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

Non-covalent protein-hyaluronic acid conjugates for long-acting ocular delivery Download PDF

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Publication number
CN116761634A
CN116761634A CN202180070826.3A CN202180070826A CN116761634A CN 116761634 A CN116761634 A CN 116761634A CN 202180070826 A CN202180070826 A CN 202180070826A CN 116761634 A CN116761634 A CN 116761634A
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China
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seq
component
therapeutic molecule
domain
binding
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CN202180070826.3A
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Chinese (zh)
Inventor
S·登格尔
R·F·凯利
H·科腾贝格
R·汉努什
S·T·汉森
P·M·休尔斯曼
S·C·梅塔
D·B·特萨
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F Hoffmann La Roche AG
Genentech Inc
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F Hoffmann La Roche AG
Genentech Inc
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Priority claimed from PCT/EP2021/078433 external-priority patent/WO2022079161A1/en
Publication of CN116761634A publication Critical patent/CN116761634A/en
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Abstract

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

Description

Non-covalent protein-hyaluronic acid conjugates for long-acting ocular delivery
Cross Reference to Related Applications
The present application claims the benefit of priority from U.S. provisional patent application No. 63/092,251, filed on 10, 15, 2020, and U.S. provisional patent application No. 63/250,782, filed on 9, 2021, both of which are commonly owned with the present application and are expressly incorporated herein in their entireties by reference as if fully set forth herein.
Technical Field
Long-acting therapies and methods of treatment employing fusion proteins and fusion protein-hyaluronic acid conjugates that bind to hyaluronic acid.
Background
Intravitreal (IVT) injections are commonly used to administer drugs to treat various ocular diseases. IVT injection allows for the administration of drugs directly after the eye, thereby eliminating the usual disorders of local and systemic administration. The direct administration of the drug in this manner allows for a higher intraocular bioavailability of the drug in posterior segment tissue, thereby more effectively treating posterior ocular disorders. Stewart, M.W., expert Opinion on Drug Metabolism & Toxicology,14 (1): 5-7 (2018). Examples of common diseases that are treated via IVT injection include age-related macular degeneration (AMD), diabetic retinal degeneration, retinal vein occlusion, and eye infections (such as endophthalmitis and retinitis). U.S. retinal expert society foundation, asrs.org/components/real-diseases/33/IVT-indexes (2017).
Despite the encouraging results in preventing disease and improving vision, IVT injections are uncomfortable and expensive and require retinal specialists to perform. IVT injections are known to cause adverse effects on some patients, such as infection, inflammation, vitreous hemorrhage, increased mosquito present in the eye, increased sensitivity to light, decreased vision, and retinal detachment. U.S. retinal expert society foundation, asrs.org/components/real-diseases/33/IVT-indexes (2017). IVT injections may also be associated with infectious endophthalmitis, aseptic endophthalmitis, porogenic retinal detachment, elevated intraocular pressure and ocular bleeding. The same applies above. The extended delivery technique via the eye can eliminate the need for repeated injections of medication, thereby improving patient compliance and clinical outcome. Methods and compositions for extending drug half-life in vitreous (e.g., maintaining drug storage capacity, low turnover in the eye, low target-retention mediated clearance, and/or quasi-stable properties in the elderly population) promote slow release of drug from the injection site to the target site, enabling higher doses to be used and reducing the number of injections required.
The vitreous half-life of a therapeutic molecule can be extended by: therapeutic molecules are bound to Hyaluronic Acid (HA) as an alternative to encapsulation or chemical modification with polymers. Cromwell, S et al, invest. Ophthalmol. Vis. Sci.59 (9): 235 (2018); ghosh, J.G. et al Nature Communications,8:14837, doi:10.1038/ncomms14837 (2017); stewart, M.W., expert Opinion on Drug Metabolism & Toxicology,14 (1): 5-7 (2018). In a specific example, the long-acting anti-VEGF antibodies are fused to the HA binding domain (HABD) of human Tumor Necrosis Factor (TNF) -stimulating gene 6 protein (TSG-6), respectively. Ghosh, J.G. et al Nature Communications,8:14837, doi:10.1038/ncomms14837 (2017). The fusion protein showed the following improvements compared to the unmodified anti-VEGF antibody: (1) 3 to 4 times increase in half-life; (2) In an animal model of neovascular retinal disease, VEGF-induced retinal changes can be attenuated over a 3 to 4-fold longer period. 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 to TSG-6 has entered clinical trials for evaluating the safety and tolerability of a single incremental dose to determine the Maximum Tolerated Dose (MTD) in neovascular age-related macular degeneration (nvAMD). clinicaltrias.gov/ct 2/show/NCT02398500 (2019). However, these tests were suspended due to serious adverse events including vitreomosquitos, inflammation and post-vitreous detachment.
