HK40093655A - Hyaluronic acid binding derivatives of versican (vg1) for long acting delivery of therapeutics - Google Patents

Description

Hyaluronic acid-binding derivatives of a multifunctional proteoglycan (VG1) for long-acting therapeutic delivery

Cross-references to related applications

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

Technical Field

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

Background Technology

Intravitreal (IVT) injections are commonly used to administer medications for a variety of eye diseases. IVT injections allow medications to be delivered directly to the posterior segment of the eye, eliminating common barriers to local and systemic administration. Direct administration of medications in this manner allows for greater intraocular bioavailability in posterior segment tissues, resulting in more effective treatment of posterior eye 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 retinopathy, retinal vein occlusion, and eye infections such as endophthalmitis and retinitis. American Society of Retina Specialists Foundation, asrs.org/patients/retinal-diseases/33/IVT-injections (2017).

Despite encouraging results in preventing disease and improving vision, IVT injections are uncomfortable, expensive, and require a retinal specialist to perform. IVT injections are known to cause adverse reactions in some patients, such as infection, inflammation, vitreous hemorrhage, increased floaters in the eye, increased light sensitivity, 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, aseptic intraocular inflammation, rhegmatogenous retinal detachment, increased intraocular pressure, and ocular hemorrhage. (Source: Ibid.) Long-acting transocular delivery technologies can eliminate the need for repeated injections, thereby improving patient compliance and clinical outcomes. Methods and compositions that prolong the half-life of drugs in the vitreous fluid (e.g., maintaining drug storage capacity, low turnover rates in the eye, low target-mediated clearance, and/or seemingly stable properties in older populations) facilitate slow release of the drug from the injection site to the target site, enabling the use of higher doses and reducing the number of injections required.

The vitreous half-life of therapeutic molecules can be extended by binding 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 one specific instance, a long-acting anti-VEGF antibody was fused to the HA-binding domain (HABD) of human tumor necrosis factor (TNF)-stimulating gene 6 protein (TSG-6). Ghosh, J.G. et al., Nature Communications, 8:14837, doi:10.1038/ncomms14837 (2017). Compared with unmodified anti-VEGF antibodies, the fusion protein showed the following improvements: (1) a 3- to 4-fold increase in half-life; (2) the ability to attenuate VEGF-induced retinal changes in animal models of neovascular retinal disease for 3 to 4 times longer. Ghosh, J.G. et al., Nature Communications, 8:14837, doi:10.1038/ncomms14837 (2017). A candidate drug, LMG324, comprising a long-acting anti-VEGF antibody fused with TSG-6, has entered clinical trials to evaluate the safety and tolerability of a single escalation dose 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 binding of antibody fragments to hyaluronic acid (HA) can reduce drug diffusion from the vitreous humor. However, this method requires chemical activation of HA; the use of non-natural linkers may result in the generation of non-natural metabolites from activated HA in the subject.

The inventors have discovered that the aforementioned drawbacks 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 containing HA; wherein each second component (a) is covalently bound to the first component and (b) is non-covalently bound to the third component. Unlike the aforementioned anti-VEGF antibody and TSG-6 fusion protein (LMG324), the second component capable of binding to HA is pre-conjugated with HA.

This application discloses materials and methods for increasing intraocular retention of therapeutic molecules comprising a fusion protein capable of binding 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.

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

Summary of the Invention

This application discloses a material and method relating to therapeutic molecules and conjugates thereof capable of binding to therapeutic targets in the eye and to hyaluronic acid. This 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 the one or more second components are covalently bound to the first component; and (c) optionally, comprising one or more third components containing hyaluronic acid, wherein, if the one or more third components are present, the one or more third components are non-covalently bound to the one or more second components.

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

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

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

Item 5 is the 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 as described in any one of items 1 to 4, wherein the third component is hyaluronic acid, wherein the hyaluronic acid (a) has the following molecular weights: (i) selected from 3 kDa to 60 kDa, 4 kDa to 30 kDa, 5 kDa to 20 kDa, or 400 Da to 200 kDa; (ii) at least 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, or 9 kDa; or (iii) at most 60 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa, 20 kDa, or 15 kDa; (b) provides a molar excess binding equivalent to one or both of these second components; and (c) has modifications to reduce 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 in _KD at 10 nM to 10 μM, 5 nM to 8 μM or 100 nM to 5 μM.

Item 8 is a therapeutic molecule as described in any one of items 1 to 7, wherein (a) the first and second components are contained in a fusion protein, particularly wherein one or both of the second components are covalently bound to the N-terminus and/or C-terminus of the first component, more particularly wherein the first component is an antibody or antigen-binding 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 one or both of the second components are directly bound to the first component or indirectly bound to the first component via a linker, the linker being particularly a linker of at least 4 amino acids and/or at most 50 or at most 25 amino acids, more particularly the linker being (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 as 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 as described in any one of items 1 to 9, wherein (a) the first component is an anti-VEGF antibody or antigen-binding fragment, particularly an anti-VEGF Fab; and/or (b) each of these one or two second components contains 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 as described in any one of items 1 to 10, wherein (i) the first component is an anti-VEGF antibody or an antigen-binding fragment, one or both of these second components contain a CD44 domain, and the third component is hyaluronic acid with a molecular weight of 5 kDa to 20 kDa; (ii) the first component is an anti-VEGF antibody or an antigen-binding fragment, one or both of these second components contain a TSG-6 domain, and the third component is hyaluronic acid with a molecular weight of 5 kDa to 20 kDa; or (iii) the first component is an anti-VEGF antibody or an antigen-binding fragment, one or both of these second components contain a VG1 domain, and the third component is hyaluronic acid with a molecular weight of 5 kDa to 20 kDa.

Item 12 is a conjugate as described in any one of items 1 to 11, wherein (a) the first component comprises (i) the VH domain of SEQ ID NO: 97, 99, 105, 109 or 144; and (ii) the 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 as described in any one of items 1 to 11, wherein (a) the first component comprises (i) the VH domain of SEQ ID NO: 97, 99, 105, 109 or 144; and (ii) the 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 as described in any one of items 1 to 11, wherein (a) the first component comprises (i) the VH domain of SEQ ID NO: 97, 99, 105, 109 or 144; 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.

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

Item 16 is the therapeutic molecule described in Item 15, wherein these second components comprise at least two linker domains of a multifunctional proteoglycan that binds 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 the therapeutic molecule to be in the range of 1.5:1 to 1:1.

Item 18 is the therapeutic molecule of 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 the 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 the therapeutic molecule of any one of items 14 to 19, wherein the second component contains at least one, at least two, at least three, at least four, or at least five mutations.

Item 21 is the therapeutic molecule of any one of items 14 to 20, wherein the second component comprises one to three mutations, wherein the one to three mutations comprise a single amino acid substitution, a double amino acid substitution, and a truncation.

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

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

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

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

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

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

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

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

Item 30 is the therapeutic molecule of any one of items 14 to 29, wherein the second component comprises, relative to SEQ ID NO:29, two, three, four, five, or six mutations at the following positions: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326, and R327.

Item 31 is the therapeutic molecule described in any one of items 14 to 30, wherein the second component comprises, relative to SEQ ID NO:29, at least one 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 32 is the 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 the therapeutic molecule described in any one of items 14 to 32, wherein the second component comprises, relative to SEQ ID NO:29, 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, Y20 8A, 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 the therapeutic molecule of any one of items 14 to 33, wherein the second component comprises, relative to SEQ ID NO:29, at least 2, 3, 4, 5, or 6 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 35 is the therapeutic molecule described in any one of items 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:4 3. 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.

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

Item 38 is the therapeutic molecule described in Item 37, wherein the cysteine-binding 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 the therapeutic molecule 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 the therapeutic molecule described in any one of items 1 to 39, wherein the first component binds to VEGF.

Item 41 is the therapeutic molecule described in Item 40, wherein the first component binding to VEGF comprises ranibizumab, aflibercept, broxolizumab-dbll, and bevacizumab.

Item 42 is the therapeutic molecule described in item 37, wherein the aptamer is polyethylene glycol-treated.

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

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

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

Item 46 is the therapeutic molecule described in item 45, wherein the cysteine binding peptide is linked to one or both of these second components via a linker comprising the sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).

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

Item 48 is a composition for use as a medicine, the composition comprising a therapeutic molecule as described in any one of items 1 to 47 and optionally a pharmaceutical excipient, diluent or carrier.

Item 49 is a composition for treating eye or brain diseases, the composition comprising a conjugate as described in any one of items 1 to 47 and optionally a pharmaceutical excipient, diluent, or carrier.

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

Item 51 is the composition used in any one of items 48 to 50, wherein (a) the composition is applied at most once every three months, particularly at most once every four months, and even 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 the 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 of treating an eye disease in a subject, the method comprising applying to the subject any one of items 1 to 47 or any one of items 48 to 52 a therapeutic molecule.

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

Item 55 is the method of Item 54, which further includes binding the therapeutic molecule to hyaluronic acid prior to the application step.

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

Item 57 is the method described in Item 56, wherein the mixture includes a container.

Item 58 is the method described in 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 prepared for administration to a subject.

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

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

Item 62 is the 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 unmodified bioactive agents.

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 containing hyaluronic acid, wherein each second component is covalently bound to the first component but not covalently bound to the third component.

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

Aspect 65 is the conjugate described in 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 an Fc domain, especially wherein the fragment is or includes 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 hyaluronic acid receptor CD44 (CD44) domain, a brain-specific connectin (BRAL1) domain, a tumor necrosis factor-stimulated gene 6 (TSG-6) domain, a lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1) domain, or a hyaluronic acid binding protein (HABP) domain, an agglutinin G1 (AG1) domain, or a multifunctional proteoglycan G1 (VG1) domain.

Aspect 67 is a conjugate as described in any one of aspects 63 to 66, wherein the conjugate comprises one or two second components, particularly 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 modifications that reduce the degradation of hyaluronic acid in the eye.

Aspect 69 is a conjugate as described in any one of Aspects 63 to 67, wherein (a) the first and second components are contained in a fusion protein, particularly wherein one or both of the second components are covalently bound to the N-terminus and/or C-terminus of the first component, more particularly wherein the first component is an antibody or an 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, the linker being particularly a linker of at least 4 amino acids and/or at most 50 or at most 25 amino acids, more particularly the linker being (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 a conjugate as described in 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, particularly VEGF.

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

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

Aspect 73 is a composition for use as a medicine, comprising a conjugate as described in any one of aspects 63 to 72 and optionally a pharmaceutical excipient, diluent, or carrier.

Aspect 74 is a composition for treating eye diseases, the composition comprising a conjugate as described in any one of aspects 63 to 73 and optionally a pharmaceutical excipient, diluent or carrier.

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

Aspect 76 is the composition used in any one of aspects 73 to 75, wherein (a) the composition is applied at most once every three months, particularly at most once every four months, and 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 the composition used in any one of aspects 73 to 76, 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).

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

Example 79 is a therapeutic molecule targeting a patient's tissue, the therapeutic molecule comprising a hyaluronic acid binding domain and a therapeutic agent, wherein the hyaluronic acid binding domain comprises at least two linker domains of a multifunctional proteoglycan.

Example 80 is a therapeutic molecule targeting a patient's tissue. The therapeutic molecule comprises a hyaluronic acid binding domain and a therapeutic agent, wherein the hyaluronic acid binding domain comprises at least two linker domains of a multifunctional proteoglycan that are bound to hyaluronic acid via the HA binding domain.

Example 81 is the therapeutic molecule described in Example 79 or 80, wherein the hyaluronic acid ranges from 400 Da to 200 kDa.

Example 82 is the therapeutic molecule described in Example 81, wherein the hyaluronic acid has a content of at least 5 kDa.

Example 83 is the therapeutic molecule described in Example 81 or 82, wherein the hyaluronic acid is 10 kDa.

Example 84 is the therapeutic molecule described in any one of Examples 79 to 83, wherein the hyaluronic acid provides a molar excess binding equivalent to the linker domains of these multifunctional proteoglycans.

Example 85 is the therapeutic molecule described in any one of Examples 79 to 84, wherein the hyaluronic acid allows the ratio of hyaluronic acid to the therapeutic molecule to be in the range of 1.5:1 to 1:1.

Example 83 is the therapeutic molecule described in any one of Examples 79 to 85, wherein the hyaluronic acid binding domain has a _KD of 10 nM to 10 μM.

Example 87 is the therapeutic molecule described in any one of Examples 79 to 86, wherein the hyaluronic acid binding domain has a _KD of 5 nM to 8 μM.

Example 88 is the therapeutic molecule described in any one of Examples 79 to 87, wherein the hyaluronic acid binding domain has a _KD of 100 nM to 5 μM.

Example 89 is the therapeutic molecule described in any one of Examples 79 to 88, wherein the hyaluronic acid 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.

Example 90 is the therapeutic molecule described in any one of Examples 79 to 89, wherein the hyaluronic acid binding domain is at least 95% identical to that of Examples 86, 60, 32 or 29.

Example 91 is the therapeutic molecule described in any one of Examples 79 to 90, wherein the hyaluronic acid binding domain contains at least one, at least two, at least three, at least four, or at least five mutations.

Example 92 is the therapeutic molecule described in any one of Examples 79 to 91, wherein the hyaluronic acid binding domain contains one to three mutations, wherein these one to three mutations include single amino acid substitution, double amino acid substitution, and truncation.

Example 93 is the therapeutic molecule described in any one of Examples 79 to 92, wherein the hyaluronic acid binding domain contains one to five mutations, wherein these one to five mutations include single amino acid substitution, double amino acid substitution, and truncation.

Example 94 is the therapeutic molecule described in any one of Examples 79 to 93, wherein the hyaluronic acid binding domain has a truncated mutation relative to SEQ ID NO:29.

Example 95 is the therapeutic molecule described in Example 94, wherein the truncated mutation comprises a truncation of 1 to 129 amino acids at the N-terminus.

Example 96 is the therapeutic molecule described in any one of Examples 79 to 95, wherein the hyaluronic acid binding domain is a truncated sequence and the Ig domain of the wild-type multifunctional proteoglycan is absent.

Example 97 is the therapeutic molecule described in any one of Examples 79 to 96, wherein the hyaluronic acid 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.

Example 98 is the therapeutic molecule described in any one of Examples 79 to 97, wherein the hyaluronic acid 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.

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

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

Example 101 is the therapeutic molecule described in any one of Examples 79 to 100, wherein the hyaluronic acid binding domain contains two, three, four, five, or six mutations at 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.

Example 102 is the therapeutic molecule described in any one of Examples 79 to 101, wherein the hyaluronic acid 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.

Example 103 is the therapeutic molecule described in any one of Examples 79 to 102, wherein the hyaluronic acid binding domain comprises at least one of Y208A and H306A.

Example 104 is the therapeutic molecule described in any one of Examples 79 to 103, wherein the hyaluronic acid binding domain 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, D19 7S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A, and LYR325LFK.

Example 105 is the therapeutic molecule described in any one of Examples 79 to 104, wherein the hyaluronic acid 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.

Example 106 is the therapeutic molecule described in any one of Examples 79 to 105, wherein the hyaluronic acid 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:39. D 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 N O: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.

Example 107 is the therapeutic molecule described in any one of Examples 79 to 106, wherein the therapeutic active agent comprises an oligopeptide, a protein, or a nucleic acid.

Example 108 is a therapeutic molecule as described in any one of Examples 79 to 107, wherein the therapeutic active agent comprises an antibody, an antigen-binding fragment, a cysteine binding peptide, a growth factor, or an aptamer.

Example 109 is the therapeutic molecule described in Example 108, wherein the therapeutic active agent is capable of binding to an antigen.

Example 110 is the therapeutic molecule described in Example 109, wherein the therapeutic active 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.

Example 111 is a therapeutic molecule as described in any one of Examples 109 or 110, wherein the therapeutic agent is an antibody or its antigen-binding fragment (including but not limited to Fab fragment, F(ab')2 fragment, Fab' fragment, VhH fragment, scFv fragment, scFv-Fc fragment or microantibody).

Example 112 is the therapeutic molecule described in any one of Examples 109 or 110, wherein the therapeutic active agent is an oligopeptide or a protein.

Example 113 is the therapeutic molecule described in Example 102, wherein the oligopeptide or protein is a cysteine-bound peptide or enzyme.

Example 114 is the therapeutic molecule described in Example 103, wherein the cysteine-binding peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:92.

Example 115 is the therapeutic molecule described in any one of Examples 79 to 108, wherein the therapeutic 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).

Example 116 is the therapeutic molecule described in Example 110, wherein the therapeutic active agent binding to VEGF includes ranibizumab, aflibercept, broxolizumab-dbll, and bevacizumab.

Example 117 is the therapeutic molecule described in any one of Examples 79 to 110, wherein the therapeutic active agent is a nucleic acid.

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

Example 119 is the therapeutic molecule described in Example 118, wherein the aptamer binds to VEGF.

Example 120 is the therapeutic molecule of any one of Examples 108, 118 or 119, wherein the aptamer is polyethylene glycol-treated.

Example 121 is the therapeutic molecule of any one of Examples 108 or 118 to 120, wherein the aptamer is

Example 122 is the therapeutic molecule described in any one of Examples 79 to 121, wherein the therapeutic agent is covalently linked to the hyaluronic acid binding domain via a connector.

Example 123 is the therapeutic molecule described in Example 122, wherein the linker is at least 4 amino acids.

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

Example 125 is the therapeutic molecule described in any one of Examples 122 to 124, wherein the linker consists of 4 to 25 amino acids.

Example 126 is the therapeutic molecule described in any one of Examples 122 to 125, wherein the linker comprises (GxS)n or (GxS)nGm, where 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).

Example 127 is the therapeutic molecule of any one of Examples 122 to 126, wherein the connector comprises GGGS (SEQ ID NO: 84) or a polymer thereof, and more particularly comprises (GGGGS)3 (SEQ ID NO: 85).

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

Example 129 is the therapeutic molecule of any one of Examples 122 to 125 or 128, wherein the connector comprises GSGSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).

Example 130 is the therapeutic molecule described in any one of Examples 79 to 107, wherein the therapeutic active agent comprises an anti-VEGF antigen-binding moiety and a cysteine knot peptide.

Example 131 is the therapeutic molecule described in Example 130, wherein the cysteine binding peptide is linked to the hyaluronic acid binding domain via a linker, the linker comprising the sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).

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

Example 133 is a therapeutic molecule as described in any one of Examples 79 to 132, wherein the hyaluronic acid binding domain can be non-covalently bound to hyaluronic acid.

