CN116761634A - Non-covalent protein-hyaluronic acid conjugates for long-acting ocular delivery - Google Patents

Abstract

A conjugate may comprise a first component capable of binding to a therapeutic target in an eye, one or more second components capable of binding to hyaluronic acid, and one or more third components comprising hyaluronic acid, wherein each second component is covalently bound to the first component and non-covalently bound to the third component; a composition comprising the conjugate for use as a medicament or in the treatment of an eye disease; and a method of treating an ocular disease in a subject. Furthermore, a therapeutic molecule targeted to a tissue of a patient may comprise a hyaluronic acid binding moiety and a therapeutically active agent, wherein the hyaluronic acid binding moiety comprises at least two multifunctional proteoglycan attachment domains. A therapeutic molecule that targets a tissue of a patient may comprise a hyaluronic acid binding moiety and a therapeutically active agent, wherein the hyaluronic acid binding moiety comprises at least two multifunctional proteoglycan linking domains that bind to (i.e., pre-complex with) hyaluronic acid. A method of delivering a therapeutic molecule targeted to a tissue of a patient comprises administering any of the therapeutic molecules described herein to the patient and allowing the therapeutic molecule to provide a long-lasting delivery of the therapeutically active agent to the target tissue.

Description

Non-covalent protein-hyaluronic acid conjugates for long-term ocular delivery

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/092,251, filed on October 15, 2020, and U.S. Provisional Patent Application No. 63/250,782, filed on September 20, 2021, both of which are commonly owned with the present case, and the entire contents of both are expressly incorporated herein by reference as if fully set forth herein.

Technical Field

Long-acting therapies and treatment methods employing fusion proteins that bind to hyaluronic acid and fusion protein-hyaluronic acid conjugates.

Background Art

Intravitreal (IVT) injections are commonly used to administer drugs to treat various eye diseases. IVT injections allow drugs to be applied directly to the back of the eye, eliminating common obstacles to local and systemic administration. Direct application of drugs in this way allows drugs to have higher intraocular bioavailability in posterior segment tissues, thereby more effectively treating posterior ocular diseases. Stewart, M.W., Expert Opinion on Drug Metabolism & Toxicology, 14 (1): 5-7 (2018). Examples of common diseases treated via IVT injections include age-related macular degeneration (AMD), diabetic retinal degeneration, retinal vein occlusion, and eye infections (such as endophthalmitis and retinitis). American Society of Retinal Specialists Foundation, asrs.org/patients/retinal-diseases/33/IVT-injections (2017).

Despite promising results in stopping the disease and improving vision, IVT injections are uncomfortable, expensive, and require retinal specialists to perform. IVT injections are known to cause adverse reactions in some patients, such as infection, inflammation, vitreous hemorrhage, increased presence of floaters in the eye, increased sensitivity to light, decreased vision, and retinal detachment. American Society of Retina Specialists Foundation, asrs.org/patients/retinal-diseases/33/IVT-injections (2017). IVT injections may also be associated with infectious endophthalmitis, sterile intraocular inflammation, rhegmatogenous retinal detachment, increased intraocular pressure, and ocular hemorrhage. Ibid. Transocular long-acting delivery technologies may eliminate the need for repeated injections of drugs, thereby improving patient compliance and clinical outcomes. Methods and compositions that extend the half-life of drugs in the vitreous humor (e.g., maintaining drug storage capacity, low turnover in the eye, clearance mediated by low retention targets, and/or seemingly stable properties in the elderly population) promote slow release of drugs from the injection site to the target site, thereby enabling the use of higher doses and reducing the number of injections required.

The vitreous half-life of therapeutic molecules can be extended by conjugating the therapeutic molecules to hyaluronic acid (HA) as an alternative to encapsulation or chemical modification with polymers. Cromwell, S et al., Invest. Ophthalmol. Vis. Sci. 59(9):235 (2018); Ghosh, J. G. et al., Nature Communications, 8:14837, doi:10.1038/ncomms14837 (2017); Stewart, M. W., Expert Opinion on Drug Metabolism & Toxicology, 14(1):5-7 (2018). In a specific example, the long-acting anti-VEGF antibody is fused to the HA binding domain (HABD) of the human tumor necrosis factor (TNF)-stimulated gene 6 protein (TSG-6), respectively. Ghosh, J.G. et al., Nature Communications, 8:14837, doi:10.1038/ncomms14837 (2017). The fusion protein showed the following improvements compared to the unmodified anti-VEGF antibody: (1) a 3- to 4-fold increase in half-life; and (2) the ability to attenuate VEGF-induced retinal changes in an animal model of neovascular retinal disease for a 3- to 4-fold longer period of time. Ghosh, J.G. et al., Nature Communications, 8:14837, doi:10.1038/ncomms14837 (2017). A drug candidate, LMG324, comprising a long-acting anti-VEGF antibody fused to TSG-6, has entered clinical trials to evaluate the safety and tolerability of single ascending doses to determine the maximum tolerated dose (MTD) in neovascular age-related macular degeneration (nvAMD). clinicaltrials.gov/ct2/show/NCT02398500 (2019). However, these trials were suspended due to serious adverse events, including vitreous floaters, inflammation, and posterior vitreous detachment.

Chemical conjugation of antibody fragments to hyaluronic acid (HA) can reduce the diffusion rate of drugs from the vitreous. However, this approach requires chemical activation of HA; the use of non-natural linkers may result in the generation of non-natural metabolites of activated HA in subjects.

The inventors have discovered that the above disadvantages can be avoided by providing a conjugate comprising: (1) a first component capable of binding to a therapeutic target in the eye, (2) one or more second components capable of binding to HA, and (3) one or more third components comprising HA; wherein each second component is (a) covalently bound to the first component and (b) non-covalently bound to the third component. Unlike the above-mentioned anti-VEGF antibody and TSG-6 fusion protein (LMG324), the second component capable of binding to HA is pre-complexed with HA.

The present application discloses materials and methods for increasing the intraocular retention of therapeutic molecules comprising fusion proteins capable of binding to hyaluronic acid (HA). In some embodiments, the fusion protein comprises: (1) a first component capable of binding to a therapeutic target in the eye, and (2) one or more second components capable of binding to HA; wherein each second component is covalently bound to the first component.

The present application also discloses conjugates, wherein the fusion further comprises one or more third components, the third components comprising HA, wherein each second component is further non-covalently bound to the third component. Further, the second component capable of binding to HA can be pre-complexed with HA. The conjugate is compatible with vitreous and has binding affinity for HA. These materials and methods provide a platform technology for the design of longer-acting drugs.

Summary of the invention

The present application discloses a material and method, which relates to therapeutic molecules capable of binding to therapeutic targets in the eye and capable of binding to hyaluronic acid and conjugates thereof. The present application provides the following items, aspects and embodiments.

Item 1 is a therapeutic molecule comprising: (a) a first component capable of binding to a therapeutic target in the eye; (b) one or more second components capable of binding to hyaluronic acid, wherein these one or more second components are covalently bound to the first component; and (c) optionally, one or more third components comprising hyaluronic acid, wherein, if these one or more third components are present, these one or more third components are non-covalently bound to these one or more second components.

Item 2 is a therapeutic molecule as described in Item 1, wherein the first component is a protein, a peptide, a receptor or a fragment thereof, a receptor ligand, a darpin, a nucleic acid, RNA, DNA or an aptamer.

Item 3 is a conjugate as described in item 1 or 2, wherein the first component is selected from antibodies, antigen-binding fragments, in particular antibody fragments, more particularly antibody fragments lacking at least the Fc domain, in particular wherein the fragment is or comprises a (Fab') ₂ fragment, a Fab' fragment or a Fab fragment, a VhH fragment, a scFv fragment, a scFv-Fc fragment and a mini antibody, more particularly a Fab fragment.

Item 4 is a therapeutic molecule described in any one of Items 1 to 3, wherein the second component comprises a hyaluronan receptor CD44 (CD44) domain, a brain-specific connexin (BRAL1) domain, a tumor necrosis factor-stimulated gene 6 (TSG-6) domain, a lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) domain, or a hyaluronan binding protein (HABP) domain, an aggrecan G1 (AG1) domain, or a versican G1 (VG1) domain.

Item 5 is a therapeutic molecule of any one of items 1 to 4, wherein the conjugate comprises one second component or two second components that are identical to each other.

Item 6 is a therapeutic molecule of any one of items 1 to 4, wherein the third component is hyaluronic acid, wherein the hyaluronic acid (a) has a molecular weight of: (i) selected from 3kDa to 60kDa, 4kDa to 30kDa, 5kDa to 20kDa or 400Da to 200kDa; (ii) is at least 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa or 9kDa; or (iii) is at most 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa or 15kDa; (b) provides a molar excess of binding equivalents to one or both of these second components; and (c) has a modification that reduces hyaluronic acid degradation in the eye.

Item 7 is the therapeutic molecule of any one of items 1 to 6, wherein the second component is capable of binding to hyaluronic acid with a _KD of 10 nM to 10 μM, 5 nM to 8 μM or 100 nM to 5 μM.

Item 8 is a therapeutic molecule according to any one of items 1 to 7, wherein (a) the first and second components are contained in a fusion protein, in particular wherein one or both of the second components are covalently bound to the N-terminus and/or C-terminus of the first component, more in particular wherein the first component is an antibody or antigen-binding fragment, and wherein the one or both second components are covalently bound to the C-terminus of the first component; and/or (b) the one or both second components are directly bound to the first component or indirectly bound to the first component via a linker, in particular a linker of at least 4 amino acids and/or at most 50 or at most 25 amino acids, more in particular the linker is (GxS) _n or (GxS) _nGm , wherein G=glycine, S=serine, (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 _or 5, and m=0, 1, 2 or 3).

Item 9 is a therapeutic molecule described in any one of Items 1 to 8, wherein the therapeutic target is VEGF, C2, C3a, C3b, C5, C5a, Htra1, IL-33, Factor P, Factor D, EPO, EPOR, IL-1β, IL-17A, IL-10, TNFα, FGFR2, PDGF or ANG2, especially VEGF.

Item 10 is a therapeutic molecule according to any one of items 1 to 9, wherein (a) the first component is an anti-VEGF antibody or antigen-binding fragment, in particular an anti-VEGF Fab; and/or (b) each of these one or two second components comprises a CD44 domain or a TSG-6 domain or a VG1 domain; and/or (c) the third component is hyaluronic acid with a molecular weight of 5 kDa to 20 kDa.

Item 11 is a therapeutic molecule of any one of items 1 to 10, wherein (i) the first component is an anti-VEGF antibody or antigen binding fragment, one or both of the second components contain a CD44 domain, and the third component is a hyaluronic acid with a molecular weight of 5 kDa to 20 kDa; (ii) the first component is an anti-VEGF antibody or antigen binding fragment, one or both of the second components contain a TSG-6 domain, and the third component is a hyaluronic acid with a molecular weight of 5 kDa to 20 kDa; or (iii) the first component is an anti-VEGF antibody or antigen binding fragment, one or both of the second components contain a VG1 domain, and the third component is a hyaluronic acid with a molecular weight of 5 kDa to 20 kDa.

Item 12 is a conjugate of any one of items 1 to 11, wherein (a) the first component comprises (i) a VH domain of SEQ ID NO: 97, 99, 105, 109 or 144; and (ii) a VL domain of SEQ ID NO: 98, 100, 106, 110 or 115; and (b) the second component comprises SEQ ID NO: 2.

Item 13 is a conjugate of any one of items 1 to 11, wherein (a) the first component comprises (i) a VH domain of SEQ ID NO: 97, 99, 105, 109 or 144; and (ii) a VL domain of SEQ ID NO: 98, 100, 106, 110 or 115; and (b) the second component comprises SEQ ID NO: 4.

Item 14 is a conjugate of any one of items 1 to 11, wherein (a) the first component comprises (i) a VH domain of SEQ ID NO: 97, 99, 105, 109 or 144; and (ii) a VL domain of SEQ ID NO: 98, 100, 106, 110 or 115; and (b) the second component comprises SEQ ID NO: 86, 60, 32 or 29.

Item 15 is a therapeutic molecule of any one of items 1 to 11, wherein the second components comprise at least two linking domains of versican.

Item 16 is the therapeutic molecule of item 15, wherein the second components comprise at least two linking domains of versican bound to hyaluronic acid.

Item 17 is the therapeutic molecule of any one of items 1 to 22, wherein the hyaluronic acid allows the ratio of hyaluronic acid to therapeutic molecule to range from 1.5:1 to 1:1.

Item 18 is a therapeutic molecule described in any one of Items 14 to 17, wherein the second component is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:86, 60, 32 or 29.

Item 19 is a therapeutic molecule of any one of items 14 to 18, wherein the second component is at least 95% identical to SEQ ID NO:86, 60, 32 or 29.

Item 20 is a therapeutic molecule described in any one of items 14 to 19, wherein the second component comprises at least 1, at least 2, at least 3, at least 4 or at least 5 mutations.

Item 21 is a therapeutic molecule of any one of items 14 to 20, wherein the second component comprises 1 to 3 mutations, wherein these 1 to 3 mutations comprise single amino acid substitutions, double amino acid substitutions and truncations.

Item 22 is a therapeutic molecule of any one of items 14 to 21, wherein the second component comprises 1 to 5 mutations, wherein these 1 to 5 mutations comprise single amino acid substitutions, double amino acid substitutions and truncations.

Item 23 is a therapeutic molecule of any one of Items 14 to 22, wherein the second component has a truncated mutation relative to SEQ ID NO: 29.

Item 24 is a therapeutic molecule as described in Item 23, wherein the truncation mutation comprises a truncation of 1 to 129 amino acids at the N-terminus.

Item 25 is a therapeutic molecule of any one of items 14 to 24, wherein the second component is a truncated sequence in which the Ig domain of wild-type versican is absent.

Item 26 is a therapeutic molecule of any one of items 14 to 25, wherein the second component comprises at least one of the following amino acids relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, Y230, F261, D295 and R233.

Item 27 is a therapeutic molecule described in any one of Items 14 to 26, wherein the second component comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the following amino acids relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, Y230, F261, D295 and R233.

Item 28 is a therapeutic molecule described in any one of Items 14 to 27, wherein the second component comprises a mutation in at least one of the following positions relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.

Item 29 is a therapeutic molecule described in any one of items 14 to 28, wherein the second component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 mutations in the following positions relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.

Item 30 is a therapeutic molecule described in any one of Items 14 to 29, wherein the second component comprises mutations in 2, 3, 4, 5 or 6 of the following positions relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.

Item 31 is a therapeutic molecule described in any one of items 14 to 30, wherein the second component comprises at least one of the following mutations relative to SEQ ID NO: 29: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A and LYR325LFK.

Item 32 is a therapeutic molecule of any one of items 14 to 31, wherein the second component comprises at least one of Y208A and H306A.

Item 33 is a therapeutic molecule according to any one of items 14 to 32, wherein the second component is relative to SEQ ID NO:29 comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the following mutations: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A and LYR325LFK.

Item 34 is a therapeutic molecule described in any one of items 14 to 33, wherein the second component comprises at least 2, 3, 4, 5 or 6 of the following mutations relative to SEQ ID NO: 29: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A and LYR325LFK.

Item 35 is a therapeutic molecule described in any of Item 14 or 18, wherein the second component is SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 or SEQ ID NO:59.

Item 36 is a therapeutic molecule according to any one of items 1 to 35, wherein the first component comprises an oligopeptide, a protein or a nucleic acid.

Item 37 is a therapeutic molecule described in any one of items 1 to 36, wherein the first component comprises a therapeutic drug, an antibody, an antigen binding fragment, an enzyme, a growth factor, an oligopeptide, a cysteine knot peptide, a growth factor, an antisense oligonucleotide, a locked nucleic acid or an aptamer.

Item 38 is a therapeutic molecule as described in Item 37, wherein the cysteine knot peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:92.

Item 39 is a therapeutic molecule as described in Item 37, wherein the growth factor comprises fibroblast growth factor, platelet-derived growth factor, nerve growth factor (NGF), VEGF, fibroblast growth factor (FGF) and insulin-like growth factor I (IGF-I).

Item 40 is a therapeutic molecule of any one of items 1 to 39, wherein the first component binds to VEGF.

Item 41 is a therapeutic molecule as described in Item 40, wherein the first component that binds to VEGF comprises ranibizumab, aflibercept, brolucizumab-dbll and bevacizumab.

Item 42 is the therapeutic molecule of Item 37, wherein the aptamer is pegylated.

Item 43 is a therapeutic molecule as described in Item 37 or 42, wherein the aptamer is

Item 44 is a therapeutic molecule according to any one of items 1 to 43, wherein the linker comprises GGGGS (SEQ ID NO: 27) or a multimer thereof, more particularly (GGGGS) 3 (SEQ ID NO: 28).

Item 45 is a therapeutic molecule of any one of items 1 to 42, wherein the linker comprises GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).

Item 46 is a therapeutic molecule of item 45, wherein the cysteine knot peptide is connected to one or both of the second components via a linker, and the linker comprises the sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).

Item 47 is a therapeutic molecule as described in Item 45 or 46, wherein the sequence comprises (a) an anti-VEGF antigen binding fragment; and (b) a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO:93 or SEQ ID NO:94.

Item 48 is a composition for use as a medicament, comprising a therapeutic molecule as described in any one of Items 1 to 47 and an optional pharmaceutically acceptable excipient, diluent or carrier.

Item 49 is a composition for treating an eye disease or a brain disease, which comprises the conjugate described in any one of Items 1 to 47 and an optional pharmaceutically acceptable excipient, diluent or carrier.

Item 50 is a composition for use in Item 49, which is formulated for intraocular delivery, particularly for intravitreal injection.

Item 51 is a composition used in any one of items 48 to 50, wherein (a) the composition is administered at most once every three months, particularly at most once every four months, more particularly at most once every six months; and/or (b) the elimination half-life of the first component in the conjugate is extended by at least 3 times, at least 4 times or at least 5 times compared to the unbound first component.

Item 52 is a composition used in any one of items 48 to 51, wherein the eye disease is age-related macular degeneration (AMD), particularly wet AMD or neovascular AMD; diabetic macular edema (DME); diabetic retinopathy (DR), particularly proliferative DR or non-proliferative DR; retinal vein occlusion (RVO); or geographic atrophy (GA).

Item 53 is a method for treating an eye disease in a subject, the method comprising administering to the subject a therapeutic molecule as described in any one of items 1 to 47 or a composition as defined in any one of items 48 to 52.

Item 54 is a method for delivering a therapeutic molecule targeted to a patient's tissue, the method comprising administering to the patient the therapeutic molecule described in any one of items 1 to 47 or the composition described in any one of items 48 to 52, and allowing the therapeutic molecule to provide long-acting delivery of the first component to the target tissue.

Item 55 is the method of item 54, further comprising conjugating the therapeutic molecule to hyaluronic acid prior to the administering step.

Item 56 is the method of Item 55, further comprising mixing a first solution comprising the therapeutic molecule with a second solution comprising the hyaluronic acid.

Item 57 is the method of Item 56, wherein the mixing includes a container.

Item 58 is the method of Item 57, wherein the container is a two-chamber syringe.

Item 59 is the method of any one of items 56 to 58, wherein the mixing produces a therapeutic molecule bound to hyaluronic acid, the therapeutic molecule being ready for administration to a subject.

Item 60 is the method of any one of items 54 to 59, wherein the administering step is a single injection.

Item 61 is the method of any one of items 54 to 60, wherein the target tissue comprises the eye or the brain.

Item 62 is a method of any one of items 54 to 61, wherein the therapeutic molecule provides improved vitreous compatibility, longer vitreous residence time, longer vitreous half-life and/or improved duration of pharmacological action compared to an unmodified bioactive agent.

Aspect 63 is a conjugate comprising (a) a first component capable of binding to a therapeutic target in the eye; (b) one or more second components capable of binding to hyaluronic acid; and (c) one or more third components comprising hyaluronic acid, (d) wherein each second component is covalently bound to the first component and not covalently bound to the third component.

Aspect 64 is the conjugate of aspect 63, wherein the first component is a protein, a peptide, a receptor or a fragment thereof, a ligand of a receptor, a darpin, a nucleic acid, RNA, DNA or an aptamer.

Aspect 65 is the conjugate of aspect 63 or 64, wherein the first component is an antibody or an antigen-binding antibody fragment, particularly an antibody fragment, more particularly an antibody fragment lacking at least the Fc domain, particularly wherein the fragment is or comprises a (Fab')2 fragment, a Fab' fragment or a Fab fragment, more particularly a Fab fragment.

Aspect 66 is the conjugate of any one of aspects 63 to 65, wherein the second component comprises a hyaluronan receptor CD44 (CD44) domain, a brain-specific connexin (BRAL1) domain, a tumor necrosis factor-stimulated gene 6 (TSG-6) domain, a lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) domain, or a hyaluronan binding protein (HABP) domain, an aggrecan G1 (AG1) domain, or a versican G1 (VG1) domain.

Aspect 67 is the conjugate of any one of aspects 63 to 66, wherein the conjugate comprises one or two second components, in particular comprises two identical second components.

Aspect 68 is the conjugate of any one of aspects 63 to 66, wherein the third component is hyaluronic acid, wherein the hyaluronic acid (a) has a molecular weight of 3 kDa to 60 kDa, particularly 4 kDa to 30 kDa, more particularly 5 kDa to 20 kDa; and/or (b) has a molecular weight of at least 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa or 9 kDa; and/or (c) has a molecular weight of at most 60 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa, 20 kDa or 15 kDa; and/or (d) has a modification that reduces degradation of hyaluronic acid in the eye.

Aspect 69 is the conjugate of any one of aspects 63 to 67, wherein (a) the first and second components are comprised in a fusion protein, in particular wherein one or both of the second components are covalently bound to the N-terminus and/or C-terminus of the first component, more in particular wherein the first component is an antibody or antigen-binding antibody fragment, and wherein one or both of the second components are covalently bound to the C-terminus of the first component; and/or (b) the second components are directly bound to the first component or indirectly bound to the first component via a linker, in particular a linker of at least 4 amino acids and/or at most 50 or at most 25 amino acids, more in particular the linker is (GxS)n or (GxS)nGm, wherein G=glycine, S=serine, (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3).

Aspect 70 is the conjugate of any one of aspects 63 to 69, wherein the therapeutic target is VEGF, C5, Factor P, Factor D, EPO, EPOR, IL-1β, IL-17A, IL-10, TNF, FGFR2, PDGF or ANG2, in particular VEGF.

Aspect 71 is the conjugate of any one of aspects 63 to 70, wherein (a) the first component is an anti-VEGF antibody or antigen-binding antibody fragment, particularly an anti-VEGF Fab; and/or (b) each of these one or two second components comprises a CD44 domain or a TSG-6 domain or a VG1 domain; and/or (c) the third component is a hyaluronic acid with a molecular weight of 5 kDa to 20 kDa, (d) particularly wherein (e) the first component is an anti-VEGF Fab, and wherein each of these one or two second components comprises a CD44 domain, and wherein the third component is a hyaluronic acid with a molecular weight of 5 kDa to 20 kDa; or (f) the first component is an anti-VEGF Fab, and wherein each of these one or two second components comprises a TSG-6 domain, and wherein the third component is a hyaluronic acid with a molecular weight of 5 kDa to 20 kDa; or (g) the first component is an anti-VEGF Fab, and wherein each of the one or two second components comprises a VG1 domain, and wherein the third component is hyaluronic acid having a molecular weight of 5 kDa to 20 kDa.

Aspect 72 is the conjugate of any one of aspects 63 to 71, the first component being an antibody having a VH domain comprised in SEQ ID NO:5 and a VL domain comprised in SEQ ID NO:6, and the second component comprises or consists of SEQ ID NO:4.

Aspect 73 is a composition for use as a medicament, comprising the conjugate according to any one of aspects 63 to 72 and optionally a pharmaceutically acceptable excipient, diluent or carrier.

Aspect 74 is a composition for treating an eye disease, comprising the conjugate of any one of aspects 63 to 73 and an optional pharmaceutically acceptable excipient, diluent or carrier.

Aspect 75 is the composition for use in aspect 73 or 74, which is formulated for intraocular delivery, particularly for intravitreal injection.

Aspect 76 is a composition for use in any one of Aspects 73 to 75, wherein (a) the composition is administered at most once every three months, particularly at most once every four months, more particularly at most once every six months; and/or (b) the elimination half-life of the first component in the conjugate is extended by at least 3 times, at least 4 times or at least 5 times compared to the unbound first component.

Aspect 77 is a composition for use according to any one of aspects 73 to 76, wherein the eye disease is age-related macular degeneration (AMD), in particular wet AMD or neovascular AMD; diabetic macular edema (DME); diabetic retinopathy (DR), in particular proliferative DR or non-proliferative DR; retinal vein occlusion (RVO); or geographic atrophy (GA).

Aspect 78 is a method of treating an eye disease in a subject, the method comprising administering to the subject the conjugate of any one of aspects 63 to 72 or the composition defined in any one of aspects 73 to 77.

Embodiment 79 is a therapeutic molecule targeted to a tissue of a patient, the therapeutic molecule comprising a hyaluronan binding domain and a therapeutically active agent, wherein the hyaluronan binding domain comprises at least two linking domains of versican.

Embodiment 80 is a therapeutic molecule targeted to a patient's tissue, the therapeutic molecule comprising a hyaluronic acid binding domain and a therapeutically active agent, wherein the hyaluronic acid binding domain comprises at least two connecting domains of a multifunctional proteoglycan, and the at least two connecting domains of the multifunctional proteoglycan are bound to hyaluronic acid via the HA binding domain.

Embodiment 81 is the therapeutic molecule of embodiment 79 or 80, wherein the hyaluronic acid ranges from 400Da to 200kDa.

Embodiment 82 is the therapeutic molecule of embodiment 81, wherein the hyaluronic acid is at least 5 kDa.

Embodiment 83 is the therapeutic molecule of embodiment 81 or 82, wherein the hyaluronic acid is 10 kDa.

Embodiment 84 is the therapeutic molecule of any one of embodiments 79 to 83, wherein the hyaluronic acid provides a molar excess of binding equivalents to the linking domains of the versicans.

