairment is visual impairment secondary to diabetic macular edema (DME).

16. The method, pharmaceutical composition, or use of any one of claims 1-13, wherein the ocular disorder is diabetic macular edema (DME).

17. The method, pharmaceutical composition, or use of any one of claims 1-13, wherein the ocular disorder is neovascular age-related macular degeneration (NV AMD).

18. The method, pharmaceutical composition, or use of any one of claims 1-17, wherein the subject’s Best Corrected Visual Acuity (BCVA) is increased after being treated.

19. The method, pharmaceutical composition, or use of claim 18, wherein the BCVA is increased about 10 to about 15 letters.

20. The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the subject’s retinal thickness is decreased after administration.

21. The method, pharmaceutical composition, or use of claim 20, wherein the subj ect’ s central subfield thickness (CST) of the retina is reduced after administration.

22. The method, pharmaceutical composition, or use of claim 21, wherein the CST is reduced by about 100 pm to about 150 pm.

23. The method, pharmaceutical composition, or use of claims 21 or 22, wherein the CST is measured by optical coherence tomography.

24. The method, pharmaceutical composition, or use of any one of claims 1-23, wherein the subject is being treated for vascular leakage, retinal vascular leakage, and / or endothelial cell leakage, and / or disorders characterized by reduced retinal or brain endothelial cell barrier functions, a compromised blood-brain barrier (BBB) or blood-retinal barrier (BRB), or diabetic retinopathy, retinopathy of prematurity, Coat’s disease, familial exudative vitreoretinopathy (FEVR), Norrie disease, macular degeneration, diabetic macular edema, or pediatric vitreoretinopathy.

25. The method, pharmaceutical composition, or use of any one of claims 1-13, wherein the ocular disorder is NV AMD, and the subject is administered the antibody in combination with aflibercept.

26. The method, pharmaceutical composition, or use of claim 25, wherein the subject has an increase in BCVA.

27. The method, pharmaceutical composition, or use of claim 26, wherein the BCVA is increased from about 5 to about 10 letters.

28. The method, pharmaceutical composition, or use of any one of claims 25-27, wherein the subject’s CST thickness is reduced by at least about 50% after administration.

29. The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the subject has an increase in best-corrected visual acuity (BCVA) using the standardized ETDRS (Early Treatment of Diabetic Retinopathy Study) chart after administration.

30. The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the subject has an improvement in visual outcomes after administration.

31. The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the subject has an improvement in at least one symptom after about 1 year of being treated with the antibody.

32. The method, pharmaceutical composition, or use of any one of the preceding claims, wherein the subject has an improvement in at least one symptom after about 2 years of being treated with the antibody.

33. The method, pharmaceutical composition, or use of any one of the preceding claims wherein the antibody is a tetravalent antibody.

34. The method, pharmaceutical composition, or use of claim 33, wherein the tetravalent antibody comprises: a first polypeptide comprising:(a) a first heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1, a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and a CORED having the amino acid sequence of SEQ ID NO: 3;(b) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10, a CDR-L2 having the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 12; and(c) a second VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 17, a CDR-H2 having the amino acid sequence of SEQ ID NO: 18 and a CDR-H3 having the amino acid sequence of SEQ ID NO: 19; a second polypeptide comprising:(a) a first heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 7, a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and a CORED having the amino acid sequence of SEQ ID NO: 9;(b) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4, a CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6;(c) a second VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 17, a CDR-H2 having the amino acid sequence of SEQ ID NO: 18 and a CDR-H3 having the amino acid sequence of SEQ ID NO: 19; and a third polypeptide comprising a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 20, a CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 22; and a fourth polypeptide comprising a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 20, a CDR-L2 having the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 22.

35. The method, pharmaceutical composition, or use of claim 34, wherein: the first polypeptide further comprises a Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3), and the first polypeptide further comprises a constant heavy chain domain 1 (CHI); the second polypeptide further comprises a Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3), and the second polypeptide further comprises a constant heavy chain domain 1 (CHI); the third polypeptide further comprises a constant light chain domain 1 (CLI); andthe fourth polypeptide further comprises a constant light chain domain 1 (CL1).

36. The method, pharmaceutical composition, or use of claim 35, wherein the Fc region of the first polypeptide and the second polypeptide are different and the CL1 domain of the third polypeptide and the fourth polypeptide are the same.

37. The method, pharmaceutical composition, or use of any one of claims 34-36, wherein: the first VH of the first polypeptide interacts with the VL of the second polypeptide to form a domain that binds LRP5; the first VH of the second polypeptide interacts with the VL of the first polypeptide to form a domain that binds LRP5; the second VH of the first polypeptide interacts with the VL of the third polypeptide to form a domain that binds FZD4; and the second VH of the second polypeptide interacts with the VL of the fourth polypeptide to form a domain that binds FZD4.

38. The method, pharmaceutical composition, or use of claim 37, wherein the first polypeptide and the second polypeptide form a heterodimer.

39. The method, pharmaceutical composition, or use of claim 38, wherein the first polypeptide and the second polypeptide form a heterodimer through the Fc region of the first polypeptide and the second polypeptide.