Example 134 is a method for delivering a therapeutic molecule to a patient’s tissue, comprising administering the therapeutic molecule as described in any one of Examples 79 to 133 to the patient, and allowing the therapeutic molecule to deliver the therapeutic active agent to the target tissue in a long-term manner.

Example 135 is the method described in Example 134, which further includes binding the therapeutic molecule to hyaluronic acid prior to the application step.

Example 136 is the method described in Example 135, which further includes mixing a first solution containing the therapeutic molecule with a second solution containing the hyaluronic acid.

Example 137 is the method described in Example 136, wherein the mixing includes a container.

Example 138 is the method described in Example 137, wherein the container is a two-chamber syringe.

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

Example 140 is the method of any one of Examples 134 to 139, wherein the administration step is a single injection.

Example 141 is the method of any one of Examples 134 to 140, wherein the target tissue comprises an eye or a brain.

Example 142 is the method of any one of Examples 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 therapeutic agent.

Additional objects and advantages will be apparent in part from the following description, and in part from it, or may be learned by practice. These objects and advantages will be realized and obtained by means of the elements and combinations specifically pointed out in the appended claims.

It should be understood that the foregoing general description and the following detailed description are merely illustrative and explanatory, and do not limit the scope of the claims.

The accompanying drawings, which are incorporated in and form a part of this specification, illustrate one or more embodiments and, together with this description, serve to explain certain principles described herein.

Attached Figure Description

Figure 1 shows the size exclusion chromatography (SEC; TSKgel UP-SW3000, 2 μm, 4.6 × 150 mm; running buffer 0.2 MkpH, 0.25 M KCl, pH 6.2) results of the Fab-HABD fusion protein VPDF-2xCD44 with or without 10 kDa hyaluronic acid (HA) pre- or unpre-conjugated. Fab-HABD was prepared as described in Example 1 and tested as described in Example 2.

Figures 2A and 2B show the intravitreal pharmacokinetics (PK) of rabbit anti-c-MetFab (RabFab) and rabbit anti-c-Met Fab-VG1 Fab-HABD (RabFab-Fab-HABD) after dose-normalized intravitreal (IVT) injection in New Zealand white rabbits. Figure 2A shows the change in the amount of RabFab or RabFab-Fab-HABD present in the vitreous over time after IVT. Data for the RabFab fusions are shown, namely ^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 shows the 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 fluorescence spectrophotometry.

Figure 3 shows histopathological images of the eyes of OS rabbits, illustrating retinal degeneration 4 days after administration of TSG6 (SEQ ID NO:32) via IVT.

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

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

Figures 6A to 6F show porcine vitreous bodies 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; + Slight precipitation; - Clear vitreous body.

Figures 7A to 7C show the vitreous heterogeneity throughout the pig eye after injection of the specified VPDF-2xCD44 sample. Figure 7A: Buffer control. Figure 7B: Unreinforced VPDF-2xCD44. Figure 7C: VPDF-2xCD44 reinforced with HA.

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

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

Figures 10A and 10B show the precipitation of RabFab-TSG6 in porcine vitreous humor, while RabFab-VG1 did not precipitate. TSG6 or VG1 were each recombined to RabFab and chemically bonded to Alexa488 via N-hydroxysuccinimide (NHS) primary amine labeling. Figure 10A shows RabFab-TSG6. Figure 10B shows RabFab-VG1.

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

Figure 12 shows the fluorescence correlation spectroscopy (FCS) measurements of the interaction between VG1 and ex vivo vitreous fluid. Measurements showing slow diffusion indicate protein interaction with vitreous fluid, while rapid diffusion indicates no interaction. The dilution factors of the vitreous fluid are shown at the top of the heatmap – the leftmost column shows undiluted controls/samples; the rightmost column shows phosphate-buffered saline (PBS), pH 7.4; the middle columns show dilution factors increasing from left to right. Measurements for the unbound controls are shown in the top two rows. Measurements for the following samples are shown in rows 3–8: free VG1, PigFab-VG1, PigFab-VG1 + 10 kDa HA (1:1), free VG1, RabFab-VG1, and RabFab-VG1 + 10 kDa HA (1:1). The unbound controls show the fastest diffusion (Figure 12, rows 1 and 2). Although all samples showed significant delayed diffusion relative to the control, free VG1, PigFab-VG1, and RabFab-VG1 all exhibited delayed diffusion until vitreous dilutions exceeded 6000-fold (Fig. 12, rows 3, 4, 6, and 7; from undiluted to dilution factor 6,561). Slow diffusion was observed in samples co-prepared with 10 kDa HA, but this effect disappeared when the dilution factor was greater than 729-fold (Fig. 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 thermal stress (i.e. protein stability) analysis of HtrA1-VG1 at 37°C. T0 = unincubated control. T4wk = after 4 weeks of incubation.

Figure 14 shows the average concentration of pigFab-VG1 in the swine flu water. Concentrations were measured by mass spectrometry after intravenous infusion of 1.8 mg pigFab-VG1 alone or pre-complexed with an equivalent mass concentration of 10 kDa HA via IVT. Averages for several animals are shown, and error bars represent standard deviations.

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

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

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

Figure 18 shows the crystal structure of the 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, and the Link2 domain is shown at the bottom left. The binding of HA is represented by the smaller HA molecule to the lower right of the VG1 molecule.

Figure 19 shows the alignment of VG1 variants SEQ ID NO:29 and SEQ ID NO:33-59. The first 20 amino acids before the N-terminus are the signal sequence of a multifunctional proteoglycan (shown with *). Amino acids within boxes are conserved residues. All of these proteins have a C-terminal His tag for purification.

Sequence Description

Table 1 provides a list of some of the sequences cited in this paper. The amino acid sequences provided are from the N-terminus to the C-terminus.

Detailed Implementation

I. Definition

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

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

As used herein, the term "antibody" refers to a whole (complete or intact) antibody. Antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (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 (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 complementarity-determining regions (CDRs), interspersed with more conserved regions called backbone regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., 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 fragments of the complete antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment of an antibody" include, but are not limited to, examples of the following antibody fragments, including but not limited to: Fab, Fab', Fab'-SH, F(ab') ₂ ; bispecific antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of 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). Furthermore, although the two domains VL and VH of an Fv fragment are encoded by separate genes, they can be linked via artificial peptide linkers using recombinant methods, enabling them to become a single protein chain where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). These single-chain antibodies can 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 usability of the fragments is screened in the same manner as for intact antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, large antibodies, micro antibodies, intracellular antibodies, bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, v-NARs, and bis-scFvs. Antigen-binding fragments can be incorporated into a single-chain molecule containing a pair of tandem Fv fragments (VH-CH1-VH-CH1), forming a pair of antigen-binding regions together with a complementary light chain polypeptide. The term "antibody" includes both polyclonal and monoclonal antibodies.

Aptamers are oligonucleotide or peptide molecules that bind to specific target molecules. Aptamers are typically created by selecting them from a large pool of random sequences, but natural aptamers also exist in riboswitch molecules. Aptamers can be used as macromolecular drugs for basic research and clinical purposes. Aptamers can bind to ribozymes and self-cleave in the presence of the target molecule. These compound molecules have additional research, industrial, and clinical uses. More specifically, aptamers can be classified as DNA, RNA, or XNA aptamers, which consist of (typically short) oligonucleotide chains, and peptide aptamers, composed of one (or more) short, variable peptide domains, are attached to protein scaffolds at both ends. Both DNA and RNA aptamers exhibit stable binding affinity to a wide variety of targets. DNA and RNA aptamers have been selected for the same targets. These targets include lysozyme, thrombin, interferon-γ, vascular endothelial growth factor (VEGF), and dopamine. In the case of, for example, VEGF, the DNA aptamer is an analogue of the RNA aptamer, in which thymine is replaced by uracil.

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

As used herein, the term "DARPin" (an abbreviation for "designed ankyrin repeat protein") refers to antibody-mimicking proteins that typically exhibit high specificity and high affinity for target proteins. They are usually genetically engineered, derived from natural ankyrin, and consist of at least three, typically four, or five repeat motifs of these proteins. For four- or five-repeated DARPins, their molecular weights are approximately 14 kDa and 18 kDa, respectively. Examples of DARPins can be found, for example, in U.S. Patent 7,417,130.

The "therapeutic effective amount" of a pharmaceutical preparation, such as a pharmaceutical composition, refers to the amount that effectively achieves the desired therapeutic or preventative effect within the required dosage and time period.

As used herein, the term “eye disease” includes any eye condition 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 fusion proteins containing both a Fab and a hyaluronan-binding domain. These terms are synonymous and may be used interchangeably throughout this disclosure.

As used in this article, “hyaluronan”, “hyaluronic acid”, “hyaluronate”, and “HA” refer to non-sulfated glucosamine polysaccharides and their salts with the chemical formula ( _{C14H21NO11 ) n .}

As used herein, “hyaluronic acid binding domain,” “hyaluronic acid binding moiety,” “HA binding domain,” or “HABD” refers to any part of the hyaluronic acid that can bind to hyaluronic acid. In some cases, HABD can 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, the ligand is typically a molecule that generates a signal by binding to a site on a target protein. This binding usually results in a conformational change in the target protein. In DNA-ligand binding studies, the ligand can be a small molecule, ion, or protein that binds to the DNA double helix. The relationship between the ligand and its binding partner is a function of charge, hydrophobicity, and molecular structure. Binding occurs over an infinitesimally small temporal and spatial range, therefore the rate constant is usually a very small number. Ligands can be naturally occurring or non-natural. Furthermore, it can be an agonist, partial agonist, antagonist, or inverse agonist.

The difference between a "non-covalent interaction" and a covalent bond is that it does not involve the sharing of electrons, but rather a more dispersed variation of electromagnetic interactions between or within molecules. Non-covalent interactions can be categorized into different types, such as electrostatics, π-effects, van der Waals forces, and hydrophobic effects. Preferably, the conjugate is provided in a separate form. The first and second components can be covalently bonded to each other via a linker or directly.

Nucleic acids are biological polymers composed of nucleotides, which are monomers formed from three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. The term "nucleic acid" is a general term for both DNA and RNA. If the sugar is a 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, preferably of synthetic origin.

Proteins are large biomolecules (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 for cells and organisms, and transporting molecules from one location to another. Proteins are primarily distinguished by their amino acid sequences, which are determined by the nucleotide sequence of their genes and typically result in protein folding into a specific three-dimensional structure that determines its activity. Short polypeptides containing fewer than 20-30 residues are generally called peptides.

As used herein, “protein conjugate” or “conjugate” refers to a protein that is non-covalently bound to hyaluronic acid.

Receptors are chemical structures, typically composed of proteins, that receive and transduce signals that may integrate into biological systems. These signals are usually chemical messengers (ligands) that bind to the receptor, causing some form of cellular/tissue response, such as changes in cell activity. Receptor action can be broadly categorized into three modes: signal relay, amplification, or integration. Relay forwards the signal, 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 this invention, receptors or fragments containing ligand-binding sites and their ligands are suitable binding counterparts (first component and therapeutic target).

As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of a person being treated, and may be for prevention or in the course of clinicopathology. Ideal therapeutic effects include preventing the occurrence or recurrence of the disease or its symptoms, alleviating the symptoms of the disease, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or alleviating the disease state, and achieving remission or improved prognosis. In some aspects, the antibodies of the present invention are used to delay the development of a disease or slow its progression.

Numerical ranges include the numbers defining the range. Taking into account significant figures and errors associated with measurement, measured and measurable values should be understood as approximate values. Furthermore, the use of "comprise," "contain," and "include" is not intended to be restrictive. It should be understood that the foregoing general and detailed descriptions are illustrative and explanatory only and do not limit the teaching.

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

Unless otherwise specified in the specification, embodiments in the specification that refer to "comprising various components" are also considered to "compose of the referenced components" or "consistently composed of the referenced components"; embodiments in the specification that refer to "comprising various components" are also considered to "comprising the referenced components" or "consistently composed of the referenced components"; and embodiments in the specification that refer to "consistently composed of various components" are also considered to "comprising the referenced components" or "comprising the referenced 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, i.e., equivalent to "and/or".

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings, examples, and embodiments. It should be understood that the drawings, examples, and embodiments (unless specifically stated otherwise) are not intended to limit the scope of the invention to the particular methods, schemes, and reagents described herein, as they can vary. The invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined by the appended claims and the included embodiments. Furthermore, the techniques used herein are for the purpose of disclosing particular embodiments only and are not intended to limit the scope of this disclosure. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used herein and in the appended claims include plural indicators. Similarly, the words “comprising,” “containing,” and “covering” should be interpreted as inclusive rather than exclusive.

The section headings used herein are for organizational purposes only and should not be construed as limiting the desired objectives in any way. All publications (scientific and patent publications) cited herein are incorporated by reference. If any material incorporated by reference contradicts any terminology defined in this specification or any other express content herein, this specification shall prevail. While this teaching is described in conjunction with various embodiments, it is not intended to limit this teaching to those embodiments. Rather, as will be understood by those skilled in the art, this teaching encompasses various alternatives, modifications, and equivalents.

II. Therapeutic molecules comprising a therapeutic active agent (i.e., the first component) and a hyaluronic acid-binding domain (HABD; i.e., the second component).

This application provides therapeutic molecules that target patient tissues, these therapeutic molecules comprising a therapeutic active agent and an HA-binding domain (HABD). Each therapeutic molecule comprises 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 comprise an HA-binding domain (HABD).

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

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

In some embodiments, the therapeutic molecule further comprises one or more third components (in addition to the first and second 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 linked to the second component) may be pre-complexed with HA (i.e., the third component). In some of these embodiments, the first, second, and third components form a conjugate.

This article provides non-limiting examples of the first, second, and third components.

A. First Component – Therapeutic Active Agent

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

Preferably, the specific binding component or binding domain has an affinity of at least ^10⁶ l/mol for its corresponding target molecule. More preferably, the specific binding domain has an affinity of ^10⁷ /mol, or even more preferably ^10⁸ /mol, or even most preferably ^10⁹ /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 stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its collateral Y is typically expressed by the dissociation constant (K_{_D} ). Affinity can be measured using conventional methods known in the art. As those skilled in the art will understand, the term "specific" is used to indicate a specific binder to which other biomolecules do not significantly bind. Preferably, the binding level with biomolecules other than the target molecule results in binding affinity of only 10% or less of that with the target molecule, more preferably only 5% or less. Preferred specific binders will meet the aforementioned minimum standards 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-bound peptide, an enzyme, or DARpin. In some embodiments, the size of the protein may range from small to large. In some embodiments, the protein is a peptide containing 2 to 20 amino acids. In some embodiments, the protein is a polypeptide containing 21 to 50 amino acids. In some embodiments, the protein is a polypeptide containing 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 contain any number of amino acids. In some embodiments, the first component is not 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. This nucleic acid may be complementary to a target-associated nucleic acid (e.g., the nucleic acid is complementary to the target mRNA 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. Numerous therapeutic targets may be present in the eye. As therapies effectively targeting these molecules and pathways are developed, there will be a need to provide improved visual outcomes while reducing the treatment burden and risks associated with frequent IVT injections.

a) Pro-angiogenesis, inflammation, and growth factor mediators

In some embodiments, the first component binds to a therapeutic target that is a mediator of angiogenesis, inflammation, and/or growth factors. Mediators of angiogenesis, inflammation, and growth factors 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), angiopoietin, 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 proteins that are generally associated with an increased risk of age-related macular degeneration (AMD). In some embodiments, the first component binds to complement pathway components 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 those associated with diabetic retinopathy or macular edema. (See Section III below.)

The term "VEGF" refers to vascular endothelial growth factor (VEGF) of the 165-amino acid group, related 121-, 189-, and 206-amino acid groups, and the naturally occurring pairs and processed forms of those growth factors. VEGF can refer to VEGF proteins from any species.

VEGF is essential for both normal development and pathological angiogenesis. Hypoxia-induced VEGF secretion by stellate cells is a key factor 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 a major strategy in designing treatments for pathological ocular angiogenesis, including age-related macular degeneration, proliferative retinopathy, and retinopathy of prematurity.

The term "VEGF-mediated disorder" refers to any condition whose symptoms or disease state require the involvement of VEGF for occurrence, progression, or persistence. Exemplary VEGF-mediated disorders include, but are not limited to: age-related macular degeneration, neovascular glaucoma, diabetic retinopathy, macular edema, pathological myopia, retinal vein occlusion, retinopathy of prematurity, abnormal angiogenesis 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-VEGFFab. This disclosure provides VEGF antibodies and antigen-binding fragments. Other usable anti-VEGF antibodies, VEGF antagonists, and VEGF receptor antagonists include, for example, ranibizumab, bevacizumab, aflibercept, phenagatanib, 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 medicament targeting VEGF, such as those disclosed in Section II.A.2.a) below.

d) Erythropoietin (EPO)

In some embodiments, the first component binds to erythropoietin (EPO). In some embodiments, the first component binds to the erythropoietin receptor (EPOR). In many species, EPO refers to the erythropoietin protein. Protein sequences of human, macaque, mouse, rat, and rabbit EPO are publicly available. Human EPO can also undergo hyperglycosylation. The terms "EPO receptor" or "EPOR" are used interchangeably to refer to the erythropoietin receptor protein in different species.

e) Angiopoietin

In some embodiments, the first component binds to angiopoietin, such as angiopoietin 2 (ANG2). ANG2 is known as a candidate treatment 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 the severity of wet AMD. Elevated intraocular ANG2 levels have also been detected in patients with diabetic retinopathy and retinal vein occlusion, suggesting potential medical significance in targeting intraocular ANG2. ANG2 refers to a protein found in various species. 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 interleukins, 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 (a potentially blinding inflammatory disease). Interleukins can be derived from any species.

g) Platelet-derived growth factor (PDGF)

In some embodiments, the first component binds to a therapeutic target, which 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 both 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 both VEGF and PDGF.

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

i)HtrA protein

In some embodiments, the first component binds to a member of the HtrA family of serine proteases. HtrA proteins have a catalytic domain containing at least one C-terminal PDZ domain and ATP-independent protease chaperones associated with protein metabolism and cell fate. Clausen et al., Molecular Cell 10(3):443-445 (2002). Four HtrA proteins exist 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 containing trypsin-like folds, and a C-terminal PDZ domain. Human genetic studies have found a strong correlation between the progression of age-related macular degeneration (AMD) and single nucleotide polymorphisms (SNPs) in the HtrA1 promoter region, leading to 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 is bound to HtrA1. In some embodiments, the first component is bound to HtrA2. In some embodiments, the first component is bound to HtrA3. In some embodiments, the first component is bound 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 epithelial-derived factor, endostatin, ciliary neurotrophic factor, complement factor 1 inhibitor, complement factor-like 1, complement factor I, etc.