Embodiment 85 is the therapeutic molecule of any one of embodiments 79 to 84, wherein the hyaluronic acid allows the ratio of hyaluronic acid to therapeutic molecule to range from 1.5:1 to 1:1.

Embodiment 86 is the therapeutic molecule of any one of embodiments 79 to 85, wherein the hyaluronan binding domain has a _KD of 10 nM to 10 μM.

Embodiment 87 is the therapeutic molecule of any one of embodiments 79 to 86, wherein the hyaluronan binding domain has a _KD of 5 nM to 8 μM.

Embodiment 88 is the therapeutic molecule of any one of embodiments 79 to 87, wherein the hyaluronan binding domain has a _KD of 100 nM to 5 μM.

Embodiment 89 is the therapeutic molecule of any one of embodiments 79 to 88, wherein the hyaluronan binding domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:86, 60, 32 or 29.

Embodiment 90 is the therapeutic molecule of any one of embodiments 79 to 89, wherein the hyaluronan binding domain is at least 95% identical to 86, 60, 32 or 29.

Embodiment 91 is the therapeutic molecule of any one of embodiments 79 to 90, wherein the hyaluronan binding domain comprises at least 1, at least 2, at least 3, at least 4 or at least 5 mutations.

Embodiment 92 is the therapeutic molecule of any one of embodiments 79 to 91, wherein the hyaluronan binding domain comprises 1 to 3 mutations, wherein these 1 to 3 mutations comprise single amino acid substitutions, double amino acid substitutions and truncations.

Embodiment 93 is the therapeutic molecule of any one of embodiments 79 to 92, wherein the hyaluronan binding domain comprises 1 to 5 mutations, wherein these 1 to 5 mutations comprise single amino acid substitutions, double amino acid substitutions and truncations.

Embodiment 94 is the therapeutic molecule of any one of embodiments 79 to 93, wherein the hyaluronan binding domain has a truncation mutation relative to SEQ ID NO:29.

Embodiment 95 is the therapeutic molecule of embodiment 94, wherein the truncation mutation comprises a truncation of 1 to 129 amino acids at the N-terminus.

Embodiment 96 is the therapeutic molecule of any one of embodiments 79 to 95, wherein the hyaluronan binding domain is a truncated sequence in which the Ig domain of wild-type versican is absent.

Embodiment 97 is the therapeutic molecule of any one of embodiments 79 to 96, wherein the hyaluronan binding domain comprises at least one of the following amino acids relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, Y230, F261, D295, and R233.

Embodiment 98 is the therapeutic molecule of any one of embodiments 79 to 97, wherein the hyaluronan binding domain comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the following amino acids relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, Y230, F261, D295 and R233.

Embodiment 99 is the therapeutic molecule of any one of embodiments 79 to 98, wherein the hyaluronan binding domain comprises a mutation at at least one of the following positions relative to SEQ ID NO:29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326, and R327.

Embodiment 100 is the therapeutic molecule of any one of embodiments 79 to 99, wherein the hyaluronan binding domain comprises mutations at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the following positions relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326, and R327.

Embodiment 101 is the therapeutic molecule of any one of embodiments 79 to 100, wherein the hyaluronan binding domain comprises mutations at 2, 3, 4, 5 or 6 of the following positions relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.

Embodiment 102 is the therapeutic molecule of any one of embodiments 79 to 101, wherein the hyaluronan binding domain comprises at least one of the following mutations relative to SEQ ID NO: 29: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A, and LYR325LFK.

Embodiment 103 is the therapeutic molecule of any one of embodiments 79 to 102, wherein the hyaluronan binding domain comprises at least one of Y208A and H306A.

Embodiment 104 is the therapeutic molecule of any one of embodiments 79 to 103, wherein the hyaluronan binding domain is relative to SEQ ID NO:29 comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the following mutations: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A and LYR325LFK.

Embodiment 105 is the therapeutic molecule of any one of embodiments 79 to 104, wherein the hyaluronan binding domain comprises at least 2, 3, 4, 5 or 6 of the following mutations relative to SEQ ID NO: 29: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A and LYR325LFK.

Embodiment 106 is the therapeutic molecule of any one of embodiments 79 to 105, wherein the hyaluronan binding domain is SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 or SEQ ID NO:59.

Embodiment 107 is the therapeutic molecule of any one of embodiments 79 to 106, wherein the therapeutically active agent comprises an oligopeptide, a protein, or a nucleic acid.

Embodiment 108 is the therapeutic molecule of any one of embodiments 79 to 107, wherein the therapeutically active agent comprises an antibody, an antigen binding fragment, a cysteine knot peptide, a growth factor, or an aptamer.

Embodiment 109 is the therapeutic molecule of embodiment 108, wherein the therapeutically active agent is capable of binding to an antigen.

Embodiment 110 is the therapeutic molecule of embodiment 109, wherein the therapeutic agent is capable of binding to VEGF, HtrA1, IL-33, C5, Factor P, Factor D, EPO, EPOR, IL-1β, IL-17A, IL-10, TNFα, FGFR2, PDGF or ANG2.

Embodiment 111 is the therapeutic molecule described in any of embodiments 109 or 110, wherein the therapeutically active agent is an antibody or an antigen-binding fragment thereof (including but not limited to a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a VhH fragment, a scFv fragment, a scFv-Fc fragment or a mini antibody).

Embodiment 112 is the therapeutic molecule of any one of embodiments 109 or 110, wherein the therapeutically active agent is an oligopeptide or a protein.

Embodiment 113 is the therapeutic molecule described in embodiment 102, wherein the oligopeptide or protein is a cysteine knot peptide or an enzyme.

Embodiment 114 is the therapeutic molecule of embodiment 103, wherein the cysteine knot peptide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:92.

Embodiment 115 is a therapeutic molecule of any one of embodiments 79 to 108, wherein the therapeutically active agent is a growth factor comprising fibroblast growth factor, platelet-derived growth factor, nerve growth factor (NGF), VEGF, fibroblast growth factor (FGF) and insulin-like growth factor I (IGF-I).

Embodiment 116 is the therapeutic molecule of embodiment 110, wherein the therapeutic agent that binds VEGF comprises ranibizumab, aflibercept, brolucizumab-dbll and bevacizumab.

Embodiment 117 is the therapeutic molecule of any one of embodiments 79 to 110, wherein the therapeutically active agent is a nucleic acid.

Embodiment 118 is the therapeutic molecule described in embodiment 117, wherein the nucleic acid is an aptamer, an antisense oligonucleotide and/or a locked nucleic acid.

Embodiment 119 is the therapeutic molecule of embodiment 118, wherein the aptamer binds VEGF.

Embodiment 120 is the therapeutic molecule of any one of embodiments 108, 118 or 119, wherein the aptamer is pegylated.

Embodiment 121 is a therapeutic molecule according to any one of Embodiments 108 or Embodiments 118 to 120, wherein the aptamer is

Embodiment 122 is the therapeutic molecule of any one of embodiments 79 to 121, wherein the therapeutically active agent is covalently linked to the hyaluronic acid binding domain via a linker.

Embodiment 123 is the therapeutic molecule of embodiment 122, wherein the linker is at least 4 amino acids.

Embodiment 124 is the therapeutic molecule of embodiment 122 or 123, wherein the length of the linker does not exceed 50 amino acids.

Embodiment 125 is the therapeutic molecule of any one of embodiments 122 to 124, wherein the linker is 4 to 25 amino acids.

Embodiment 126 is a therapeutic molecule described in any one of embodiments 122 to 125, wherein the linker comprises (GxS)n or (GxS)nGm, wherein G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3).

Embodiment 127 is the therapeutic molecule of any one of embodiments 122 to 126, wherein the linker comprises GGGS (SEQ ID NO: 84) or a multimer thereof, more particularly comprises (GGGGS) 3 (SEQ ID NO: 85).

Embodiment 128 is the therapeutic molecule of any one of embodiments 122 to 125, wherein the linker comprises (GxS)n, wherein G=glycine, S=serine, and (n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

Embodiment 129 is the therapeutic molecule of any one of embodiments 122 to 125 or 128, wherein the linker comprises GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).

Embodiment 130 is the therapeutic molecule of any one of embodiments 79 to 107, wherein the therapeutic agent comprises an anti-VEGF antigen binding portion and a cysteine knot peptide.

Embodiment 131 is the therapeutic molecule of embodiment 130, wherein the cysteine knot peptide is linked to the hyaluronic acid binding domain via a linker, and the linker comprises the sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).

Embodiment 132 is the therapeutic molecule of embodiment 130 or 131, wherein the sequence comprises (a) an anti-VEGF antigen binding portion; and (b) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:93 or SEQ ID NO:94.

Embodiment 133 is the therapeutic molecule of any one of embodiments 79 to 132, wherein the hyaluronic acid binding domain can non-covalently bind to hyaluronic acid.

Embodiment 134 is a method of delivering a therapeutic molecule targeted to a tissue of a patient, comprising administering to the patient a therapeutic molecule as described in any one of embodiments 79 to 133, and allowing the therapeutic molecule to provide long-acting delivery of the therapeutically active agent to the target tissue.

Embodiment 135 is the method of embodiment 134, further comprising conjugating the therapeutic molecule to hyaluronic acid prior to the administering step.

Embodiment 136 is the method of embodiment 135, further comprising mixing a first solution comprising the therapeutic molecule with a second solution comprising the hyaluronic acid.

Embodiment 137 is the method of embodiment 136, wherein the mixing includes a container.

Embodiment 138 is the method of embodiment 137, wherein the container is a two-chamber syringe.

Embodiment 139 is the method of any one of embodiments 136 to 138, wherein the mixing produces a therapeutic molecule bound to hyaluronic acid, the therapeutic molecule being ready for administration to a subject.

Embodiment 140 is the method of any one of embodiments 134-139, wherein the administering step is a single injection.

Embodiment 141 is the method of any one of embodiments 134-140, wherein the target tissue comprises the eye or the brain.

Embodiment 142 is the method of any one of embodiments 134 to 141, wherein the therapeutic molecule provides improved vitreous compatibility, longer vitreous residence time, longer vitreous half-life, and/or improved duration of pharmacological action compared to an unmodified therapeutically active agent.

Additional objects and advantages will be described in part in the following description and in part will be obvious from the description or may be learned through practice. These objects and advantages will be realized and obtained by the elements and combinations particularly pointed out in the appended claims.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiments and, together with the description, serve to explain certain principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the size exclusion chromatography (SEC; TSKgel UP-SW3000, 2 μm, 4.6 × 150 mm; running buffer 0.2M KPh, 0.25M KCl, pH 6.2) analysis results of Fab-HABD fusion protein VPDF-2xCD44 pre-complexed or not pre-complexed with 10 kDa hyaluronic acid (HA). Fab-HABD was prepared as described in Example 1 and tested as described in Example 2.

Figures 2A to 2B show the vitreous pharmacokinetics (PK) of rabbit anti-c-MetFab (RabFab) and rabbit anti-c-Met Fab-VG1 Fab-HABD (RabFab-Fab-HABDs) after dose-normalized intravitreal (IVT) injection in New Zealand white rabbits. Figure 2A shows the amount of RabFab or RabFab-Fab-HABD present in the vitreous over time after IVT. Data are shown for RabFab fusions, i.e., ^125I -RabFab-2xTSG6 (SEQ ID NO: 15 and SEQ ID NO: 16; 0.5 mg/eye) and RabFab-1xTSG6 (SEQ ID NO: 13 and SEQ ID NO: 14; 0.3 mg/eye), RabFab (SEQ ID NO: 61 and SEQ ID NO: 62; 0.3 mg/eye), and ^125I -ranibizumab Control (0.5 mg/eye). Data points are dose normalized. Figure 2B Vitreous pharmacokinetics of RabFab (0.15 mg/eye) or RabFab-2xTSG6 (0.026 mg/eye, 0.15 mg/eye or 2.5 mg/eye) monitored by fluorophotometry.

FIG. 3 shows histopathological images of OS rabbit eyes showing retinal degeneration 4 days after administration of TSG6 (SEQ ID NO: 32) via IVT.

Figures 4A-B show the IVT pharmacokinetic (PK) profiles (mean drug concentration over time) of VPDF (unmodified; Figure 4A) and VPDF-2xCD44+10 kDa HA (Figure 4B) in aqueous and vitreous humor.

Figures 5A-C show different mixtures with porcine vitreous. Figure 5A shows porcine vitreous mixed with unmodified anti-VEGF/anti-PDGFFab fragment (VPDF), which is homogeneous (transparent). Figure 5B shows porcine vitreous mixed with VPDF-2xCD44, which is inhomogeneous (precipitated). Figure 5C shows porcine vitreous mixed with 1% (w/v) HA 10kDa VPDF-2xCD44 pre-complex, which is homogeneous (transparent).

Figures 6A to 6F show porcine vitreous mixed with different concentrations of VPDF-2xCD44. Figure 6A: 37.5 mg/mL VPDF-2xCD44. Figure 6B: 9.4 mg/mL VPDF-2xCD44. Figure 6C: 2.4 mg/mL VPDF-2xCD44. Figure 6D: 0.6 mg/mL VPDF-2xCD44. Figure 6E: 0.15 mg/mL VPDF-2xCD44. Figure 6F: 0.04 mg/mL VPDF-2xCD44. +++ Severe precipitation; ++ Moderate precipitation; + Mild precipitation; - Clear vitreous.

Figures 7A-C show vitreous inhomogeneity throughout a pig eye following injection of the indicated VPDF-2xCD44 samples. Figure 7A: Buffer control. Figure 7B: Uncomplexed VPDF-2xCD44. Figure 7C: VPDF-2xCD44 complexed with HA.

Figures 8A-B show the domain structure of versican and the amino acid sequence of the linking domain. Versican is an endogenous substance in the vitreous humor. Figure 8A shows the domains of versican: VG1 domain, GAG attachment domain and G3 domain. The VG1 domain (WT VG1; SEQ ID NO:29) comprises an Ig-like domain followed by two linking domains (i.e., Link1 and Link2), which are responsible for HA binding. Figure 8B shows a sequence alignment of the linking domains including TSG6 LD (SEQ ID NO:4), VG1 Link1 (SEQ ID NO:30) and VG1 Link2 (SEQ ID NO:31).

Figures 9A-B show precipitation of TSG6 but not WT VG1 in porcine vitreous humor. Turbidity was observed after mixing TSG6 (but not WT VG1) with porcine vitreous diluted 1:4 (PBS). The final concentration of TSG6 and WT VG1 in the vitreous was approximately 1 mg/mL. Figure 9A shows TSG6 vs. control - a precipitate was observed after centrifugation. Figure 9B shows WT VG1 vs. control - no precipitate was observed after centrifugation.

Figures 10A-B show that RabFab-TSG6 precipitates in porcine vitreous, while RabFab-VG1 does not. TSG6 or VG1 were each recombinantly attached to RabFab and conjugated to Alexa488 via N-hydroxysuccinimide (NHS) primary amine labeling chemistry. Figure 10A shows RabFab-TSG6. Figure 10B shows RabFab-VG1.

Figures 11A-C show that VG1 and RabFab-VG1 did not precipitate in rabbit vitreous humor. Figure 11A shows about 40 g/L of VG1. Figure 11B shows about 40 g/L of RabFab-VG1. Figure 11C shows about 17 g/L of RabFab-VG1 + 10 kDa HA. No precipitation was observed under any conditions.

Figure 12 shows fluorescence correlation spectroscopy (FCS) measurements of VG1 interacting with the vitreous humor in vitro. Measurements showing slow diffusion indicate that the protein interacts with the vitreous humor, while rapid diffusion indicates that no interaction occurs. The dilution factor of the vitreous humor is shown at the top of the heat map - the leftmost column shows the undiluted control/sample; the rightmost column shows phosphate buffered saline (PBS), pH 7.4; the middle column shows the dilution factor increasing from left to right. The measurements of the unbound control are shown in the top two rows. The measurements of the following samples are shown in rows 3-8: free VG1, PigFab-VG1, PigFab-VG1+10kDa HA (1:1), free VG1, RabFab-VG1, and RabFab-VG1+10kDa HA (1:1). The unbound control shows the fastest diffusion (Figure 12, rows 1 and 2). While all samples showed significantly delayed diffusion relative to the control, free VG1, PigFab-VG1, and RabFab-VG1 all showed delayed diffusion until the vitreous was diluted more than 6000-fold (Figure 12, rows 3, 4, 6, and 7; from undiluted to dilution factor 6,561). Slow diffusion was observed for samples co-formulated with 10 kDa HA, but this effect disappeared when the dilution factor was greater than 729-fold (Figure 12, row 5: PigFab-VG1 + 10 kDa HA (1:1), and row 8: RabFab-VG1 + 10 kDa HA (1:1); from dilution factor 729 to PBS). These results suggest that VG1 can interact with endogenous HA.

Figure 13 shows the results of heat stress (ie protein stability) analysis of anti-HtrA1-VG1 at 37°C. T0 = unincubated control. T4wk = after 4 weeks of incubation.

Figure 14 shows the average concentration of pigFab-VG1 in porcine aqueous humor. Concentrations were measured by mass spectrometry after injection of 1.8 mg pigFab-VG1 alone or pre-complexed with an equal mass concentration of 10 kDa HA via IVT. Mean values of several animals are shown, with error bars representing standard deviations.

FIG. 15 shows the percent inhibition of neovascularization by VPDF VG1 in rat laser-induced choroidal neovascularization (rat laser CNV).

Figures 16A-C show histopathology of test article treated rabbit eyes 30 days after treatment.Figure 16A shows WTVG1, Figure 16B shows RabFab-VG1, and Figure 16C shows RabFab-VG1 bound to HA.

Figures 17A-B show brain levels after mice received intraventricular injections. Figure 17A shows the amount of protein retained in the brain over time. Figure 17B shows exposure levels in the brain as measured by the area under the curve (AUC). ** indicates p<0.01, and *** indicates p<0.001 for comparisons between groups. Anti-gD = anti-herpes simplex virus-1 glycoprotein D. BRD = anti-gDFab-VG1.

Figure 18 shows the crystal structure of WT VG1 and HA conjugate. The Ig domain of VG1 is shown at the top of the figure, the Link1 structure is shown at the bottom right of the figure, and the Link2 domain is shown at the bottom left of the figure. The binding of HA is represented by the smaller HA molecule to the right below the VG1 molecule.

Figure 19 shows the alignment of VG1 variants SEQ ID NO: 29, SEQ ID NO: 33-59. The 20 amino acids before the N-terminus are the versican signal sequence (shown with an *). The amino acids in the box are conserved residues. All of these proteins carry a C-terminal His tag for purification.

Sequence Description

A listing of certain sequences cited herein is provided in Table 1. The amino acid sequences are provided from N-terminus to C-terminus.

DETAILED DESCRIPTION

1. Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention, preferred methods and materials are now described.

Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings:

As used herein, the term "antibody" refers to a full (complete or intact) antibody. An antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into highly variable regions, called complementary determining regions (CDRs), with more conservative regions interspersed in the middle, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to a given antigen (e.g., a therapeutic target in the eye, such as VEGF) and thereby exhibit the desired antigen-binding activity. The antigen-binding function of an antibody can be performed by a fragment of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment of an antibody" include, but are not limited to, the following examples of antibody fragments, including, but not limited to: Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dAb); and multispecific antibodies formed from antibody fragments; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; single domain antibody (dAb) fragments consisting of either a VH domain or a VL domain; and isolated complementarity determining regions (CDRs). For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by an artificial peptide linker using recombinant methods to enable them to become a single protein chain, in which the VL and VH regions are paired to form a monovalent molecule (called a single-chain Fv (scFv)). These single-chain antibodies may include one or more antigen-binding fragments of an antibody. These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding fragments may also be incorporated into single-domain antibodies, large antibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. Antigen-binding fragments may be incorporated into a single-chain molecule comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), forming a pair of antigen-binding regions together with complementary light chain polypeptides. The term "antibodies" includes polyclonal antibodies and monoclonal antibodies.

Aptamers are oligonucleotides or peptide molecules that bind to specific target molecules. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers are also present in riboswitches. Aptamers can be used as macromolecular drugs for basic research and clinical purposes. Aptamers can be combined with ribozymes and self-cleave in the presence of target molecules. These compound molecules have additional research, industrial and clinical uses. More specifically, aptamers can be divided into DNA or RNA or XNA aptamers, which are composed of (usually short) oligonucleotide chains, and peptide aptamers composed of one (or more) short variable peptide domains are attached to protein scaffolds at both ends. DNA and RNA aptamers both show stable binding affinity to various targets. DNA and RNA aptamers have been selected for the same target. These targets include lysozyme, thrombin, interferon gamma, vascular endothelial growth factor (VEGF), dopamine. In the case of VEGF, for example, DNA aptamers are analogs of RNA aptamers, in which thymine replaces uracil.

A covalent bond, also known as a molecular bond, is a type of chemical bond that involves the sharing of electron pairs between atoms. These electron pairs are called shared electron pairs or bonding electron pairs, and when they share electrons, the stable balance of attractive and repulsive forces between atoms is called a covalent bond.

As used herein, the term "DARPin" (abbreviation for designed ankyrinrepeat protein) refers to antibody-mimicking proteins that typically exhibit high specificity and high affinity binding to a target protein. They are typically genetically engineered, derived from natural ankyrins, and consist of at least three, typically four or five, repeating motifs of these proteins. For four or five repeat DARPins, their molecular weight is approximately 14 kDa or 18 kDa, respectively. Examples of DARPins can be found, for example, in U.S. Patent No. 7,417,130.

A "therapeutically effective amount" of a pharmaceutical agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic effect.

As used herein, the term "eye disease" includes any eye disease associated with pathological angiogenesis and/or atrophy. The term "eye disease" is synonymous with the terms "eye condition," "eye disorder," "ocular condition," "ocular disease," and "ocular disorder."

As used herein, "Fab-hyaluronan-binding domain" and "Fab-HABD" refer to a fusion protein comprising a Fab and a hyaluronan-binding domain. These terms are synonymous and can be used interchangeably throughout this disclosure.

As used herein, "hyaluronan", "hyaluronic acid", "hyaluronate" and "HA" refer to a non-sulfated glycosaminoglycan having the chemical formula (C ₁₄ H ₂₁ NO ₁₁ ) _n and its salts.

As used herein, "hyaluronic acid binding domain", "hyaluronic acid binding moiety", "HA binding domain" or "HABD" refers to any portion capable of binding hyaluronic acid. In some cases, HABD may be a domain of an HA-binding protein.

A ligand is a substance that forms a complex or conjugate with a biomolecule to achieve its biological purpose. In protein-ligand binding, a ligand is usually a molecule that generates a signal by binding to a site on a target protein. This binding usually causes a change in the conformation of the target protein. In DNA-ligand binding studies, a ligand can be a small molecule, ion, or protein that binds to the DNA double helix. The relationship between a ligand and a binding partner is a function of charge, hydrophobicity, and molecular structure. The binding occurs within an infinitesimal time and space range, so the rate constant is usually a very small number. A ligand can be a naturally occurring ligand or a non-naturally occurring ligand. In addition, it may be an agonist, a partial agonist, an antagonist, or an inverse agonist.

"Non-covalent interaction" differs from a covalent bond in that it does not involve the sharing of electrons, but rather involves a more diffuse variation of electromagnetic interactions between or within molecules. Non-covalent interactions can be divided into different categories, such as electrostatics, π-effects, van der Waals forces, and hydrophobic effects. Preferably, the conjugate is provided in an isolated form. The first component and the second component may be covalently bound to each other via a linker or directly.

Nucleic acids are biopolymers composed of nucleotides, which are monomers formed of three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. The term "nucleic acid" is a general term for DNA and RNA. If the sugar is the complex ribose, the polymer is RNA (ribonucleic acid); if the sugar is deoxyribose derived from ribose, the polymer is DNA (deoxyribonucleic acid).

As used herein, the term "peptide linker" refers to a peptide containing an amino acid sequence, which is preferably of synthetic origin.

Proteins are macromolecular biopolymers (polypeptides) composed of long chains of one or more amino acid residues. Proteins perform a range of functions in living organisms, including catalyzing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ primarily in their amino acid sequence, which is determined by the nucleotide sequence of their genes and usually results in the protein folding into a specific three-dimensional structure that determines its activity. Short polypeptides containing fewer than 20-30 residues are often called peptides.

As used herein, "protein conjugate" or "conjugate" refers to a protein non-covalently bound to hyaluronic acid.

Receptors are chemical structures, usually composed of proteins, that receive and transduce signals that may be integrated into biological systems. These signals are usually chemical messengers (ligands), which bind to receptors and cause some form of cell/tissue response, such as changes in cell activity. The mode of action of receptors can be mainly divided into three types: signal relay, amplification or integration. Relay sends the signal forward, amplification increases the effect of a single ligand, and integration allows the signal to be incorporated into another biochemical pathway. In this sense, a receptor is a protein molecule that recognizes and responds to endogenous chemical signals. Therefore, in the context of the present invention, a receptor or fragment comprising a ligand binding site and its ligand are suitable binding counterparts (first component and therapeutic target).

As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual to be treated, and can be performed for prevention or during the course of clinical pathology. Desirable therapeutic effects include preventing the occurrence or recurrence of the disease or its symptoms, alleviating the symptoms of the disease, alleviating any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, ameliorating or alleviating the disease state, and achieving the purpose of alleviating or improving prognosis. In some aspects, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

Numerical ranges include the numbers defining the range. Taking into account significant figures and errors associated with the measurements, measured values and measurable values should be understood as approximate values. In addition, the use of "comprise/comprises/comprising", "contain/contains" and "include/includes/including" is not intended to be limiting. It should be understood that the foregoing general description and detailed description are only exemplary and illustrative and do not limit the teaching.

All numbers in the specification and claims are modified by the term "about." This means that each number includes minor variations, which are defined as 10% of the stated value or range.