Chemical binding of the antibody fragment to Hyaluronic Acid (HA) can reduce the diffusivity of the drug from the vitreous. However, this method requires chemical activation of HA; the use of non-native linkers may result in the production of non-native metabolites by activated HA in the subject.
The inventors have found that the above disadvantages can be avoided by providing a conjugate comprising: (1) a first component capable of binding to a therapeutic target in the eye, (2) one or more second components capable of binding to HA, and (3) one or more third components comprising HA; wherein each second component is (a) covalently bound to the first component and (b) non-covalently bound to the third component. Unlike the anti-VEGF antibodies and TSG-6 fusion proteins (LMG 324) described above, the second component capable of binding to HA is pre-complexed with HA.
Materials and methods for increasing intraocular retention of therapeutic molecules comprising fusion proteins capable of binding Hyaluronic Acid (HA) are disclosed. In some embodiments, the fusion protein comprises: (1) A first component capable of binding to a therapeutic target in the eye, and (2) one or more second components capable of binding to HA; wherein each second component is covalently bound to the first component.
The application also discloses conjugates wherein the fusion further comprises one or more third components comprising HA, wherein each second component is further non-covalently bound to a third component. Further, the second component capable of binding to HA may be pre-complexed with HA. The conjugate is vitreous compatible and HAs binding affinity for HA. The materials and methods provide a platform technology for longer drug-in-life designs.
Disclosure of Invention
The present application discloses a material and method relating to therapeutic molecules and conjugates thereof capable of binding to therapeutic targets in the eye and capable of binding to hyaluronic acid. The 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, one or more third components comprising hyaluronic acid, wherein the one or more third components, if present, are non-covalently bound to the one or more second components.
Item 2 is the therapeutic molecule of item 1, wherein the first component is a protein, peptide, receptor or fragment thereof, ligand for a receptor, darpin, nucleic acid, RNA, DNA, or aptamer.
Item 3 is the conjugate of item 1 or 2, whereinThe first component is selected from the group consisting of an antibody, an antigen binding fragment, particularly an antibody fragment, more particularly an antibody fragment lacking at least an Fc domain, particularly wherein the fragment is or comprises (Fab') 2 Fragments, fab' fragments or Fab fragments, vhH fragments, scFv-Fc fragments and miniantibodies, more particularly Fab fragments.
Item 4 is the therapeutic molecule of any one of items 1 to 3, wherein the second component comprises a hyaluronic acid receptor CD44 (CD 44) domain, a brain-specific junction protein (BRAL 1) domain, a tumor necrosis factor stimulating gene 6 (TSG-6) domain, a lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1) domain, or a Hyaluronic Acid Binding Protein (HABP) domain, an aggrecan G1 (AG 1) domain, or a multifunctional proteoglycan G1 (VG 1) 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 the therapeutic molecule of any one of items 1 to 4, 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) Is at least 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa or 9kDa; or (iii) is at most 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa or 15kDa; (b) Providing a molar excess of binding equivalents to one or both of the second components; and (c) has a modification that reduces degradation of hyaluronic acid in the eye.
Item 7 is the therapeutic molecule of any one of items 1 to 6, wherein the second component is capable of K at 10nM to 10. Mu.M, 5nM to 8. Mu.M, or 100nM to 5. Mu.M D Binds to hyaluronic acid.
Item 8 is the therapeutic molecule of any one of items 1 to 7, wherein (a) the first and second components are comprised 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 the one or both of the second components are covalently bound to the C-terminus of the first component; and/or (b)) These one or two second components are bound directly to the first component or indirectly to the first component via a linker, in particular a linker of at least 4 amino acids and/or of at most 50 or at most 25 amino acids, more in particular the linker is (GxS) n Or (GxS) n G m 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).
Item 9 is the therapeutic molecule of any one of items 1 to 8, wherein the therapeutic target is VEGF, C2, C3a, C3b, C5a, htra1, IL-33, factor P, factor D, EPO, EPOR, IL-1 β, IL-17A, IL-10, tnfα, FGFR2, PDGF or ANG2, in particular VEGF.