The term "factor D" refers to the 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). Rhesus factor P can be purified from rhesus monkey serum (protocol adapted from Nakano et al., 1986, J Immunol Methods 90:77-83). Factor P is also known in this field 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 eye diseases may be used as the first component (as described in Section III below). In some embodiments, the first component comprises a recognized therapeutic agent 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 agent for treating eye diseases.

a) Drugs targeting VEGF

In some embodiments, the first component comprises a VEGF antagonist, including, 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-angiogenic 2 bispecific antibody such as RG-7716; Roche)); (2) a soluble VEGF receptor fusion protein (e.g., aflibercept)); (3) an anti-VEGF (e.g., abicipar pegol; Molecular Partners AG/Allergan); or (4) an anti-VEGF aptamer (e.g., pilgatani sodium).

In some embodiments, the first component comprises ranibizumab, which is specifically used to treat 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 specifically 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 embodiments, the first component comprises aflibercept, which is specifically 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 specifically 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 embodiments, the first component comprises (pegaptanibsodium) which is specifically 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.

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-angiogenic 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 (pigatanib sodium; NeXstar Pharmaceuticals/OSI Pharmaceuticals)), aptamers capable of blocking VEGFR, neutralizing anti-VEGF... Aptamers of VEGFR antibodies, small molecule inhibitors of VEGFR tyrosine kinases, anti-VEGF (e.g., abicipar pegol; Molecular Partners AG/Allergan), small interfering RNAs that inhibit VEGF or VEGFR expression, VEGFR tyrosine kinase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin)-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindole-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and sunitinib (sunitinib)) and combinations thereof.

c) Anti-angiogenic agents

In some embodiments, the first component comprises an agent having anti-angiogenic activity for the treatment of eye diseases, such as an anti-inflammatory drug, a mammalian-targeted rapamycin (mTOR) inhibitor (e.g., rapamycin, everolimus, and temsirolimus), cyclosporine, a tumor necrosis factor (TNF) antagonist (e.g., an anti-TNFα antibody or its antigen-binding fragment (e.g., infliximab, adalimumab, certolizumab pegol, and golimumab) or a soluble receptor fusion protein (e.g., etanercept), an anticomplement drug, a nonsteroidal anti-inflammatory drug (NSAID), or a combination thereof.

d) Neuroprotective agents

In some embodiments, the first component comprises an agent that has neuroprotective effects and may potentially reduce disease progression. For example, the agent may reduce the progression from dry AMD to wet AMD. Examples of neuroprotective agents include a class of drugs known as “neurosteroids,” which includes drugs such as dehydroepiandrosterone (DHEA) (PRASTERA ^™ and), dehydroepiandrosterone sulfate, and thioprenone.

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) a soluble PDGFR receptor fusion protein, (4) a dual PDGF/VEGF antagonist/inhibitor (e.g., DE-120 (Santen) or X-82 (TyrogeneX)), (5) a bispecific anti-PDGF/anti-VEGF antibody, (6) an anti-PDGFR antibody, or (7) a small molecule inhibitor (e.g., squalene).

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 medications used to treat eye diseases

In some embodiments, the first component comprises a recognized therapeutic agent for treating eye diseases. Treatment of eye diseases is as described in Section III below. Examples of recognized therapeutic agents 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 vitrectomy), DNA or RNA (e.g., for gene therapy technologies), agents mediating neuroprotective effects (such as providing neurotrophic factors, blocking excessive glutamate stimulation, stabilizing Ca2 ⁺ homeostasis, preventing apoptosis, modulating immune status via vaccination, inducing endogenous neuroprotective mechanisms, antioxidants, and vitamin and mineral supplements).

In some embodiments, the first component comprises any suitable DME and/or DR therapeutic agent, specifically for the treatment of eye diseases, including but not limited to VEGF antagonists (e.g., or), corticosteroids (e.g., corticosteroid implants, dexamethasone IVT implants; or fluocinolone acetonide IVT implants) or corticosteroids formulated for administration via IVT injection (e.g., antacids), or combinations thereof. In some cases, the eye disease is DME and/or DR.

Further examples of recognized medicines suitable for use as a first-component condition for the treatment of eye diseases include, but are not limited to: (verteporfin; a photoactivated medicine that is often used in combination with photodynamic therapy using nonthermal lasers), PKC412, Endovion (NS 3728; NeuroSearch A/S), neurotrophic factors (e.g., glial neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, 9-cis-retinal, ophthalmic medicines (e.g., phospholine iodide, ethothiocholine, or carbonic anhydrase inhibitors), veovastat (AE-941; AEterna Laboratories, Inc.), Sirna-027 (AGF-745; Sima Therapeutics, Inc.) nc.), neurotrophic factors (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 by, for example, EntreMed, Inc.), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan/Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan), LYN-002 (Lynkeus Biotech), microalgal 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. Patent No. 7,771,742, and VEGFR inhibitor SUGEN (SU5416), or Pfizer's Inlyta (dacomitinib), (lalorlatinib)), NX-278-L (NeXstar Pharmaceuticals/Gilead Sciences), Opt-24 (OPTIS France SA), retinal ganglion neuroprotective agents (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; IconicTherapeutics), 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 ROBO4 antagonist, and an anti-ROBO4 antibody (e.g., DS-7080a; Daiichi Sankyo).

In some embodiments, the first component comprises tryptophan-tRNA synthetase (TrpRS), squalene, (anecocelesta acetate for long-acting suspension; Alcon, Inc.), Combretastatin A4 precursor (CA4P), (mifepristone-ru486)), subtenon triamcinolone acetonide, IVT crystalline triamcinolone, matrix metalloproteinase inhibitors (e.g., Prinomastat; AG3340; Pfizer), fluocinolone acetonide (including fluocinolone intraocular implants; 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. Antibody-antigen binding fragments

In some embodiments, the first component comprises or is derived from an antibody or an antigen-binding fragment thereof capable of binding to an antigen. The degree to which the antibody or antigen-binding fragment binds to an unrelated, non-target protein is less than about 10% of the antibody's binding to the target, as measured, for example, by surface plasmon resonance (SPR). In some aspects, the dissociation constant (K_{_D}) of the antibody or antigen-binding fragment bound 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 lower, e.g., 10 ^{^-8} M to 10^{^-13} M, e.g., 10 ^{^-9} to 10 ^{^-13} M). When the K_{_D} of the antibody is 1 μM or less, the antibody or its antigen-binding fragment is said to be "specifically bound" to the target.

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

In some embodiments, the antibody or its antigen-binding fragment binds to an antigen present in the eye. In some embodiments, the antibody or its antigen-binding fragment 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, broxolizumab-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 any bispecific anti-VEGF/anti-Ang2 bispecific antibody or a variant thereof disclosed in RG-7716 or WO 2010/069532 or WO 2016/073157. 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, for example, WO 2016/073890), siltuximab, olokizumab, clazakizumab, sirukumab, elsilimomab, OPR-003, MEDI5117, PF-04236921, or a variant thereof.

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

In some embodiments, the first component is RabFab, which is an antigen-binding Fab fragment derived from a parental monoclonal antibody (G10) produced in rabbits, targeting 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 its antigen-binding fragment.

4. Growth factors

In some embodiments, the first component comprises growth factors. In some embodiments, the growth factors comprise 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 binding 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 with SEQ ID NO:92 (cysteine knot peptide sequence).

The cysteine knot peptide may be covalently linked to another molecule to form a first component, including any of the exemplary first components described in Sections II.A.2 through 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, HABD (i.e., the second component) is covalently linked to the cysteine linker 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 with SEQ ID NO: 95. In some embodiments, the covalent linker comprises the sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95). In some embodiments, the cysteine linker is covalently linked to VG1 with a C-terminal His tag (SEQ ID NO: 29). In some embodiments, the cysteine linker is covalently linked to VG1 having an Ig domain deletion and a C-terminal His tag (SEQ ID NO: 32). In some embodiments, the cysteine linker is covalently linked to VG1 with an N-terminal His tag. In some embodiments, the cysteine linker is covalently linked to VG1 having 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 an HA-binding protein (which contains an HA-binding domain; HABD). In some embodiments, the second component comprises HABD. Examples of proteins containing HABD include CD44, tumor necrosis factor-stimulated gene-6 (TSG6), multifunctional proteoglycans, brain-specific connecton 1 (BRAL1), lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1), and aggregate proteoglycans.

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

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

The term "hyaluronan-binding protein" or "HA-binding protein" refers to proteins or families of proteins that bind to hyaluronic acid (HA). Typically, these HA-binding proteins include 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, agglutinin, multifunctional proteoglycan, Brevican, Neurocan, hyaluronic acid binding protein 1 (HABP1; also known as C1qBP/C1qR and p32), HAPLN1 (also known as connexin and CRTL1), hyaluronic acid 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, approximately 100 amino acids in length, that participates in HA binding; this domain is referred to as 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 this invention. Any HA-binding protein, such as the HABD of the exemplary proteins described above, can be included in the second component to impart the ability to bind to HA. Preferably, the second component comprises a CD44 (CD44) domain, a brain-specific linker protein (BRAL1) domain, a tumor necrosis factor-stimulated gene 6 (TSG-6) domain, a lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1) domain, a hyaluronic acid-binding protein (HABP) domain, an agglutinin G1 (AG1) domain, or a multifunctional proteoglycan G1 (VG1) domain. Exemplary and suitable HA-binding molecules (including peptide tags) for use in the eye are described in WO 2014/99997 and WO2015/19824, the contents of which are incorporated herein by reference in their entirety. Any of the sequences described herein may be used in this 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 prolonging its intraocular half-life. The first component may benefit from a longer ocular retention time and/or a longer duration of action on eye diseases.

Furthermore, the second component may non-covalently bind to a third component containing HA to form a conjugate. In some embodiments, each second component in the conjugate may bind to a separate molecule of HA. In some embodiments, two or more second components may bind to the same HA molecule.

In many embodiments, the binding affinity of HABD to HA can be within several ranges; the binding affinity can be modulated according to the mechanism of action of the therapeutic agent. For example, if the site of action is in the vitreous fluid, a high binding affinity may help the biologic remain in the vitreous fluid. Conversely, if the site of action is in the retina, a lower binding affinity may help the biologic cross 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). Not bound by theory, in some embodiments, the binding affinity (K_{_D}) 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 between 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 small volumes in the solution. Fluorescence intensity fluctuates due to molecular motion, and quantitative analysis of these fluctuations yields the diffusion time of the molecules. Diffusion in the biological matrix can be determined by using a fluorescent dye with appropriate spectral characteristics. In some embodiments, FCS observations are correlated with SPR measurements.

In some embodiments, HABD comprises a wild-type sequence relative to its source protein. In some embodiments, HABD may contain one or more mutations in its protein sequence relative to its source protein. In many embodiments, these mutations comprise single-amino acid substitutions, double-amino acid substitutions, additions, deletions, and truncations.

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

In some embodiments, HABD contains amino acids that facilitate 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 required for long-acting therapy.

In some embodiments, HABD contains amino acids that contribute to the thermal stability of HABD and/or therapeutic molecules. In some embodiments, these amino acids may be retained to maintain thermal stability. In some embodiments, these amino acids may be substituted to alter thermal stability.

In some embodiments, the HABD comprises at least one, at least two, at least three, at least four, or at least five mutations relative to a reference sequence disclosed herein. In some embodiments, the HABD comprises one to three mutations, wherein these one to three mutations independently comprise a single amino acid substitution, a double amino acid substitution, an addition, a deletion, or a truncation. In some embodiments, the HABD comprises one to five mutations, wherein these one to five mutations independently comprise a single amino acid substitution, a double amino acid substitution, an addition, a deletion, or a truncation.

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

1.CD44

In some embodiments, the second component is derived from CD44 (SEQ ID NO:1). The CD44 receptor includes a junctional domain, a GAG attachment domain, a transmembrane domain, and a cytoplasmic domain. Several isotypes with different modular compositions treated by alternative splicing are described. In some embodiments, the second component is derived from or includes a CD44 HA receptor domain. In some embodiments, the second component is derived from or includes 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-binding linker domain. In some embodiments, the second component is derived from or comprises the TSG6 HA-binding linker domain. In some embodiments, the second component is derived from or comprises SEQ ID NO:4.

3. Multifunctional proteoglycans

In some embodiments, the second component is derived from a multifunctional proteoglycan. The multifunctional proteoglycan comprises the following domains: VG1, a GAG attachment domain, and a G3 domain (Figure 8A). The VG1 domain (SEQ ID NO: 29) comprises an Ig domain, Link1, and Link2 (Figure 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 HA.

a) Wild-type VG1

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

b) Mutant VG1

In some embodiments, the HABD comprises a mutated VG1. In many embodiments, the VG1 mutation is relative to the amino acid sequence shown in SEQ ID NO:29 (WT VG1), 32 (VG1ΔIg), 60 (WT VG1 common sequence), or 86 (VG1ΔIg common 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 common sequence), or 86 (VG1ΔIg common 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 common sequence), or 86 (VG1ΔIg common sequence).

c) Truncate VG1

In some embodiments, HABD comprises a truncated mutation relative to SEQ ID NO:29 (WT VG1) or 60 (WT VG1 concordant sequence). In some embodiments, HABD comprises a truncation of 1 to 129 amino acids from the N-terminus of the multifunctional proteoglycan. In some embodiments, HABD comprises a truncated sequence in which the Ig domain of the wild-type multifunctional proteoglycan is absent. In some embodiments, 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:32 (VG1ΔIg) or SEQ ID NO:86 (VG1ΔIg concordant sequence). In some embodiments, HABD comprises a sequence that is at least 95% identical to SEQ ID NO:32 (VG1ΔIg) or SEQ ID NO:86 (VG1ΔIg concordant sequence). In some embodiments, HABD comprises SEQ ID NO:32 (VG1ΔIg).

d) Amino acid substitution

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

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

In some embodiments, HABD, relative to SEQ ID NO:29, comprises at least one 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. In some embodiments, HABD comprises at least one of Y208A and H306A.

In some embodiments, HABD, 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. In some embodiments, HABD, relative to SEQ ID NO:29, comprises at least 2, 3, 4, 5, or 6 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.

In some embodiments, 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 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.

4. Brain-specific connexin 1 (BRAL1)

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

5. Lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1)

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

6. Aggregates

In some embodiments, the second component is derived from proteoglycan. Proteoglycan comprises three globular domains: the G1 domain has a structural motif that connects to the protein and interacts with HA; the G2 domain is homologous to the G1 domain and participates in product processing; and the G3 domain forms the carboxyl terminus of the core protein. In some embodiments, the second component comprises the G1 domain derived from proteoglycan.

C. 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 and second components) is pre-complexed with HA to form a conjugate. In some embodiments, the third component is HA with a molecular weight of 5 kDa to 20 kDa.

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+ _D concentration of less than or equal to 10.0 μM. For example, the second component may bind HA with a K+ _D concentration 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 found in the extracellular matrix and on cell surfaces. HA contains repeating disaccharide units of N-acetylglucosamine (GlcNac) and glucuronic acid (GlcUA), linked by alternating β1→3-glucuronic acid and β1→4-glucosaminidic bonds to form a linear polymer. HA is further described in Necas et al., 2008, Veterinarni Medicina, 53:397-411. Glycosaminoglycans are ubiquitous in the extracellular matrix of all vertebrates and also found in the capsules of some streptococcal strains. Functionally, HA molecules are important for maintaining a highly hydrated extracellular matrix in tissues, participating in cell adhesion and supporting cell migration. The vitreous humor, besides water, is mainly composed of HA, which excels in retaining moisture and structure in the central part of the eye. It helps keep the eye lubricated and replenish any lost moisture. HA also exhibits a variety of biological functions through interactions with numerous HA-binding proteins and cell surface receptors such as CD44 and lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1). Examples of HA-binding proteins and HABDs are described in Section II.B above.

Hyaluronic acid (HA) has a wide molecular weight range from 1,000 Da to 10,000,000 Da. Naturally occurring high-molecular-weight HA in tissues is degraded into smaller molecules via metabolic pathways through the lymphatic system, lymph nodes, liver, and kidneys. While the known half-life of HA in plasma is approximately 2.5 to 5.5 minutes, its half-life in the vitreous humor of the eye has been reported to be approximately 70 days. HA's unique physicochemical properties and diverse biological functions have led to its widespread application in biomedical fields, such as drug delivery, arthritis treatment, ophthalmic surgery, and tissue engineering. In particular, the targeted, specific, and long-acting delivery of biological/pharmaceutical drugs via various delivery routes has been extensively studied. Utilizing its viscoelastic and mucosal adhesion properties, HA has been developed as an effective delivery carrier for topical ophthalmic drugs.

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

In some embodiments, unmodified, naturally occurring HA is preferably used. In these embodiments, using unmodified, naturally occurring HA reduces side effects. For example, pre-compounding HABD with 10 kDa of HA reduces in vitro precipitation in vitreous fluid and mitigates intraocular toxicity observed in pigs and rabbits. In other instances, when the HABD is TSG-6 or CD44, intraocular toxicity such as inflammation and retinal toxicity was observed when TSG-6 or CD44 was not pre-compounded with HA.

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

In some embodiments, HA may have minor chemical modifications. Chemical modifications can be used to reduce HA degradation, increase or decrease water solubility, alter HA diffusion rate, and/or HA viscosity. Two general methods for chemically modifying HA are known in the art – (1) crosslinking HA using functional chemical reagents and (2) coupling HA using monofunctional reagents. Divinyl sulfone, diepoxides, formaldehyde, and dihalides are bifunctional reagents used for crosslinking HA. Chemically modified HA formulations include, but are not limited to, aminoethyl methacrylate HA, adipic acid dihydrazide-grafted HA, dimethyl ether complex HA, HA-cysteine ethyl ester, urea-crosslinked HA, and N-acetylcysteine HA. Modifications that reduce HA degradation in the eye are of particular interest.