Unless otherwise specified in the specification, embodiments that are cited in the specification as "comprising various components" are also considered to be "consisting of the cited components" or "consisting essentially of the cited components"; embodiments that are cited in the specification as "consisting of various components" are also considered to be "consisting of the cited components" or "consisting essentially of the cited components"; and embodiments that are cited in the specification as "consisting essentially of various components" are also considered to be "consisting of the cited components" or "comprising the cited components" (this interchangeability does not apply to the use of these terms in the claims). Unless the context clearly indicates otherwise, the term "or" is used in an inclusive sense, that is, equivalent to "and/or".

Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying figures, examples and embodiments. It should be understood that the figures, examples and embodiments (unless otherwise specifically noted) are not intended to limit the scope of the present invention to the specific methods, schemes and reagents described herein, as they may vary. The present invention is intended to encompass all substitutions, modifications and equivalents that may be included in the present invention as defined by the appended claims and the included embodiments. In addition, the technology used herein is only for the purpose of disclosing specific embodiments, rather than attempting to limit the scope of the present disclosure. Unless the context otherwise clearly indicates, the singular forms "one", "a kind of" and "the" as used herein and in the appended claims include plural indicators. Similarly, the words "comprise", "contain" and "cover" should be interpreted as inclusive rather than exclusive.

The section headings used herein are only for organizational purposes and should not be interpreted as limiting the desired target in any way. All publications (scientific and patent publications) cited herein are incorporated by reference. If any material incorporated by reference conflicts with any term defined in this specification or any other explicit content of this specification, this specification shall prevail. Although this teaching is described in conjunction with various embodiments, it is not intended that this teaching is limited to these embodiments. On the contrary, as understood by those skilled in the art, this teaching encompasses various substitutions, modifications and equivalents.

11. A therapeutic molecule comprising a therapeutically active agent (ie, a first component) and a hyaluronic acid binding domain (HABD; ie, a second component)

The present application provides therapeutic molecules targeting patient's tissues, which include therapeutically active agents and HA-binding domains (HABD). Each therapeutic molecule includes a first component and one or more second components. The first component is capable of binding to a therapeutic target in the eye. These second components are capable of binding to HA and therefore include HA binding domains (HABD).

In some embodiments, the therapeutic molecule is a fusion protein comprising a first component and one or more second components. The first component and the second component are covalently bound to each other to form a fusion protein. In some embodiments, the therapeutic molecule further comprises a peptide linker.

In some embodiments, the therapeutic molecule comprises a second component. In some embodiments, the therapeutic molecule comprises two or more second components. In particular, if an antibody or an antigen-binding fragment thereof consisting of two proteins (i.e., a heavy chain or fragment thereof and a light chain or fragment thereof) is used, the therapeutic molecule may comprise two second components. In these embodiments, the first second component is connected to the heavy chain of the antibody or antigen-binding fragment, and the second second component is connected to the light chain of the antibody or antigen-binding fragment. In some embodiments, the first second component is connected to the C-terminus of the heavy chain of the Fab fragment, and the second second component is connected to the C-terminus of the light chain of the Fab fragment.

In some embodiments, the therapeutic molecule further comprises (in addition to the first component and the second component) one or more third components. The second component is covalently bound to the first component, and the second component is non-covalently bound to the third component. In some embodiments, the third component is hyaluronic acid (HA). In some of these embodiments, the second component is capable of binding HA, and the therapeutic molecule protein (i.e., the first component covalently connected to the second component) can be pre-compounded with HA (i.e., the third component). In some of these embodiments, the first component, the second component and the third component form a conjugate.

Non-limiting examples of first components, second components, and third components are provided herein.

A. First Component - Therapeutically Active Agent

In many embodiments, the first component is capable of binding to a therapeutic target, making it a bioactive agent or a therapeutically active agent. In some embodiments, the first component is capable of binding to a therapeutic target in the eye. As used herein, the term "capable of binding" refers to a substance or agent or component that can specifically bind to a target and optionally modulate the activity of the target. In other words, the first component has therapeutic activity in the eye due to binding to a therapeutic target in the eye. In some embodiments, the first component can activate, deactivate, increase or decrease the activity of the therapeutic target after binding to the therapeutic target. In some embodiments, the therapeutic target is a suitable structure in the eye, and its activity is associated with the eye disease to be treated. In some embodiments, the first component binds to a component in the signal transduction cascade that is directly upstream or downstream of the therapeutic target. In some embodiments, the first component comprises a known therapeutic drug for treating an eye disease.

Preferably, the specific binding component or binding domain has an affinity of at least 10 ⁶ l/mol for its corresponding target molecule. Preferably, the specific binding domain has an affinity of 10 ⁷ /mol, or even more preferably 10 ⁸ /mol, or even most preferably 10 ⁹ /mol for its target molecule. "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant ( _KD ). Affinity can be measured by conventional methods known in the art. As will be appreciated by the skilled artisan, the term "specific" is used to indicate a specific binding agent that does not significantly bind to a binding domain in the presence of other biological molecules. Preferably, the level of binding to biomolecules other than the target molecule results in a binding affinity that is only 10% or less, more preferably only 5% or less of the affinity to the target molecule, respectively.Preferred specific binding agents will meet the above minimum criteria for affinity and specificity.

In some embodiments, the first component comprises a protein such as a receptor or a fragment thereof, which binds to a therapeutic target, an antibody or a fragment thereof, a growth factor, a cysteine knot peptide, an enzyme or a DARpin. In some embodiments, the size of the protein may range from small to large. In some embodiments, the protein is a peptide comprising 2 to 20 amino acids. In some embodiments, the protein is a polypeptide comprising 21 to 50 amino acids. In some embodiments, the protein is a polypeptide comprising more than 50 amino acids. In some embodiments, the protein is a protein complex comprising two or more chains or amino acids, wherein each amino acid chain may comprise any number of amino acids. In some embodiments, the first component is no greater than 80 kDa. In some embodiments, the first component is greater than 80 kDa.

In some embodiments, the first component comprises a nucleic acid which may be DNA or RNA. The nucleic acid may be complementary to a nucleic acid associated with the target (e.g., the nucleic acid is complementary to an mRNA of the target or a related portion thereof). In some embodiments, the nucleic acid is an aptamer. In some embodiments, the nucleic acid comprises an antisense oligonucleotide. In some embodiments, the nucleic acid comprises a locked nucleic acid.

1. Therapeutic targets in the eye

In some embodiments, the first component binds to a therapeutic target in the eye. There may be many therapeutic targets in the eye. With the development of therapies that effectively target these molecules and pathways, there will be a need to provide improvements in visual outcomes while reducing the treatment burden and risks associated with frequent IVT injections.

a) Pro-angiogenic, inflammatory and growth factor mediators

In some embodiments, the first component binds to a therapeutic target that is a pro-angiogenic, inflammatory and/or growth factor mediator. Pro-angiogenic, inflammatory and growth factor mediators are involved in retinal diseases, such as neovascular age-related macular degeneration (AMD; wet AMD), diabetic retinopathy and retinal vein occlusion.

Examples of these pro-angiogenic, inflammatory or growth factor mediator molecules include, but are not limited to, platelet-derived growth factor (PDGF), angiogenin, S1P, integrin ανβ3, integrin ανβ5, integrin α5β1, betacellulin, apelin/APJ, erythropoietin, complement factor D and TNFα.

b) Proteins in age-related macular degeneration (AMD)

In some embodiments, the first component binds to a protein that is generally associated with an increased risk of age-related macular degeneration (AMD). In some embodiments, the first component binds to a complement pathway component such as C2, Factor B, Factor H, CFHR3, C3b, C5, C5a, and C3a. In some embodiments, the first component binds to HtrA1, ARMS2, TIMP3, HLA, IL-8, CX3CR1, TLR3, TLR4, CETP, LIPC, or COL10A1.

c) Vascular endothelial growth factor (VEGF)

In some embodiments, the first component binds to vascular endothelial growth factor (VEGF). VEGF is known to be associated with a variety of eye diseases, such as diseases or conditions associated with diabetic retinopathy or macular edema. (See Section III below.)

The term "VEGF" refers to the 165-amino acid vascular endothelial cell growth factor, the related 121-, 189- and 206-amino acid vascular endothelial cell growth factors, and naturally occurring alleles and processed forms of those growth factors. VEGF may refer to a VEGF protein from any species.

VEGF is essential in normal development and pathological angiogenesis. Hypoxia-induced stellate cells secrete VEGF, which is a key factor in guiding retinal angiogenesis. Elevated VEGF levels also induce pathological growth of new blood vessels in the retina and choroid. Inhibition of angiogenic factors (such as VEGF) has become the main strategy for the treatment of pathological ocular angiogenesis (including age-related macular degeneration, proliferative retinal degeneration and retinal degeneration of prematurity).

The term "VEGF-mediated disorder" refers to any disorder whose symptoms or disease state require VEGF for development, progression or persistence. Exemplary VEGF-mediated disorders include, but are not limited to, age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, diabetic macular edema, pathological myopia, retinal vein occlusion, retinopathy of prematurity, abnormal vascular proliferation associated with phacomatoses, edema (such as edema associated with brain tumors), Meigs syndrome, rheumatoid arthritis, psoriasis, and atherosclerosis.

In some embodiments, the first component is a VEGF receptor, such as VEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR.

In some embodiments, the first component is an anti-VEGF antibody or antigen-binding fragment, more particularly an anti-VEGF Fab. The present disclosure provides VEGF antibodies and antigen-binding fragments. Other anti-VEGF antibodies, VEGF antagonists and VEGF receptor antagonists that can be used include, for example, ranibizumab, bevacizumab, aflibercept, pegaptanib, CT-322 and anti-VEGF antibodies and fragments thereof, as described in US 2012/0014958, WO 1998/045331 and WO 2015/198243, which are incorporated herein by reference in their entirety. In some embodiments, the first component comprises a drug targeting VEGF, such as those disclosed in Section II.A.2.a) below.

d) Erythropoietin (EPO)

In some embodiments, the first component is bound to erythropoietin (EPO). In some embodiments, the first component is bound to an erythropoietin receptor (EPOR). In many species, EPO refers to erythropoietin protein. The protein sequence of human, macaque, mouse, rat, rabbit EPO is publicly available. Human EPO can also be highly glycosylated. The terms "EPO receptor" or "EPOR" are used interchangeably and refer to erythropoietin receptor proteins in different species.

e) Angiogenin

In some embodiments, the first component binds to an angiogenin such as angiopoietin 2 (ANG2). ANG2 is known to be a candidate therapeutic for wet AMD because it plays a role in both angiogenesis and immune activation, both processes involved in pathological neovascularization in the eye. In the human eye, higher levels of ANG2 are associated with disease severity in wet AMD. Elevated levels of intraocular ANG2 have also been detected in patients with diabetic retinopathy and retinal vein occlusion, indicating that targeting intraocular ANG2 has potential medical significance. ANG2 refers to proteins in different species. Some researchers have also suggested using combined inhibition of VEGF-A/ANG2 to significantly reduce vascular leakage, immune reactivity, and apoptosis.

f) Interleukins

In some embodiments, the first component binds to an interleukin, such as interleukin (IL-) 1β (IL-1β), IL-6, IL-10, IL-17A, and IL-19. Interleukins are associated with eye diseases such as uveitis, an inflammatory disease that can cause blindness. Interleukins can be derived from any species.

g) Platelet-derived growth factor (PDGF)

In some embodiments, the first component binds to a therapeutic target that is platelet-derived growth factor (PDGF) or platelet-derived growth factor subunit B (PDGF-BB). PDGF and PDGF-BB can be derived from any species. In some embodiments, the first component comprises a PDGF antagonist, such as those disclosed in Section II.A.2.e) below.

h)VPDF

In some embodiments, the first component binds to VEGF and PDGF. Various proteins, antibodies, antibody fragments, binding domains, agonists and antagonists can bind to VEGF and PDGF. As used herein, the term "anti-VP" refers to a bispecific antibody or fragment thereof that binds to VEGF and PDGF.

In some embodiments, the first component is a dual-targeting Fab, ie, dutaFab. As used herein, "anti-VPDF" refers to a dutaFab that binds to VEGF and PDGF.

i) HtrA protein

In some embodiments, the first component is bound to a member of the HtrA family of serine proteases. The HtrA protein has a catalytic domain comprising at least one C-terminal PDZ domain and a protease chaperone protein independent of ATP that is associated with protein metabolism and cell fate. Clausen et al., Molecular cell 10(3):443-445(2002). There are four HtrA proteins in the human body: HtrA1, HtrA2, HtrA3 and HtrA4. In humans, HtrA1, HtrA3 and HtrA4 share the same domain architecture: an N-terminal IGFBP-like module and a Kazal-like module, a protease domain comprising a trypsin-like fold, and a C-terminal PDZ domain. Human genetic studies have found that there is a strong correlation between the progression of age-related macular degeneration (AMD) and single nucleotide polymorphisms (SNPs) in the HtrA1 promoter region, resulting in elevated HtrA1 transcript levels. Dewan et al., Science 314:989-992 (2006); Yang et al., Science 314:992-933 (2006).

In some embodiments, the first component binds to HtrA1. In some embodiments, the first component binds to HtrA2. In some embodiments, the first component binds to HtrA3. In some embodiments, the first component binds to HtrA4.

j) Other therapeutic targets

In some embodiments, the first component binds to one of the following therapeutic targets: factor P, factor D, TNFα, FGFR, IL-6R, Tie2, S1P, integrin αvβ3, integrin αvβ5, integrin α5β1, betacellulin, apelin/APJ, complement factor D, TNFα, HtrA1, ST-2 receptor, insulin, human growth factor, complement factor H, CD35, CD46, CD55, CD59, complement receptor 1-related (CRRY), nerve growth factor, pigment epithelium-derived factor, endostatin, ciliary neurotrophic factor, complement factor 1 inhibitor, complement factor-like 1, complement factor I, etc.

The term "Factor D" refers to Factor D protein derived from any species.

The term "factor P" refers to factor P protein derived from any species. Human factor P is available from Complement Tech (Tyler, TX). Macaque factor P can be purified from macaque serum (protocol adapted from Nakano et al., 1986, J Immunol Methods 90:77-83). Factor P is also known in the art as "properdin".

The term "FGFR2" refers to fibroblast growth factor receptor 2 derived from any species.

2. Treatment drugs

Any suitable therapeutic agent for treating an eye disease can be used as the first component (as described in Section III below). In some embodiments, the first component comprises a recognized therapeutic drug that binds to a target in the eye. In some embodiments, the first component binds to a human-derived target. In some embodiments, the first component comprises a recognized therapeutic drug for treating an eye disease.

a) Drugs targeting VEGF

In some embodiments, the first component comprises a VEGF antagonist, which includes, for example but not limited to: (1) an anti-VEGF antibody (e.g., (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single-chain antibody fragment; Novartis) or a bispecific anti-VEGF antibody (e.g., an anti-VEGF/anti-angiopoietin 2 bispecific antibody such as RG-7716; Roche)); (2) a soluble VEGF receptor fusion protein (e.g., Aflibercept); (3) Anti-VEGF (e.g., abicipar pegol; Molecular Partners AG/Allergan); or (4) anti-VEGF aptamers (e.g., pegaptanib sodium).

In some embodiments, the first component comprises (Ranibizumab), which is particularly useful for treating eye diseases. In some cases, the eye disease is age-related macular degeneration (AMD; e.g., wet AMD). In some cases, the eye disease is geographic atrophy (GA). In some cases, the eye disease is diabetic macular edema (DME) and/or diabetic retinopathy (DR; e.g., non-proliferative DR (NPDR) or proliferative DR (PDR)).

In some embodiments, the first component comprises RTH-258, which is particularly useful for treating an eye disease. In some cases, the eye disease is AMD (e.g., wet AMD). In some cases, the eye disease is GA.

In some embodiments, the first component comprises (aflibercept), which is particularly used to treat eye diseases. In some cases, the eye disease is AMD (e.g., wet AMD). In some cases, the eye disease is GA. In some cases, the eye disease is DME and/or DR (e.g., NPDR or PDR).

In some embodiments, the first component comprises abicipar pegol, which is particularly useful for treating an eye disease. In some cases, the eye disease is AMD (e.g., wet AMD). In some cases, the eye disease is GA.

In some embodiments, the first component comprises (pegaptanibsodium), which is particularly useful for treating eye diseases. In some cases, the eye disease is AMD (e.g., wet AMD). In some cases, the eye disease is GA.

b) Anti-angiogenic agents

In some embodiments, the first component comprises an anti-angiogenic agent. Non-limiting examples of anti-angiogenic agents include: anti-VEGF antibodies (e.g., anti-VEGF Fab (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single-chain antibody fragment; Novartis), bispecific anti-VEGF antibodies (e.g., anti-VEGF/anti-angiopoietin 2 bispecific antibodies such as RG-7716; Roche), soluble recombinant receptor fusion proteins (e.g., (aflibercept); also known as VEGF Trap Eye; Regeneron/Aventis), VEGF variants, soluble VEGF receptor (VEGFR) fragments, aptamers capable of blocking VEGF (e.g., anti-VEGF PEGylated aptamers (pegaptanib sodium; NeXstar Pharmaceuticals/OSI Pharmaceuticals)), aptamers capable of blocking VEGFR, aptamers that neutralize anti-VEGFR antibodies, small molecule inhibitors of VEGFR tyrosine kinase, anti-VEGF (e.g., abicipar pegol; Molecular Partners AG/Allergan), small interfering RNA that inhibits VEGF or VEGFR expression, VEGFR tyrosine kinase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidinyl)-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and (sunitinib)) and combinations thereof.

c) Anti-angiogenic agents

In some embodiments, the first component comprises an agent with anti-angiogenic activity for treating an eye disease, such as an anti-inflammatory drug, a mammalian target of rapamycin (mTOR) inhibitor (e.g., rapamycin, (everolimus) and (temsirolimus), cyclosporine, tumor necrosis factor (TNF) antagonists (e.g., anti-TNFα antibodies or antigen-binding fragments thereof (e.g., infliximab, adalimumab, certolizumab pegol, and golimumab) or soluble receptor fusion proteins (e.g., etanercept)), anti-complement drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), or combinations thereof.

d) Neuroprotective agents

In some embodiments, the first component comprises a medicament that has a neuroprotective effect and can potentially reduce disease progression. For example, the medicament can reduce the progression of dry AMD to wet AMD. Examples of neuroprotectants include a class of drugs known as "neurosteroids", which include drugs such as dehydroepiandrosterone (DHEA) (PRASTERA ^TM and ), dehydroepiandrosterone sulfate and pregnenolone sulfate.

e) PDGF antagonists

In some embodiments, the first component comprises a PDGF antagonist. In some embodiments, the PDGF antagonist is (1) an anti-PDGF antibody (e.g., REGN2176-3), (2) an anti-PDGF-BB PEGylated aptamer (e.g., E10030; Ophthotech/Novartis), (3) soluble PDGFR receptor fusion proteins, (4) dual PDGF/VEGF antagonists/inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)), (5) bispecific anti-PDGF/anti-VEGF antibodies, (6) anti-PDGFR antibodies, or (7) small molecule inhibitors (e.g., squalamine).

f) Complement system antagonists

In some embodiments, the first component comprises a complement system antagonist. Examples of complement system antagonists include: complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Opthotech)), anti-C5 antibodies (e.g., LFG-316; Novartis), properdin antagonists (e.g., anti-properdin antibodies; CLG-561; Alcon), complement factor D antagonists (e.g., anti-complement factor D antibodies; lampalizumab; Roche) and C3 blocking peptides (e.g., APL-2; Appellis).

g) Recognized drugs for the treatment of eye diseases

In certain embodiments, the first component comprises a recognized therapeutic drug for the treatment of eye diseases. The treatment of eye diseases is described in Section III below. Examples of recognized drugs include: nonsteroidal anti-inflammatory drugs (NSAIDs), steroids (e.g., for reducing inflammation and/or fibrosis), antibiotics, local ophthalmic anesthetics, ophthalmic adhesives (e.g., for postoperative wound closure), enzyme preparations (for vitreous surgery), DNA or RNA (e.g., for gene therapy techniques), agents that mediate neuroprotective effects (such as providing neurotrophic factors, blocking excessive glutamate stimulation, stabilizing Ca ²⁺ constant, preventing apoptosis, regulating immune status via vaccination, inducing endogenous neuroprotective mechanisms, antioxidants, vitamins and mineral supplements).

In some embodiments, the first component comprises any suitable DME and/or DR therapeutic agent, particularly for treating eye diseases, including but not limited to VEGF antagonists (e.g., or ), corticosteroids (eg, corticosteroid implants, a dexamethasone IVT implant; or Fluocinolone acetonide IVT implant) or a corticosteroid formulated for administration by IVT injection (e.g., Amidex) or a combination thereof. In some cases, the eye disease is DME and/or DR.

Further examples of recognized drugs for treating eye diseases suitable for use as the first component condition include, but are not limited to: (verteporfin; a photoactivated drug that is often used in combination with photodynamic therapy using non-thermal lasers), PKC412, Endovion (NS 3728; NeuroSearch A/S), neurotrophic factors (e.g., glial-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, 9-cis-retinal, ocular drugs (e.g., phospholine iodide, echothiophate, or carbonic anhydrase inhibitors), veovastat (AE-941; AEterna Laboratories, Inc.), Sirna-027 (AGF-745; Sima Therapeutics, Inc.), neurotrophins (including, by way of example only, NT-4/5, Genentech), Cand5 (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals), integrin antagonists (including those from Jerini AG and Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (as used, for example, by EntreMed, Inc.), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan/Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan), LYN-002 (Lynkeus Biotech), microalgae compounds (Aquasearch/Albany, Mera Pharmaceuticals), D-9120 (Celltech Group plc), ATX-S10 (Hamamatsu Photonics), TGF-β2 (Genzyme/Celtrix), tyrosine kinase inhibitors (e.g., those described in U.S. Pat. No. 7,771,742, and the VEGFR inhibitor SUGEN (SU5416), or Pfizer's Inlyta (dacomitinib), (lalorlatinib), NX-278-L (NeXstar Pharmaceuticals/Gilead Sciences), Opt-24 (OPTIS France SA), retinal cell ganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), cyclosporine A, therapeutic agents used in photodynamic therapy (e.g., Receptor-targeted PDT, Bristol-Myers Squibb, Co.; porfimer sodium for injection with PDT; verteporfin, QLT Inc.; rostaporfin for use with PDT, Miravent Medical Technologies; talaporfin sodium for use with PDT, Nippon Petroleum; and motexafin lutetium, Pharmacyclics, Inc.), antisense oligonucleotides (including, for example, products tested by Novagali Pharma SA and ISIS-13650, Ionis Pharmaceuticals), and combinations thereof.

In some embodiments, the first component comprises a tissue factor antagonist (e.g., hI-con1; Iconic Therapeutics), an α-adrenergic receptor agonist (e.g., brimonidine tartrate; Allergan), a peptide vaccine (e.g., S-646240; Shionogi), an amyloid β antagonist (e.g., anti-β amyloid monoclonal antibody; GSK-933776), an S1P antagonist (e.g., anti-S1P antibody; iSONEP ^™ ; Lpath Inc), a ROB04 antagonist, an anti-ROBO4 antibody (e.g., DS-7080a; Daiichi Sankyo).

In some embodiments, the first component comprises tryptophan-tRNA synthetase (TrpRS), squalamine, (anecortave acetate for long-acting suspension; Alcon, Inc.), Combretastatin A4 prodrug (CA4P), (mifepristone-ru486), subtenon triamcinolone acetonide, IVT crystalline triamcinolone acetonide, matrix metalloproteinase inhibitors (e.g., Prinomastat; AG3340; Pfizer), fluocinolone acetate (including fluocinolone intraocular implant; Bausch & Lomb/controlled delivery system), linomide, inhibitors of integrin β3 function, angiostatin, and combinations thereof. These and other therapeutic agents are described, for example, in U.S. Patent Application No. US 2014/0017244, which is incorporated herein by reference in its entirety.

3. Antibodies and antigen-binding fragments

In some embodiments, the first component comprises or is derived from an antibody or antigen-binding fragment thereof that is capable of binding to an antigen. The antibody or antigen-binding fragment binds to an unrelated, non-target protein to a degree that is less than about 10% of the binding of the antibody to the target, as measured, for example, by surface plasmon resonance (SPR). In certain aspects, the dissociation constant ( _KD ) of the antibody or antigen-binding fragment binding to the target is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., ^10-8 M or less, such as ^10-8 M to ^10-13 M, such as ^10-9 to ^10-13 M). When the _KD of an antibody is 1 μM or less, the antibody or antigen-binding fragment thereof is said to "specifically bind" to a target.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a bispecific antibody, an antibody lacking at least an Fc domain, a Fab fragment, a (Fab') ₂ fragment, a Fab' fragment, a VhH fragment, a scFv fragment, a scFv-Fc fragment, or a minibody.

In some embodiments, the antibody or antigen-binding fragment thereof binds to an antigen present in the eye. In some embodiments, the antibody or antigen-binding fragment thereof may bind to VEGF, HtrA1, IL-33, C5, Factor P, Factor D, EPO, EPOR, IL-1β, IL-17A, IL-10, TNFα, FGFR2, PDGF, or ANG2.

In some embodiments, the first component is an anti-VEGF antibody or antibody binding fragment, an anti-PDGF antibody or antibody-binding fragment, an anti-ANG2 antibody or antibody binding fragment, or an anti-IL-1β antibody or antibody binding fragment. Examples of antibodies that bind to VEGF include (Ranibizumab), (Aflibercept), (brocizumab-dbll) and (bevacizumab).

In some embodiments, the antibody comprises a bispecific antibody. In some embodiments, the bispecific antibody is an anti-VEGF/anti-Ang2 bispecific antibody, such as RG-7716 or any bispecific anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO 2010/069532 or WO 2016/073157 or variants thereof. In some embodiments, the bispecific antibody is an anti-VPDF antibody, i.e., an anti-VEGF and anti-PDGF dutaFab antibody.

In some embodiments, the first component is an anti-IL-6 antibody, such as EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890), siltuximab ( ), olokizumab, clazakizumab, sirukumab, elsilimomab, OPR-003, MEDI5117, PF-04236921, or variants thereof.

In some embodiments, the first component is an anti-IL-6R antibody, such as tocilizumab; ) (see, e.g., WO 1992/019579), sarilumab, ALX-0061, SA237, or variants thereof.