40. The method, pharmaceutical composition, or use of any one of claims 34-39, wherein the Fc region of the first polypeptide comprises a serine at position 366, an alanine at position 368 and a valine at position 407; and the Fc region of the second polypeptide comprises a tryptophan at position 366, wherein the positions are according to EU numbering.

41. The method, pharmaceutical composition, or use of claim 40, wherein the Fc region of the first polypeptide further comprises an isoleucine at position 354 and a leucine at position 357, and the Fc region of the second polypeptide further comprises a methionine at position 347, a phenylalanine atposition 349, an aspartic acid at position 350 and a methionine a position 368, wherein the positions are according to EU numbering.

42. The method, pharmaceutical composition, or use of claim 40 or claim 41, wherein the first polypeptide comprises a cysteine at position 349, and the second polypeptide comprises a cysteine at position 354, wherein the positions are according to EU numbering.

43. The method, pharmaceutical composition, or use of any one of claims 34-42, wherein the Fc regions of the first polypeptide and / or the second polypeptide lack effector function or have reduced effector function.

44. The method, pharmaceutical composition, or use of claim 43, wherein the Fc regions of the first polypeptide and the second polypeptide comprise a glycine at position 397, an alanine at position 265, or both a glycine at position 397 and an alanine at position 265, wherein the positions according to EU numbering.

45. The method, pharmaceutical composition, or use of claim 43, wherein the Fc regions of the first polypeptide and the second polypeptide comprise an alanine at position 234, an alanine at position 235, or a serine at position 331, or any combination thereof, wherein the positions are according to EU numbering.

46. The method, pharmaceutical composition, or use of claim 45, wherein the Fc regions of the first polypeptide and the second polypeptide comprise an alanine at position 234 and an alanine at position 235, wherein the positions are according to EU numbering.

47. The method, pharmaceutical composition, or use of claim 45, wherein the Fc regions of the first polypeptide and the second polypeptide comprise an alanine at position 234, an alanine at position 235, and a serine a position 331, wherein the positions are according to EU numbering.

48. The method, pharmaceutical composition, or use of any one of claims 35-47, wherein: the first polypeptide, from N-terminus to C-terminus, comprises:i) the first heavy chain variable domain; ii) the light chain variable domain; iii) the Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3); iv) the second heavy chain variable domain; and v) the constant heavy chain domain 1 (CHI), wherein the light chain variable domain of the first polypeptide and the second heavy chain variable domain of the first polypeptide are each, independently, attached to the Fc region of the first polypeptide by a polypeptide linker; the second polypeptide from N-terminus to C-terminus comprises: i) the first heavy chain variable domain; ii) the light chain variable domain; iii) the Fc region comprising a constant heavy chain domain 2 (CH2) and a constant heavy chain domain 3 (CH3); iv) the second heavy chain variable domain; and v) the constant heavy chain domain 1 (CHI), wherein the light chain variable domain of the second polypeptide and the second heavy chain variable domain of the second polypeptide are each, independently, attached to the Fc region of the second polypeptide by a polypeptide linker.

49. The method, pharmaceutical composition, or use of claim 48, wherein the light chain variable domain of the of the first polypeptide and the light chain variable domain of the second polypeptide are linked to the N-terminus of the Fc region of the first polypeptide and the second polypeptide, respectively, by a polypeptide linker comprising the amino acid sequence of GGGGSGGGGSEPKSSDKTHT (SEQ ID NO: 26).

50. The method, pharmaceutical composition, or use of claim 48 or claim 49, wherein the second heavy chain variable domain of the first polypeptide and the second heavy chain variable domain of the second polypeptide are linked to the C-terminus of the Fc region of the first polypeptide and the second polypeptide, respectively, by a polypeptide linker comprising the amino acid sequence of GGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 27).

51. The method, pharmaceutical composition, or use of any one of claims 34-50, wherein the first VH of the first polypeptide is linked to the VL of the first polypeptide by a polypeptide linker comprising the acid sequence of GGGGS (SEQ ID NO: 25); and the first VH of the second polypeptide is linked to the VL of the second polypeptide by a polypeptide linker comprising the acid sequence of GGGGS (SEQ ID NO: 25).

52. The method, pharmaceutical composition, or use of claim 33, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28, the second polypeptide comprises the amino acid of SEQ ID NO: 29, and the third polypeptide and the fourth polypeptide each comprise the amino acid sequence of SEQ ID NO: 30.

53. The method, pharmaceutical composition, or use of claim 34, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28, the second polypeptide comprises the amino acid of SEQ ID NO: 29, and the third polypeptide and the fourth polypeptide each comprise the amino acid sequence of SEQ ID NO: 30.

54. The method, pharmaceutical composition, or use of claim 36, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28, the second polypeptide comprises the amino acid of SEQ ID NO: 29, and the third polypeptide and the fourth polypeptide each comprise the amino acid sequence of SEQ ID NO: 30.

55. The method, pharmaceutical composition, or use of any one of claims 1-54, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28, the second polypeptide comprises the amino acid of SEQ ID NO: 29, and the third polypeptide and the fourth polypeptide each comprise the amino acid sequence of SEQ ID NO: 30.

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