2. Therapeutic molecules are pre-complexed with hyaluronic acid (HA) to form conjugates.

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 high, leading to adverse effects, as described in Example 5 below. In some cases, these effects may be caused by the contact of free HABD with IVT HA at the injection site. Pre-complexing of HABD with HA reduces these adverse effects by allowing time for HABD to diffuse from the injection site to the rest of the vitreous. As HABD transitions from interacting with the pre-complexed HA to interacting with the IVT HA, the diffusion time slows down, and the vitreous half-life is prolonged. Therefore, in some embodiments, the therapeutic molecule is a conjugate containing the therapeutic molecule and further comprising one or more third components containing 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 a separated conjugate, i.e., the conjugate is not present 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, for example: 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 connected via covalent linkers.

Fusion proteins are proteins composed of two or more originally separate proteins or peptides linked together. This method yields a single polypeptide possessing the functional properties derived from each of the original, individual proteins. Proteins can be fused directly to each other. Proteins can also be fused via linkers, which increase the likelihood that proteins will fold independently and behave as intended. Dimeric or multimeric fusion proteins can be produced through genetic engineering by fusing with the original protein to a peptide domain that induces protein complexation (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 follows the first component directly (or vice versa), and there are no other chemical elements (atoms or groups) between the two components. In some embodiments, the last amino acid of the first component is immediately adjacent to the first amino acid of the second component.

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

In some embodiments, one or both of these 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 one or both of these 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, HABD is covalently bound to the C-terminus of Fab.

1. Peptide linker

In many embodiments, the peptide linker connects the therapeutic active agent (i.e., the first component) to 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) that allow adjacent protein domains to move freely relative to each other. Therefore, in some embodiments, the peptide linker is a glycine-serine linker, i.e., a peptide linker composed 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 is composed of GGGGS (SEQ ID NO:27) or its polymer, more particularly of (GGGGS) ₃ (SEQ ID NO:28).

In some embodiments, the peptide linker comprises (GS) _n , where 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 embodiments of therapeutic molecules and conjugates

1. VEGF

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

a)G6.31

In some embodiments, the first component is an antibody containing 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 containing a VH domain as shown in SEQ ID NO:105. In some embodiments, the first component is an antibody containing a VL domain as shown in SEQ ID NO:106.

b) PigFab

In some embodiments, the first component is an antibody comprising PigFab anti-VEGF Fab. In some embodiments, the first component is an antibody having a VH domain as shown in SEQ ID NO:66. In some embodiments, the first component is an antibody having a VL domain as shown 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 included in SEQ ID NO:77. In some embodiments, the first component is an antibody having a VL domain included 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, one or both of these second components comprise a CD44 HA receptor domain. In some embodiments, the second component comprises SEQ ID NO:2.

e)TSG6

In some embodiments, one or both of these second components comprise a TSG6 domain. In some embodiments, one or both of these 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, one or both of these second components comprise a VG1 domain. In some embodiments, one or both of these second components comprise one or both of the following SEQ ID NO: 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 bound peptide (CKP)

In some embodiments, the first component optionally further comprises a cysteine knot peptide (CKP) in addition to containing an 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 with 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 with 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 with SEQ ID NO:94.

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

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

2.NVS24

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

In some embodiments, the first component comprises an NVS24 antibody. In some embodiments, the first component is an antibody having a VH domain included in SEQ ID NO:21. In some embodiments, the first component is an antibody having a VL domain included in SEQ ID NO:22. In some embodiments, the first component is an antibody containing a VH domain as shown in SEQ ID NO:109. In some embodiments, the first component is an antibody containing 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. Dual-targeting antibodies against VEGF and PDGF (anti-VP-dutaFab; anti-VPDF)

In some embodiments, the therapeutic molecule comprises (1) a first component capable of binding VEGF and PDGF (such as bispecific antibodies or dual-targeting antibodies, dutaFab, as described in Section II.A.1.h above); and (2) one or two second components comprising 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 included in SEQ ID NO:5. In some embodiments, the first component is an antibody having a VL domain included in SEQ ID NO:6. In some embodiments, the first component is an antibody containing a VH domain as shown in SEQ ID NO:99. In some embodiments, the first component is an antibody containing a VL domain as shown in SEQ ID NO:100.

a)CD44

In some embodiments, one or both of these second components comprise a CD44 HA receptor domain. In some embodiments, one or both of these 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, one or both of these second components comprise a CD44-ko domain. In some embodiments, one or both of these 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, one or both of these second components comprise a VG1 domain. In some embodiments, one or both of these second components comprise one or both of the following SEQ ID NO: 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 RabFab VH and VL domains. In some embodiments, the RabFab antibody comprises: a VH domain, as shown in SEQ ID NO: 13; and a VL domain, as shown in SEQ ID NO: 14. In some embodiments, the RabFab antibody comprises the VH domain shown in SEQ ID NO: 107. In some embodiments, the RabFab antibody comprises the VL domain shown in SEQ ID NO: 108.

a)TSG6

In some embodiments, one or both of these second components comprise a TSG6 domain. In some embodiments, one or both of these 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, one or both of these second components comprise a VG1 domain. In some embodiments, one or both of these second components comprise one or both of the following SEQ ID NO: 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 anti-complement factor D antibody Fab (20D12v2.3). In some embodiments, the first component is an antibody having a VH domain included in SEQ ID NO:75. In some embodiments, the first component is an antibody having a VL domain included 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, one or both of these second components comprise a VG1 domain. In some embodiments, one or both of these second components comprise one or both of the following SEQ ID NO: 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 human HtrA1. In some embodiments, the first component is an antibody having a VH domain included in SEQ ID NO: 118. In some embodiments, the first component is an antibody having a VL domain included 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, one or both of these second components comprise a VG1 domain. In some embodiments, one or both of these second components comprise one or both of the following SEQ ID NO: 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 used in the treatment of eye diseases. Eye diseases can be characterized by altered or uncontrolled neovascularization and/or invasion of ocular tissues such as the retina or cornea. Eye diseases can also be characterized by atrophy of retinal tissues (photoreceptors and underlying retinal pigment epithelial cells (RPE) and choroidal capillaries). Non-restricted eye diseases include, for example, age-related macular degeneration (AMD) (e.g., wet AMD, dry AMD, intermediate AMD, late AMD, and geographic atrophy (GA)), macular degeneration, macular edema, diabetic macular edema (DME) (e.g., focal, non-central DME, and diffuse, central DME), retinal degeneration, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high-altitude DR), other ischemic retinal degenerations, ROP, retinal vein occlusion (RVO) (e.g., central (CRVO) and branch (BRVO) forms), CNV (e.g., myopic CNV), corneal angiogenesis, diseases associated with corneal angiogenesis, retinal angiogenesis, diseases associated with retinal/choroidal angiogenesis, central serous retinal degeneration (CSR), pathological myopia, von Hippel-Lindau, ocular histoplasmic disease, FEVR, and Coats' disease. Norrie's disease, retinal abnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, redness and swelling, ocular neovascularization, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinal degeneration, retinal angioma proliferation, macular telangiectasia, iris angiogenesis, intraocular angiogenesis, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis, including but not limited to CMV retinitis, and ocular tinnitus. Retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g., allergic) conjunctivitis), Leber congenital amaurosis (also known as Leber congenital amaurosis or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular tissue plaque fungal infection, blepharitis, dry eye syndrome, ocular trauma, and other ophthalmic diseases or conditions where the disease or condition is associated with angiogenesis, vascular leakage, and/or retinal edema or retinal atrophy. Other exemplary ocular diseases include those caused by abnormal proliferation of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinal degeneration.

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

Exemplary eye diseases associated with choroidal neovascularization and retinal vascular system defects (including increased vascular leakage, aneurysms, and capillary dripping) include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elastica, Paget's disease, venous occlusion, arterial occlusion, carotid artery occlusive disease, chronic uveitis/hyalitis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), and Eale's disease. Behcet's disease, infections causing retinitis or choroiditis (e.g., multifocal choroiditis), hypothetical ocular histoplasmosis, Best's disease (vitiligo macular degeneration), myopia, optic pits, Stargart's disease, pars plana inflammation of the ciliary body, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, and complications after trauma and laser treatment.

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

In some embodiments according to (or as applied to) any of the above embodiments, the eye disease is an intraocular neovascularization disease selected from the group consisting of: proliferative retinal degeneration, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemic-related retinal degeneration, diabetic macular edema, pathological myopia, Heber-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (including CRVO and BRVO), corneal neovascularization, retinal neovascularization, and retinopathy of prematurity (ROP). In a preferred embodiment of the invention, 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).

The therapeutic molecules, conjugates, and compositions disclosed herein can be used as medicines 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., human 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

This document provides a method for treating an eye disease, comprising delivering a therapeutic molecule, conjugate, or composition to a patient's tissue. In many embodiments, these methods include administering a therapeutic molecule such that it provides a sustained delivery of a therapeutic active agent to the target tissue. In many embodiments, the target tissue is in the eye.

A. Application method

Therapeutic molecules, conjugates, or compositions can be administered in any effective and convenient manner, including, for example, via local, oral, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intratracheal, or intradermal routes. Preferably, the composition is suitable for ocular application, and more specifically, the composition is suitable for IVT administration. Thus, in a preferred embodiment, the composition is formulated for intraocular delivery, particularly for IVT injection. Therapeutic molecules, conjugates, or compositions can be administered to an individual as injectable compositions (e.g., as sterile aqueous dispersions) during treatment or as a prophylactic agent.

Unbound by this theory, it is proposed that the injected conjugate promotes the diffusion of HA from pre-complexed HABD before it interacts with IVT HA. Due to the slow dissociation, the concentration of free HABD in the vitreous is lower. Compared to therapeutic molecules that are not pre-complexed with HA, the lower concentration of free HABD in the vitreous may pose less harm to the eye.

In some embodiments, the administration step is a single injection. In some embodiments, the administration step includes more than one single injection.

B. Composition

This document provides 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 are defined as preparations in a form that allows the bioactivity of the active ingredient contained therein to be effective and contains no other components that would have unacceptable toxicity to the subject to whom 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 includes pharmaceutical excipients, diluents, or carriers, such as buffering substances, stabilizers, preservatives, or other components, especially commonly known components associated with the pharmaceutical composition.

Generally, the nature of the optional ingredients or additives will depend on the specific form of the composition and the method of administration. Pharmaceutical carriers may enhance or stabilize the composition, or may be used to facilitate its preparation. These carriers may include, but are not limited to, physiologically compatible saline, buffered saline, glucose, water, glycerol, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption delay agents, and combinations thereof. The composition may additionally contain one or more other therapeutic agents, particularly those suitable for the treatment or prevention of diseases or conditions, such as those associated with eye diseases like retinal vascular diseases. The formulation should be adapted to the method of administration. For example, parenteral preparations typically contain injectable fluids, including pharmaceutically and physiologically acceptable fluids used as carriers, such as water, physiological saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers.

The composition may contain 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 present 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, the pharmaceutical compositions disclosed herein employ a therapeutically effective amount or dose of a therapeutic molecule or conjugate. The therapeutic molecule and conjugate are formulated into pharmaceutical dosage forms using conventional methods known to those skilled in the art. The dosing regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus injection may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased according to the urgency of the treatment situation. Formulation of parenteral compositions in dose units is particularly advantageous, as it facilitates administration and ensures uniformity of dosage. As used herein, dose units refer to physically discrete units suitable for use as a single dose in a subject to be treated; each unit contains a predetermined amount of the active compound, the amount of which is calculated to produce the desired therapeutic effect associated with the desired drug carrier.

The actual dose level of the active ingredient (i.e., the therapeutic molecule and conjugate) in a pharmaceutical composition can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response, composition, and non-toxic administration method for a specific patient. The selected dose level depends on a variety of pharmacokinetic factors, including: the activity of the specific composition of the invention used; the route of administration; the time of administration; the excretion rate of the specific compound used; the duration of treatment; other drugs, compounds, and/or materials used in combination with the specific composition used; the patient's age, sex, weight, disease, general health status, and medical history; and similar factors. The dose 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 begin administering the therapeutic molecule of the conjugate used in the pharmaceutical composition at a dose below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, the effective dose of the compositions described herein for treating ocular diseases depends on many different factors, including the route of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is preventative or therapeutic.

Treatment doses need to be titrated to optimize safety and efficacy. Doses for IVT administration using the conjugates of the present invention can range from 0.1 mg/eye to 10 mg/eye per injection. A single dose per eye can be administered by one or more injections per eye. For example, a single dose of 20 mg/eye can be delivered in two 10 mg injections, resulting in a total dose of 20 mg. The volume of each injection can be between 10 μL and 50 μL, while the volume of each dose can be between 10 μL and 100 μL. Consider dosages and regimens approved by the U.S. Food and Drug Administration (FDA) for Lucentis. Other dosages and regimens applicable to anti-VEGF antibodies or antigen-binding fragments are described in US 2012/0014958, the entire contents of which are incorporated herein by reference.

The composition can be administered multiple times. The interval between single doses can be weekly, monthly, or annually. Intervals can also be irregular, determined based on the patient's required retreatment, based on, for example, visual acuity or macular edema. Furthermore, alternative dosing intervals can be determined by a physician and administered monthly or as needed to achieve efficacy. 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 treatment rate, retinal thickness as measured by optical coherence tomography (OCT), and visual acuity. Dosage and frequency can vary depending on the half-life of the conjugate according to the invention in the patient and the levels of therapeutic targets (e.g., VEGF, C5, EPO, factor P, etc.). However, in a preferred embodiment of the 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 prolonged half-life of the first component in the conjugate relative to the corresponding unbound (free) first component (and thus the prolonged duration of effectiveness). Accordingly, the elimination half-life of the first component in the conjugate is at least 3, 4, or 5 times longer than that of 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 via IVT injection and measuring the remaining concentration at different time points using analytical methods known in the art, such as ELISA, mass spectrometry, Western 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)). Generally, molecules that are part of a conjugate (e.g., antibodies or fragments) exhibit a longer intraocular half-life than 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), by 50% compared to the free first component (e.g., from 5 days to 7.5 days), by 75% compared to the free first component (e.g., from 5 days to 8.75 days), by 100% compared to the free first component (e.g., from 5 days to 10 days), and in some respects, the half-life of the conjugate is expected to 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 longer; etc.).

D. Combination therapy

Combination therapy encompasses both combined administration (where two or more therapeutic agents are contained in the same or a single formulation) and single administration, in which case the administration of the therapeutic molecule and conjugate may occur before, simultaneously with, and/or after the administration of another one or more therapeutic agents. In some embodiments, the therapeutic molecule, conjugate, or composition is administered simultaneously with another compound. In some embodiments, the therapeutic molecule, conjugate, or composition is administered before or after another compound. In some embodiments, the administration of the therapeutic molecule, conjugate, or composition and the administration of another therapeutic agent occur within about one, two, three, four, or five months, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days.

Any suitable therapeutic agent for an eye disease can be used as this additional compound, particularly as a pharmaceutical agent for treating an eye disease as described in Section III above. Furthermore, any molecule described in Section II.A above as a component of a therapeutic molecule can also be used as an additional compound in combination therapy.

In some embodiments, additional compounds are the anti-angiogenic agents described in Section II.A.1.h above and Carmeliet et al. in Nature 407:249-257 (2000). Other suitable anti-angiogenic agents include corticosteroids, angiogenic inhibitory steroids, anecoxetine 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 epithelial-derived factor (PEDF), γ-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 new angiogenesis sites (e.g., anti-vascular endothelial cadherin (CD-144) antibodies and/or anti-SDF-1 antibodies), and combinations thereof.

Therapeutic molecules, conjugates, or compositions may also be administered in combination with therapeutic or surgical methods for the treatment of eye diseases (e.g., AMD, DME, DR, RVO, or GA), including, for example: laser coagulation (e.g., panretinal photocoagulation (PRP)), occult junction laser therapy, macular hole surgery, macular displacement surgery, implantable microtelescopes, PHI-motor angiography (also known as microlaser therapy and feeder vessel treatment), photon beam therapy, microstimulation therapy, retinal detachment and vitrectomy, scleral buckling, submacular surgery, transpupillary thermotherapy, and phototherapy. Therapeutic applications include: RNA interference (RNAi), in vitro rheological processes (also known as membrane filtration and rheological therapy), microchip implantation, stem cell therapy, gene replacement therapy, ribonuclease gene therapy (including gene therapy for hypoxia-responsive elements, Oxford Biomedica; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor/retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell grafts, e.g., Astellas Pharma US, Inc., ReNeuron, CHA Biotech), acupuncture, and combinations thereof.

Therapeutic molecules, conjugates, or compositions may also be administered in combination with visual cycle regulators (e.g., emixustat hydrochloride); squalene (e.g., OHR-102; Ofr 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, such as 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 CureNeurosciences); 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 co-formulated with aflibercept. In some cases, these co-formulations can be administered in combination with therapeutic molecules, conjugates, or compositions.

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

In some 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; integrin 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 some embodiments, the additional compound is an antibody or an antigen-binding fragment thereof, including examples of antibodies and antigen-binding fragments described in Section II.A.3 above.

E. Target tissue

In some embodiments, the target tissue includes the eye, brain, bone, and/or tumor. In some embodiments, the tissue includes 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 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 a longer half-life relative to the injection site compared to unmodified therapeutic agents. In some embodiments, the therapeutic molecule, conjugate, or composition may further provide a longer duration of pharmacological action in the target tissue compared to unmodified therapeutic agents.

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

F. Binding therapeutic molecules to HA

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

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

In some embodiments, the size of the HA ranges from 400 Da to 200 kDa. In some embodiments, the HA is at least 5 kDa. In some embodiments, the HA is 10 kDa. 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 binding equivalent for HABD. In some embodiments, the HA size/content allows the HA to therapeutic molecule ratio to be in the range of 1.5:1 to 1:1.

Example

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

The following examples discuss fusion proteins containing a Fab fragment or peptide and a hyaluronic acid-binding domain, known as Fab-HABD. Examples 1 through 7 involve CD44 and/or TSG6 HABD. Examples 8 through 18 involve VG1 HABD.

Example 1. Generation of Fab-hyaluronic acid binding domain fusion protein (Fab-HABD) and complexation with HA

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

Fab-HABD is 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).

Dig antibody is covalently linked to one or two CD44 HA receptor domains and used as a non-binding control molecule (SEQ ID NO: 9 to 12).

A. Materials and Methods

1. Protein expression

Various Fab-HABD expression plasmids were generated using standard molecular biology techniques for restriction selection or gene synthesis. Individual expression vectors were generated for each polypeptide chain. Expression was performed in HEK293 cells (ThermoFisher) with expression plasmids mixed at a 1:1 ratio.