In some embodiments, the first component is RabFab, which is an antigen-binding Fab fragment derived from a parent monoclonal antibody (G10) produced in rabbits, directed against a phosphorylated peptide derived from the intracellular domain of the human cMET receptor, and therefore does not bind to extracellular targets in the eye. Shatz, W. et al., Mol. Pharm., 13(9): 2996-3003 (2016).

In some embodiments, the antigen-binding fragment comprises a peptide or polypeptide that is not an antibody or antigen-binding fragment thereof.

4. Growth Factors

In some embodiments, the first component comprises a growth factor. In some embodiments, the growth factor comprises fibroblast growth factor, platelet-derived growth factor, nerve growth factor (NGF), VEGF, fibroblast growth factor (FGF), and insulin-like growth factor-I (IGF-I).

5. Cysteine-knotted peptide

In some embodiments, the first component comprises a cysteine knot peptide. In some embodiments, the cysteine knot peptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 92 (cysteine knot peptide sequence).

The cysteine knot peptide can be covalently linked to another molecule to form a first component, including any of the exemplary first components described in Sections II.A.2-II.A.4 above. In some embodiments, the first component comprises a cysteine knot peptide covalently linked to an anti-VEGF antigen-binding fragment.

In some embodiments, the HABD (i.e., the second component) is covalently linked to the cysteine knot peptide of the first component. In some embodiments, the covalent linker comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 95. In some embodiments, the covalent linker comprises the sequence GSGSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95). In some embodiments, the cysteine knot peptide is covalently linked to VG1 with a C-terminal His tag (SEQ ID NO: 29). In some embodiments, the cysteine knot peptide is covalently linked to VG1 with an Ig domain deletion and a C-terminal His tag (SEQ ID NO: 32). In some embodiments, the cysteine knot peptide is covalently linked to VG1 with an N-terminal His tag. In some embodiments, the cysteine knot peptide is covalently linked to VG1 with an Ig domain deletion and an N-terminal His tag.

B. Second component - hyaluronic acid binding domain (HABD)

In many embodiments, the second component comprises or is derived from a HA binding protein (which comprises a HA binding domain; HABD). In some embodiments, the second component comprises a HABD. Examples of proteins comprising a HABD include CD44, tumor necrosis factor stimulated gene-6 (TSG6), versican, brain-specific connexin (BRAL1), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and aggrecan.

In some embodiments, the two second components may be different or the same. For example, the therapeutic molecule may include a second component comprising two CD44 domains, two TSG-6 domains, two VG1 domains, or any combination of the foregoing domains to form a pair of two different domains.

The eye is a complex tissue with several distinct compartments, including the cornea, aqueous humor, lens, vitreous humor, retina, retinal pigment epithelium, and choroid. These compartments include extracellular macromolecules such as HA.

The term "hyaluronan-binding protein" or "HA-binding protein" refers to a protein or family of proteins that bind HA. Typically, these HA-binding proteins comprise a HABD. Various HA-binding molecules are well known in the art and can be used as a second component (see, for example, Day et al., 2002, J Bio. Chem 277:4585; and Yang et al., 1994, EMBO J 13:286-296). Exemplary HA binding proteins include CD44, LYVE-1, aggrecan, versican, Brevican, Neurocan, hyaluronan binding protein 1 (HABP1; also known as C1qBP/C1qR and p32), HAPLN1 (also known as connexin and CRTL1), hyaluronan and proteoglycan connexin 4 (HAPLN4; also known as brain connexin 2), Layilin, Stabilin-1, Stabilin-2, brain-specific connexin (BRAL1) or tumor necrosis factor-stimulated gene 6 (TSG-6), RHA M, bacterial HA synthase and type VI collagen.

Many HA binding proteins and peptide fragments contain a common domain, which is about 100 amino acids in length and is involved in HA binding; this domain is called the "LINK Domain" (Yang et al., EMBO J 13:2, 286-296 (1994) and Mahoney et al., J Bio. Chem 276:25, 22764-22771 (2001)). Any of these proteins can be used in the present invention. Any HA binding protein such as the HABD of the above exemplary proteins can be included in the second component to give the ability to bind to HA. Preferably, the second component comprises a CD44 (CD44) domain, a brain-specific connexin (BRAL1) domain, a tumor necrosis factor-stimulated gene 6 (TSG-6) domain, a lymphatic endothelial hyaluronan receptor 1 (LYVE-1) domain, a hyaluronan binding protein (HABP) domain, aggrecan G1 (AG1) domain or a versican G1 (VG1) domain. Exemplary and suitable HA binding molecules for use in the eye (including peptide tags) are described in WO 2014/99997 and WO 2015/19824, the contents of which are incorporated herein by reference in their entirety. Any of the sequences described therein may be used in the present invention.

In some embodiments, the second component is covalently linked to the first component to reduce the clearance of the first component from the eye, thereby extending its intraocular half-life. The first component may benefit from longer eye retention and/or longer duration of action on eye diseases.

In addition, the second component can be non-covalently bound to a third component comprising HA to form a conjugate. In some embodiments, each second component in the conjugate can be bound to a separate molecule of HA. In some embodiments, two or more second components can be bound to the same HA molecule.

In many embodiments, the binding affinity of the HABD to HA can be within several ranges; the binding affinity can be adjusted according to the mechanism of action of the therapeutic agent. For example, if the site of action is in the vitreous humor, a high binding affinity may help the biologic to remain in the vitreous humor. Conversely, if the site of action is in the retina, a lower binding affinity may help the biologic to pass through the vitreous to reach the retina.

In many embodiments, the binding affinity of HABD to HA can be measured using methods including surface plasmon resonance (SPR). Without being bound by theory, in some embodiments, the binding affinity ( _KD ) of HABD to HA ranges from 10 nM to 10 μM, 5 nM to 10 nM, and 100 nM to 5 μM.

In many embodiments, the interaction of HABD and HA can be observed. In some embodiments, the interaction is observed using a method including fluorescence correlation spectroscopy (FCS). In FCS, the diffusion of molecules can be determined by monitoring the fluorescence intensity of a small volume portion in the solution. Fluorescence intensity fluctuates due to the movement of molecules, and quantitative analysis of these fluctuations can derive the diffusion time of molecules. By using a fluorescent dye with appropriate spectral characteristics, diffusion in a biological matrix can be determined. In some embodiments, the observed value of FCS is related to the measured value of SPR.

In some embodiments, HABD comprises a wild-type sequence compared to the protein from which it is derived. In some embodiments, HABD may comprise one or more mutations in its protein sequence compared to the protein from which it is derived. In many embodiments, these mutations comprise single amino acid substitutions, double amino acid substitutions, additions, deletions, and truncations.

In some embodiments, the HABD comprises a single amino acid substitution or a double amino acid substitution. In many instances, the substitution may comprise a conservative mutation, wherein the amino acid substitution changes the original amino acid to a different amino acid with similar biochemical properties. In other instances, the substitution may comprise a non-conservative mutation, wherein the amino acid substitution changes the original amino acid to a different amino acid with different biochemical properties.

In some embodiments, the HABD comprises amino acids that contribute to HA binding. In some embodiments, these amino acids may be conserved to maintain HA binding affinity. In some embodiments, these amino acids may be substituted to alter HA binding affinity, depending on the affinity and duration desired for long-acting treatment.

In some embodiments, the HABD comprises amino acids that contribute to the thermostability of the HABD and/or therapeutic molecule. In some embodiments, these amino acids may be retained to maintain thermostability. In some embodiments, these amino acids may be substituted to alter thermostability.

In some embodiments, the HABD comprises at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to a reference sequence disclosed herein. In some embodiments, the HABD comprises 1 to 3 mutations, wherein these 1 to 3 mutations independently comprise single amino acid substitutions, double amino acid substitutions, additions, deletions, and truncations. In some embodiments, the HABD comprises 1 to 5 mutations, wherein these 1 to 5 mutations independently comprise single amino acid substitutions, double amino acid substitutions, additions, deletions, and truncations.

In some embodiments, the second component comprises or is derived from CD44, TSG6, or versican. In some embodiments, the second component comprises a CD44 domain, a TSG6 domain, or a versican domain.

1.CD44

In some embodiments, the second component is derived from CD44 (SEQ ID NO: 1). The CD44 receptor comprises a connecting domain, a GAG attachment domain, a transmembrane domain, and a cytoplasmic domain. Several isoforms with different modular compositions processed by alternative splicing are described. In some embodiments, the second component is derived from or comprises a CD44 HA receptor domain. In some embodiments, the second component is derived from or comprises SEQ ID NO: 2.

2. Tumor necrosis factor-stimulated gene 6 (TSG6)

In some embodiments, the second component is derived from TSG6. TSG-6, also known as TNFAIP6, consists of an HA binding linker domain followed by a CUB domain. In some embodiments, the second component is derived from or comprises a TSG6 HA binding linker domain. In some embodiments, the second component is derived from or comprises SEQ ID NO: 4.

3. Versatile proteoglycan

In some embodiments, the second component is derived from versican. Versican comprises the following domains: VG1, GAG attachment domain and G3 domain (FIG. 8A). The VG1 domain (SEQ ID NO: 29) comprises an Ig domain, Link1 and Link2 (FIG. 8A). In some embodiments, the second component comprises Link1 (SEQ ID NO: 30) and/or Link2 (SEQ ID NO: 31), wherein Link1 and/or Link2 are capable of binding to HA.

a) Wild-type VG1

In some embodiments, the HABD comprises wild-type (WT) VG1, the amino acid sequence of which is set forth in SEQ ID NO: 29. In some embodiments, the HABD comprises the amino acid sequence set forth in Link1 (SEQ ID NO: 30) and/or Link2 (SEQ ID NO: 31).

b) Mutation of VG1

In some embodiments, the HABD comprises a mutant VG1. In many embodiments, the VG1 mutation is relative to the amino acid sequence shown as SEQ ID NO: 29 (WT VG1), 32 (VG1ΔIg), 60 (WT VG1 consensus sequence) or 86 (VG1ΔIg consensus sequence). In some embodiments, the HABD comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 29 (WT VG1), 32 (VG1ΔIg), 60 (WT VG1 consensus sequence) or 86 (VG1ΔIg consensus sequence). In some embodiments, the HABD comprises a sequence that is at least 95% identical to SEQ ID NO: 29 (WT VG1), 32 (VG1ΔIg), 60 (WT VG1 consensus sequence) or 86 (VG1ΔIg consensus sequence).

c) Truncated VG1

In some embodiments, the HABD comprises a truncation mutation relative to SEQ ID NO: 29 (WT VG1) or 60 (WT VG1 consensus sequence). In some embodiments, the HABD comprises a truncation of 1 to 129 amino acids from the N-terminus of versican. In some embodiments, the HABD comprises a truncated sequence in which the Ig domain of wild-type versican is absent. In some embodiments, the HABD comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32 (VG1ΔIg) or SEQ ID NO: 86 (VG1ΔIg consensus sequence). In some embodiments, the HABD comprises a sequence at least 95% identical to SEQ ID NO: 32 (VG1ΔIg) or SEQ ID NO: 86 (VG1ΔIg consensus sequence). In some embodiments, the HABD comprises SEQ ID NO: 32 (VG1ΔIg).

d) Amino acid substitution

In some embodiments, the HABD comprises at least one of the following amino acids relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, Y230, F261, D295, and R233. In some embodiments, the HABD comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the following amino acids relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, Y230, F261, D295, and R233.

In some embodiments, the HABD comprises a sequence in which an amino acid can be mutated relative to wild type to increase or decrease HA binding affinity. In some embodiments, the HABD comprises a mutation at at least one of the following positions relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326, and R327. In some embodiments, the HABD comprises mutations at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the following positions relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326, and R327. In some embodiments, the HABD comprises mutations at 2, 3, 4, 5, or 6 of the following positions relative to SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326, and R327.

In some embodiments, the HABD comprises at least one of the following mutations relative to SEQ ID NO: 29: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A, and LYR325LFK. In some embodiments, the HABD comprises at least one of Y208A and H306A.

In some embodiments, the HABD comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the following mutations relative to SEQ ID NO: 29: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A, and LYR325LFK. In some embodiments, the HABD comprises at least 2, 3, 4, 5, or 6 of the following mutations relative to SEQ ID NO: 29: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A, and LYR325LFK.

In some embodiments, the HABD is SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 , SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 or SEQ ID NO:59.

4. Brain-specific connexin (BRAL1)

In some embodiments, the second component is derived from BRAL1. BRAL1 comprises an immunoglobulin domain, a connecting domain module 1 and a connecting domain module 2. Connecting domain modules 1 and 2 are capable of binding to HA. In some embodiments, the second component comprises a connecting domain module 1 and/or a connecting domain module 2 from BRAL1.

5. Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1)

In some embodiments, the second component is derived from LYVE-1. LYVE-1 is a homolog of CD44 that comprises a linking domain that binds to HA. In some embodiments, the second component comprises a linking domain from LYVE-1.

6. Aggrecan

In some embodiments, the second component is derived from aggrecan. Aggregate proteoglycan contains three globular domains: the G1 domain has a structural motif of the linker protein and interacts with HA; the G2 domain is homologous to the G1 domain and is involved in product processing; the G3 domain constitutes the carboxyl terminus of the core protein. In some embodiments, the second component comprises the G1 domain from aggrecan.

C. The third component - hyaluronic acid (HA)

In some embodiments, the therapeutic molecule further comprises one or more third components. In some embodiments, the third component comprises HA. In some embodiments, the therapeutic molecule (comprising the first component and the second component) is pre-compounded with HA to form a conjugate. In some embodiments, the third component is HA with a molecular weight of 5kDa to 20kDa.

In some embodiments, the second component of the therapeutic molecule is non-covalently bound to the third component to form a conjugate. In some embodiments, the second component of the therapeutic molecule is covalently bound to the third component to form a conjugate.

Preferably, the second component covalently linked to the first component binds to the third component (i.e., hyaluronic acid) with a _K of less than or equal to 10.0 μM. For example, the second component can bind to HA with a _K of less than or equal to 9.0 μM, 8.0 μM, 7.0 μM, 6.0 μM, 5.0 μM, 4.0 μM, 3.0 μM, 2.0 μM, 1.5 μM, 1.0 μM, or 0.5 μM.

1. Hyaluronic acid (HA)

Hyaluronic acid (HA) is a linear glycosaminoglycan present in the extracellular matrix and cell surface. HA contains repeated disaccharide units of N-acetylglucosamine (GlcNac) and glucuronic acid (GlcUA), which are connected by alternating β1→3 glucuronic acid and β1→4 glucosamine (glucosaminidic) bonds to form a linear polymer. HA is further described in the following literature: Necas et al., 2008, Veterinarni Medicina, 53: 397-411. Glycosaminoglycans are ubiquitous in the extracellular matrix of all vertebrates and are also present in the capsule of some streptococcal strains. Functionally, HA molecules are important for maintaining highly hydrated extracellular matrix in tissues, which participate in cell adhesion and support cell migration. The vitreous humor, in addition to water, is mainly composed of HA, which excels in retaining moisture and structure in the central part of the eye. It helps to keep the eye lubricated and replenish any lost moisture. HA also exhibits a variety of biological functions by interacting with a number of HA binding proteins and cell surface receptors such as CD44 and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Examples of HA binding proteins and HABDs are described above in Section II.B.

HA has a wide molecular weight range of 1000Da to 10000000Da. The natural high molecular weight HA in the tissue is degraded into small molecules in the metabolic pathways through the lymphatic system, lymph nodes, liver and kidneys. Although the half-life of HA in plasma is known to be about 2.5 minutes to 5.5 minutes, it is reported that its half-life in the vitreous body of the eye is about 70 days. The unique physicochemical properties and various biological functions of HA make it widely used in biomedical fields (such as drug delivery, arthritis treatment, ophthalmic surgery and tissue engineering). In particular, HA has been widely studied for the targeting specificity and long-acting delivery of biological/drugs through various delivery routes. Utilizing its viscoelasticity and mucoadhesion properties, HA has been developed as a delivery vehicle for effective local ophthalmic drugs.

It has been shown that HA of defined size is suitable for the present invention. Accordingly, HA may have a molecular weight of at least 2, 3, 4, 5, 6, 7, 8 or 9 kDa and/or a molecular weight of at most 60, 50, 40, 30, 25, 20 or 15 kDa. In particular, a suitable range of molecular weight is 3 kDa to 60 kDa, in particular 4 kDa to 30 kDa, more in particular 5 kDa to 20 kDa.

In some embodiments, unmodified naturally occurring HA is preferably used. In these embodiments, the use of unmodified naturally occurring HA reduces side effects. For example, pre-complexing the HABD with 10 kDa HA can reduce in vitro precipitation in the vitreous humor and reduce intraocular toxicity observed in pigs and rabbits. In other examples, when the HABD is TSG-6 or CD44, intraocular toxicity such as inflammation and retinal is observed when TSG-6 or CD44 is not pre-complexed with HA.

In some embodiments, HA is a hyaluronate salt, including but not limited to potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.

In some embodiments, HA may have minor chemical modifications. Chemical modifications can be used to reduce HA degradation, increase or decrease water solubility, change HA diffusion rate and/or HA viscosity. Two general methods for chemically modifying HA are known in the art - (1) cross-linking HA using functional chemical reagents and (2) coupling HA using monofunctional reagents. Divinyl sulfone, diepoxide, formaldehyde and dihalides are bifunctional reagents used to cross-link HA. Chemically modified HA preparations include, but are not limited to, aminoethyl methacrylate HA, adipic acid dihydrazide grafted HA, dimethyl ether composite HA, HA-cysteine ethyl ester, urea cross-linked HA and N-acetyl cysteine HA. Of particular interest are modifications that reduce HA degradation in the HA eye.

2. Pre-complexation of therapeutic molecules with hyaluronic acid (HA) to form a conjugate

In some embodiments, the therapeutic molecule is pre-complexed with HA to form a conjugate. The initial concentration of free HABD contained in the therapeutic molecule at the injection site may be very high, resulting in adverse effects, as described in Example 5 below. In some cases, these effects may be caused by contact of free HABD with IVT HA at the injection site. The pre-complexation of HABD with HA reduces these adverse effects by giving HABD time to diffuse from the injection site to the rest of the vitreous. When HABD changes from interacting with pre-complexed HA to interacting with IVT HA, the diffusion time slows down and the vitreous half-life is extended. Therefore, in some embodiments, the therapeutic molecule is a conjugate, comprising the therapeutic molecule, and further comprising one or more third components comprising HA.

In some embodiments, the conjugate comprises a non-covalent interaction between the therapeutic molecule and HA. In some embodiments, the conjugate comprises a covalent interaction between the therapeutic molecule and HA.

In some embodiments, the conjugate may be an isolated conjugate, i.e., the conjugate is not in the individual to be treated. In some aspects, the conjugate is purified to a purity greater than 95% or 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J Chromatogr B 848:79-87 (2007).

D. Fusion protein

In some embodiments, the first component and the second component are proteins, more preferably contained in a fusion protein; the first component and the second component are linked via a covalent linker.

Fusion proteins are proteins formed by connecting two or more originally separated proteins or peptides. The method results in a single polypeptide with functional properties derived from each of the original, separate proteins. Proteins can be fused directly to each other. Proteins can also be fused via a linker that increases the likelihood that the proteins fold independently of each other and behave as expected. Dimeric or multimeric fusion proteins can be produced by genetic engineering by fusing the original protein with a peptide domain that induces protein complexing (such as with an antibody domain).

In some embodiments, the second component is directly bound to the first component. This means that the second component directly follows the first component (or vice versa), and there is no other chemical element (atom or group) between these two components. In some embodiments, the last amino acid of the first component is adjacent to the first amino acid of the second component. In some embodiments, the last amino acid of the second component is adjacent to the first amino acid of the first component.

In some embodiments, the second component is indirectly bound to the first component via a joint (particularly a peptide joint). In some embodiments, this means that the peptide joint is between the first component and the second component. In some embodiments, the peptide joint is between the last amino acid of the first component and the first amino acid of the second component. In some embodiments, the peptide joint is between the last amino acid of the second component and the first amino acid of the first component.

In some embodiments, these one or two second components are covalently bound to the N-terminus and/or C-terminus of the first component. In some embodiments, the first component is an antibody or antigen binding fragment, and these one or two second components are covalently bound to the C-terminus of the first component (directly or via a peptide linker). In embodiments where the fusion protein is Fab-HABD, the HABD is covalently bound to the C-terminus of the Fab.

1. Peptide linker

In many embodiments, the peptide linker connects the therapeutically active agent (i.e., the first component) to the HABD (i.e., the second component). In some embodiments, the linker comprises at least 4 amino acids. In some embodiments, the linker comprises 4 to 25 amino acids. In some embodiments, the linker comprises 5 to 100 amino acids. In some embodiments, the linker comprises 10 to 50 amino acids. In some embodiments, the length of the linker does not exceed 25 amino acids. In some embodiments, the length of the linker does not exceed 50 amino acids.

In some embodiments, the peptide linker comprises flexible residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. Thus, in some embodiments, the peptide linker is a glycine-serine linker, i.e., a peptide linker consisting of a pattern of glycine and serine residues. In one embodiment, the peptide linker is (GxS) _n or (GxS) _nGm , where G=glycine and S=serine. In these embodiments, x=3; n=3, 4, 5 _or 6; and m=0, 1, 2 or 3. In other embodiments, x=4; n=2, 3, 4 or 5; and m=0, 1, 2 or 3. In some embodiments, x=4 and n=2 or 3. In some embodiments, x=4 and n=2.

In some embodiments, the peptide linker consists of GGGGS (SEQ ID NO: 27) or a multimer thereof, more particularly consists of (GGGGS) ₃ (SEQ ID NO: 28).

In some embodiments, the peptide linker comprises (GS) _n , wherein G is glycine and S is serine. In these embodiments, n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n = 10. In some embodiments, the linker is SEQ ID NO:95.

E. Certain Examples of Therapeutic Molecules and Degradants

1. VEGF

In some embodiments, the therapeutic molecule comprises (1) a first component comprising an anti-VEGF antibody, antibody fragment, antigen binding fragment or Fab; (2) one or two second components, wherein the second component comprises a CD44 HA receptor domain, a TSG6 domain and/or a VG1 domain.

In some embodiments, the conjugate comprises (1) a first component comprising an anti-VEGF antibody, antigen-binding fragment, antibody fragment, or Fab; (2) one or two second components; and (3) HA having a molecular weight range of 5 kDa to 20 kDa.

a) G6.31

In some embodiments, the first component is an antibody comprising G6.31 anti-VEGF Fab. In some embodiments, the first component is an antibody having a VH domain contained in SEQ ID NO: 17. In some embodiments, the first component is an antibody having a VL domain contained in SEQ ID NO: 18. In some embodiments, the first component is an antibody comprising a VH domain as shown in SEQ ID NO: 105. In some embodiments, the first component is an antibody comprising a VL domain as shown in SEQ ID NO: 106.

b) PigFab

In some embodiments, the first component is an antibody comprising a PigFab anti-VEGF Fab. In some embodiments, the first component is an antibody having a VH domain contained in SEQ ID NO: 66. In some embodiments, the first component is an antibody having a VL domain contained in SEQ ID NO: 65. In some embodiments, the first component is an antibody comprising a VH domain as shown in SEQ ID NO: 97. In some embodiments, the first component is an antibody comprising a VL domain as shown in SEQ ID NO: 98.

c) Ranibizumab

In some embodiments, the first component is an antibody comprising ranibizumab. In some embodiments, the first component is an antibody having a VH domain contained in SEQ ID NO: 77. In some embodiments, the first component is an antibody having a VL domain contained in SEQ ID NO: 76. In some embodiments, the first component is an antibody comprising a VH domain as shown in SEQ ID NO: 114. In some embodiments, the first component is an antibody comprising a VL domain as shown in SEQ ID NO: 115.

d) CD44

In some embodiments, these one or both second components comprise a CD44 HA receptor domain. In some embodiments, the second component comprises SEQ ID NO:2.

e)TSG6

In some embodiments, these one or both second components comprise a TSG6 domain. In some embodiments, these one or both second components comprise SEQ ID NO:4.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and/or SEQ ID NO:20.

f)VG1

In some embodiments, these one or both second components comprise a VG1 domain. In some embodiments, these one or both second components comprise one or both of the following SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:76 and/or SEQ ID NO:77.

g) Cysteine knot peptide (CKP)

In some embodiments, the first component optionally further comprises a cysteine knot peptide (CKP) in addition to the anti-VEGF antigen-binding fragment. In some embodiments, the CKP has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 92.

In some embodiments, the therapeutic molecule has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 93. In some embodiments, the anti-VEGF antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 94.

In some embodiments, the therapeutic molecule comprises a first component comprising an anti-VEGF antigen binding fragment, and the cysteine knot peptide may further comprise a second component comprising a HABD as described in Section II.B above.

In some embodiments, the conjugate comprises (1) a first component comprising an anti-VEGF antigen-binding fragment, (2) one or two second components comprising a HABD, and (3) HA having a molecular weight range of 5 kDa to 20 kDa.

2. NVS24

In some embodiments, the therapeutic molecule comprises (1) a first component comprising the anti-VEGF antibody NVS24, and (2) a second component comprising a TSG6 (Lava12) domain.

In some embodiments, the first component comprises the NVS24 antibody. In some embodiments, the first component is an antibody having a VH domain contained in SEQ ID NO: 21. In some embodiments, the first component is an antibody having a VL domain contained in SEQ ID NO: 22. In some embodiments, the first component is an antibody comprising a VH domain as shown in SEQ ID NO: 109. In some embodiments, the first component is an antibody comprising a VL domain as shown in SEQ ID NO: 110.

a)TSG6(Lava12)

In some embodiments, the second component comprises a TSG6 (Lava12) domain. In some embodiments, the second component comprises SEQ ID NO:113.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:21 and/or SEQ ID NO:22.

3. Anti-VEGF and anti-PDGF dual-targeting antibodies (anti-VP-dutaFab; anti-VPDF)

In some embodiments, the therapeutic molecule comprises (1) a first component that is capable of binding to VEGF and PDGF (such as a bispecific antibody or a dual-targeting antibody, dutaFab), as described in Section II.A.1.h) above; and (2) one or two second components that comprise a CD44 HA receptor domain, a TSG6 domain, and/or a VG1 domain.

In some embodiments, the first component is an antibody having a VH domain contained in SEQ ID NO: 5. In some embodiments, the first component is an antibody having a VL domain contained in SEQ ID NO: 6. In some embodiments, the first component is an antibody comprising a VH domain as shown in SEQ ID NO: 99. In some embodiments, the first component is an antibody comprising a VL domain as shown in SEQ ID NO: 100.

a) CD44

In some embodiments, these one or both second components comprise a CD44 HA receptor domain. In some embodiments, these one or both second components comprise SEQ ID NO:2.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8.

In some embodiments, these one or both second components comprise a CD44-ko domain. In some embodiments, these one or both second components comprise a CD44-ko domain as shown in SEQ ID NO: 25 and/or SEQ ID NO: 26.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:25 and/or SEQ ID NO:26.

b)TSG6(Lava12)

In some embodiments, the second component comprises a TSG6 (Lava12) domain. In some embodiments, the second component comprises SEQ ID NO:113.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:23 and/or SEQ ID NO:24.

c)VG1

In some embodiments, these one or both second components comprise a VG1 domain. In some embodiments, these one or both second components comprise one or both of the following SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:72 and/or SEQ ID NO:73.

4. RabFab

In some embodiments, the therapeutic molecule comprises (1) a first component comprising a RabFab antibody (as described in Section II.A.3 above); and (2) one or two second components comprising a TSG6 domain and/or a VG1 domain.

In some embodiments, the RabFab antibody comprises a RabFab VH and VL domain. In some embodiments, the RabFab antibody comprises: a VH domain, which is contained in SEQ ID NO: 13; and a VL domain, which is contained in SEQ ID NO: 14. In some embodiments, the RabFab antibody comprises a VH domain as shown in SEQ ID NO: 107. In some embodiments, the RabFab antibody comprises a VL domain as shown in SEQ ID NO: 108.

a)TSG6

In some embodiments, these one or both second components comprise a TSG6 domain. In some embodiments, these one or both second components comprise SEQ ID NO:4.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and/or SEQ ID NO:16.

b) VG1

In some embodiments, these one or both second components comprise a VG1 domain. In some embodiments, these one or both second components comprise one or both of the following SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:63 and/or SEQ ID NO:64.

5.20D12v2.3

In some embodiments, the first component is an antibody comprising an anti-complement factor D antibody Fab (20D12v2.3). In some embodiments, the first component is an antibody having a VH domain contained in SEQ ID NO: 75. In some embodiments, the first component is an antibody having a VL domain contained in SEQ ID NO: 74. In some embodiments, the first component is an antibody comprising a VH domain as shown in SEQ ID NO: 111. In some embodiments, the first component is an antibody comprising a VL domain as shown in SEQ ID NO: 112.

a) VG1

In some embodiments, these one or both second components comprise a VG1 domain. In some embodiments, these one or both second components comprise one or both of the following SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:74 and/or SEQ ID NO:75.

6.HtrA1

In some embodiments, the first component is an antibody comprising an antibody or antibody fragment capable of binding to human HtrA1. In some embodiments, the first component is an antibody having a VH domain contained in SEQ ID NO: 118. In some embodiments, the first component is an antibody having a VL domain contained in SEQ ID NO: 119. In some embodiments, the first component is an antibody comprising a VH domain as shown in SEQ ID NO: 116. In some embodiments, the first component is an antibody comprising a VL domain as shown in SEQ ID NO: 117.

a) VG1

In some embodiments, these one or both second components comprise a VG1 domain. In some embodiments, these one or both second components comprise one or both of the following SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.

In some embodiments, the therapeutic molecule comprises SEQ ID NO:118 and/or SEQ ID NO:119.

III. Treatment of eye diseases

Materials and methods are used in the treatment of eye diseases. Eye diseases can be characterized by altered or unregulated neovascularization and/or invasion into structures of eye tissues such as the retina or cornea. Eye diseases can be characterized by atrophy of retinal tissues (photoreceptors and underlying retinal pigment epithelial cells (RPE) and choriocapillaris). Non-limiting eye diseases include, for example, age-related macular degeneration (AMD) (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, diabetic macular edema (DME) (e.g., focal, non-central DME and diffuse, centrally involved DME), retinal degeneration, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other retinal degenerations associated with ischemia, ROP, retinal vein occlusion (RVO) (e.g., central (CRVO) and branch (BRVO) forms), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal/choroidal neovascularization, central serous retinal degeneration (CSR), pathological myopia, von Hippel-Lindau, ocular histoplasmosis, FEVR, Coats' disease, Norrie's disease, retinal abnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, erythema, ocular angiogenesis disease, angiogenic glaucoma, retinitis pigmentosa (RP), hypertensive retinal degeneration, retinal angiomatous proliferation, macular telangiectasia, iris angiogenesis, intraocular angiogenesis, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis, including but not limited to CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g., allergic) conjunctivitis), Leber's congenital amaurosis (also known as Leber's congenital amaurosis or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, ocular trauma, Diseases and other ophthalmic diseases, wherein the disease or condition is associated with angiogenesis, vascular leakage and/or retinal edema or retinal atrophy. Other exemplary eye diseases include diseases caused by abnormal proliferation of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinal degeneration.

Exemplary diseases associated with corneal neovascularization (iris neovascularization, corner neovascularization, or skin redness) include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, excessive contact lens wear, atopic keratitis, superior marginal keratitis, pterygium sicca, sjogrens, rosacea, phlyctenulosis, syphilis, mycobacterial infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infection, herpes zoster infection, protozoan infection, Kaposi's sarcoma, molluscum corneal ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis, trauma, Wegeners sarcoidosis, scleritis, Steven's Johnson syndrome, disease), pemphigoid radial keratotomy, and corneal transplant rejection.

Exemplary ocular diseases associated with choroidal neovascularization and defects in the retinal vasculature, including increased vascular leakage, aneurysms, and capillary dripping, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinal degeneration of prematurity, retinal edema (including macular edema), Eales disease, Bechets disease, infections causing retinitis or choroiditis (e.g., multifocal choroiditis), presumed ocular histoplasmosis, Bests disease (vitelliform macular degeneration), myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications.

Exemplary eye diseases associated with retinal tissue (photoreceptors and underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or advanced dry AMD), macular atrophy (e.g., atrophy and/or geographic atrophy associated with neovascularization), diabetic retinopathy, Stargardt's disease, Skorsby fundus atrophy, retinoschisis (abnormal splitting of the retinal neurosensory layer), and retinitis pigmentosa.

In certain embodiments according to (or as applied to) any of the above embodiments, the eye disease is an intraocular neovascular disease selected from the group consisting of proliferative retinal degeneration, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemic-related retinal degenerations, diabetic macular edema, pathological myopia, Feng Heber-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (including CRVO and BRVO), corneal neovascularization, retinal neovascularization and retinal degeneration of prematurity (ROP). In a preferred embodiment of the present invention, the eye disease is age-related macular degeneration (AMD), in particular wet AMD or neovascular AMD; diabetic macular edema (DME); diabetic retinopathy (DR), in particular proliferative DR or non-proliferative DR; retinal vein occlusion (RVO); or geographic atrophy (GA).

The therapeutic molecules, conjugates and compositions disclosed herein can be used as drugs for treating eye diseases in mammalian subjects. Examples of mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human. In some embodiments, the therapeutic target of the therapeutic molecules, conjugates, and compositions is a target in the human eye.

IV. Treatment Methods

Provided herein are methods for treating eye diseases, the methods comprising delivering a therapeutic molecule, conjugate, or composition to a patient's tissue. In many embodiments, the methods comprise administering a therapeutic molecule such that the therapeutic molecule provides a long-acting delivery of a therapeutically active agent to a target tissue. In many embodiments, the target tissue is in the eye.

A. Application Method

The therapeutic molecule, conjugate or composition can be administered in any effective and convenient manner, including, for example, administration by topical, oral, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intratracheal or intradermal routes. Preferably, the composition is suitable for administration to the eye, and more specifically, the composition may be suitable for IVT administration. Thus, in a preferred embodiment, the composition is formulated for intraocular delivery, particularly for IVT injection. In therapy or as a prophylactic, the therapeutic molecule, conjugate or composition may be administered to an individual as an injectable composition (e.g., as a sterile aqueous dispersion).

Without being bound by this theory, it is believed that the injection of the conjugate promotes diffusion of HA from the pre-complexed HABD before interaction with the IVT HA. Due to the slow dissociation, the concentration of free HABD in the vitreous is lower. The lower concentration of free HABD in the vitreous may be less harmful to the eye than the therapeutic molecule that is not pre-complexed with HA.

In some embodiments, the administering step is a single injection. In some embodiments, the administering step comprises more than one single injection.

B. Composition

Provided herein are compositions for use as medicines, particularly for the treatment of eye diseases. Compositions may be referred to as pharmaceutical compositions because they are intended for use in the pharmaceutical field or as medicines, and refer to preparations that are in a form that allows the biological activity of the active ingredients contained therein to be effective and do not contain other components that are unacceptably toxic to the subject to which the composition will be administered.

In some embodiments, the composition comprises a therapeutic molecule. In some embodiments, the composition comprises a conjugate.

In some embodiments, the composition optionally comprises a pharmaceutically acceptable excipient, diluent or carrier, such as a buffer substance, stabilizer, preservative or other ingredients, especially those commonly known in connection with pharmaceutical compositions.

In general, the properties of the optional ingredients or additional ingredients will depend on the specific form of the composition and the mode of administration adopted. Pharmaceutical carriers can enhance or stabilize the composition, or can be used to promote the preparation of the composition. These carriers may include, but are not limited to, physiological compatibility saline, buffered saline, glucose, water, glycerol, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc. and combinations thereof. The composition may additionally include one or more other therapeutic agents, particularly including those suitable for treating or preventing diseases or conditions associated with eye diseases such as retinal vascular diseases. The preparation should adapt to the mode of administration. For example, parenteral preparations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc., used as carriers. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain a small amount of nontoxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, etc.

The composition may include a stabilizer. The term "stabilizer" refers to a substance that protects the composition from adverse conditions (such as those occurring during heating or freezing) and/or prolongs the stability or shelf life of the conjugate of the invention under certain conditions or states. Examples of stabilizers include, but are not limited to, sugars such as sucrose, lactose, and mannose; sugar alcohols such as mannitol; amino acids such as glycine or glutamic acid; and proteins such as human serum albumin or gelatin.

C. Effective dose

Typically, a therapeutically effective amount or effective dose of a therapeutic molecule or conjugate is used in the pharmaceutical composition of the present disclosure. The therapeutic molecule and conjugate are formulated into a pharmaceutical dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. It is particularly advantageous to prepare parenteral compositions in dosage unit form, which is convenient for administration and ensures uniformity of dosage. As used herein, the dosage unit form refers to a physically discrete unit suitable for use as a single dose of a subject to be treated; each unit contains a predetermined amount of active compound, the amount of which is calculated to produce the desired therapeutic effect associated with the desired drug carrier.

The actual dosage level of the active ingredients (i.e., therapeutic molecules and conjugates) in the pharmaceutical composition can be varied to obtain an amount of active ingredients that is effective to achieve the desired therapeutic response for a particular patient, the composition, and the mode of administration that is non-toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including: the activity of the particular composition of the invention employed; the route of administration; the time of administration; the excretion rate of the particular compound employed; the duration of treatment; other drugs, compounds and/or materials used in combination with the particular composition employed; the age, sex, weight, disease, general health and past medical history of the patient being treated; and similar factors. The dosage level can be selected and/or adjusted to achieve a therapeutic response determined using one or more ocular/visual assessments described herein. A physician or veterinarian may start administering the therapeutic molecule of the conjugate employed in the pharmaceutical composition at a dose lower than the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. In general, the effective dosage of the composition for treating eye diseases described herein depends on many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is preventive or therapeutic.

The therapeutic dose needs to be titrated to optimize safety and efficacy. The dose for IVT administration using the conjugate of the present invention may be in the range of 0.1 mg/eye to 10 mg/eye per injection. A single dose per eye may be implemented by one or more injections per eye. For example, a single dose of 20 mg/eye may be delivered in 2 divided doses, 10 mg per injection, for a total dose of 20 mg. The volume of each injection may be between 10 microliters and 50 microliters, and the volume of each dose may be between 10 microliters and 100 microliters. Consider doses and regimens approved by the U.S. Food and Drug Administration (FDA) for Lucentis. Other doses and regimens suitable for anti-VEGF antibodies or antigen-binding fragments are described in US 2012/0014958, and are incorporated herein by reference in their entirety.

The composition can be administered multiple times. The administration interval between single doses can be weekly, monthly or annual. The interval can also be irregular, determined according to the patient's required retreatment, based on, for example, visual acuity or macular edema. In addition, alternative dosing intervals can be determined by a physician and administered once a month or when necessary to exert the effect. Effectiveness is based on the condition of the eye and the type and severity of the eye disease, for example, characterized by pathological growth, anti-VEGF therapeutic rate, retinal thickness and visual acuity measured by optical coherence tomography (OCT). The dosage and frequency can vary according to the half-life of the conjugate of the present invention in the patient and the level of the therapeutic target (e.g., VEGF, C5, EPO, factor P, etc.). However, in a preferred embodiment of the present invention, the composition is administered at most once every three months, particularly at most once every four months, and more particularly once every six months. This reflects the extended half-life of the first component in the conjugate compared to the corresponding unbound (free) first component (and therefore the extended duration of effectiveness). Accordingly, the elimination half-life of the first component in the conjugate is extended by at least 3 times, at least 4 times, or at least 5 times compared to the unbound first component. The relative increase in the elimination half-life of the first component in the conjugate compared to the free first component can be determined by administering these molecules by IVT injection and measuring the residual concentration at different time points using analytical methods known in the art, such as ELISA, mass spectrometry, protein blotting, radioimmunoassay, or fluorescent labeling. Blood concentrations can also be measured and used to calculate the rate of clearance from the eye (Xu L et al., invest Ophthalmol Vis ScL, 54 (3): 1816-24 (2013)). In general, molecules (e.g., antibodies or fragments) that are part of the conjugate exhibit an intraocular half-life that is longer than that of the free molecule. The half-life of the conjugate in the eye can be increased by 25% compared to the free first component (e.g., from 5 days to 6.25 days), its half-life can be increased by 50% compared to the free first component (e.g., from 5 days to 7.5 days), its half-life can be increased by 75% compared to the free first component (e.g., from 5 days to 8.75 days), its half-life can be increased by 100% compared to the free first component (e.g., from 5 days to 10 days), and in certain aspects, it is expected that the half-life of the conjugate can be increased by more than 100% compared to the free first component (e.g., from 5 days to 15 days, 20 days or 30 days; from 1 week to 3 weeks, 4 weeks or more; etc.).

D. Combination therapy

Combination therapy encompasses co-administration (where two or more therapeutic agents are contained in the same or separate formulations) as well as separate administration, in which case the administration of the therapeutic molecule and the conjugate may occur before, simultaneously with, and/or after the administration of the additional therapeutic agent or agents. In certain embodiments, the therapeutic molecule, conjugate, or composition is administered simultaneously with the additional compound. In certain embodiments, the therapeutic molecule, conjugate, or composition is administered before or after the additional compound. In some embodiments, the administration of the therapeutic molecule, conjugate, or composition and the administration of the additional therapeutic agent are within about one, two, three, four, or five months of each other, or within about one, two, three, four, five, or six days.

Any suitable therapeutic agent for treating an eye disease can be used as the additional compound, in particular as an agent for treating an eye disease. Eye diseases are as described in Section III above. In addition, any molecule described in Section II.A above as a component of a therapeutic molecule can also be used as an additional compound used in the combination therapy.

In some embodiments, the additional compound is an anti-angiogenic agent described in Section II.A.1.h) above and in Carmeliet et al., Nature 407: 249-257 (2000). Other suitable anti-angiogenic agents include corticosteroids, angiostatic steroids, anecortave acetate, angiostatin, endostatin, tyrosine kinase inhibitors, matrix metalloproteinase (MMP) inhibitors, insulin-like growth factor binding protein 3 (IGFBP3), stromal cell-derived factor (SDF-1) antagonists (e.g., anti-SDF-1 antibodies), pigment epithelium-derived factor (PEDF), gamma-secretase delta-like ligand 4, integrin antagonists, hypoxia-inducible factor (HIF)-1α antagonists, protein kinase CK2 antagonists, drugs that inhibit stem cells (e.g., endothelial progenitor cells) located at the site of new blood vessel formation (e.g., anti-vascular endothelial cadherin (CD-144) antibodies and/or anti-SDF-1 antibodies), and combinations thereof.

The therapeutic molecule, conjugate or composition can also be administered in conjunction with a therapeutic or surgical method for treating an eye disease (e.g., AMD, DME, DR, RVO or GA), including, for example: laser photocoagulation (e.g., panretinal photocoagulation (PRP)), occult laser action, macular hole surgery, macular shift surgery, implantable microtelescope, PHI-motion angiography (also known as microlaser therapy and feeder vessel treatment), photon beam therapy, microstimulation therapy, retinal detachment and vitreous surgery, scleral buckle, submacular surgery, transpupillary thermotherapy, photosystem I therapy, use of RNA interference (RNAi), in vitro rheological process (also known as membrane filtration and rheotherapy), microchip implantation, stem cell therapy, gene replacement therapy, ribonuclease gene therapy (including gene therapy for hypoxia response element, Oxford Biomedica; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor/retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplants, e.g., Astellas Pharma US, Inc., ReNeuron, CHA Biotech), acupuncture, and combinations thereof.

The therapeutic molecule, conjugate or composition can also be administered in combination with a visual cycle regulator (e.g., emixustat hydrochloride); squalamine (e.g., OHR-102; Ohr Pharmaceutical); vitamin and mineral supplements (e.g., those disclosed in Age-Related Eye Disease Study 1 (AREDS1; zinc and/or antioxidants) and Study 2 (AREDS2; zinc, antioxidants, lutein, zeaxanthin and/or omega-3 fatty acids); cell-based therapies, e.g., NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (suspension of RPE cells; Cell Cure Neurosciences) or MA09-hRPE cell transplantation (Ocata Therapeutics).

In some embodiments, the additional therapeutic agent is an AMD therapeutic agent. For example, the anti-PDGFR antibody REGN2176-3 can be combined with aflibercept Co-formulation. In some cases, these co-formulations may be administered in combination with a therapeutic molecule, conjugate, or composition.

In some embodiments, the additional compound comprises a lentiviral vector expressing endostatin and angiostatin (eg, RetinoStat).

In certain embodiments, the additional compound binds to a second biomolecule selected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R; PDGF; angiopoietin; Ang2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D; TNFα; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with AMD risk, such as complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. In certain embodiments, the additional compound is an antibody or antigen-binding fragment thereof, including the examples of antibodies and antigen-binding fragments described in Section II.A.3 above.

E. Target tissue

In some embodiments, the target tissue comprises the eye, brain, bone and/or tumor. In some embodiments, the tissue comprises the retina. In some embodiments, the therapeutic molecule, conjugate or composition is injected into the eye, brain, bone or tumor. In some embodiments, the therapeutic molecule, conjugate or composition is injected into the vitreous fluid, cerebrospinal fluid or synovial fluid. In some embodiments, the therapeutic molecule, conjugate or composition is injected subcutaneously.

In some embodiments, the therapeutic molecule, conjugate or composition provides improved compatibility, longer residence time and/or longer half-life relative to the injection site compared to an unmodified therapeutic agent. In some embodiments, the therapeutic molecule, conjugate or composition may further provide a longer duration of pharmacological action in the target tissue compared to an unmodified therapeutic agent.

In some embodiments, the therapeutic molecule, conjugate or composition provides improved vitreous compatibility, longer vitreous residence time, longer vitreous half-life and/or improved duration of pharmacological action compared to an unmodified therapeutic agent. In some embodiments, the therapeutic molecule, conjugate or composition provides improved compatibility, longer residence time, longer half-life and/or longer duration of pharmacological action in the brain compared to an unmodified therapeutic agent.

F. Binding of Therapeutic Molecules to HA

In some embodiments, the method includes binding the therapeutic molecule to HA prior to the administering step (i.e., pre-complexing the therapeutic molecule with HA to form a conjugate). In these embodiments, the pre-complex allows the therapeutic molecule to bind to HA. In some of these embodiments, HA is bound to the HABD of the therapeutic molecule. Examples of HABDs are described above in Section II.B.

In some embodiments, the method comprises mixing a first solution comprising a therapeutic molecule with a second solution comprising HA. In some embodiments, the mixing comprises a container. Examples of containers include vials, single chamber syringes, and two chamber syringes. In some embodiments, the mixing produces a therapeutic molecule bound to HA that is ready for administration to a subject.

In some embodiments, the size of HA ranges from 400Da to 200kDa. In some embodiments, HA is at least 5kDa. In some embodiments, HA is 10kDa. In some embodiments, the HA size/content allows a molar excess of HA relative to the HA binding site to be present in the binding or pre-complex mixture. In some embodiments, the HA size/content provides a molar excess of binding equivalents for the HABD. In some embodiments, the HA size/amount allows a ratio of HA to therapeutic molecule in the range of 1.5:1 to 1:1.

Examples

The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments may be implemented, given the general description given above.

The following examples discuss fusion proteins comprising a Fab fragment or peptide and a hyaluronic acid binding domain, i.e., Fab-HABD. Examples 1 to 7 relate to CD44 and/or TSG6 HABD. Examples 8 to 18 relate to VG1 HABD.

Example 1. Generation of Fab-hyaluronan binding domain fusion protein (Fab-HABD) and complexing with HA

Fusion proteins (hereinafter referred to as Fab-HABD) of Fab fragments and hyaluronic acid binding domains were generated (Table 2). HABD was recombinantly fused to the C-terminus of the heavy chain of the Fab fragment via a linker sequence containing Gly-Ser to form Fab-HABD (referred to herein as "1x version"). In some cases, additional HABD was fused to the C-terminus in the light chain of the Fab fragment (referred to herein as "2x version").

eFab-HABD was generated using Fab fragments that specifically bind to VEGF and PDGF (referred to as "VPDF"), digoxigenin (referred to as "Dig"), and VEGF (clone "G6.31").

HABD is derived from CD44 (SEQ ID NO: 2) or TSG6 (SEQ ID NO: 4).

The Dig antibody was covalently linked to one or both CD44 HA receptor domains and used as a non-binding control molecule (SEQ ID NOs: 9 to 12).

A. Materials and Methods

1. Protein Expression

Expression plasmids for various Fab-HABDs were generated by restriction cloning or gene synthesis using standard molecular biology techniques. Separate expression vectors were generated for each polypeptide chain. Expression was performed in HEK293 cells (ThermoFisher) and expression plasmids were mixed at a 1:1 ratio.

In some cases, TSG6 is expressed in E. coli.

In some cases, RabFab-1xTSG6 and RabFab-2xTSG6 are secreted from stably transfected Chinese hamster ovary (CHO) cells.

2. Protein Purification

The supernatant was collected by centrifugation at 4000 rpm, 4° C. for 20 min. Thereafter, the cell-free supernatant was filtered through a 0.22 μm bottle-top filter and stored in a refrigerator (−20° C.).

Fab-HABD was purified from cell culture supernatant by affinity chromatography using anti-Ckappa and anti-CH1 resins and size exclusion chromatography (SEC).

Briefly, sterile filtered cell culture supernatants were captured on KappaSelect resin (GE Healthcare) equilibrated with 1 x PBS buffer (10 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO ₄ , 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer, and eluted with 100 mM sodium citrate (pH 2.8). The eluted antibody fractions were combined and the pH was adjusted to 7.5. Proteins were then captured on CaptureSelect IgG-CH1 resin (Life Technologies) equilibrated with 1 x PBS buffer (10 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO ₄ , 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with elution buffer, and eluted with 100 mM sodium citrate (pH 2.8). The concentration of protein samples was determined at 280 nm on a Nanodrop 800 spectrophotometer (Thermo Scientific).

Analytical SEC was performed via a HiLoad 16/60 Superdex 200 preparative grade column (GE Healthcare) using 20 mM histidine, 140 mM NaCl (pH 6.0) buffer at a flow rate of 1.5 mL/min.

Pooled antibody-containing fractions from size exclusion chromatography were frozen at -80°C and stored for further use.

In some cases, TSG6 was purified from E. coli. Briefly, E. coli cells were extracted using a buffer consisting of 7 M guanidine hydrochloride, 50 mM Tris-HCl, 100 mM sodium tetrathioate, and 20 mM sodium sulfite. After homogenization, centrifugation and filtration of the supernatant, the His-tagged protein was captured on a Ni-NTA column (GE Healthcare) equilibrated with 6M guanidine hydrochloride and 25mM Tris-HCl (pH 8.6). The column was washed with 25mM Tris-HCl (pH 8.6), 0.1% Triton X-114, and eluted with a buffer containing 250mM imidazole. TSG6 eluted from the column was refolded by diluting it to 1.5mg/mL and then dialyzed overnight with a solution of 0.5M guanidine hydrochloride, 0.5M L-arginine, 1mM reduced glutathione (GSH) and 1mM oxidized glutathione (GSSG) at 4°C. After the buffer was exchanged for 25mM sodium acetate (pH 5.0), the refolded material was purified by cation exchange chromatography on SP-Sepharose ^TM (GE Healthcare).

In some cases, RabFab-1xTSG6 and RabFab-2xTSG6 are secreted from stably transfected Chinese hamster ovary (CHO) cells and purified from cell culture medium. These proteins do not need to be refolded. RabFab-1xTSG6 has the Fab fused to TSG via a gly-gly-gly-gly-ser linker; the HABD is at the C-terminus of the HC. RabFab-2xTSG6 has the Fab fused to TSG6 via a gly-gly-gly-gly-ser linker; one HABD is at the C-terminus of the HC and the other is at the C-terminus of the LC. Both proteins have a His tag at the C-terminus of the heavy chain, which is used for purification. These Fab-HABDs were purified from CHO supernatant using three column chromatography steps, including: (1) capture on an antigen affinity column as described in Shatz, W. et al., Mol. Pharm., 13(9): 2996-3003 (2016), (2) separation of His-tagged material on a Nickel-NTA column, and then (3) cation exchange chromatography on SP-Sepharose.