In some cases, TSG6 is expressed in Escherichia coli.

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

2. Protein purification

The supernatant was collected by centrifugation at 4000 rpm and 4 °C for 20 minutes. The cell-free supernatant was then filtered through a 0.22 μm top filter and stored in a refrigerator (-20 °C).

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

In short, sterile filtered _cell culture supernatant was captured on KappaSelect resin (GE Healthcare) equilibrated with 1x PBS buffer (10 mM _Na₂HPO₄ , 1 mM _KH₂PO₄ , 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 1x PBS buffer (10 mM _Na₂HPO₄ , 1 mM _KH₂PO₄ , 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 the protein sample was determined at 280 nm using a Nanodrop 800 spectrophotometer (ThermoScientific).

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

The antibody-containing fractions obtained by size exclusion chromatography were frozen at -80°C and stored for further use.

In some cases, TSG6 is purified from *E. coli*. Briefly, *E. coli* cells are extracted using a buffer consisting of 7M guanidine hydrochloride, 50 mM Tris-HCl, 100 mM sodium tetrathionate, and 20 mM sodium sulfite. After homogenization, centrifugation, and filtration of the supernatant, the His-tagged protein is captured on a Ni-NTA column (GE Healthcare) equilibrated with 6M guanidine hydrochloride and 25 mM Tris-HCl (pH 8.6). The column is washed with 25 mM Tris-HCl (pH 8.6) and 0.1% Triton X-114, and eluted with a buffer containing 250 mM imidazole. The TSG6 eluted from the column was refolded by diluting it to 1.5 mg/mL and then dialyzing overnight at 4°C with a solution of 0.5 M guanidine hydrochloride, 0.5 M arginine, 1 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). After the buffer was replaced with 25 mM sodium acetate (pH 5.0), the refolded material was purified by cation exchange chromatography on an SP-Sepharose ^™ (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 require refolding. RabFab-1xTSG6 has the Fab fused to TSG via a gly-gly-gly-gly-ser linker; 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, while 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, which included: (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. With hyaluronic acid (HA) and complex

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

Example 2. Molecular characteristics of Fab-HABD

Example 2.1. Interaction with HA

A. Materials and Methods

The binding ability of Fab-HABD to HA was examined. The binding of Fab-CD44 and Fab-TSG6 Fab-HABD to HA was tested by SPR using a Biacore T200 instrument (GE Healthcare) (Table 3). In short, Fab-CD44 Fab-HABD was injected into HA-coated chips (SCBS HY, Xantect Bioanalytics GmbH, Germany) over 80 or 120 seconds at concentrations ranging from 3.7 nM to 300 nM. In some experiments, HA-coated chips were prepared by indirectly coupling biotin-HA (Sigma-Aldrich, St. Louis, Missouri US) to S-series sensor chips (SA) coated with streptavidin (GE Healthcare). The dissociated phase was monitored for 600 seconds. Subsequently, the surface was regenerated by injecting 10 mM glycine (pH 1.5) for 60 seconds or injecting 3 M _MgCl2 for 30 seconds. Bulk refractive index difference was corrected by subtracting the reaction 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 between VDPF-2xCD44 and HA was tested using isothermal titration calorimetry (ITC). Briefly, _the Fab-CD44 fusion was dialyzed with PBS (10 mM _Na₂HPO₄ , 1 mM _KH₂PO₄ , 137 mM NaCl, 2.7 mM KCl _, pH 7.4). After dialyzing, HA was dissolved using the remaining buffer dose, ensuring all molecules were under identical buffer conditions to avoid any buffer-related mismatches. HA molecules were loaded into the sample cell at concentrations of 10 μM (10 kDa HA) or 2 μM (50 kDa HA), respectively. Deionized water was loaded into the reference cell. The syringe was filled with 150 μM of the Fab-CD44 fusion protein. Titration experiments were performed at 25 °C. The affinity constant K and stoichiometry N were calculated using a set of site models in Origin 7.0 (OriginLab Corporation).

Similarly, the interaction between TSG6 and 10 kDa HA was measured using an 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 measurements were taken at 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 at 1.0, and the HA concentration and affinity constant K were variable parameters.

B. Result

Table 3 shows the _KD of Fab-CD44s and Fab-TSG6s bound to HA, as measured by SPR.

The strength of the interaction is determined by the HABD sequence and the binding affinity of the interaction (i.e., the 2x version exhibits higher functional affinity by binding closely to HA).

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

Regarding the stoichiometry of interactions, it was found that on average, each 10 kDa HA molecule could bind 1.5 VPDF-2xCD44 molecules, while each 50 kDa HA molecule could bind 5 VPDF-2xCD44 molecules.

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

Example 2.2. Stability of Fab-HABD

For long-term delivery of Fab-HABD into the eye, the protein needs to remain stable at body temperature over a range of several months. A prerequisite is that Fab-HABD has thermal stability above 37°C.

A. Materials and Methods

The thermal stability of VPDF-2xCD44 and TSG6 was assessed using static light scattering and protein autofluorescence. Samples were diluted to approximately 1 mg/mL, and the temperature was increased from 25 °C to 80 °C using an Optim instrument (Avacta, Inc.) at a heating rate of 0.1 °C/min. During this process, light scattering and fluorescence data after irradiation with a 266 nm laser were recorded. The aggregation initiation temperature was determined to be approximately 75 °C, which was defined as the temperature at which scattering intensity increased. Simultaneously, fluorescence emission spectra were recorded.

For VPDF-CD44, two transitions were detected at approximately 56 °C and 79 °C when plotting the centroid mean of the fluorescence spectrum against temperature. These transitions indicate protein denaturation (likely of the Fab and CD44 domains). Therefore, any scattering or spectral changes associated with thermal defolding occurred well above 37 °C, suggesting good stability of this Fab-HABD.

B. Result

The two transformations of VPDF-2xCD44 were measured at 56 °C and 79 °C, respectively. These two T_{_m values} indicate the denaturation transformations of VPDF-2xCD44, with the CD44 domain denaturing at 56 °C and the Fab domain denaturing at 79 °C.

For TSG6, the observed _{initial temperature of} Tm was 35℃, while the actual measured temperature of _Tm was 43℃.

Example 3. In vivo efficacy of laser-induced choroidal neovascularization (CNV) in rats.

A. Materials and Methods

Fab-HABD was investigated 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 binding to IVT HA (i.e., Fab-HABD inhibits neovascularization); and (2) Fab-HABD has a longer-lasting in vivo efficacy compared to the corresponding unmodified Fab fragment.

To this end, rats received IVT injections of a protein preparation one or three weeks before laser injury (six laser burns per eye). One week after the laser injury was initiated, fluorescence angiography (FA) was used to analyze vascular lesions.

Fab-HABD was compared with the corresponding unmodified Fab fragment. To test the durable efficacy of Fab-HABD, the dose of unmodified Fab was titrated to the “lowest effective dose” (i.e., a low degree of detectable inhibition of angiogenesis compared to the vector over the duration of the rat model), indicating that Fab-HABD had a more durable efficacy at the same dose and duration in the model.

B. Result

All tested Fab-HABDs exhibited inhibitory effects on angiogenesis in vivo (Table 5). This result indicates that although Fab-HABD binds to HA in the vitreous body, it reaches the relevant tissues to exert its pharmacological effects.

Within the same model settings and at the same dosage, all tested Fab-HABD fragments exhibited a longer duration of pharmacological effect compared to the unmodified Fab fragment (Table 5). This suggests that the ability to bind to IVT HA prolongs the in vivo pharmacological effect.

Despite significant differences in affinity for HA, the resolution of the in vivo model was insufficient to distinguish the persistence of efficacy among the different molecules. No tolerability issues were detected at the low non-therapeutic doses applied in the model. All eyes of rats receiving the Fab-HABD dose were completely normal, without any signs of disturbance, comparable to eyes receiving the buffer only in vivo.

Example 4. Rabbit pharmacokinetic (PK) studies using RabFab, RabFab-1xTSG6, and RabFab-2xTSG6

A. Materials and Methods

Proteins used in animal studies were prepared by dialysis in 20 mM histidine-acetate, 150 mM NaCl (pH 5.5), or phosphate-buffered saline (PBS) (pH 7.4). The formulations were isotonic, with osmolality between 300 mOsm/kg and 340 mOsm/kg as measured by freezing point. Size exclusion chromatography (SEC) analysis showed that all proteins in these formulations had a monomer content ≥95%. Endotoxin levels at the final administered concentrations were assessed to be less than 0.1 EU per eye.

In the vitreous half-life study, additional pharmacokinetic studies were conducted in which female rabbits were administered bilaterally via ITV injection of either eye a 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), for a total volume of 50 μL. The concentrations of the test sample in the vitreous and aqueous humor were measured at specified time points using a fluorometric assay, 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 via non-compartmental analysis using Phoenix WinNonlin (Certara Inc., Mountain View, CA). For concentration-time profiles generated using fluorescence intensity methods, samples taken within the first 48 hours after administration were excluded from PK analysis due to high variability, which could be attributed to individual differences at the application site and subsequent diffusion of the test sample through the vitreous humor. (Dickmann, L.J. et al., Invest. Ophthalmol. Vis. Sci., 56(11):6991-6999 (2015)). PK analysis was performed using non-compartmental methods, with clearance (CL) calculated as CL = dose/AUC, where the dose was known and the AUC was determined using a linear trapezoidal method. Steady-state volume of distribution was calculated as V = CL/kel, using the clearance value and the elimination rate constant obtained from the terminal slope. Elimination half-life was calculated as t1/2 = ln(2)/kel.

B. Result

The pharmacokinetic (PK) assays of New Zealand white rabbits were initially used to examine the ability of HA binding to affect intraocular residence time. Although the HA concentration in rabbit vitreous fluid (approximately 65 μg/mL) is much lower than in human vitreous fluid (100–400 μg/mL) or other preclinical species such as pigs (with vitreous HA of approximately 180 μg/mL) or cynomolgus monkeys (with vitreous HA of approximately 150 μg/mL), rabbits are frequently used in early PK studies following in vitro transluminal (IVT) administration of the test substance. The study was designed to administer 0.3 mg/eye RabFab, 0.3 mg/eye RabFab-1xTSG6, or 0.5 mg/eye RabFab-2xTSG6 via IVT. Recovery assays using ex vivo added vitreous fluid proteins demonstrated that RabFab and RabFab-1xTSG6 could be quantified using ELISA with a previously described anti-genotypic detection antibody (Shatz et al., 2016 Molecular Pharmaceutics). However, the recovery rate obtained by ELISA using RabFab-2xTSG6 was poor, making radiochemical determination of the vitreous concentration of this material necessary. In the PK study, RabFab-2xTSG6 was radiolabeled with iodine ^-125 .

As shown in Figure 2A, the PK parameters are summarized in Table 6. Both RabFab-1xTSG6 and RabFab-2xTSG6 exhibited longer vitreous retention times compared to free RabFab. The half-life of RabFab-1xTSG6 was 1.4 times longer than that of RabFab, while the half-life of RabFab-2xTSG6 was increased by 2.2 times. These results indicate that the fusion of Fab with HABD can increase the retention time of these molecules in the ocular ventricle. Given the higher vitreous HA concentrations in other species, it is expected that the half-life of Fab-HABD will be extended even more significantly in those animals.

Furthermore, vitreous half-life studies showed that the vitreous half-life of RabFab-2xTSG6 was approximately 3 to 4 times longer than that of RabFab as observed by fluorescence spectrophotometry, with no significant dose dependence within the assessed range (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 eye tolerance of RabFab-1xTSG6 and TSG6

A. Materials and Methods

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

B. Result

Generally, animals receiving RabFab-1xTSG-6 had less severe outcomes than those receiving free TSG6. Animals receiving free TSG6 showed significant clinical findings. Although four animals were scheduled for necropsy on day 4, four other animals were prematurely sacrificed on day 12 or 17 instead of day 30 due to significant clinical findings and animal welfare concerns. These clinical findings included eyelid and conjunctival swelling and redness, eyes remaining closed when staff approached, and ocular inflammation and irritation. By day 3 post-administration, animals receiving free TSG6 exhibited marked early posterior cataracts and variable retinal vascular attenuation, results correlated with microscopic findings of lens and external to complete retinal degeneration. Similar, but less severe, outcomes were observed in animals receiving RabFab-1xTSG6. From day 7 post-administration, all animals showed marked, predominantly mononuclear, inflammation. Inflammation and retinal degeneration were multifocally associated with evidence of retinal detachment, hypertrophy, peripheral migration of vimentin, glial fibrillary acidic protein (GFAP), and glutamine synthase-positive Müller cells. Histopathological images of retinal degeneration 4 days after IVT administration of TSG6 are shown in Figure 3.

Two animals that received RabFab-1xTSG6 and underwent necropsy on day 4 showed evidence of antidrug antibodies (ADA) in their serum on day 4. However, one of the animals received pre-administration of ADA, while the other three animals in the treatment group did not. The animal in this group that underwent later necropsy was ADA-negative on days 4 and 8, but became ADA-positive on day 15. Due to the poor sensitivity of this assay, the analysis of serum ADA response in animals treated with free TSG6 remains inconclusive.

Generally, animals receiving RabFab-1xTSG6 had less severe outcomes than those receiving free TSG6 (Table 8). Cataracts were present in every animal, but they were punctate in nature and not correlated in microscopic sections. There was no clinical evidence of retinal degeneration, but very slight to mild microscopic evidence of outer retinal degeneration was present in a single eye. Similar moderate to severe vitreous and aqueous humor cell abnormalities appeared from day 8. Animals were euthanized on days 4 and 17.

The assessment of anti-RabFab response became complicated because 3/8 of the animals had values above the critical value before administration (2 animals received free TSG6, and 1 animal received RabFab-1xTSG6). However, after administration of the test product, 3/4 of the animals receiving RabFab-1xTSG6 developed new or increased ADA titers: one animal was euthanized on day 4, and the other two were euthanized on day 17. In contrast, animal 1, which received only free TSG6 (and was euthanized on day 4), had an elevated ADA titer compared to before administration.

Early onset of clinical signs and microscopic lesions indicated a direct role of TSG6 in retinal and lens degeneration; however, later findings were confounded by the unexpectedly strong ADA response. Peripheral migration of Müller cells was determined to be a nonspecific response following retinal injury or detachment in rabbits.

Example 6. Pharmacokinetics (PK) of therapeutic doses in miniature pigs

The aim of this study was to determine the intraocular and systemic PK parameters of Fab-HABD and Fab-HABD-HAs, and the resulting prolongation of the intraocular half-life (t _1/2 ), in which the drugs were administered once via IVT to miniature pigs. In addition, anti-drug antibodies (ADA), intraocular tolerance, and ophthalmic pathology (in some study subjects) were investigated.

A. Materials and Methods

Fourteen SPF miniature pigs were given the following experimental product in both eyes at a therapeutic dose (50 μL/eye) (Table 9).

Following IVT administration, blood and aqueous humor samples were collected periodically throughout the study period (up to 9 weeks) from animals that received the test product, and vitreous fluid was collected shortly after planned euthanasia to track systemic and intraocular pharmacokinetic (PK) levels of the test product. Test product concentrations in plasma, aqueous humor, and vitreous fluid were analyzed, and the presence of ADA in plasma and vitreous samples was further analyzed.

B. Results: In the in vivo phase of this study, gross observations of the eyes of two out of five animals receiving VPDF-2xCD44 indicated that the pig eyes could not tolerate the IVT injection of the test substance, leading to the premature sacrifice of these animals. One eye from each animal was provided for histopathological evaluation. In summary, these gross observations were: vitreous opacities, lower than normal vitreous viscosity, and behavioral signs of vision loss. Histopathological findings in the eyes included moderate mixed cellular inflammation around the vessels/vessels, primarily mononuclear cell infiltration in the iris, ciliary body, trabecular meshwork, and retina. Retinal degeneration included ganglion cell degeneration, cell loss in the INL, photoreceptor aggregation, and nuclear translocation in the PR layer. In addition, eosinophilic proteinaceous material, mixed cellular infiltration, and fibrous filaments were observed in the vitreous. No abnormalities were found in the optic nerve.

Compared to VPDF-2xCD44, the gross observation results of the eyes of at least one of the five animals receiving VPDF-2xCD44 + 10kDa HA were significantly less severe. A brief observation of reddish/white membranes accumulating behind the cornea in the anterior chamber of both eyes was noted, but early termination was not considered necessary.

In summary, the combination of VPDF-2xCD44 with HA (i.e., occupying the CD44 HA binding site with HA before IVT injection) does improve the intraocular tolerability of VPDF-2xCD44.

No visual observations or tolerability issues were observed in the animal group that received the unmodified VPDF.

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

While the 5.8-day IVT t1 _/2 of unmodified VPDF is within the expected range for this type of molecule, the 48-day IVT t1 _/2 of VPDF-2xCD44+10kDa HA corresponds to an approximately 8-fold increase in intraocular residence time compared to unmodified VPDF. In summary, VPDF-2xCD44+10kDa exhibits significantly improved tolerability compared to VPDF-2xCD44 not conjugated with HA, and a significantly improved intraocular half-life compared to unmodified VPDF.

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

Macroscopic observations from in vivo miniature pig studies (i.e., vitreous opacity) indicate that the incompatibility of VPDF-2xCD44 with the porcine vitreous (i.e., precipitation) can be mitigated by pre-complexing VPDF-2xCD44 with pure HA. To further examine these effects and test whether these observations are limited to VPDF-2xCD44 molecules or can also be detected against other Fab-HABDs, we developed an in vitro testing system to detect vitreous degeneration.

An in vitro “droplet” test was developed to evaluate the vitreous compatibility of several Fab-HABDs pre-complexed with HA. This example demonstrates that Fab-HABDs pre-complexed with HA (i.e., conjugates) are vitreous compatible in in vitro experiments. Previously observed vitreous incompatibility may have been caused by free HABD, which is mitigated by HA pre-complexation. Results show that the incompatibility of Fab-HABD with free HABD depends on concentration. Furthermore, CD44ko (a Fab-HABD mutant containing a point mutation that disables HA binding) is vitreous compatible in both pre-complexed and dissociated forms.

Example 7.1. Pre-complexation of VPDF-2xCD44 with 10kDa HA improves in-vitreous (IVT) tolerance.