3. Compound with hyaluronic acid (HA)

Fab-HABD was mixed with 10kDa sodium hyaluronate (Lifecore, Biomedical) in a ratio of 1: 1 (w/w) to form Fab-HABD-HA conjugates (hereinafter referred to as Fab-HABD-HAs). After mixing, the conjugate was concentrated and rebuffered with an AmiconUltra10kDa cutoff concentrator (Millipore). The final formulation was 20mM histidine pH 6.0, 260mM sucrose, 140mM NaCl, 0.02% Tween 20. Finally, the conjugate was filtered with a 0.22μm filter (Ultrafree-MC, Centri nine gal Units 0.22μm, GV Durapore). The formation of protein-HA conjugates was monitored by shifting to a shorter retention time in SEC compared to the corresponding Fab-HABD (see Figure 1).

Example 2. Molecular properties of Fab-HABD

Example 2.1. Interaction with HA

A. Materials and Methods

The ability of HABD of Fab-HABD to bind to HA is checked.Biacore T200 instrument (GE Healthcare) is used to test the combination of Fab-CD44 and Fab-TSG6 Fab-HABD and HA by SPR (Table 3).In short, Fab-CD44 Fab-HABD is injected into the chip (SCBS HY, Xantect Bioanalytics GmbH, Germany) coated by HA in 80 seconds or 120 seconds, and its concentration range is 3.7nM to 300nM respectively.In some experiments, the chip coated with HA is prepared by indirectly coupling biotin-HA (Sigma-Aldrich, St.Louis, Missouri US) to the S series sensing chip SA coated with streptavidin (GE Healthcare).Dissociation phase is monitored for 600 seconds.Subsequently, 10mM glycine (pH1.5) is injected and continued for 60 seconds or 3M _MgCl is injected and continued for 30 seconds to regenerate the surface. Bulk refractive index differences were corrected by subtracting the response obtained from buffer injection. All experiments were performed at 25°C using PBS-T (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl pH 7.4, 0.05% Tween-20). The resulting curves were fitted to a 1:1 Langmuir binding model using BIAevaluation software. All experiments were performed at 25°C using PBS-T (10 mM _Na2HPO4 , ₁ mM _KH2PO4 , 137 mM NaCl, 2.7 mM KCl pH 7.4, _0.05 % Tween-20).

In addition, the interaction of VDPF-2xCD44 and HA was tested by isothermal titration calorimetry (ITC). In brief, Fab-CD44 fusions were dialyzed with PBS (10mM Na ₂ HPO ₄ , 1mM KH ₂ PO ₄ , 137mM NaCl, 2.7mM KCl pH 7.4). After dialysis, the remaining buffer was used to dissolve HA, so all molecules were under exactly the same buffer conditions to avoid any mismatch associated with the buffer. HA molecules were loaded into the sample pool at a concentration of 10 μM (10kDa HA) or 2 μM (50kDa HA), respectively. Deionized water was loaded into the reference pool. The syringe was filled with Fab-CD44 fusion proteins at a concentration of 150 μM. The titration experiment was carried out at 25 ° C. Affinity constants K and stoichiometric ratios N were calculated using a set of site models in Origin 7.0 (OriginLab Corporation).

Similarly, the interaction of TSG6 with 10 kDa HA was measured using ITC (Table 4), except that in these experiments, TSG6 (20 μM) was placed in the calorimeter cell and titrated with HA (50 μM) in a syringe. Solutions containing PBS were prepared as described above, and the measurement temperature was 25°C or 37°C. These measurements were performed on an Auto PEAQ ITC instrument (Malvern Instruments). Data analysis was as described in the previous paragraph, except that N was fixed to 1.0, and HA concentration and affinity constant K were variable parameters.

B. Results

The K _D of Fab-CD44s and Fab-TSG6s binding to HA measured by SPR is shown in Table 3.

The strength of the interaction is determined by the HABD sequence and the binding nature of the interaction (ie, the 2x version exhibits higher functional affinity via tighter binding to HA).

ITC analysis (Table 4) yields the HA affinity shown below, with a calculated binding site concentration of 400-745 μM, indicating an estimated stoichiometry of 8-15 TSG6 molecules per 10 kDa HA chain. A similar experiment using 50 μM VPDF-2xCD44 in the pool and 150 μM 10 kDa HA in the syringe yielded a _KD of 25 μM and an apparent stoichiometry of 4.5 VPDF-2xCD44 per 10 kDa HA chain. The weaker HA binding affinity of CD44 requires the use of higher concentrations in ITC experiments. The binding stoichiometry of 10 kDa is 2-3 times greater for 1xTSG6 than for 2xCD44, as expected from the bivalent HA binding properties of the 2xCD44 fusion and the higher molecular weight of CD44 compared to TSG6. The strength of the interaction is determined by the HABD sequence and the binding properties of the interaction (i.e., the 2x version exhibits a higher functional affinity via close binding to HA).

In terms of the stoichiometry of the interaction, it was found that on average each 10 kDa HA molecule could bind to 1.5 VPDF-2xCD44, while each 50 kDa HA molecule could bind to 5 VPDF-2xCD44.

According to SPR measurements, VPDF-2xCD44 is able to bind to both VEGF and PDGF ligands simultaneously. The binding of VEGF and PDGF to VPDF-2xCD44 was compared to their binding to unmodified VPDF. Briefly, PDGF was coupled to an S-series sensor chip CM5 (GE Healthcare) using standard coupling chemistry to obtain a surface density of approximately 4000 resonance units (RU). After injection of 3 μg/mL of VPDF-2xCD44 fusion and unmodified VPDF control, VEGF was injected at a concentration of 5 μg/mL to demonstrate that the Fab binds to the ligands PDGF and VEGF simultaneously. Subsequently, the surface was regenerated by injecting 10 mM glycine pH 2.0 for 60 seconds. SPR measurements confirmed that the fusion of HABD to the C-terminus of the heavy chain of the VPDF Fab fragment did not interfere with the interaction of the ligand with the target protein.

Example 2.2. Stability of Fab-HABD

The use of Fab-HABD for long-term delivery in the eye requires that the protein remain stable at body temperature for up to several months. A prerequisite for this is that the thermal stability of Fab-HABD is above 37°C.

A. Materials and Methods

The thermal stability of VPDF-2xCD44 and TSG6 was tested by static light scattering and protein autofluorescence. The sample was diluted to about 1 mg/mL and the temperature was raised from 25°C to 80°C at a heating rate of 0.1°C/min using an Optim instrument (Avacta Inc.). During this process, light scattering and fluorescence data after 266nm laser irradiation were recorded. The aggregation onset temperature was measured to be about 75°C, which was defined as the temperature at which the scattering intensity increased. At the same time, the fluorescence emission spectrum was recorded.

For VPDF-CD44, when the centroid mean of the fluorescence spectrum was plotted versus temperature, two transitions were measured at approximately 56°C and 79°C. These transitions indicate denaturation of the protein (probably the Fab and CD44 domains). Therefore, any scattering or spectral changes associated with thermal unfolding are well above 37°C, indicating good stability of this Fab-HABD.

B. Results

Two transitions of VPDF-2xCD44 were measured at 56° C. and 79° C. These two T _m indicate the transitions of VPDF-2xCD44 denaturation, where the CD44 domain denatures at 56° C. and the Fab denatures at 79° C.

For TSG6, the observed T _monset was measured to be 35°C and the measured T _m was 43°C.

Example 3. In vivo efficacy in rat laser choroidal neovascularization (CNV)

A. Materials and Methods

Fab-HABD was studied in an in vivo rat model of laser-induced choroidal neovascularization (rat laser CNV) to test the following hypotheses: (1) Fab-HABD is effective in vivo despite being bound to IVT HA (i.e., Fab-HABD can inhibit neovascularization); and (2) Fab-HABD has longer-lasting in vivo efficacy compared to the corresponding unmodified Fab fragment.

To this end, rats received IVT injections of the protein formulation either 1 or 3 weeks before laser injury (six laser burns per eye). One week after laser injury, lesions were analyzed for vascular growth using fluorescence angiography (FA) imaging.

Fab-HABD was compared to the corresponding unmodified Fab fragment. To test the sustained efficacy of Fab-HABD, the dose of unmodified Fab was titrated to the "lowest effect dose" (i.e., only a low degree of inhibition of detectable neovascularization compared to vehicle over the duration of the rat model), indicating that at the same dose and duration of the model, Fab-HABD had a more sustained efficacy.

B. Results

All tested Fab-HABDs showed an inhibitory effect on neovascularization in vivo (Table 5). This result indicates that Fab-HABD, although bound to HA in the vitreous body, reaches relevant tissues to exert pharmacological effects.

In the same model setting and at the same dose, all tested Fab-HABDs showed longer lasting pharmacological effects than unmodified Fab fragments (Table 5). This suggests that the ability to bind to IVT HA can prolong the pharmacological effects in vivo.

Despite significant differences in affinity for HA, the resolution of the in vivo model was insufficient to distinguish the durability of efficacy of the different molecules. At the low, non-therapeutic doses applied in the model, no tolerability issues were detected. All eyes of rats that received the Fab-HABD dose were completely normal without any signs of interference, comparable to eyes that received buffer only during the in vivo phase.

Example 4. Rabbit Pharmacokinetic (PK) Study Using RabFab, RabFab-1xTSG6, and RabFab-2xTSG6

A. Materials and Methods

Proteins for animal studies were formulated in 20 mM histidine-acetate, 150 mM NaCl (pH 5.5) or phosphate buffered saline (PBS) (pH 7.4) via dialysis. The formulations were isotonic, with osmotic pressures between 300 mOsm/kg and 340 mOsm/kg as measured by the freezing point method. Size exclusion chromatography (SEC) analysis showed that the monomer ratio of all proteins in these formulations was ≥95%. Endotoxin levels were assessed to be less than 0.1 EU per eye at the final dosing concentration.

In the vitreous half-life study, an additional pharmacokinetic study was performed in which control (PBS, n=1), 0.15 mg/eye of AlexaFluor-488 labeled RabFab (n=2), or AlexaFluor-488 (AF-488) labeled RabFab-2xTSG6 at doses of 0.05 mg/eye (n=2), 0.15 mg/eye (n=2), and 2.5 mg/eye (n=4) were administered to both eyes of female rabbits by 1TV injection in a total volume of 50 μL. Test article concentrations in the vitreous and aqueous humor at designated time points were measured using fluorophotometry as previously described. Dickmann, L.J. et al., Invest. Ophthalmol. Vis. Sci., 56(11): 6991-6999 (2015). Pharmacokinetic parameters were estimated using concentration-time curves by non-compartmental analysis using Phoenix WinNonlin (Certara Inc., Mountain View, CA). For concentration-time profiles generated using the fluorescence brightness method, sampling results within the first 48 hours after administration were excluded from the PK analysis due to high variability, which may be attributed to individual differences in the site of administration and subsequent diffusion of the test article through the vitreous. Dickmann, L.J. et al., Invest. Ophthalmol. Vis. Sci., 56(11): 6991-6999 (2015). PK analysis was performed using a non-compartmental analysis method, and the clearance (CL) was calculated as CL = dose/AUC, where the dose was known and the AUC was measured using the linear trapezoidal method. The steady-state distribution volume was calculated as V = CL/kel, using the clearance value and the elimination rate constant obtained from the slope of the terminal phase. The elimination half-life was calculated as t1/2 = ln(2)/kel.

B. Results

Initially, pharmacokinetic (PK) experiments using New Zealand white rabbits were used to examine the ability of HA binding to affect intraocular residence time. Although the HA concentration in rabbit vitreous (approximately 65 μg/mL) is much lower than that in human vitreous (100-400 μg/mL) or other preclinical species such as pigs (whose vitreous HA is approximately 180 μg/mL) or cynomolgus monkeys (whose vitreous HA is approximately 150 μg/mL), rabbits are often used for early PK studies after IVT administration of test articles. The study design was to use IVT to inject 0.3 mg/eye of RabFab, 0.3 mg/eye of RabFab-1xTSG6, or 0.5 mg/eye of RabFab-2xTSG6. Recovery experiments using proteins in ex vivo added vitreous humor showed that RabFab and RabFab=1xTSG6 could be quantified using ELISA with previously described anti-idiotypic detection antibodies (Shatz et al., 2016 Molecular Pharmaceutics). However, the poor recovery obtained by ELISA with RabFab-2xTSG6 necessitated radiochemical determination of vitreous concentrations of this material. In the PK studies, RabFab-2xTSG6 was radiolabeled with ¹²⁵ iodine.

As shown in Figure 2A and the PK parameters are summarized in Table 6, both RabFab-1xTSG6 and RabFab-2xTSG6 exhibited longer vitreous residence times compared to free RabFab. The half-life of RabFab-1xTSG6 was 1.4-fold longer than that of RabFab, while the half-life of RabFab-2xTSG6 was increased 2.2-fold. These results suggest that fusion of Fab to HABD increases the retention time of these molecules in the ocular chamber. Given the higher vitreous HA concentrations in other species, it is expected that the half-life of Fab-HABD would be extended to a greater extent in those animals.

In addition, vitreous half-life studies demonstrated that the vitreous half-life of RabFab-2xTSG6 was increased approximately 3- to 4-fold compared with that observed for RabFab by fluorophotometry, with no apparent dose dependence within the range evaluated (Figure 2B); however, the 21-day study duration was insufficient to reliably determine pharmacokinetic parameters, and it was estimated that approximately 40% of RabFab-2xTSG6 remained in the vitreous at the end of the study.

Example 5. Rabbit ocular tolerance of RabFab-1xTSG6 and TSG6

A. Materials and Methods

The toxicity of a single ITV administration of free TSG6 and RabFab-1xTSG6 was evaluated in New Zealand white rabbits. A single IVT administration study lasting 4 weeks was designed and performed (Table 7). Anti-drug antibodies (ADA) against RabFab-1xTSG6 or free TSG6 in serum were measured by ELISA. The plates were coated with RabFab-1xTSG-6 or free TSG6, incubated with serum collected from the study animals, and then anti-drug antibodies were detected with HRP-conjugated goat anti-rabbit Fc antibodies.

B. Results

In general, animals receiving RabFab-1xTSG-6 had less severe results than animals receiving free TSG6 administration. Animals receiving free TSG6 administration had significant clinical observations. Although 4 animals were scheduled for autopsy on day 4, the other 4 animals were sacrificed early on day 12 or 17 instead of day 30 due to significant clinical observations and animal welfare issues. These clinical observations included swelling and redness of the eyelids and conjunctiva, animals keeping their eyes closed when staff approached, and ocular inflammation and irritation. As of 3 days after administration, animals receiving free TSG6 administration showed significant posterior incipient cataracts and variable retinal vascular weakening, which were associated with microscopic findings of lens and outer to complete retinal degeneration. Similar but less severe results were also present in animals receiving RabFab-1xTSG6 administration. Starting from the 7th day after administration, all animals showed significant inflammation mainly of mononuclear cells. Inflammation and retinal degeneration were multifocally associated with evidence of retinal detachment, hypertrophy and peripheral migration of vimentin, glial fibrillary acid protein (GFAP) and glutamine synthetase positive Müller cells. Histopathological images showing retinal degeneration 4 days after TSG6 administration via IVT are shown in FIG3 .

Both animals that received RabFab-1xTSG6 and underwent necropsy on day 4 had evidence of anti-drug antibodies (ADA) in their serum on day 4. However, one of the animals was pre-dosed with ADA, whereas the remaining three animals in this treatment group were not. The animal in this group that underwent a late necropsy was serum ADA negative on days 4 and 8, but became ADA positive on day 15. Analysis of serum ADA responses in animals treated with free TSG6 was inconclusive due to the poor sensitivity of the assay.

In general, animals receiving RabFab-1xTSG6 had less severe outcomes than animals receiving free TSG6 administration (Table 8). Cataracts were present in every animal, but were punctate in nature and no correlation was found in microscopic sections. There was no clinical evidence of retinal degeneration, but there was microscopic evidence of minimal to mild outer retinal degeneration in one eye. Similar moderate to severe vitreous and aqueous humor cells appeared starting on day 8. Animals were euthanized on days 4 and 17.

The evaluation of the anti-RabFab response was complicated by the fact that 3/8 animals had values above the cutoff before dosing (2 animals received free TSG6 and 1 animal received RabFab=1xTSG6). However, after the test article was administered, 3/4 animals that received RabFab-1xTSG6 had new or increased ADA titers: 1 of them was euthanized on day 4 and the other two animals were euthanized on day 17. In contrast, animal 1 (autopsy performed on day 4) that received only free TSG6 had an elevated ADA titer compared to before dosing.

The early onset of clinical signs and microscopic lesions suggested a direct role for TSG6 in retinal and lens degeneration; however, findings at later time points were confounded by unexpectedly strong ADA responses. The peripheral migration of Müller cells was concluded to be a nonspecific response after rabbit retinal injury or retinal detachment.

Example 6. Pharmacokinetics (PK) of Therapeutic Doses in Minipigs

The aim of this study was to determine the intraocular and systemic PK parameters and the resulting extension of intraocular half-life (t1 _/2 ) of Fab-HABD and Fab-HABD-HAs administered once by IVT injection (IVT) to minipigs. In addition, anti-drug antibodies (ADA), intraocular tolerance and ocular pathology (in some study subjects) were investigated.

A. Materials and Methods

14 SPF minipigs received therapeutic doses of the following test articles (50 μL/eye) in both eyes (Table 9).

Following IVT dosing, blood and aqueous humor samples were collected from animals receiving test article at regular intervals throughout the study (up to 9 weeks), and vitreous humor was collected shortly after planned euthanasia to follow systemic and intraocular PK of the test article. Test article concentrations in plasma, aqueous humor, and vitreous humor were analyzed, and plasma and vitreous samples were further analyzed for the presence of ADA.

B. Results: During the in vivo phase of the study, macroscopic observations of the eyes of 2 of the 5 animals that received VPDF-2xCD44 indicated that the porcine eye could not tolerate the IVT injection of the test article, leading to the premature sacrifice of these animals. One eye of each animal was provided for histopathological evaluation. In brief, these macroscopic observations were: vitreous opacities, lower than normal vitreous viscosity, and finally behavioral signs of visual loss. Histopathological findings in the eyes included moderate mixed cellular inflammation of the perivascular/vascular, predominantly mononuclear cells infiltrates of the iris, ciliary body, trabecular meshwork, and retina. Retinal degeneration included ganglion cell degeneration, cell loss in the INL, photoreceptor aggregation, and nuclear displacement of the PR layer. In addition, eosinophilic proteinaceous material was observed in the vitreous as well as mixed cellular infiltrates and filaments. No abnormal findings were found in the optic nerve.

Macroscopic findings were significantly less severe in at least 1 of the 5 animals receiving VPDF-2xCD44+10 kDa HA compared to VPDF-2xCD44. Red/white film in the anterior chamber of both eyes with accumulation behind the cornea was briefly noted but was not considered necessary for early termination.

In conclusion, complexing of VPDF-2xCD44 with HA (ie, occupation of CD44 HA binding sites with HA prior to IVT injection) did improve the intraocular tolerability of VPDF-2xCD44.

No macroscopic observations or tolerability issues were noted in the group of animals receiving unmodified VPDF.

PK results for the test articles VPDF-2xCD44 and VPDF-2xCD44+10 kDa HA from aqueous humor and vitreous were calculated from individual concentration-time data by non-compartmental analysis and are presented graphically in Figures 4A-4B.

While the IVT t _1/2 of 5.8 days for unmodified VPDF is within the expected range for this type of molecule, the IVT t _1/2 of 48 days for VPDF-2xCD44+10 kDa HA corresponds to an approximately 8-fold increase in intraocular residence time compared to unmodified VPDF. In summary, VPDF-2xCD44+10 kDa exhibits significantly improved tolerability compared to VPDF-2xCD44 not complexed with HA, and significantly improved intraocular half-life compared to unmodified VPDF.

Example 7. Pre-compounding with HA to improve vitreous compatibility

Macroscopic observations from the in vivo minipig studies (i.e., vitreous opacities) suggested that incompatibility of VPDF-2xCD44 with porcine vitreous (i.e., precipitate formation) could be mitigated by pre-complexing VPDF-2xCD44 with pure HA. To further examine these effects and test whether these observations were limited to the VPDF-2xCD44 molecule or could also be detected for other Fab-HABDs, we developed an ex vivo test system to detect vitreous degeneration.

An in vitro "droplet" test was developed to evaluate the vitreous compatibility of several Fab-HABDs when pre-compounded with HA. This example illustrates that Fab-HABD (i.e., conjugate) pre-compounded with HA is compatible with vitreous in an in vitro experiment. The vitreous incompatibility observed previously may be caused by free HABD, which is alleviated by pre-compounding with HA. The results show that the incompatibility of Fab-HABD with free HABD depends on the concentration. In addition, CD44ko (a Fab-HABD mutant containing a point mutation that invalidates HA binding) is compatible with vitreous in both pre-compounded and separated forms.

Example 7.1. Pre-complexation of VPDF-2xCD44 with 10 kDa HA improves intravitreal (IVT) tolerability

A. Materials and Methods

In the first test, porcine vitreous was homogenized 10x in a Dounce homogenizer and centrifuged at 10000 g for 2 minutes to remove debris. A 2 μl drop of homogenized vitreous was then applied to a glass microscope slide. In addition, 2 μl of the test sample (i.e., Fab-HABD or Fab-HABD-HA at a specified concentration) was added to the top of the vitreous drop without further mixing. One minute after the droplets merged, the samples were examined for inhomogeneity and precipitation by optical microscopy at 40x magnification in bright field mode.

B. Results

The porcine vitreous mixed with unmodified VPDF at a concentration of 200 mg/mL in 20 mM histidine, 140 mM NaCl (pH 6.0) was homogeneous and transparent ( FIG. 5A ), whereas the porcine vitreous mixed with VPDF-2xCD44 at a concentration of 20 mg/mL in 20 mM histidine, 140 mM NaCl (pH 6.0) was heterogeneous and exhibited signs of transparent precipitation ( FIG. 5B ).

This result suggests that VPDF-2xCD44 is also incompatible with porcine vitreous after in vivo IVT injection. Therefore, vitreous incompatibility may be a root cause of VPDF-2xCD44 in vivo tolerability issues.

Pre-complexation of VPDF-2xCD44 with 1% (w/v) HA (10 kDa, Lifecore, Biomedical) at a concentration of 20 mg/mL in 20 mM histidine, 140 mM NaCl (pH 6.0) resulted in vitreous compatibility (Figure 5C). This result mirrors the results of the above-mentioned minipig in vivo study, which showed that pre-complexation of VPDF-2xCD44 with 10 kDa HA improved IVT tolerance.

Example 7.2. Vitreous incompatibility of VPDF-2xCD44 is concentration dependent

A. Materials and Methods

To test the concentration dependence of vitreous incompatibility of VPDF-2xCD44, 2 μl of porcine vitreous was mixed with a 1:4 dilution of VPDF-2xCD44 in 20 mM histidine, 140 mM NaCl, pH 6.0 at a starting concentration of 37.5 mg/mL. Vitreous inhomogeneities in vitreous and protein mixtures were examined by light microscopy.

B. Results

The detected heterogeneity depended on the protein concentration (Table 10; Figures 6A to 6F). Vitreous compatibility of VPDF-2xCD44 was achieved between 0.6 and 0.15 mg/mL. Correlating these results with the results of the above-mentioned in vivo minipig study (VPDF-2xCD44 concentration = 17.4 mg/mL), it is suggested that similar heterogeneity that may result from IVT injection of a VPDF-2xCD44 solution at a concentration of 17.4 mg/mL may be the root cause of the observed tolerability issues.

Example 7.3. Vitreous incompatibility of VPDF-2xCD44 is associated with its interaction with intravitreal (IVT) HA

A. Materials and Methods

To test whether the vitreous incompatibility of VPDF-2xCD44 was caused by the interaction of VPDF-2xCD44 with IVT HA, we designed a variant of the molecule (VPDF2xCD44-ko) that contained a point mutation within the HA binding site of CD44 that abolished binding to HA while leaving the rest of the protein binding intact (referred to herein as the “ko variant”).

B. Results

The CD44ko variant exhibited identical behavior in terms of transient expression, purification and biophysical characterization (analytical size analysis, denaturing SDS capillary electrophoresis), and its identity was confirmed by mass spectrometry. The introduction of the HA binding site mutation resulted in a complete loss of affinity, as shown by SPR results (tested using the same method as in Example 2).

When the VPDF-2xCD44-ko variant was tested for vitreous compatibility at the same concentration as 2x VPDF as described in Example 7.2 above, no vitreous inhomogeneity was detected, indicating vitreous compatibility (Table 11).

Example 7.4. VPDF-2xCD44 is compatible with vitreous after hyaluronidase pretreatment

A. Materials and Methods

In addition, we tested the vitreous compatibility of VPDF-2xCD44 in porcine vitreous pretreated with hyaluronidase to degrade HA. To this end, hyaluronidase from porcine testis (Sigma) was dissolved in PBS at a concentration of 2 mg/mL (>1.5 U/μL). 1 μL of hyaluronidase solution was added to 50 μL of porcine vitreous and incubated at 37°C for 2 hours. Control samples were treated with PBS buffer only.

B. Results

As a result, VPDF-2xCD44 showed no inhomogeneity when mixed with hyaluronidase pre-treated vitreous at a concentration of 20 mg/mL, which may be due to the degradation of high molecular weight HA.

Taken together, these results suggest that vitreous incompatibility may be the underlying cause of VPDF-2xCD44 in vivo tolerability issues related to the interaction of CD44-HABD with high molecular weight IVT HA.

Example 7.5. Vitreous incompatibility of Fab-HABD is related to the interaction of HABD with vitreous HA at specific concentrations

A. Materials and Methods

To test whether the vitreous incompatibility was a feature of VPDF-2xCD44 alone, we tested other Fab-HABDs or HABD alone for vitreous inhomogeneity as described in Examples 7.2 and 7.3 above. The proteins tested were as described in Example 1.

B. Results

VPDF-1xCD44 exhibited comparable vitreous inhomogeneity to VPDF-2xCD44. The results suggest that the increased binding and potential cross-linking of the 2x version of the HA polymer is not associated with vitreous incompatibility. The results suggest that the interaction between the CD44 HABD and the IVT HA is associated with vitreous incompatibility (Table 12).

Fab-HABD containing the TSG6 domain showed comparable vitreous inhomogeneity to Fab-HABD containing CD44. The Fab component G6.31 showed no vitreous inhomogeneity at the same concentration, whereas the isolated TSG6 domain did. This result again supports the idea that vitreous incompatibility is related to the interaction of HABD with vitreous HA at a specific concentration.

Example 7.6. Vitreous incompatibility can be resolved by pre-compounding with HA

A. Materials and Methods

To test whether the detected vitreous incompatibility of VPDF-1xCD44 and TSG6 variants could be resolved by pre-complexing with HA, we generated the conjugates shown in Table 13 containing 1% (w/v) HA (10 kDa, Lifecore, Biomedical).

B. Results

Vitreous inhomogeneities were resolved for all Fab-HABDs tested by pre-complexing with 10 kDa HA (Table 13). These results suggest that pre-complexing of HA binding proteins with pure HA may serve as a method to improve vitreous compatibility and thereby potential tolerability issues of these molecules.

Example 7.7. Vitreous incompatibility of Fab-HABD is not unique to porcine vitreous

A. Materials and Methods

To test whether the detected vitreous incompatibility of Fab-HABD containing CD44 and TSG6 was an effect that occurred only in porcine vitreous, we used rabbit vitreous instead of porcine vitreous and performed compatibility tests as described in Examples 7.1 to 7.6 above.

B. Results

For all Fab-HABDs tested, the same vitreous incompatibilities as with porcine vitreous were detected. In addition, all vitreous incompatibilities detected in rabbit vitreous were resolved by pre-complexation of the Fab-HABD with 10 kDa HA.

These results suggest that vitreous incompatibility is not specific to porcine vitreous.

Example 7.8. In vivo injection-induced vitreous inhomogeneities

A. Materials and Methods

To correlate the ex vivo vitreous compatibility test results with the tolerability results from the in vivo minipig study, we tested whether vitreous inhomogeneities could be detected in whole pig eyes and resolved by HA pre-compounding.

To this end, whole pig eyes were collected immediately after slaughter and injected with 50 μl of a solution of 17.4 mg/mL VPDF-2xCD44 in 20 mM histidine, 140 mM NaCl (pH 6.0) +/- 1% (w/v) HA 10 kDa. Eyes (same as in the in vivo minipig studies described above). Eyes were then transferred to HBSS (Lonza, Biowhittaker) and stored at 37°C for 4 hours. After incubation, eyes were opened and vitreous was removed to examine for inhomogeneities.

B. Results

As shown in Figures 7A to 7C, vitreous incompatibility during injection can be resolved by pre-complexation of Fab-HABD with HA. Injection of buffer did not result in IVT inhomogeneity, resulting in a transparent vitreous (Figure 7A). Injection of VPDF-2xCD44 resulted in the appearance of dense white inhomogeneities (precipitate-like) in the vitreous around the injection side (Figure 7B). The vitreous of eyes injected with VPDF pre-complexed with pure HA showed significant differences (Figure 7C): although inhomogeneities were detected, the density and thickness of the inhomogeneities were significantly reduced throughout the vitreous.

These results suggest that VPDF-2xCD44-induced heterogeneity also occurs near the injection site throughout the pig eye. Without being bound by this theory, we believe that the same heterogeneity is induced upon injection in vivo and that this heterogeneity may be the root cause of the observed tolerability issues.

Furthermore, pre-complexation of VPDF-2xCD44 with HA reduced the heterogeneity observed around the injection site. We believe that the same effect is responsible for the improved tolerability of VPDF-2xCD44-HA observed in vivo. Combined with the observations in Examples 7.5 and 7.6 that vitreous incompatibility also occurred with another TSG6 HABD and was similarly resolved by pre-formulation with pure HA, we believe this approach is a general principle for improving vitreous compatibility and, thereby, IVT tolerability of proteins containing HABDs.

Example 8. Versican VG1 and VG1ΔIg HABD are able to bind HA

Versican HABDs were investigated to determine if they could be used as HABDs that provide superior ocular tolerability and intraocular residence time to those of TSG6 and CD44 HABDs.

Versican has been identified to have a tandemly repeated linking module. As shown in FIG8A , the amino acid sequence of versican encodes an Ig-like domain, which is connected to two linking modules, such that the N-terminal fragment of versican (herein named WT VG1) comprises an N-terminal Ig-like domain and two linking domains. In this example, we obtained WT VG1 and a truncated variant VG1ΔIg without an Ig domain and tested whether they can bind to HA. In addition, in the following examples, WT VG1 and a Fab-HABD composed of Fab and WT VG1 were tested for in vitro vitreous compatibility and tolerance during IVT injection into rabbits and minipigs.

A. Materials and Methods

Expression plasmids for each protein were generated by restriction cloning and/or gene synthesis using standard molecular biology techniques. Expression was performed in CHO or HEK293 cells.

The supernatant was collected by centrifugation at 4000 rpm, 4° C. for 20 min. Thereafter, the cell-free supernatant was filtered through a 0.22 μm bottle-top filter and stored in a refrigerator (−20° C.).

WT VG1 and His-tagged mutants of VG1ΔIg were purified from cell culture supernatants by affinity chromatography using Ni-NTA resin and SEC. Briefly, sterile filtered cell culture supernatants were captured on HisTrap resin, washed and eluted using a buffer containing a high concentration of imidazole. The eluted protein fractions were combined and concentrated and then subjected to SEC analysis using 20 mM histidine-acetate, 150 mM NaCl (pH 5.5) as the running buffer.

Binding of WT VG1 and VG1ΔIg to HA was tested by SPR using a Biacore T200 instrument (GE Healthcare). Briefly, WT VG1 and VG1ΔIg were injected over an S-series CM5 chip (GE Healthcare Life Science Solutions) that was indirectly coated with biotin-HA (Creative PEGWorks, North Carolina) via immobilized streptavidin over a period of 80 or 120 seconds. The injection concentration range was 0.5 nM to 1 μM. The dissociation phase was monitored for 300 to 600 seconds. Subsequently, the surface was regenerated by injecting 1 M MgCl ₂ for 15 seconds. All experiments were performed at 25°C using PBS (10 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO ₄ , 137 mM NaCl, 2.7 mM KCl, pH 7.4). The resulting HA binding curves were fit to a 1:1 Langmuir binding model using BIAevaluation software.

B. Results

Versican HABD is able to bind HA. The HA binding _KD of various proteins is shown in Table 14.

Example 9. Glycosaminoglycan binding patterns of WT VG1 and TSG6 proteins

A. Materials and Methods

The glycosaminoglycan (GAG) binding morphology of WT VG1 and TSG6 was determined by SPR measurements of binding to heparin sulfate and chondroitin sulfate using a Biacore T200 instrument (GE Healthcare). In brief, the protein was injected into an S-series CM5 chip (GE Healthcare Life Science Solutions) coated with biotin-heparin sulfate or biotin-chondroitin sulfate indirectly by streptavidin within 180 seconds. The injection concentration range was approximately 5nM to 1000nM, respectively. The dissociation phase was monitored for 120 seconds. Subsequently, 1M MgCl _was injected and continued for 30 seconds to regenerate the surface.

B. Results

The results showed that WT VG1 was more selective than TSG6 in binding (Table 15). WT VG1 was not observed to bind to heparin sulfate or chondroitin sulfate, while TSG6 bound tightly to both heparin and chondroitin sulfate.

Example 10. Fab-HABD containing VG1 HABD is able to bind antigen and HAA. Materials and Methods

A.1. Construct design

Fab-HABD is produced by or can be produced by recombinant fusion of WT VG1 sequence to the C-terminus of the heavy chain of the Fab fragment or the N-terminus of the IgG1 heavy chain. For peptide-VG1 fusions, a peptide (EETI) attached to both the N-terminus of WT VG1 (EETI-VG1) and the C-terminus of WT VG1 (VG1-EETI) is produced or can be produced. Two additional constructs were also produced in which a TEV cleavage site was introduced between EETI and WT VG1 (EETI-TEV-VG1 and VG1-TEV-EETI). The linkers used or may be used are shown in Table 16.

A.2. Generation of species-matched surrogate porcine anti-VEGF Fab

Since a search of the abYsis database did not yield known examples of heavy and light chain pairings for porcine (Sus scrofa) IgG, we attempted to generate antibodies that actively bind to known antigens by transplanting CDRs from anti-VEGF Fab (G6.31.AARR). The NCBI sequence tag (EST) database was searched for porcine mRNA ESTs with high sequence identity to the G6.31 framework ( _VH 4/ _VL K2). Several sequences were selected and the CDRs from G6.31.AARR were transplanted into the appropriate framework regions to generate "porcine" G6.31.AARR. The heavy and light chain sequences were randomly paired and expressed in 293Expi or CHO cells in 30 mL culture. Purification was performed on Capto L resin, followed by size exclusion chromatography analysis, and the purified cells were examined by SDS-PAGE, mass spectrometry, and evaluated for binding to human and porcine VEGF. A sequence with good affinity for VEGF was selected for amplification and subsequent tox/PK analysis and recombinantly fused to VG1 to generate PigFab-VG1.

A.3. Protein expression and purification

Protein expression was performed by cationic lipid transfection of DNA constructs into CHO or 293 Expi cells. The culture volume was 30 mL to 35 L. For some constructs, rapid stable cell lines were generated to increase protein production per unit culture volume.

Purification was performed by affinity chromatography using Ni-NTA resin for molecules with a 6x-histidine tag or Gamma bind Plus resin for Fab fusions. In some cases, a secondary ion exchange step was performed before a final size selection step on Sephadex resin.

A.4. HA binding

To confirm that VG1 retained its HA binding properties as a Fab-HABD, SPR was used as previously described in Example 2.1. Experiments were performed using single cycle kinetics and dissociation was monitored for up to 600 seconds. The protein concentration tested varied between different proteins but ranged between 500 nM and 6.25 nM.

A.5. Antigen Binding

Antigen binding was tested by immobilizing the corresponding antigens directly on S-series CM5 chips (GE Healthcare) and measuring binding by SPR as described in Example 2.1. Different protein concentrations were used based on the known affinity of the interaction.

B. Results

B.1. Hyaluronic acid (HA) binding

All HA binding data were fitted to a 1:1 Langmuir binding model using BIAevaluation software. The _KD for each protein is shown in Table 17.

B.2. Antigen Binding

Table 18 shows the proteins analyzed for their antigen binding ability and the _KD measured. Fusion of VG1 to the C-terminus of various Fab heavy chains did not affect antigen binding. For EETI-VG1 fusions, flexibility of the linker and attachment site affected antigen binding. More flexible linkers and C-terminal fusions were preferred.

Example 11. In vitro vitreous compatibility of HABD

A. Materials and Methods

This example describes testing of the solubility of the VG1 domain in vitreous humor. Vitreous humor was prepared using a Dounce homogenizer and then centrifuged at 10,000 x g for 2 minutes to remove debris and was used for these studies.

Additional experiments utilized Alexa 488 labeled proteins, allowing both bright field and fluorescence microscopy to monitor precipitation in the vitreous humor ex vivo. Equal volumes of the test article were mixed with the vitreous humor by sequential injection into each of the two channels of a 3-in-1 3-channel-slide (ibidi, USA, Inc. Cat#80316), and the mixing interface was visually monitored by microscopy.

B. Results

After TSG6 was mixed with porcine vitreous humor previously diluted 1:4 with PBS (pH 7.4), the solution became turbid (FIG. 9A), and a precipitate was observed after centrifugation of the mixture. In contrast, after VG1 was mixed with porcine vitreous humor at a ratio of 1:4 and 1:1, the solution remained clear (FIG. 9B), and no precipitate was observed after centrifugation.

Further, precipitation of RabFab-TSG6 was observed in the ex vivo porcine vitreous (FIG. 10A), whereas no precipitation was observed for RabFab-VG1 (FIG. 10B). Similarly, no precipitation was observed in the ex vivo rabbit vitreous when comparing VG1 (FIG. 11A), RabFab-VG1 (FIG. 11B), or a formulation containing equal concentrations (based on mass) of RabFab-VG1 and 10 kDa HA (FIG. 11C).

In contrast to TSG6, preformulation of VG1 with 10 kDa HA was not always required to prevent precipitation in the vitreous humor in vitro.

Example 12. In vitro interaction of VG1 with vitreous humor

A. Materials and Methods

Fluorescence correlation spectroscopy (FCS) was used to examine the interaction of isolated VG1 and Fab-VG1 Fab-HABD with ex vivo vitreous humor. VG1 and Fab-VG1 were covalently labeled on lysine residues using PEG4-DY647-N-hydroxysuccinimide ester. The fluorescence emission of DY647 can be excited by lasers at 594nm or 633nm and detected at longer wavelengths. The reaction chemistry was controlled so that the labeling level per molecule was no greater than 1 fluorescent dye. Porcine vitreous humor was collected from the eyes of freshly slaughtered animals and homogenized using a Dounce homogenizer. This material was serially diluted 1:3 with phosphate buffered saline (PBS) pH 7.4. The labeled test article was added to each diluted aliquot to a final concentration of 20nM. The test articles were (1) free VG1, (2) pigFab-VG1, (3) pigFab-VG1 mixed with 10 kDa HA at a weight ratio of 1:1, (4) RabFab-VG1, and (5) RabFab-VG1 mixed with 10 kDa HA at a weight ratio of 1:1. After incubation at room temperature for 2 hours, FCS was performed.

B. Results

The results of the FCS measurements are shown in FIG12 . All samples showed significantly delayed diffusion when incubated with undiluted or slightly diluted vitreous compared to incubation in buffer (PBS) alone. For free VG1, PigFab-VG1, and RabFab-VG1, this delayed diffusion persisted until the vitreous was diluted more than 6000-fold ( FIG12 , rows 3, 4, 6, and 7; from undiluted to a dilution factor of 6561). Slow diffusion was also observed for samples co-formulated with 10 kDa HA, but this effect disappeared when the vitreous was diluted ≥ 729-fold ( FIG12 , row 5: PigFab-VG1+10 kDa HA (1:1), and row 8: RabFab-VG1+10 kDa HA (1:1); from a dilution factor of 729 to PBS). These results indicate a strong interaction between vitreous components (most likely endogenous high molecular weight HA in the vitreous) and the test articles containing VG1. Even in the presence of low molecular weight HA, VG1 was able to interact with endogenous HA at low dilutions of the vitreous humor (Figure 12, row 5: PigFab-VG1 + 10 kDa HA (1:1), and row 8: RabFab-VG1 + 10 kDa HA (1:1)). This suggests that VG1 and Fab-VG1 can dissociate from 10 kDa HA and bind to HA present in the vitreous humor. However, once the vitreous humor is greatly diluted, there is no high molecular weight HA in sufficient concentration to compete with the binding of low molecular weight HA to VG1 (Figure 12, row 5: PigFab-VG1 + 10 kDa HA (1:1), and row 8: RabFab-VG1 + 10 kDa HA (1:1); from a dilution factor of 729 to PBS). VG1 bound to low MW HA experienced little or negligible diffusion slowing compared to unbound material ( FIG. 12 , PBS control for row 5: PigFab-VG1+10 kDa HA (1:1), and row 8: RabFab-VG1+10 kDa HA (1:1); these samples had 10 kDa HA without added vitreous).

Example 13. Effect of pre-complexation with 10 kDa HA on the thermal stress stability of Fab-VG1

A. Materials and Methods

The effect of pre-complexation of Fab-VG1 with 10 kDa HA on thermal stress stability was tested using anti-HtrA1-VG1 protein. In these experiments, anti-HtrA1-VG1 was prepared at a concentration of 3 mg/mL with phosphate buffered saline (PBS) pH 7.4, and 1.8 mg/mL of 10 kDa HA was added or not. The concentration of 1.8 mg/mL (180 μM) of 10 kDa HA was 5 times molar excess relative to the anti-HtrA1-VG1 concentration (35 μM). These preparations were incubated at a temperature of 37°C for 4 weeks and then analyzed by non-reducing capillary electrophoresis sodium dodecyl sulfate (NR CE-SDS), as described by Michels et al. 2007 (Anal. Chem. 79, 5963). In addition to monomeric species and fragments, aggregates resistant to SDS denaturation can be detected by NR CE-SDS.

B. Results

As shown in Figure 13 and summarized in Table 19, pre-complexation with 10 kDa HA inhibited the formation of SDS-stable aggregates in anti-HtrA1-VG1. The formation rate of high molecular weight forms (HMWF) decreased from 1.2% per week to 0.1% per week. The presence of 10 kDa HA also appeared to affect fragmentation, although less than the effect on aggregation, but the formation rate of low molecular weight forms (LWMF) was reduced by about 2 times when complexed with HA. These results indicate that at neutral pH, the inclusion of 10 kDa HA in the formulation stabilizes anti-HtrA1-VG1 under heat stress conditions.

Example 14. Intraocular tolerability of VG1 and VG1 Fab-HABD

A. Materials and Methods

A.1. Intravitreal (IVT) injection and endpoint assessment

use The tolerability of IVT injections of VG1 and Fab-VG1 Fab-HABD was evaluated. The design of this study is shown in Table 20.

Each minipig received a single injection of 50 μL via IVT in both eyes. Based on historical data, this volume is well tolerated in minipigs. The IVT injection procedure was performed by a board-certified veterinary ophthalmologist. Group 1 minipigs received vehicle control injections. Group 2 minipigs received isolated WT VG1 (produced as described in Example 8 above). Group 3 minipigs received pigFab-VG1 (produced as described in Example 10 above) treatment Group 4 minipigs received pigFab-VG1 preformulated with an equal weight of 10kDa HA. All test products were formulated with 20mM histidine-acetate, 150mM NaCl (pH 5.5) to a specified protein concentration. The dose of pigFab-VG1 in Groups 3 and 4 represents the maximum feasible dose to keep the total endotoxin level at less than 0.05 endotoxin units (EU) per eye. This level of endotoxin has been previously found to be tolerable in minipig intraocular studies. Given the molecular weight difference between WT VG1 (approximately 30 kDa) and pigFab-VG1 (approximately 80 kDa), the dose levels in Group 2 compared to Groups 3 and 4 represent 1.6 HA binding molar equivalents per dose.

The following parameters and endpoints were assessed in this study: mortality, clinical signs, body weight, ophthalmology (examination, intraocular pressure measurement, wide-field color fundus imaging, OCT imaging, and electroretinogram [ERG]), bioanalysis, toxicokinetic parameters, antidrug antibody assessment, gross necropsy findings, and histopathological examination.

Ophthalmoscopic examinations were performed on both eyes of all surviving animals by a board-certified veterinary ophthalmologist via indirect ophthalmoscope and slit-lamp biomicroscope. All animals underwent ophthalmological examinations prior to treatment and on days 1 (post-dose), 3, 5, 8, 15, 17, 22, and 29.

Intraocular pressure (IOP) was measured in both eyes of all surviving animals by applanation tonometry during ophthalmological examination by a board-certified ophthalmologist. IOP was measured in all animals before treatment and on days 1 (after dosing), 3, 5, 8, 15, 17, 22, and 29.

Wide-field color fundus imaging was performed on all surviving animals using the Clarity RetCam Shuttle on day 29. Attempts were made to photograph the administered test article if visible.

On day 29, optical coherence tomography was performed using a Heidelberg Spectralis HRA/OCT system; a single, perpendicular, high-resolution line scan was performed through the optoderm.

All surviving animals were evaluated for ERG on day 29. Animals were dark-adapted for a minimum of 1 hour before ERG. Full-field flash ERG with Ganzfeld dome stimulation (flash intensity conformed to ISCEV standard parameters and light adaptation time was 5 minutes (Retiport Gamma, Roland Consult)); amplitude and latency values were measured from the tracings.

Blood samples (approximately 0.5 mL) were collected from all surviving animals via the anterior vena cava through a thoracic inlet for determination of serum concentrations of the test article. Animals were not fasted prior to blood collection, except for intervals consistent with other procedures for fasting. Blood was collected once before treatment, on day 1 (6 hours and 12 hours after dosing), and on days 2, 3, 5, 8, 12, 15, 22, and 29.

Blood samples were collected into serum separator tubes and allowed to clot at controlled room temperature until centrifuged at 1300 g for 10 minutes at controlled room temperature within 60 minutes of collection. Within 30 minutes of the start of centrifugation, the resulting serum was aliquoted into pre-labeled 0.50 mL 2D barcoded Matrix tubes (Thermo Cat 3744). All aliquots were snap frozen on dry ice and stored frozen at -60°C to -90°C.

Serum samples were tested for the presence of anti-drug antibodies (ADA) in an ELISA assay. The test article was immobilized on the assay plate, incubated with the serum and washed, and the immune complexes were then detected with an anti-porcine IgG reagent that has an Fc portion conjugated to horseradish peroxidase for enzymatic detection.

On day 15, aqueous humor was collected from all animals by a board-certified veterinary ophthalmologist. The tip of a 31-gauge needle was inserted obliquely into the sclera behind the limbus at an approximately 90-degree angle while the eye was positioned using conjunctival forceps for aqueous humor collection. The angle of the needle was then shallowed before the needle entered the anterior chamber between the iris and cornea. The syringe plunger was slowly withdrawn to aspirate the maximum obtainable volume of aqueous humor, which could be up to 50 μL. The needle was removed, and the episcleral tissue was brought close to the puncture site and grasped with conjunctival forceps. The same sample collection procedure was performed on the contralateral eye. The collected samples were stored in 1.0 mL glass matrix trakmates 2D barcoded storage tubes, which were then capped with TPE caps. The samples were frozen in liquid nitrogen and stored frozen at -60°C to -90°C. The test article content in aqueous humor was determined using a mass spectrometry-based assay.

A.2. Sample preparation

PigFab standard calibration curves were made by adding different amounts of PigFab to porcine aqueous matrix diluted with 25 mM ammonium bicarbonate. The standards/samples were then treated as follows: disulfide bonds from cysteinyl residues were reduced with 10 mM DTT at 60°C for 1 hour, followed by alkylation of thiol groups with 55 mM iodoacetamide at room temperature in the dark for 45 minutes. The standards/samples were then digested with 36 μg/mL trypsin (sequencing grade trypsin, V5111, Promega) and incubated overnight at 37°C. After digestion, heavy peptides were added to the standard and sample solutions. A linear calibration curve was obtained over the 0.5-12 μg/mL concentration range.

A.3. Labeled peptides

Peptide standards containing heavy isotope labels in amino acids R (LLIYSASFLYSGVPSR m/z: 891.98+2) were purchased (New England Peptide, Gardner, MA, USA). Characterization and concentration data were provided by the manufacturer. The labeled peptides were stored in 1 mL of water at -80°C.

A.4. Analysis by mass spectrometry (MS)

The peptides were analyzed on an Acquity UPLC (Waters Corporation, Milford, MA) using an ACQUITY UPLC CSHC18 Peptide Column (130 1.7μm, 1mm×100mm) was used to separate the digest from PigFab under gradient elution conditions. The column was maintained at 50°C and the autosampler sample tray was maintained at 8°C. The mobile phases were water (A) containing 0.1% FA and acetonitrile (B) containing 0.1% FA at a flow rate of 0.04mL/min. The sample was eluted with the following gradient: B increased from 2% to 90% in 2 minutes and then decreased to 2% B in 2 minutes to re-equilibrate the column. The injection volume was 10μL.

A Triple Quad 6500 mass spectrometer (Ab Sciex, Framington, MA) was operated in positive ion multiple reaction monitoring (MRM) mode and equipped with Turbo V ion source. The monitored PigFab precursor (Q1) ion was LLIYSASFLYSGVPSR (m/z: 886.98+2) with a declustering potential of 90 V, and the monitored product (Q3) ion was 359.20 m/z with a collision energy of 29 eV. Two other product ions (765.39 m/z and 602.33 m/z) were also monitored as qualifier ions with collision energies of 37 eV and 30 eV, respectively. The MS/MS setting parameters were as follows: ion spray voltage, 4500 V; curtain, 30 psi; nebulizer gas (GS1), 25 psi; temperature, 300 °C; dwell time, 50 ms. The heavy peptide of PigFab (891.97 m/z) was also generated and quantified using the transition at 369.204 m/z with a collision energy of 29 eV.

Data acquisition was performed using Sciex Analyst software version 1.7.1 (TripleTOF) and raw data were displayed using PeakView 2.2.

B. Results

All animals survived until the planned termination date. Therefore, no unplanned euthanasia was required. Ophthalmic findings related to the test article included vitreous opacities in the area of test article injection and minimal posterior uveitis.

The ophthalmological examination results were as follows:

(1) Group 2 (WT VG1) - On Day 1, very mild opacities were observed in the temporal vitreous in 2 of 6 animals. This finding resolved on Day 3 and remained absent until study termination. One of 4 animals developed very mild posterior uveitis on Day 15, which resolved on Day 17. The very mild posterior uveitis was considered to be of no clinical significance.

(2) Group 3 (pigFab-VG1) - On day 1, all 6 animals exhibited vitreous opacities in the temporal vitreous in the area of test article injection. On day 3, this finding persisted in all 6 animals, and on day 5, 4/4 animals exhibited diffuse signs involving the central vitreous. From days 8 to 22, we observed a decrease in the area of vitreous affected, and on day 29, only 2 of 4 animals developed mild opacities in the temporal vitreous. The regional vitreous opacities did not appear to be inflammatory in nature, but appeared to have a local effect on vitreous consistency.

(3) Group 4 (pigFab-VG1+10 kDa HA) – No ophthalmological examination results related to the test article were obtained during the duration of the study.

IOP values were within normal range in all animals at all time points throughout the study period.