A. Materials and Methods

In the first test, porcine vitreous humor was homogenized 10x in a Dounce homogenizer and centrifuged at 10,000g for 2 minutes to remove debris. A 2μl drop of homogenized vitreous humor was then applied to a glass microscope slide. Additionally, 2μl of the test sample (i.e., a specified concentration of Fab-HABD or Fab-HABD-HA) was added to the top of the vitreous humor drop without further mixing. One minute after droplet coalescence, the sample was examined for inhomogeneity and precipitation using an optical microscope at 40x magnification in bright-field mode.

B. Result

Porcine vitreous humors mixed with unmodified VPDF at a concentration of 200 mg/mL in 20 mM histidine and 140 mM NaCl (pH 6.0) were homogeneous and transparent (Fig. 5A), while porcine vitreous humors mixed with VPDF-2xCD44 at a concentration of 20 mg/mL in 20 mM histidine and 140 mM NaCl (pH 6.0) were heterogeneous and exhibited transparent precipitate characteristics (Fig. 5B).

These results indicate that VPDF-2xCD44 is also incompatible with porcine vitreous humor after in vivo IVT injection. Therefore, vitreous humor incompatibility may be a fundamental cause of in vivo tolerance problems with VPDF-2xCD44.

Pre-complexation of 20 mg/mL VPDF-2xCD44 with 1% (w/v) HA (10 kDa, Lifecore, Biomedical) in 20 mM histidine and 140 mM NaCl (pH 6.0) resulted in vitreous compatibility (Figure 5C). This result reflects the findings of the aforementioned in vivo studies in miniature pigs, which demonstrated 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-dependent nature of vitreous incompatibility of VPDF-2xCD44, 2 μl of porcine vitreous humor was mixed with a 1:4 dilution of VPDF-2xCD44 in 20 mM histidine and 140 mM NaCl (pH 6.0) at an initial concentration of 37.5 mg/mL. Vitreous inhomogeneity in the mixture of vitreous humor and protein was examined using an optical microscope.

B. Result

The detected heterogeneity depended on the protein concentration (Table 10; Figures 6A to 6F). Vitreous compatibility of VPDF-2xCD44 ranged from 0.6 to 0.15 mg/mL. Correlating these results with those from the aforementioned in vivo miniature pig study (VPDF-2xCD44 concentration = 17.4 mg/mL) suggests that similar heterogeneity resulting from IVT injection of a 17.4 mg/mL VPDF-2xCD44 solution may be the underlying cause of the observed tolerance problems.

Example 7.3. The vitreous incompatibility of VPDF-2xCD44 is related to its interaction with the intravitreal (IVT) HA.

A. Materials and Methods

To test whether the vitreous incompatibility of VPDF-2xCD44 is caused by the interaction between VPDF-2xCD44 and IVT HA, we designed a variant of the molecule (VPDF2xCD44-ko) that contains a point mutation within the HA binding site of CD44 that eliminates the binding to HA while retaining the complete binding of the rest of the protein (referred to as the "ko variant" in this paper).

B. Result

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

When the vitreous compatibility of this VPDF-2xCD44-ko variant was tested 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 vitreous-compatible after hyaluronidase pretreatment.

A. Materials and Methods

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

B. Result

As a result, VPDF-2xCD44 did not show any heterogeneity when mixed with hyaluronidase-pretreated vitreous at a concentration of 20 mg/mL, possibly due to the degradation of high molecular weight HA.

In summary, these results suggest that vitreous incompatibility may be the root cause of in vivo tolerance issues of VPDF-2xCD44 and the interaction between CD44-HABD and high molecular weight IVT HA. Example 7.5. Vitreous incompatibility of Fab-HABD is related to the interaction between HABD and vitreous HA at specific concentrations.

A. Materials and Methods

To test whether vitreous incompatibility is unique to VPDF-2xCD44, we tested the vitreous inhomogeneity of other Fab-HABDs or standalone HABDs as described in Examples 7.2 and 7.3 above. The proteins tested were as described in Example 1.

B. Result

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

Fab-HABD containing the TSG6 domain exhibits vitreous inhomogeneity comparable to that containing CD44. Fab component G6.31 did not exhibit vitreous inhomogeneity at the same concentration, while isolated TSG6 domains did. This result further supports the view that vitreous incompatibility is related to the interaction between HABD and vitreous HA at specific concentrations.

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

A. Materials and Methods

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

B. Result

Vitreous inhomogeneity in all tested Fab-HABDs was resolved by pre-complexation with 10 kDa HA (Table 13). These results suggest that pre-complexation of HA-binding proteins with pure HA can serve as a method to improve vitreous compatibility and thereby mitigate potential tolerance issues associated with 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 is an effect that occurs only in the 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. Result

For all tested Fab-HABDs, the same vitreous incompatibilities as in porcine vitreous were detected. Furthermore, all vitreous incompatibilities detected in rabbit vitreous were resolved by pre-compositing Fab-HABD with a 10kDa HA.

These results indicate that vitreous incompatibility is not unique to the porcine vitreous.

Example 7.8. In vivo injection to induce vitreous heterogeneity

A. Materials and Methods

To establish a correlation between in vitro vitreous compatibility test results and in vivo tolerance results in miniature pigs, we tested whether vitreous inhomogeneity could be detected in the entire pig eye and whether this inhomogeneity could be resolved by HA pre-complexation.

For this purpose, whole pig eyes were collected immediately after slaughter, and 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 was injected into the eyes (same as in the in vivo miniature pig studies described above). The eyes were then transferred to HBSS (Lonza, Biowhittaker) and stored at 37°C for 4 hours. After incubation, the eyes were opened, and the vitreous humor was removed to examine for heterogeneity.

B. Result

As shown in Figures 7A to 7C, vitreous incompatibility during injection can be resolved by pre-compositing Fab-HABD with HA. Injection of the buffer does not cause IVT inhomogeneity, resulting in a clear vitreous (Figure 7A). Injection of VPDF-2xCD44 resulted in the appearance of dense white inhomogeneous material (precipitate) in the vitreous surrounding the injection side (Figure 7B). The vitreous of eyes injected with VPDF pre-composited with pure HA showed significant differences (Figure 7C): although inhomogeneity was detected, its density and thickness throughout the vitreous were significantly reduced.

These results indicate that the heterogeneity induced by VPDF-2xCD44 also occurs near the injection site throughout the pig eye. Unbound by this theory, we believe that the same heterogeneity is induced upon in vivo injection, and that this heterogeneity may be the root cause of the observed tolerance problems.

Furthermore, pre-compounding VPDF-2xCD44 with HA reduced the heterogeneity observed around the injection site. We believe the same effect led to 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 could similarly be resolved by pre-mixing with pure HA, we consider this approach a general principle for improving vitreous compatibility and thereby improving IVT tolerance of HABD-containing proteins.

Example 8. Multifunctional proteoglycans VG1 and VG1ΔIg HABD can bind HA

The study investigated HABDs of multifunctional proteoglycans to determine whether they could be used as HABDs that provide superior intraocular tolerability and intraocular residence time compared to TSG6 and CD44 HABDs.

The multifunctional proteoglycan was identified as having tandemly repeated linker modules. As shown in Figure 8A, the amino acid sequence of the multifunctional proteoglycan encodes an Ig-like domain, which is connected to two linker modules, such that the N-terminal fragment of the multifunctional proteoglycan (named WT VG1 in this paper) contains an N-terminal Ig-like domain and two linker domains. In this example, we obtained WT VG1 and a truncated variant, VG1ΔIg, without the Ig domain, and tested their ability to bind to HA. Furthermore, in the following examples, the in vitro vitreous compatibility and tolerability of WT VG1 and the Fab-HABD composed of Fab and WT VG1 when injected via IVT into rabbits and miniature pigs were tested.

A. Materials and Methods

Expression plasmids for various proteins were generated using restriction cloning and/or gene synthesis employing standard molecular biology techniques. Expression was then performed in CHO or HEK293 cells.

The supernatant was collected by centrifugation at 4000 rpm and 4 °C for 20 minutes. The cell-free supernatant was then filtered through a 0.22 μm top filter and stored in a refrigerator (-20 °C).

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

The binding of WT VG1 and VG1ΔIg to HA was tested by SPR using a Biacore T200 instrument (GE Healthcare). In short, WT VG1 and VG1ΔIg were injected into an S-series CM5 chip (GE Healthcare Life Science Solutions) over 80 or ₁₂₀ seconds. This chip indirectly coated with biotin-HA via immobilized streptavidin (Creative PEGWorks, North Carolina). The injection concentration ranged from 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 _MgCl2 for 15 seconds. All experiments were performed at 25°C using PBS (10 mM _Na₂HPO₄ , 1 mM _KH₂PO₄ , 137 mM NaCl, 2.7 _mM KCl, pH 7.4). The resulting HA binding curves were fitted to a 1:1 Langmuir binding model using BIAevaluation software.

B. Result

The multifunctional proteoglycan HABD can 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 modulus of WT VG1 and TSG6 was determined using a Biacore T200 instrument (GE Healthcare) via SPR measurement of binding to heparin sulfate and chondroitin sulfate. In short, the proteins were injected over 180 seconds onto an S-series CM5 chip (GE Healthcare Life Science Solutions) indirectly coated with biotin-heparin sulfate or biotin-chondroitin sulfate via streptavidin. Injection concentrations ranged from approximately 5 nM to 1000 nM. The dissociation phase was monitored for 120 seconds. Subsequently, surface regeneration was achieved by injecting 1 M _MgCl2 for 30 seconds.

B. Result

The results showed that WT VG1 was more selective than TSG6 in binding (Table 15). No binding of WT VG1 to heparin sulfate or chondroitin sulfate was observed, while TSG6 bound tightly to both heparin and chondroitin sulfate.

Example 10. Fab-HABD containing VG1 HABD can bind antigen and HA.

A. Materials and Methods

A.1. Component Design

The Fab-HABD system is generated through recombination fusion of the WT VG1 sequence with the C-terminus of the Fab fragment heavy chain or the N-terminus of the IgG1 heavy chain. For peptide-VG1 fusions, a peptide (EETI) is generated or can be generated that simultaneously attaches to the N-terminus of WT VG1 (EETI-VG1) and the C-terminus of WT VG1 (VG1-EETI). Two additional constructs are also generated in which a TEV cleavage site is introduced between EETI and WT VG1 (EETI-TEV-VG1 and VG1-TEV-EETI). The linkers used or potentially used are shown in Table 16.

A.2. Development of species-matched alternative porcine anti-VEGF Fab

Since a search of the abYsis database yielded no known instances of heavy and light chain pairing of porcine (Sus scrofa) IgG, we attempted to generate antibodies that actively bind to known antigens by transplanting a CDR from an anti-VEGF Fab (G6.31.AARR). We searched the NCBI Sequence Tag (EST) database for porcine mRNA ESTs with high sequence identity to the G6.31 backbone ( _VH4 / _VLK2 ). Several sequences were selected, and a CDR from G6.31.AARR was transplanted into the appropriate backbone region to generate “porcine” G6.31.AARR. Heavy and light chain sequences were randomly paired and expressed in 293Expi or CHO cells in 30 mL cultures. The cells were purified on Capto L resin and analyzed by size exclusion chromatography. The purified cells were examined by SDS-PAGE and mass spectrometry, and their binding to human and porcine VEGF was evaluated. A sequence with good affinity for VEGF was selected for amplification and subsequent tox/PK analysis, and this sequence was recombinated and fused with VG1 to generate PigFab-VG1.

A.3. Protein Expression and Purification

Protein expression was achieved by transfecting the cationic lipids of the DNA construct into CHO or 293Expi cells. Culture volumes ranged from 30 mL to 35 L. For some constructs, rapid and stable cell lines were generated to increase protein yield per unit culture volume.

Purification is performed by affinity chromatography, where Ni-NTA resin is used for molecules labeled with 6x-histidine, or Gamma-bind Plus resin is used for Fab fusion. In some cases, a secondary ion exchange step is performed prior to the final particle size sieving step on Sephadex resin.

A.4.HA combination

To confirm that VG1 retains 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 concentrations tested varied among different proteins, but ranged between 500 nM and 6.25 nM.

A.5. Antigen binding

Antigen binding was tested by directly immobilizing the corresponding antigen onto the S-series CM5 chip (GE Healthcare) and binding using SPR measurements as described in Example 2.1. Different protein concentrations were used based on known interaction affinities.

B. Result

B.1. Hyaluronic acid (HA) binding

Using BIAevaluation software, all HA binding data were fitted to a 1:1 Langmuir binding model. The KD _values for various proteins are shown in Table 17.

B.2. Antigen binding

Table 18 shows the proteins analyzed for their antigen-binding ability and the measured _KD . VG1 fusion with the C-terminus of various Fab heavy chains does not affect antigen binding. For EETI-VG1 fusion, the flexibility of the linker and attachment site affects antigen binding. More flexible linkers and C-terminal fusions are preferred.

Example 11. Live external vitreous compatibility of HABD

A. Materials and Methods

This example describes the testing of the solubility of the VG1 domain in vitreous fluid. Vitreous fluid was prepared using a Dounce homogenizer and then centrifuged at 10000 x g for 2 minutes to remove debris; this was then used in these studies.

Additional experiments utilized Alexa488-labeled proteins, enabling both bright-field and fluorescence microscopy to monitor precipitation in ex vivo vitreous fluid. Equal volumes of the sample were mixed with vitreous fluid by sequentially injecting into each of the two channels of a three-in-one three-channel □ slide (ibidi, USA, Inc. Cat#80316), and the mixing interface was visually monitored using a microscope.

B. Result

When TSG6 was mixed with porcine vitreous fluid pre-diluted with PBS (pH 7.4) at a ratio of 1:4, the solution became cloudy (Fig. 9A), and precipitation was observed after centrifugation of the mixture. In contrast, when VG1 was mixed with porcine vitreous fluid at ratios of 1:4 and 1:1, the solutions remained clear (Fig. 9B), and no precipitation was observed after centrifugation.

Furthermore, RabFab-TSG6 precipitation was observed in isolated porcine vitreous bodies (Fig. 10A), while no precipitation was observed for RabFab-VG1 (Fig. 10B). Similarly, no precipitation was observed in isolated rabbit vitreous bodies when comparing VG1 (Fig. 11A), RabFab-VG1 (Fig. 11B), or formulations containing equal concentrations (based on mass) of RabFab-VG1 and 10 kDa HA (Fig. 11C).

Compared to TSG6, VG1 with 10 kDa HA preforms is not always necessary to prevent precipitation in in vitro vitreous fluids.

Example 12. In vitro interaction between VG1 and vitreous fluid

A. Materials and Methods

The interaction between isolated VG1 and Fab-VG1 Fab-HABD and ex vivo vitreous fluid was examined using fluorescence correlation spectroscopy (FCS). VG1 and Fab-VG1 were covalently labeled with lysine residues using PEG4-DY647-N-hydroxysuccinimide ester. The fluorescence emission of DY647 could be detected by laser excitation at 594 nm or 633 nm and at longer wavelengths. The reaction chemistry was controlled so that the labeling level of each molecule was no greater than one fluorescent dye. Porcine vitreous fluid 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 sample was added to each diluted aliquot to bring the final concentration to 20 nM. The test samples were (1) free VG1, (2) pigFab-VG1, (3) pigFab-VG1 mixed with 10kDa HA at a 1:1 weight ratio, (4) RabFab-VG1, and (5) RabFab-VG1 mixed with 10kDa HA at a 1:1 weight ratio. After incubation at room temperature for 2 hours, FCS was performed.

B. Result

The FCS measurements are shown in Figure 12. Compared to incubation alone in buffered saline (PBS), all samples exhibited significantly delayed diffusion when incubated with undiluted or slightly diluted vitreous solution. For free VG1, PigFab-VG1, and RabFab-VG1, this delayed diffusion persisted until the vitreous solution was diluted more than 6000-fold (Figure 12, rows 3, 4, 6, and 7; from undiluted to a dilution factor of 6561). Slow diffusion was also observed in samples co-prepared with 10 kDa HA, but this effect disappeared when the vitreous solution was diluted ≥729-fold (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). These results indicate a strong interaction between the vitreous components (most likely endogenous high molecular weight HA from the vitreous solution) and the VG1-containing samples. Even in the presence of low molecular weight HA, VG1 can interact with endogenous HA at low vitreous fluid dilutions (Fig. 12, row 5: PigFab-VG1 + 10 kDa HA (1:1), and row 8: RabFab-VG1 + 10 kDa HA (1:1)). This indicates that VG1 and Fab-VG1 can dissociate from 10 kDa HA and bind to HA present in the vitreous fluid. However, once the vitreous fluid is significantly diluted, there will be no sufficiently high concentration of high molecular weight HA to compete with VG1 for binding to low molecular weight HA (Fig. 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). Compared to unbound materials, VG1 bound to low MW HA exhibits a small or negligible slowdown in diffusion (Fig. 12, PBS controls for row 5: PigFab-VG1+10kDa HA (1:1), and row 8: RabFab-VG1+10kDa HA (1:1); these samples have 10kDa HA without added glass).

Example 13. Effect of precomposite with 10kDa 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 in phosphate-buffered saline (PBS) pH 7.4, with or without the addition of 1.8 mg/mL 10 kDa HA. The concentration of 1.8 mg/mL (180 μM) of 10 kDa HA was a 5-fold molar excess relative to the concentration of anti-HtrA1-VG1 (35 μM). These preparations were incubated at 37 °C for 4 weeks and then analyzed by non-reducing capillary electrophoresis with sodium dodecyl sulfate (NR CE-SDS), as described by Michels et al. 2007 (Anal. Chem. 79, 5963). In addition to monomeric species and fragments, anti-SDS denatured aggregates could be detected by NR CE-SDS.

B. Result

As shown in Figure 13 and summarized in Table 19, pre-compounding with 10 kDa HA inhibited the formation of SDS-stabilized aggregates in anti-HtrA1-VG1. The formation rate of the high molecular weight form (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 its effect on aggregation; however, the formation rate of the low molecular weight form (LWMF) decreased by approximately two-fold when compounded with HA. These results suggest that the inclusion of 10 kDa HA in the formulation stabilizes anti-HtrA1-VG1 under thermal stress conditions at neutral pH.

Example 14. Intraocular Tolerance of VG1 and VG1Fab-HABD A. Materials and Methods

A.1. Intravitreal Transfusion (IVT) and Endpoint Assessment

The study was designed to evaluate the tolerability of IVT injections using VG1 and Fab-VG1Fab-HABD. The study design is shown in Table 20.

Each miniature pig received a single 50 μL injection via IVT in both eyes. This volume was well tolerated in miniature pigs based on historical data. The IVT procedure was performed by a certified veterinary ophthalmologist. Group 1 miniature pigs received a mediator control injection. Group 2 miniature pigs received isolated WT VG1 (produced as described in Example 8 above). Group 3 miniature pigs received pigFab-VG1 (produced as described in Example 10 above). Group 4 miniature pigs received pre-prepared pigFab-VG1 with an equal weight of 10 kDa HA. All test samples were prepared to the specified protein concentration using 20 mM histidine-acetate and 150 mM NaCl (pH 5.5). The doses of pigFab-VG1 in Groups 3 and 4 represent the maximum feasible dose to maintain total endotoxin levels at less than 0.05 endotoxin units (EU) per eye. This level of endotoxin has been found to be tolerated in previous intraocular studies in miniature pigs. Given the molecular weight difference between WT VG1 (approximately 30 kDa) and pigFab-VG1 (approximately 80 kDa), the dose levels in Group 2 represent 1.6 HA-binding molar equivalents per dose, compared to Groups 3 and 4.

This study evaluated the following parameters and endpoints: mortality, clinical signs, weight, ophthalmological examination (examination, intraocular pressure measurement, wide field color fundus imaging, OCT imaging and electroretinography [ERG]), bioanalysis, toxicokinetics, anti-drug antibody assessment, overall autopsy results and histopathological examination.

Ophthalmoscopy was performed on both eyes of all surviving animals by a certified veterinary ophthalmologist using indirect ophthalmoscopy and slit-lamp biomicroscopy. Ophthalmological examinations were performed on all animals before treatment and on day 1 (after drug administration), and on days 3, 5, 8, 15, 17, 22, and 29.

Intraocular pressure (IOP) was measured in both eyes of all surviving animals by a certified ophthalmologist during an ophthalmic examination using the applanation method. IOP was measured in all animals before treatment and on day 1 (after drug administration), and on days 3, 5, 8, 15, 17, 22, and 29.

On day 29, wide-field color fundus imaging was performed on all surviving animals using a Clarity RetCam Shuttle. If visible, attempts were made to photograph the administered test specimen.

On day 29, optical coherence tomography was performed using the Heidelberg Spectralis HRA/OCT system; single, vertical, high-resolution line scans were performed via optical nerves.

On day 29, all surviving animals underwent ERG assessment. Animals were acclimatized in darkness for at least 1 hour prior to ERG. Full-field flash ERG with Ganzfeld dome stimulation (flash intensity conforming to ISCEV standard parameters and light acclimatization time of 5 minutes (Retiport Gamma, Roland Consult)) was used; amplitude and delay values were measured during recording.

Blood samples (approximately 0.5 mL) were collected from all surviving animals via the thoracic inlet and anterior vena cava to determine the serum concentration of the test substance. Animals were not fasted prior to blood collection, except at intervals consistent with other procedural fasting. Blood samples were collected once before treatment, on day 1 (6 and 12 hours after administration), and on days 2, 3, 5, 8, 12, 15, 22, and 29.

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

The presence of anti-drug antibodies (ADAs) in serum samples is detected in an ELISA assay. The test sample is immobilized on an assay plate, incubated with serum and washed, and then the immune complex is detected using an anti-pig IgG reagent, which has an Fc moiety that binds to horseradish peroxidase for enzymatic detection.

On day 15, aqueous humor was collected from all animals by a certified veterinary ophthalmologist. The eyeball was positioned using conjunctival forceps for aqueous humor collection, while the tip of a 31-gauge needle was inserted at approximately a 90-degree angle into the sclera behind the limbus. The angle 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 available volume of aqueous humor, up to 50 μL. The needle was removed, and the extrascleral tissue was brought close to the puncture site and clamped with conjunctival forceps. The same sampling procedure was performed on the other eye. The collected samples were stored in 1.0 mL glass-based tramates 2D barcode storage tubes and capped with TPE. The samples were frozen in liquid nitrogen and stored at -60°C to -90°C. The concentration of the test sample in the aqueous humor was determined using a mass spectrometry-based assay.

A.2. Sample Preparation

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

A.3. Labeled peptides

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

A.4. Analysis by mass spectrometry (MS)

Digestants from PigFab were separated using an ACQUITY UPLC CSHC18 peptide analysis column (1.7 μm, 1 mm × 100 mm) on an Acquity UPLC (Waters Corporation, Milford, MA) under gradient elution conditions. The column was maintained at 50 °C, and the autosampler sample tray was maintained at 8 °C. The mobile phase consisted of water (A) containing 0.1% FA and acetonitrile (B) containing 0.1% FA, at a flow rate of 0.04 mL/min. The sample was eluted with the following gradient: B was increased from 2% to 90% over 2 minutes, and then decreased to 2% B over 2 minutes to reequilibrate the column. The injection volume was 10 μL.

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

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

B. Result

All animals survived to the planned termination date. Therefore, unplanned euthanasia was not necessary. Ophthalmological findings related to the test subjects included vitreous opacities and very mild posterior uveitis in the injection area.

The ophthalmological examination results are as follows:

(1) Group 2 (WT VG1) – On day 1, very slight opacities were observed in the temporal vitreous in 2 of the 6 animals. This result subsided on day 3 and remained until the end of the study. One of the 4 animals developed very slight posterior uveitis on day 15, which subsided on day 17. The very slight 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 at the injection site of the test substance. This result persisted in all 6 animals on day 3, and on day 5, 4/4 of the animals showed signs of diffusion involving the central vitreous. From day 8 to day 22, we observed a reduction in the affected vitreous area, and on day 29, only 2 out of 4 animals had mild opacities in the temporal vitreous. The regional vitreous opacities did not appear to be inflammatory in nature, but appeared to have a localized effect on vitreous consistency.

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

At all points throughout the study period, the intraocular pressure of all animals remained within the normal range.

At any point in time, none of the animals exhibited anti-drug antibodies (ADA) against the test substance.

Color fundus photographs and optical coherence tomography (OCT) images taken on day 29 showed that all animals in groups 2 and 4 had normal vitreous and retinal morphology. All animals in group 3 had very slight localized vitreous opacities on fundus photographs and very slight posterior vitreous hypersensitivity on OCT, consistent with ophthalmic examination findings.

Electroretinography (ERG) was performed on all animals on day 29. At a Scotopic light intensity of 0.01, animal number 3006 showed a moderate decrease in b-wave amplitude in both eyes. All other light intensities were within the normal range, indicating a background abnormality affecting dim retinal function. Animals 2005 and 4003 showed some asymmetry between eyes, with a slight decrease in OD amplitude relative to OS. This result is suspected to be related to recording conditions, including off-center eye localization OD. No results indicating a case-effect were found in any animal.

No visual findings related to the test sample were found in the eye or optic nerve.

All gross observations of animals treated with the test agent were either background findings for the species or considered incidental and unrelated to the test agent. These observations were infrequent, lacked a clear dose relationship in terms of incidence or severity, and/or had no relevant microscopic findings related to the test agent.

No microscopic findings related to the test sample were found in the eye or optic nerve.

All microscopic observations were considered incidental and unrelated to the test sample. These observations were known background findings for the species and/or had similar incidence and severity for animals receiving control and test sample treatments.

The level of PigFab-VG1 in aqueous humor samples was determined by mass spectrometry. High aqueous humor levels were obtained and maintained for 30 days after intravitreal injection of 1.8 mg/eye of PigFab-VG1 or PigFab-VG1 pre-complexed with an equal mass of 10 kDa HA into miniature pig eyes (Figure 14). These results indicate the presence of measurable levels of the test sample in miniature pig eyes during the 4-week study period. The concentration at 30 days was at least an order of magnitude higher than the measured values for unmodified Fab (Figure 4A).

In Gottingen miniature pigs, single IVT administration of various dose levels of WT VG1 (1.13 mg/eye), pigFab-VG1 (1.8 mg/eye), or pigFab-VG1 + 10 kDa HA (1.8 mg/eye) was well tolerated. All animals survived to the planned termination time without any abnormal clinical findings and without impact on body weight. The lack of detectable immune responses to the test article with single injections during the study allowed for assessment of the test article's direct effects. Ophthalmological findings were limited to: transient, very mild vitreous opacities at the test article injection site that resolved by day 3 (WT VG-1); and vitreous opacities near the test article injection site that did not completely resolve by the end of the study (pigFab-VG1). No ophthalmoscopic findings were found with pigFab-VG1 + 10 kDa HA, and IOP, OCT, and ERG results were normal in all animals. There are no visual or microscopic effects related to the test sample in the eye or optic nerve.

Example 15. Efficacy of VPDF-VG1 in laser-induced choroidal neovascularization (laser CNV) in rats

Fab-HABD was investigated 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 inhibits neovascularization); and (2) the duration of in vivo efficacy of Fab-HABD is equivalent to or better than that of the unmodified Fab fragment.

A. Materials and Methods

Rats received intravitreal injections of a protein preparation one or three weeks prior to laser lesioning (six laser burns per eye). One week after the laser lesion was initiated, vascular lesions were analyzed using fluorescence angiography (FA).

Fab-HABD was compared with the corresponding unmodified Fab fragment. To test the durable efficacy of Fab-HABD, the dose of unmodified Fab was titrated to the “lowest effective dose” (i.e., a low degree of detectable inhibition of angiogenesis compared to the vector over the duration of the rat model), indicating that Fab-HABD had a more durable efficacy at the same dose and duration in the model.

B. Result

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

Example 16. Intraocular tolerance of VG1 and VG1 Fab-HABD in New Zealand white rabbits

A. Materials and Methods

The aim of this study was to determine the intraocular tolerability of the test products WT VG1, RabFab-VG1, and RabFab-VG1 pre-formulated with 1:1 (w/w) 10 kDa HA in male New Zealand white rabbits during a 30-day observation period following a single bilateral IVT injection. The study design is shown in Table 21.

This study evaluated the following parameters and endpoints: mortality, clinical signs, body weight, food consumption, ophthalmological (i.e., examination, intraocular pressure measurement, wide field color fundus imaging, OCT and ERG), bioanalysis, toxicokinetics, anti-drug antibody assessment, overall autopsy results, and histopathological examination.

B. Result

Based on assessments of body weight and food consumption, no systemic test-related effects were found. No macroscopic autopsy findings were also present, and all tissues were considered to be within normal ranges.

The presence of anti-drug antibodies (ADA) in rabbit serum was measured before administration and on days 8, 15, 22, and 29 of the study. Prior to administration, three out of six animals designated to receive WT VG1 had measurable serum ADA. Similarly, one-sixth and zero-sixths of animals, respectively, treated with RabFab-VG1 or RabFab-VG1 + 10kDa HA, had pre-existing serum ADA against the test substance. On day 8, all animals in the WT VG1 and RabFab-VG1 groups showed serum ADA against the test substance and remained positive throughout the study, while two-thirds of the animals in the RabFab-VG1 + 10kDa HA group had serum ADA. On day 15, all animals were positive for serum ADA, which remained positive until the end of the study.

In animals treated with WT VG1, RabFab-VG1, and RabFab-VG1 + 10kDa HA, clinical ophthalmoscopy findings were consistent with the development of anterior and posterior uveitis, but the severity varied between treatment groups. For example, WT VG1 resulted in moderate anterior and posterior uveitis by day 22, including posterior subcapsular cataracts in some eyes. In contrast, most animals treated with RabFab-VG1 showed mild uveitis at the same time point. Furthermore, animals treated with RabFab-VG1 + 10kDa HA showed only very mild to mild anterior and posterior uveitis. Within each treatment group, signs of uveitis improved by day 29 after initiation of systemic anti-inflammatory therapy on day 18 (which also included topical ocular treatment for animals receiving WT VG1). Decreased intraocular pressure was consistent with active uveitis, while changes in vitreous opacities corresponded to the severity of uveitis in different groups. In these cases, vitreous opacities were limited to mild in animals treated with RabFab-VG1 + 10kDa HA, while WT VG1 animals exhibited moderate opacities and posterior pole cataracts. The severity of treatment-dependent effects was also observed by OCT and ERG, with decreased scotopic and photopic amplitudes indicating severe alterations and degeneration of retinal function in eyes treated with WT VG1.

Compared to other experimental agents (where the RabFab-VG1 effect (Fig. 16B) was less severe and the RabFab-VG1+10kDaHA effect (Fig. 16C) was limited to the vitreous and only very slight to mild in severity), the endoscopic effect was most significant in eyes treated with WT VG1 (Fig. 16A). WT VG1-associated vitreous inflammation included the area near the optic disc, retinal detachment and varying degrees of necrosis, and an inflammatory cell layer adjacent to the posterior lens capsule. Furthermore, 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 very slight 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 very mild to moderate diffuse photoreceptor lesions, typically associated with reactive Muller cells. However, RabFab-VG1 photoreceptor layer lesions differ from WT VG1-associated retinal necrosis because the latter is selective only for the photoreceptor layer, while retinal necrosis involves multiple retinal layers. Furthermore, both WT VG1 and RabFab-VG1-treated eyes exhibited fibrovascular membrane features in the vitreous. In these cases, the membrane consisted of fibroblasts, abundant new blood vessels, and early collagen deposition. Traction bands detached from the membrane were also observed. Compared to WT VG1 and RabFab-VG1, the RabFab-VG1+10kDa HA-related effects were limited to very mild to mild mononuclear inflammation confined to the vitreous and internal limiting membrane.

In summary, a single IVT administration of WT VG1, RabFab-VG1, or RabFab-VG1 + 10kDa HA to New Zealand white rabbits led to the development of anterior and posterior uveitis, most severely in animals administered WT VG1, moderately in animals administered RabFab-VG1, and mild to moderately in animals administered RabFab-VG1 + 10kDa HA. Microscopic examination revealed significant retinal necrosis and retinal degeneration, respectively, associated with decreased ERG amplitude, in addition to inflammation, with WT VG1 and RabFab-VG1 administration. At the end of the study, no signs of active anterior uveitis were observed in the eyes administered RabFab-VG1 + 10kDa HA, and only very mild chronic posterior uveitis was observed. Although the interpretation of these results is confounded with the ADA observations for all test samples on day 15, the pre-complexation or binding of RabFab-VG1 with 10kDa HA does appear to improve the tolerability of this Fab-HABD in rabbit eyes.

Brain preservation of Fab-VG1

The ability to bind to HA via VG1 was demonstrated through intracerebroventricular injection in mice to achieve retention in the brain. For these purposes, the non-target binding antibody against herpes simplex virus-1 glycoprotein D (anti-gD) was used as the Fab fragment (anti-gD Fab), the complete 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 population. The protocol for using live animals (AUS 75-15; approved January 25, 2021) was approved by the University of Kansas Institutional Animal Care and Use Committee (IACUC). All animals were cared for by Animal Care Unit (ACU) personnel and veterinarians in a temperature-controlled environment with a 12-hour dark/light cycle, and with unrestricted access to food and water.

A.2. Binding of antibody to IRDye800CW NHS ester

According to the manufacturer's instructions, the antibody was conjugated with IRDye800. In short, the antibody was reacted with IRDye800 in PBS containing 10% potassium phosphate buffer at 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 conjugated 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 with 1.5-2% isoflurane and placed in a stereotaxic apparatus (Stoelting Co.). A midline sagittal incision was made to expose the skull and mark the anterior fontanelle. A small incision was made 1.0 mm on the right side of the skull and 0.3 mm anterior to the anterior fontanelle. A 10 μL Hamilton syringe (serial number 7762-06) with a detachable 33-gauge needle was fitted 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. 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 sample was kept on ice until centrifuged at 10,000 × g for 3 minutes, and the plasma was stored at -80°C until analysis. Mice were sacrificed by deep anesthesia with 4-5% isoflurane followed by cardiac perfusion with ice-cold HBSS containing 0.1% Tween-20 at different time points. Brain, heart, lung, liver, spleen, and kidney were collected and stored on ice until analysis.

A.4. Antibody Organ Quantification

The isolated organs were weighed and mechanically homogenized in 1 mL PBS. Standard near-infrared fluorescence (NIRF) antibody solutions were prepared by diluting the stock solution with different volumes of PBS. Calibration curves were then generated for each organ in a 96-well plate by adding 10 μL of the standard solution to 100 μL of homogenized blank organ and scanning each well using an Odyssey Clx scanner. The fluorescence intensity of each well was plotted against the antibody concentration per gram of organ to obtain a linear curve. Organs from mice that received intraventricular injection were compared with the calibration curve to determine antibody deposition. Similarly, plasma analysis was performed using blank plasma first diluted 5-fold. Then, 10 μL of antibody standard was added to an aliquot of 100 μL of diluted plasma to generate a standard curve, which was compared with plasma samples from mice that received intraventricular injection.

B. Result

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

The crystallization conditions for HA-bound WT VG1 were determined using commercial crystallization screening (Hampton Research and Qiagen). The structure of HA-bound VCAN was obtained by soaking crystals in HA6-mer. Crystals were harvested and rapidly frozen in liquid nitrogen without cryoprotectant. Diffraction data were collected using beamlines 12-2 or 14-1 of the Stanford Synchrotron Radiation Facility (SSRL) on Pilatus 6M or Eiger 16M detectors (Dectris), respectively. The structure was iteratively refined by modeling in COOT and then further refined using REFMAC5, BUSTER, or Phenix-Refine. Adams, P.D. et al., Acta Crystallogr. D. Biol. Crystallogr., 66(Pt 2):213-221 (2010); Blanc, E. et al., Acta Crystallogr. D. Biol. Crystallogr., 60:2210-2221 (2004); Emsley, P. et al., Acta Crystallogr. D. Biol. Crystallogr., 66(Pt 2):213-221 (2010); l.Crystallogr.,66:486-501(2010); Emsley and Cowtan, ActaCrystallogr.D Biol.Crystallogr.,66:2 126-2132 (2004); Murshudov, G.N. et al., ActaCrystallogr. D Biol. Crystallogr., 67: 355-367, (2011).

The structure of the WT VG1-HA conjugate was analyzed using PyMol and/or Chimera (Figure 18), and the residues that interact 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 fluorometry (DSF). Briefly, 0.1 mg/mL of purified protein was mixed with Sypro Orange dye in PBS. Each sample was treated in increments of 0.05°C/sec over a temperature gradient from 25°C to 95°C, and the increase in fluorescence was monitored at 585 nm. The original fluorescence units were plotted as negative derivatives using a custom Excel macro, and Tm was calculated.