None of the animals showed anti-drug antibodies (ADA) against the test article at any time point.

Color fundus photographs and optical coherence tomography images taken on day 29 showed normal vitreous and retinal morphology in all animals in Groups 2 and 4. All animals in Group 3 had minimal focal vitreous opacities on fundus photographs and minimal posterior vitreous hypersensitivity on OCT, consistent with the findings of ophthalmologic examination.

Electroretinogram analysis was performed on all animals on day 29. Animal 3006 had a moderate reduction in b-wave amplitude in both eyes at Scotopic 0.01 light intensity. All other light intensities were within normal range, suggesting that this animal had background abnormalities affecting dim retinal function. Animals 2005 and 4003 showed some asymmetry between eyes, with a mild reduction in amplitude OD compared to OS. This result is suspected to be related to the recording conditions, including the non-central eye positioning OD. No results suggesting a test article effect were found in any animal.

There were no test article-related macroscopic findings in the eye or optic nerve.

All macroscopic observations in animals treated with the test article were background findings for the species or were considered incidental and unrelated to the test article. These observations were of low incidence, lacked a clear dose relationship in incidence or severity, and/or there were no relevant microscopic findings related to the test article.

There were no test article-related microscopic findings in the eye or optic nerve.

All microscopic observations were considered incidental findings and unrelated to the test article. These observations were known background findings for the species and/or were of similar incidence and severity in animals receiving control and test article treatment.

The levels of PigFab-VG1 in aqueous humor samples were determined by mass spectrometry. After IVT injection of 1.8 mg/eye of PigFab-VG1 or PigFab-VG1 pre-complexed with an equal mass of 10 kDa HA in minipig eyes, high aqueous humor levels were obtained and maintained for 30 days (Figure 14). These results indicate that measurable levels of the test article were present in minipig eyes over the 4-week study period. The concentrations at 30 days were at least an order of magnitude higher than those observed for the unmodified Fab (Figure 4A).

In Gottingen minipigs, administration of WT VG1 (1.13 mg/eye), pigFab-VG1 (1.8 mg/eye), or pigFab-VG1+10 kDa HA (1.8 mg/eye) at various dose levels by single IVT was well tolerated. All animals survived to the planned termination time with no abnormal clinical observations, and body weight was unaffected. The lack of detectable immune response to the test article during the study using a single injection allowed the assessment of the direct effect of the test article. Ophthalmic findings were limited to transient, minimal vitreous opacities at the site of test article injection, which resolved on day 3 (WT VG-1); and vitreous opacities near the site of test article injection, which did not completely resolve at the time of study termination (pigFab-VG1). There were no ophthalmoscopic findings related to pigFab-VG1+10 kDa HA, and IOP, OCT, and ERG results were normal in all animals. There were no macroscopic or microscopic effects related to the test article in the eye or optic nerve.

Example 15. Efficacy of VPDF-VG1 in Rat Laser-Induced Choroidal Neovascularization (Rat Laser CNV)

Fab-HABD was studied in an in vivo rat model of laser-induced choroidal neovascularization (rat laser CNV) to test the following hypotheses: (1) Fab-HABD is effective in vivo (i.e., Fab-HABD can inhibit neovascularization); and (2) the duration of in vivo efficacy of Fab-HABD is comparable to or superior to that of unmodified Fab fragments.

A. Materials and Methods

Rats received IVT injections of the protein formulations 1 or 3 weeks before laser injury (six laser burns per eye). One week after laser injury, lesions were analyzed for vascular growth using fluorescence angiography (FA) imaging.

Fab-HABD was compared to the corresponding unmodified Fab fragment. To test the sustained efficacy of Fab-HABD, the dose of unmodified Fab was titrated to the "lowest effect dose" (i.e., only a low degree of inhibition of detectable neovascularization compared to vehicle over the duration of the rat model), indicating that Fab-HABD had a more sustained efficacy at the same dose and duration of the model.

B. Results

As shown in Figure 15, administration of VPDF-VG1 7 or 21 days before laser treatment effectively inhibited CNV lesions. In this study, the durability of the effect of VPDF-VG1 was comparable to that of the unmodified Fab.

Example 16. Intraocular Tolerance of VG1 and VG1 Fab-HABD in New Zealand White Rabbits

A. Materials and Methods

The purpose of this study was to determine the intraocular tolerance of the test articles WT VG1, RabFab-VG1, and RabFab-VG1 preformulated with 1:1 (w/w) 10 kDa HA after a single bilateral IVT injection into male New Zealand white rabbits over a 30-day observation period. The study design is shown in Table 21.

The following parameters and endpoints were evaluated in this study: mortality, clinical signs, body weight, food consumption, ophthalmology (i.e., examination, intraocular pressure measurement, wide-field color fundus imaging, OCT, and ERG), bioanalysis, toxicokinetic parameters, anti-drug antibody assessment, gross necropsy findings, and histopathological examination.

B. Results

Based on evaluation of body weight and food consumption, there were no systemic test article-related effects. There were no macroscopic necropsy findings and all tissues were considered to be within normal limits.

The presence of anti-drug antibodies (ADA) in rabbit serum was determined prior to dosing and on days 8, 15, 22, and 29 of the study. Prior to dosing, 3 of the 6 animals assigned to receive WT VG1 had measurable serum ADA. Similarly, 1/6 and 0/6 animals assigned to receive RabFab-VG1 or RabFab-VG1+10kDa HA treatment, respectively, had pre-existing serum ADA against the test article. On day 8, all animals in the WT VG1 and RabFab-VG1 groups showed ADA against the test article in their serum and remained positive during the study, while in the RabFab-VG1+10kDa HA group, 2/3 of the animals had serum ADA. On day 15, all animals were positive for serum ADA and remained so until the end of the study.

Clinical ophthalmoscopic findings were consistent with the development of anterior and posterior uveitis in animals treated with WT VG1, RabFab-VG1, and RabFab-VG1 + 10 kDa HA, but the severity varied between treatment groups. For example, WTVG1 resulted in moderate anterior and posterior uveitis, including posterior subcapsular cataracts in some eyes, at day 22. In contrast, most animals treated with RabFab-VG1 exhibited mild uveitis at the same time point. In addition, animals treated with RabFab-VG1 + 10 kDa HA exhibited only minimal to mild anterior and posterior uveitis. In each treatment group, after systemic anti-inflammatory treatment was initiated on day 18 (which also included topical ocular treatment for animals receiving WT VG1), improvement in signs of uveitis occurred on day 29. Decreases in intraocular pressure were consistent with active uveitis, and the degree of change in vitreous opacities also corresponded to the severity of uveitis in the different groups. Under these conditions, vitreous opacities were limited to mild vitreous opacities in animals treated with RabFab-VG1 + 10 kDa HA, whereas WT VG1 animals exhibited moderate opacities and posterior pole cataracts. Treatment-dependent severity of effects was also observed by OCT and ERG, with reduced scotopic and photopic amplitudes indicating severe alterations and degeneration of retinal function in eyes treated with WT VG1.

Endoscopic effects were also most pronounced in eyes treated with WT VG1 (Fig. 16A) compared to the other test articles, in which the RabFab-VG1 effect (Fig. 16B) was less severe and the RabFab-VG1+10kDaHA effect (Fig. 16C) was limited to the vitreous and was only minimal to mild in severity. WT VG1-associated vitreous inflammation included areas near the optic nerve head, retinal detachment and varying degrees of necrosis, and an inflammatory cell layer adjacent to the posterior lens capsule. In addition, the anterior chamber contained homogeneous eosinophilic material consistent with serum proteins. In the retina, WT VG1-associated inflammation was characterized by mixed cell types extending into the retinal parenchyma, accompanied by minimal to significant necrosis and vascular and perivascular inflammation. Reactive Muller cells were observed in the central retina.

Similar to WT VG1, eyes treated with RabFab-VG1 exhibited minimal to moderate diffuse photoreceptor lesions that were often associated with reactive Muller cells. However, RabFab-VG1 photoreceptor layer lesions differed from WT VG1-associated retinal necrosis in that the lesions were selective only to the photoreceptor layer, whereas retinal necrosis involved multiple retinal layers. In addition, both WT VG1- and RabFab-VG1-treated eyes presented features of fibrovascular membranes in the vitreous. In these cases, the membranes were composed of fibroblasts, numerous new vessels, and early collagen deposition. Traction cords detached from the membranes were also observed. In contrast to WTVG1 and RabFab-VG1, RabFab-VG1+10kDa HA-associated effects were limited to minimal to mild mononuclear inflammation that was confined to the vitreous and internal limiting membrane.

In conclusion, a single IVT administration of WT VG1, RabFab-VG1, or RabFab-VG1 + 10 kDa HA to New Zealand white rabbits resulted in the development of anterior and posterior uveitis that was most severe in animals administered WT VG1, moderate in animals administered RabFab-VG1, and mild to moderate in animals administered RabFab-VG1 + 10 kDa HA. On microscopic examination, in addition to inflammation, there was significant retinal necrosis and retinal degeneration in WT VG1 and RabFab-VG1, respectively, which correlated with decreased ERG amplitude. At the end of the study, there were no signs of active anterior uveitis in the RabFab-VG1 + 10 kDa HA eyes, and only minimal chronic posterior uveitis was observed. Although the interpretation of these results is confounded by the ADA observations for all test articles at day 15, pre-complexation or conjugation of RabFab-VG1 with 10 kDa HA does appear to improve the tolerability of this Fab-HABD in rabbit eyes.

Example 17. Brain Retention of Fab-VG1

The ability to achieve brain retention through HA binding by VG1 was tested by intracerebroventricular injection in mice. For these purposes, the non-target binding antibody anti-herpes simplex virus-1 glycoprotein D (anti-gD) was used as a Fab fragment (anti-gD Fab), intact IgG (anti-gD IgG) or as a fusion protein with VG1 (anti-gD Fab-VG1; BRD).

A. Materials and Methods

A.1. Animals

Wild-type C57BL/6 mice used in these studies were obtained from the University of Kansas breeding colony. The protocol for the use of live animals (AUS 75-15; approval date: January 25, 2021) was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Kansas. All animals were cared for by the Animal Care Unit (ACU) staff and veterinarians in a temperature-controlled environment with a 12-hour dark/light cycle, and the animals had unrestricted access to feed and water.

A.2. Binding of Antibodies to IRDye800CW NHS Ester

The antibodies were conjugated to IRDye800 according to the manufacturer's instructions. Briefly, the antibodies were reacted with IRDye800 in PBS containing 10% potassium phosphate buffer pH 9 (v/v) at 25°C for 2 hours. Excess dye was removed using a ZebaSpin desalting column (Fisher Scientific) with a molecular weight cutoff of 7 kDa. The purity of the bound antibody was assessed using SDS-PAGE. The SDS-PAGE gel was scanned at 800 nm using an Odyssey CLx NIR scanner to confirm that all excess dye had been removed.

A.3. Intraventricular injection

Healthy C57BL/6 mice aged 5-10 weeks were anesthetized using 1.5-2% isoflurane and placed in a stereotaxic apparatus (Stoelting Co.). A midline sagittal incision was made to expose the skull of the mouse and mark the bregma. A small hole was made on the right side of the skull 1.0 mm and 0.3 mm anterior to the bregma. A 10 μL Hamilton syringe (No. 7762-06) with a 33-gauge detachable needle was assembled to the stereotaxic apparatus and used to inject 5 μL of an antibody solution at a concentration of 1 mg/mL into the lateral ventricle of the mouse to a depth of 2.25 mm. The antibody was infused at a rate of 1 μL/min. Immediately before euthanasia, a blood sample (approximately 100 μL) from the submandibular vein was collected into a frozen plasma collection tube containing lithium heparin as an anticoagulant. The samples were kept on ice until centrifuged at 10,000×g for 3 minutes, and the plasma was stored at -80°C until analysis. After various time points, mice were sacrificed via transcardial perfusion with ice-cold HBSS containing 0.1% Tween-20 while deeply anesthetized with 4-5% isoflurane. Brain, heart, lung, liver, spleen, and kidney were collected and kept on ice until analysis.

A.4. Antibody Organ Quantification

The separated organs are weighed and mechanically homogenized in 1mL PBS. Standard near-infrared fluorescence (NIRF) antibody solution is prepared by diluting the stock solution with different amounts of PBS. Then in 96-well plates, by adding 10 μL standard solution to 100 μL homogenized blank organs and using OdysseyClx scanner to scan each hole, a calibration curve is generated for each organ. The fluorescence intensity of each hole and the antibody concentration of each gram of organ are mapped to obtain a linear curve. The organs from mice receiving intraventricular injection are compared with the calibration curve to determine antibody deposition. Similarly, plasma analysis is carried out with blank plasma diluted 5 times at first. Then, 10 μL antibody standards are added to aliquots of 100 μL diluted plasma to generate a standard curve, compared with the plasma samples of mice receiving intraventricular injection.

B. Results

As shown in Figure 17, anti-gDFab-VG1 (BRD; SEQ ID NO: 121 and SEQ ID NO: 124) persisted longer in the brain and provided greater exposure levels (expressed as area under the curve (AUC)) than equivalent doses of anti-gD Fab (SEQ ID NO: 120 and SEQ ID NO: 121) or anti-gD IgG (SEQ ID NO: 121 and SEQ ID NO: 122). These differences in exposure levels were statistically significant, with p values of less than 0.01 for anti-gD Fab-VG1 compared to anti-gD Fab and p values of less than 0.001 for anti-gD Fab-VG1 compared to anti-gD IgG.

Example 18. Generation of VG1 affinity variants

A. Materials and Methods

A.1. Crystallization of WT VG1 and identification of HA-binding residues

Crystallization conditions for WT VG1 bound to HA were determined using commercial crystallization screens (Hampton Research and Qiagen). The structure of HA-bound VCAN was obtained by soaking crystals with HA6-mer. Crystals were harvested and flash frozen in liquid nitrogen without cryoprotectants. Diffraction data were collected using Stanford Synchrotron Radiation Light Source (SSRL) beamlines 12-2 or 14-1 on a Pilatus6M or Eiger 16M detector (Dectris), respectively. The structure was iteratively refined by building a model in COOT and then refined with REFMAC5, BUSTER, or Phenix-Refine. Adams, P.D. et al., ActaCrystallogr.D Biol.Crystallogr., 66 (Pt 2): 213-221 (2010); Blanc, E. et al., ActaCrystallogr.D Biol.Crystallogr., 60: 2210-2221 (2004); Emsley, P. et al., ActaCrystallogr.D Biol.C Crystallogr., 66: 486-501 (2010); Emsley and Cowtan, ActaCrystallogr. D Biol. Crystallogr., 66: 2126-2132 (2004); Murshudov, G.N. et al., ActaCrystallogr. D Biol. Crystallogr., 67: 355-367 (2011).

The WT VG1-HA conjugate structure ( FIG. 18 ) was analyzed using PyMol and/or Chimera, and the residues interacting with HA were identified based on hydrogen bonding, electrostatic, and hydrophobic interaction potentials.

A.2. Differential Scanning Fluorescence (DSF)

The thermal stability of WT VG1 and single amino acid variants was measured using differential scanning fluorescence (DSF). Briefly, 0.1 mg/mL purified protein was mixed with Sypro Orange dye in PBS. Each sample was treated in a temperature gradient from 25°C to 95°C in increments of 0.05°/sec and the increase in fluorescence was monitored at 585nm. The raw fluorescence units were plotted as negative derivatives and Tm was calculated using a custom excel macro.

A.3. Design of VG1 variants based on the crystal structure of WT VG1-HA conjugate

According to the WT VG1-HA conjugate crystal structure (Figure 18), HA was found to bind only to the link1 domain. Therefore, modeling was used to predict link 2 residues that may be involved in HA binding. In order to verify the crystal structure and identify mutants that weaken the HA binding affinity of WT VG1, the residues in crystal contact were mutated to alanine, or in some cases, mutated to alternative amino acids. In addition, some WT VG1 residues do not make crystal contacts with HA but are important for HA binding in TSG6 (based on the sequence alignment between the VG1 linker domain structure and the TSG-6 linker domain; Figure 8B), and they are also mutated to alanine. To examine the combined effects of mutations, several double-site mutants were formed, for example, Lys260 and Phe261 were changed to Arg and Tyr (KF260RY), respectively, and the HA binding ability was tested. Table 22 lists the VG1 variants produced by the method described in Example 10. The amino acid sequence alignment of the VG1 variants produced and tested is shown in Figure 19.

A.4. Molecular properties

HA binding was measured by SPR as described in Example 10. Mutants were injected for 120 seconds and dissociation was monitored for 180 seconds.

B. Results

B.1. VG1 variants have reduced HA binding levels

Mutants R160A, Y161A and D197A showed reduced HA binding in the range of 2 to 7 μM. Table 23 shows the measured _ka (M ^-1 s ^-1 ), _kd (s ^-1 ) and _KD (M) for each VG1 variant as measured by SPR.

B.2. Stability of VG1 variants

The generated VG1 mutants and the measured Tm (melting temperature; °C) of each mutant are shown in Table 24. Although most mutations slightly reduced or had no effect on thermal stability compared to WT VG1, Y208A and H306A showed improvements of 2.16°C and 2.81°C in Tm, respectively.

Equal form

It is believed that the foregoing written description is sufficient to enable those skilled in the art to practice the present embodiment. The foregoing description and examples detail certain embodiments and describe the best mode contemplated by the inventor. However, it should be understood that no matter how detailed the foregoing content is in the text, the embodiments can be practiced in many ways and should be interpreted according to the attached claims and any equivalent forms thereof.

As used herein, the term "about" refers to numerical values (including, for example, integers, fractions, and percentages), whether or not explicitly stated. The term "about" generally refers to a numerical range (e.g., +/- 5-10%) that one of ordinary skill in the art would consider equivalent to the stated value (e.g., having the same function or result). When terms such as "at least" and "about" precede a list of numerical values or ranges, these terms will modify all values or ranges provided in the list. In some cases, the term "about" may include numerical values rounded to the nearest significant figure.

Claims (31)

1. A therapeutic molecule comprising: a. A first component capable of binding to a therapeutic target in the eye, b. one or more second components capable of binding to hyaluronic acid, wherein said one or more second components are covalently bound to said first component, and c. Optionally, one or more third components comprising hyaluronic acid, Wherein, where present, the one or more third components are non-covalently bound to the one or more second components. 2. The therapeutic molecule of claim 1, wherein the first component is a protein, a peptide, a receptor or a fragment thereof, a ligand of a receptor, a darpin, a nucleic acid, RNA, DNA or an aptamer. 3. Therapeutic molecule according to claim 1 or 2, wherein said first component is selected from the group consisting of antibodies, antigen-binding fragments, in particular antibody fragments, more particularly antibody fragments lacking at least an Fc domain, in particular Wherein said fragment is or comprises a (Fab') ₂ fragment, a Fab' fragment, a Fab fragment, a VhH fragment, a scFv fragment, a scFv-Fc fragment and a minibody, more particularly a Fab fragment. 4. The therapeutic molecule of any one of claims 1 to 3, wherein the second component comprises a hyaluronic acid receptor CD44 (CD44) domain, a brain-specific connexin (BRAL1) domain, a tumor Necrosis factor-stimulated gene 6 (TSG-6) domain, lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1) domain, or hyaluronic acid-binding protein (HABP) domain, aggrecan G1 (AG1) domain or versican G1 (VG1) domain. 5. The therapeutic molecule according to any one of claims 1 to 4, wherein the conjugate comprises one second component or two second components that are identical to each other. 6. The therapeutic molecule of any one of claims 1 to 4, wherein the third component is hyaluronic acid, wherein the hyaluronic acid a.Have i. A molecular weight selected from 3kDa to 60kDa, 4kDa to 30kDa, 5kDa to 20kDa or 400Da to 200kDa; ii. A molecular weight of at least 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa or 9kDa; or iii. A molecular weight of up to 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa or 15kDa; b. Provide a molar excess of binding equivalents to the one or both second components; and c. Has modifications that reduce the degradation of hyaluronic acid in the eye. 7. The therapeutic molecule of any one of claims 1 to 6, wherein the second component is capable of binding to hyaluronic acid with a _KD of 10 nM to 10 μM, 5 nM to 8 μM, or 100 nM to 5 μM. 8. The therapeutic molecule according to any one of claims 1 to 7, wherein a. Said first and second components are comprised in a fusion protein, in particular wherein one or both of said second components are covalently bound to the N-terminus of said first component and /or the C-terminus, more particularly wherein said first component is an antibody or antigen-binding fragment, and wherein said one or both second components are covalently bound to the C-terminus of said first component; and / or b. The one or both second components are bound to the first component directly or indirectly via a linker, in particular at least 4 amino acids and/or A linker of up to 50 or up to 25 amino acids, more particularly said linker is (GxS) _n or (GxS) _n G _m , where G = glycine and S = serine, (x=3, n=3, 4, 5, or 6, and m=0, 1, 2, or 3) or (x=4, n=2, 3, 4, or 5, and m=0, 1, 2 or 3). 9. The therapeutic molecule according to any one of claims 1 to 8, wherein the therapeutic target is VEGF, C2, C3a, C3b, C5, C5a, HtrA1, IL-33. Factor P, Factor D, EPO , EPOR, IL-1β, IL-17A, IL-10, TNFα, FGFR2, PDGF or ANG2. 10. The therapeutic molecule according to any one of claims 1 to 9, wherein a. The first component is an antibody or antigen-binding fragment directed against VEGF; and/or b. Each of the one or two second components comprises a CD44 domain or a TSG-6 domain or a VG1 domain; and/or c. The third component is hyaluronic acid with a molecular weight of 5 kDa to 20 kDa. 11. The therapeutic molecule according to any one of claims 1 to 10, wherein a. The first component is an anti-VEGF antibody or antigen-binding fragment, the one or two second components comprise a CD44 domain, and the third component is hyaline with a molecular weight of 5 kDa to 20 kDa. acid; b. The first component is an anti-VEGF antibody or antigen-binding fragment, the one or two second components comprise a TSG-6 domain, and the third component has a molecular weight of 5 kDa to 20 kDa. hyaluronic acid; or c. The first component is an anti-VEGF antibody or antigen-binding fragment, the one or two second components comprise a VG1 domain, and the third component is hyaline with a molecular weight of 5 kDa to 20 kDa. acid. 12. The therapeutic molecule according to any one of claims 1 to 11, wherein a. The first component contains i. The VH domain of SEQ ID NO: 97, 99, 105, 109 or 114; and ii. The VL domain of SEQ ID NO: 98, 100, 106, 110 or 115; and b. The second component contains SEQ ID NO:2. 13. The therapeutic molecule according to any one of claims 1 to 11, wherein a. The first component contains i. The VH domain of SEQ ID NO: 97, 99, 105, 109 or 114; and ii. The VL domain of SEQ ID NO: 98, 100, 106, 110 or 115; and b. The second component contains SEQ ID NO:4. 14. The therapeutic molecule according to any one of claims 1 to 11, wherein a. The first component contains i. The VH domain of SEQ ID NO: 97, 99, 105, 109 or 114; and ii. The VL domain of SEQ ID NO: 98, 100, 106, 110 or 115; and b. The second component comprises SEQ ID NO: 86, 60, 32 or 29. 15. The therapeutic molecule of claim 14, wherein the second component is at least 90%, 91%, 92%, 93%, 94%, 95%, SEQ ID NO: 86, 60, 32 or 29, 96%, 97%, 98%, 99% or 100% identical. 16. The therapeutic molecule of claim 14 or 15, wherein the second component comprises 1 to 5 mutations, wherein the 1 to 5 mutations comprise single amino acid substitutions, double amino acid substitutions and/or truncation. 17. The therapeutic molecule of any one of claims 14 to 16, wherein the second component has a truncating mutation relative to SEQ ID NO:29. 18. The therapeutic molecule of claim 17, wherein the truncating mutation comprises a truncation of 1 to 129 amino acids at the N-terminus. 19. The therapeutic molecule of any one of claims 14 to 18, wherein the second component is a truncated sequence in which the Ig domain of wild-type versican is absent. 20. The therapeutic molecule according to any one of claims 14 to 19, wherein the second component comprises 1, 2, 3, 4, 5 of the following positions relative to SEQ ID NO: 29 Mutations in one or six: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327. 21. The therapeutic molecule according to any one of claims 14 to 20, wherein the second component comprises at least 1, 2, 3, 4 of the following mutations relative to SEQ ID NO: 29, 5 or 6 pcs: R160A, Y161A, D197A, D197S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A and LYR325LFK. 22. The therapeutic molecule of any one of claims 14 to 21, wherein the second component comprises at least one of Y208A and H306A. 23. The therapeutic molecule according to claim 14 or 15, wherein the second component is SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 or SEQ ID NO:59. 24. The therapeutic molecule of any one of claims 1 to 23, wherein the first component further comprises a cysteine knot peptide. 25. The therapeutic molecule of claim 24, wherein the cysteine knot peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% identity. 26. The therapeutic molecule according to claim 24 or 25, wherein the amino acid sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96 of SEQ ID NO:93 or SEQ ID NO:94 %, 97%, 98%, 99% or 100% identity. 27. A composition for use as a medicament, said composition comprising a therapeutic molecule according to any one of claims 1 to 26 and optionally a pharmaceutically acceptable excipient, diluent or carrier. 28. A composition for treating eye disease or brain disease, said composition comprising a therapeutic molecule according to any one of claims 1 to 26 and optionally a pharmaceutical excipient, diluent or carrier . 29. The composition for use according to claim 28, formulated for intraocular delivery, in particular for intravitreal injection. 30. The composition for use according to claim 28 or 29, wherein the eye disease is age-related macular degeneration (AMD), in particular wet AMD or neovascular AMD, diabetic macular edema (DME), Diabetic retinopathy (DR), in particular proliferative DR or non-proliferative DR, retinal vein occlusion (RVO) or geographic atrophy (GA). 31. A method of delivering a therapeutic molecule targeted to tissue of a patient, comprising combining a therapeutic molecule according to any one of claims 1 to 26 or a composition according to any one of claims 27 to 30 is administered to the patient and allows the therapeutic molecule to provide long-lasting delivery of the first component to the target tissue.