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

Based on the crystal structure of the WT VG1-HA conjugate (Figure 18), it was found that HA binds only to the link1 domain. Therefore, modeling was used to predict link 2 residues that might be involved in HA binding. To verify the crystal structure and identify mutants that weaken the HA binding affinity of WT VG1, the crystal contact residues were mutated to alanine, or in some cases, to a substitute amino acid. In addition, some WT VG1 residues that do not form crystal contacts with HA but are important for HA binding in TSG6 (based on sequence alignment between the VG1 linker domain structure and the TSG-6 linker domain; Figure 8B) were also mutated to alanine. To examine the combinatorial effects of the mutations, several two-site mutants were formed, such as Lys260 and Phe261 becoming Arg and Tyr (KF260RY), respectively, and HA binding ability was tested. Table 22 lists the VG1 variants generated as described in Example 10. The amino acid sequence alignments of the generated and tested VG1 variants are shown in Figure 19.

A.4. Molecular Properties

As described in Example 10, HA binding was measured by SPR. The mutant was injected 120 seconds later and dissociation was monitored for 180 seconds.

B. Result

The B.1.VG1 variant has reduced HA binding levels.

The mutants R160A, Y161A, and D197A exhibited weakened HA binding in the range of 2 to 7 μM. Table 23 shows the measured k _{_a} (M ^{^-1}s^{^-1} ), k _{_d} (s^{^-1} ), and k_{_D} (M) of each VG1 variant as determined by SPR.

B.2. Stability of VG1 variants

Table 24 shows the generated VG1 mutants and the measured Tm (heat-breaking temperature; °C) for each mutant. Although most mutants had a slight decrease or no effect on thermal stability compared to WT VG1, Y208A and H306A showed improvements in Tm of 2.16 °C and 2.81 °C, respectively.

equal form

The foregoing written description is considered sufficient to enable those skilled in the art to practice these embodiments. The foregoing description and examples detail certain embodiments and describe the best mode envisioned by the inventors. However, it should be understood that, regardless of the level of detail in the foregoing text, the embodiments can be practiced in various ways and should be interpreted in accordance with the appended claims and any equivalents thereof.

As used herein, the term "about" refers to index values (including, for example, integers, fractions, and percentages), whether explicitly stated or not. The term "about" generally refers to a range of values (e.g., +/- 5-10%) that a person skilled 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 values or ranges, these terms modify all values or ranges provided in the list. In some cases, the term "about" may include a value rounded to the nearest significant figure.

Claims (95)

1. A therapeutic molecule targeting a patient's tissue, comprising a hyaluronic acid binding domain and a therapeutic agent, wherein the hyaluronic acid binding domain comprises at least two linker domains of a multifunctional proteoglycan. 2. A therapeutic molecule targeting a patient's tissue, comprising a hyaluronic acid binding domain and a therapeutic agent, wherein the hyaluronic acid binding domain comprises at least two linker domains of a multifunctional proteoglycan bound to hyaluronic acid via an HA-binding domain. 3. The therapeutic molecule according to claim 1 or 2, wherein the hyaluronic acid ranges from 400 Da to 200 kDa. 4. The therapeutic molecule according to claim 3, wherein the hyaluronic acid has a content of at least 5 kDa. 5. The therapeutic molecule according to claim 3 or 4, wherein the hyaluronic acid is 10 kDa. 6. The therapeutic molecule according to any one of claims 1 to 5, wherein the hyaluronic acid provides a molar excess binding equivalent to the linker domain of the multifunctional proteoglycan. 7. The therapeutic molecule according to any one of claims 1 to 6, wherein the hyaluronic acid is permitted to be in a ratio of hyaluronic acid to therapeutic molecule ranging from 1.5:1 to 1:1. 8.86 The therapeutic molecule according to any one of claims 1 to 7, wherein the hyaluronic acid binding domain has a _KD of 10 nM to 10 μM. 9. The therapeutic molecule according to any one of claims 1 to 8, wherein the hyaluronic acid binding domain has a _KD of 5 nM to 8 μM. 10. The therapeutic molecule according to any one of claims 1 to 9, wherein the hyaluronic acid binding domain has a _KD of 100 nM to 5 μM. 11. The therapeutic molecule according to any one of claims 1 to 10, wherein the hyaluronic acid 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. 12. The therapeutic molecule according to any one of claims 1 to 11, wherein the hyaluronic acid binding domain is at least 95% identical to 86, 60, 32 or 29. 13. The therapeutic molecule according to any one of claims 1 to 12, wherein the hyaluronic acid binding domain comprises at least one, at least two, at least three, at least four, or at least five mutations. 14. The therapeutic molecule according to any one of claims 1 to 13, wherein the hyaluronic acid binding domain comprises one to three mutations, wherein the one to three mutations comprise a single amino acid substitution, a double amino acid substitution, and a truncation. 15. The therapeutic molecule according to any one of claims 1 to 14, wherein the hyaluronic acid binding domain comprises one to five mutations, wherein the one to five mutations comprise a single amino acid substitution, a double amino acid substitution, and a truncation. 16. The therapeutic molecule according to any one of claims 1 to 15, wherein the hyaluronic acid binding domain has a truncated mutation relative to SEQ ID NO:29. 17. The therapeutic molecule according to 16, wherein the truncated mutation comprises a truncation of 1 to 129 amino acids at the N-terminus. 18. The therapeutic molecule according to any one of claims 1 to 17, wherein the hyaluronic acid binding domain is a truncated sequence, wherein the Ig domain of the wild-type multifunctional proteoglycan is absent. 19. The therapeutic molecule according to any one of claims 1 to 18, wherein the hyaluronic acid 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. 20. The therapeutic molecule according to any one of claims 1 to 19, wherein the hyaluronic acid 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. 21. The therapeutic molecule according to any one of claims 1 to 20, wherein the hyaluronic acid binding domain 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. 22. The therapeutic molecule according to any one of claims 1 to 21, wherein the hyaluronic acid binding domain comprises mutations in 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. 23. The therapeutic molecule according to any one of claims 1 to 22, wherein the hyaluronic acid binding domain comprises 2, 3, 4, 5, or 6 mutations relative to the following positions in SEQ ID NO: 29: R160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326, and R327. 24. The therapeutic molecule according to any one of claims 1 to 23, wherein the hyaluronic acid 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. 25. The therapeutic molecule according to any one of claims 1 to 24, wherein the hyaluronic acid binding domain comprises at least one of Y208A and H306A. 26. The therapeutic molecule according to any one of claims 1 to 25, wherein the hyaluronic acid binding domain 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, D197 S, Y208A, Y208F, R214K, M222A, Y230A, Y230F, R233A, K260A, K260R, F261Y, KF260RY, D 295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L325A, Y326A, R327A and LYR325LFK. 27. The therapeutic molecule according to any one of claims 1 to 26, wherein the hyaluronic acid 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. 28. The therapeutic molecule according to any one of claims 1 to 27, wherein the hyaluronic acid 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:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:30 ...40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 D 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 N O: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. 29. The therapeutic molecule according to any one of claims 1 to 28, wherein the therapeutic active agent comprises an oligopeptide, a protein, or a nucleic acid. 30. The therapeutic molecule according to any one of claims 1 to 29, wherein the therapeutic active agent comprises an antibody, an antigen-binding fragment, a cysteine-binding peptide, a growth factor, or an aptamer. 31. The therapeutic molecule according to claim 30, wherein the therapeutic active agent is capable of binding an antigen. 32. The therapeutic molecule according to 31, wherein the therapeutic active agent is capable of binding VEGF, HtrA1, IL-33, C5, factor P, factor D, EPO, EPOR, IL-1β, IL-17A, IL-10, TNFα, FGFR2, PDGF or ANG2. 33. The therapeutic molecule according to claim 31 or 32, wherein the therapeutic active agent is an antibody or an antigen-binding fragment thereof (including but not limited to Fab fragment, F(ab')2 fragment, Fab' fragment, VhH fragment, scFv fragment, scFv-Fc fragment or microantibody). 34. The therapeutic molecule according to claim 31 or 32, wherein the therapeutic active agent is an oligopeptide or a protein. 35. The therapeutic molecule according to claim 29 or 34, wherein the oligopeptide or protein is a cysteine-bound peptide or an enzyme. 36. The therapeutic molecule according to 113, 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. 37. The therapeutic molecule according to any one of claims 1 to 108, wherein the therapeutic active agent is a growth factor, the 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). 38. The therapeutic molecule according to 110, wherein the therapeutic active agent binding to VEGF comprises ranibizumab, aflibercept, broxolizumab-dbll, and bevacizumab. 39. The therapeutic molecule according to any one of claims 1 to 110, wherein the therapeutic active agent is a nucleic acid. 40. The therapeutic molecule according to 117, wherein the nucleic acid is an aptamer, an antisense oligonucleotide, and/or a locked nucleic acid. 41. The therapeutic molecule according to claim 118, wherein the aptamer binds to VEGF. 42. The therapeutic molecule according to any one of claims 108, 118 or 119, wherein the aptamer is polyethylene glycol-treated. 43. The therapeutic molecule according to any one of claims 108 or 118 to 120, wherein the aptamer is 44. The therapeutic molecule according to any one of claims 1 to 121, wherein the therapeutic agent is covalently linked to the hyaluronic acid binding domain via a connector. 45. The therapeutic molecule according to 122, wherein the linker is at least 4 amino acids. 46. The therapeutic molecule according to 122 or 123, wherein the linker is no longer than 50 amino acids. 47. The therapeutic molecule according to any one of claims 122 to 124, wherein the linker is 4 to 25 amino acids. 48. The therapeutic molecule according to any one of claims 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). 49. The therapeutic molecule according to any one of claims 122 to 126, wherein the connector comprises GGGS (SEQ ID NO: 84) or a polymer thereof, more particularly comprising (GGGGS)3 (SEQ ID NO: 85). 50. The therapeutic molecule according to any one of claims 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). 51. The therapeutic molecule according to any one of claims 122 to 125 or 128, wherein the connector comprises GGSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95). 52. The therapeutic molecule according to any one of claims 1 to 107, wherein the therapeutic active agent comprises an anti-VEGF antigen-binding moiety and a cysteine knot peptide. 53. The therapeutic molecule according to claim 30, 35, 36 or 52, wherein the cysteine binding peptide is connected to the hyaluronic acid binding domain via a linker comprising the sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95). 54. The therapeutic molecule according to claim 52 or 53, wherein the sequence comprises (a) an anti-VEGF antigen binding portion; and (b) has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:93 or SEQ ID NO:94. 55. The therapeutic molecule according to any one of claims 1 to 54, wherein the hyaluronic acid binding domain is capable of non-covalently binding to hyaluronic acid. 56. A method of delivering a therapeutic molecule to a patient’s tissue, comprising administering the therapeutic molecule according to any one of claims 1 to 133 to the patient, and allowing the therapeutic molecule to provide long-lasting delivery of a therapeutically active agent to the target tissue. 57. The method of claim 56, further comprising binding the therapeutic molecule to hyaluronic acid prior to the application step. 58. The method of claim 57, further comprising mixing a first solution containing the therapeutic molecule with a second solution containing the hyaluronic acid. 59. The method of claim 58, wherein the mixing comprises a container. 60. The method of claim 59, wherein the container is a two-chamber syringe. 61. The method according to any one of claims 58 to 60, wherein the mixing produces a therapeutic molecule bound to hyaluronic acid, the therapeutic molecule being prepared for application to a subject. 62. The method according to any one of claims 56 to 61, wherein the application step is a single injection. 63. The method according to any one of claims 56 to 62, wherein the target tissue includes the eye or the brain. 64. The method according to any one of claims 56 to 63, 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 therapeutic agent. 65. A therapeutic molecule comprising: a. A first component capable of binding to therapeutic targets in the eye. b. One or more second components capable of binding to hyaluronic acid, wherein the one or more second components are covalently bound to the first component, and c. Optionally, it comprises one or more third components containing hyaluronic acid. Wherein, if one or more third components are present, the one or more third components are non-covalently bound to the one or more second components. 66. The therapeutic molecule of claim 65, wherein the first component is a protein, peptide, receptor or fragment thereof, receptor ligand, darpin, nucleic acid, RNA, DNA or aptamer. 67. The therapeutic molecule according to claim 65 or 67, wherein the first component is selected from antibodies, antigen-binding fragments, particularly antibody fragments, more particularly antibody fragments lacking at least an Fc domain, especially wherein the fragment is or comprises (Fab') ₂ fragments, Fab' fragments, Fab fragments, VhH fragments, scFv fragments, scFv-Fc fragments, and microantibodies, more particularly Fab fragments. 68. The therapeutic molecule according to any one of claims 65 to 68, wherein the second component comprises a hyaluronic acid receptor CD44 (CD44) domain, a brain-specific connecton (BRAL1) domain, a tumor necrosis factor-stimulated gene 6 (TSG-6) domain, a lymphatic endothelial hyaluronic acid receptor-1 (LYVE-1) domain or a hyaluronic acid binding protein (HABP) domain, an agglutinin G1 (AG1) domain or a multifunctional proteoglycan G1 (VG1) domain. 69. The therapeutic molecule according to any one of claims 65 to 69, wherein the conjugate comprises one second component or two second components that are identical to each other. 70. The therapeutic molecule according to any one of claims 65 to 69, wherein the third component is hyaluronic acid, wherein the hyaluronic acid a. Has the following molecular weight: i. Selected from 3kDa to 60kDa, 4kDa to 30kDa, 5kDa to 20kDa, or 400Da to 200kDa; ii. It is at least 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, or 9kDa; or iii. It is at most 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa, or 15kDa; b. Providing a molar excess of binding equivalent to one or both of the second components; and c. It has modifications that reduce the degradation of hyaluronic acid in the eye. 71. The therapeutic molecule according to any one of claims 65 to 69, wherein the second component is capable of binding to hyaluronic acid at a _KD of 10 nM to 10 μM, 5 nM to 8 μM, or 100 nM to 5 μM. 72. The therapeutic molecule according to any one of claims 65 to 71, wherein a. The first component and the second component are contained in a fusion protein, particularly wherein one or both of the second components are covalently bound to the N-terminus and/or C-terminus of the first component, more particularly wherein the first component is an antibody or antigen-binding 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 one or both second components are directly bound to the first component or indirectly bound to the first component via a linker, wherein the linker is particularly a linker of at least 4 amino acids and/or at most 50 or at most 25 amino acids, more particularly the linker is (GxS) _n or (GxS) _{nGm ,} where 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). 73. The therapeutic molecule according to any one of claims 65 to 72, 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. 74. The therapeutic molecule according to any one of claims 65 to 73, wherein a. The first component is an antibody or antigen-binding fragment against VEGF; and/or b. Each of the one or two second components comprises a CD44 domain, a TSG-6 domain, or a VG1 domain; and/or c. The third component is hyaluronic acid with a molecular weight of 5kDa to 20kDa. 75. The therapeutic molecule according to any one of claims 65 to 74, wherein a. The first component is an anti-VEGF antibody or an antigen-binding fragment, one or two second components contain a CD44 domain, and the third component is hyaluronic acid with a molecular weight of 5 kDa to 20 kDa; b. The first component is an anti-VEGF antibody or an antigen-binding fragment, one or both of the second components contain a TSG-6 domain, and the third component is hyaluronic acid with a molecular weight of 5 kDa to 20 kDa; or c. The first component is an anti-VEGF antibody or an antigen-binding fragment, one or two second components contain a VG1 domain, and the third component is hyaluronic acid with a molecular weight of 5 kDa to 20 kDa. 76. The therapeutic molecule according to any one of claims 65 to 75, wherein a. The first component contains i. VH domains of SEQ ID NO: 97, 99, 105, 109 or 114; and ii. VL domains of SEQ ID NO: 98, 100, 106, 110, or 115; and b. The second component comprises SEQ ID NO:2. 77. The therapeutic molecule according to any one of claims 65 to 75, wherein a. The first component contains i. VH domains of SEQ ID NO: 97, 99, 105, 109 or 114; and ii. VL domains of SEQ ID NO: 98, 100, 106, 110, or 115; and b. The second component comprises SEQ ID NO:4. 78. The therapeutic molecule according to any one of claims 65 to 75, wherein a. The first component contains i. VH domains of SEQ ID NO: 97, 99, 105, 109 or 114; and ii. VL domains of SEQ ID NO: 98, 100, 106, 110, or 115; and b. The second component comprises SEQ ID NO: 86, 60, 32 or 29. 79. The therapeutic molecule of claim 78, 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. 80. The therapeutic molecule of claim 78 or 79, wherein the second component comprises one to five mutations, wherein the one to five mutations comprise a single amino acid substitution, a double amino acid substitution, and/or truncation. 81. The therapeutic molecule according to any one of claims 78 to 80, wherein the second component has a truncated mutation relative to SEQ ID NO:29. 82. The therapeutic molecule of claim 81, wherein the truncated mutation comprises a truncation of 1 to 129 amino acids at the N-terminus. 83. The therapeutic molecule according to any one of claims 78 to 82, wherein the second component is a truncated sequence in which the Ig domain of the wild-type multifunctional proteoglycan is absent. 84. The therapeutic molecule according to any one of claims 78 to 83, wherein the second component comprises a mutation in one, two, three, four, five, or six 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. 85. The therapeutic molecule according to any one of claims 78 to 84, wherein the second component comprises at least 1, 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. 86. The therapeutic molecule according to any one of claims 78 to 85, wherein the second component comprises at least one of Y208A and H306A. 87. The therapeutic molecule according to claim 78 or 79, 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:4 3. 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. 88. The therapeutic molecule according to any one of claims 65 to 87, wherein the first component further comprises a cysteine knot peptide. 89. The therapeutic molecule of claim 88, wherein the cysteine binding peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:92. 90. The therapeutic molecule according to claim 88 or 89, wherein the amino acid sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:93 or SEQ ID NO:94. 91. A composition for use as a medicament, said composition comprising a therapeutic molecule according to any one of claims 65 to 90 and optionally a pharmaceutical excipient, diluent, or carrier. 92. A composition for treating an eye disease or a brain disease, said composition comprising a therapeutic molecule according to any one of claims 65 to 90 and optionally a pharmaceutical excipient, diluent, or carrier. 93. The composition used according to claim 92, formulated for intraocular delivery, particularly for intravitreal injection. 94. The composition used according to claim 92 or 93, 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). 95. A method of delivering a therapeutic molecule to a patient’s tissue, comprising administering the therapeutic molecule according to any one of claims 65 to 90 or the composition according to any one of claims 91 to 94 to the patient, and allowing the therapeutic molecule to provide long-term delivery of a first component to the target tissue.