Item 10 is the therapeutic molecule of any one of claims 1 to 9, wherein (a) the first component is an antibody or antigen binding fragment against VEGF, in particular an anti-VEGF Fab; and/or (b) each of these one or two second components comprises a CD44 domain or a TSG-6 domain or a VG1 domain; and/or (c) the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa.
Item 11 is the therapeutic molecule of any one of items 1 to 10, wherein (i) the first component is an anti-VEGF antibody or antigen-binding fragment, the one or two second components comprise a CD44 domain, and the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa; (ii) The first component is an anti-VEGF antibody or antigen-binding fragment, the one or two second components comprise a TSG-6 domain, and the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa; or (iii) the first component is an anti-VEGF antibody or antigen-binding fragment, the one or two second components comprise VG1 domains, and the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa.
Item 12 is the conjugate of any one of items 1 to 11, wherein (a) the first component comprises (i) a VH domain of SEQ ID No. 97, 99, 105, 109 or 144; and (ii) a VL domain of SEQ ID NO 98, 100, 106, 110 or 115; and (b) the second component comprises SEQ ID NO. 2.
Item 13 is the conjugate of any one of items 1 to 11, wherein (a) the first component comprises (i) a VH domain of SEQ ID No. 97, 99, 105, 109 or 144; and (ii) a VL domain of SEQ ID NO 98, 100, 106, 110 or 115; and (b) the second component comprises SEQ ID NO. 4.
Item 14 is the conjugate of any one of items 1 to 11, wherein (a) the first component comprises (i) a VH domain of SEQ ID No. 97, 99, 105, 109 or 144; and (ii) a VL domain of SEQ ID NO 98, 100, 106, 110 or 115; and (b) the second component comprises SEQ ID NO 86, 60, 32 or 29.
Item 15 is the therapeutic molecule of any one of items 1 to 11, wherein the second components comprise at least two linking domains of a multifunctional proteoglycan.
Item 16 is the therapeutic molecule of item 15, wherein the second components comprise at least two linking 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 a ratio of hyaluronic acid to therapeutic molecule ranging from 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 comprises at least 1, at least 2, at least 3, at least 4, or at least 5 mutations.
Item 21 is the therapeutic molecule of any one of items 14 to 20, wherein the second component comprises 1 to 3 mutations, wherein the 1 to 3 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 1 to 5 mutations, wherein the 1 to 5 mutations comprise a single amino acid substitution, a double amino acid substitution, and a truncation.
Item 23 is the therapeutic molecule of any one of items 14 to 22, wherein the second component has a truncation mutation relative to SEQ ID No. 29.
Item 24 is the therapeutic molecule of item 23, wherein the truncation 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, wherein no Ig domain of a wild-type multifunctional proteoglycan is present.
Item 26 is the therapeutic molecule of any one of items 14 to 25, wherein the second component comprises at least one of the following amino acids relative to SEQ ID No. 29: r160, Y161, E194, D197, Y208, R214, Y230, F261, D295, and R233.
Item 27 is the therapeutic molecule of any one of items 14 to 26, wherein the second component comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the following amino acids relative to SEQ ID No. 29: r160, Y161, E194, D197, Y208, R214, Y230, F261, D295, and R233.
Item 28 is the therapeutic molecule of any one of items 14 to 27, wherein the second component comprises a mutation in at least one of the following positions relative to SEQ ID No. 29: r160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.
Item 29 is the therapeutic molecule of any one of items 14 to 28, wherein the second component comprises a mutation 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.
Item 30 is the therapeutic molecule of any one of items 14 to 29, wherein the second component comprises a mutation in 2, 3, 4, 5 or 6 of the following positions relative to SEQ ID No. 29: r160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.
Item 31 is the therapeutic molecule of any one of items 14 to 30, wherein the second component comprises at least one of the following mutations relative to SEQ ID No. 29: R160A, Y161A, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a 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 of any one of items 14 to 32, wherein the second component 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, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a and LYR325LFK.
Item 34 is the therapeutic molecule of any one of items 14 to 33, wherein the second component comprises at least 2, 3, 4, 5 or 6 of the following mutations relative to SEQ ID No. 29: R160A, Y161A, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a and LYR325LFK.
The therapeutic molecule of 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: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.
Item 36 is the therapeutic molecule of any one of items 1 to 35, wherein the first component comprises an oligopeptide, protein, or nucleic acid.
Item 37 is the therapeutic molecule of any one of items 1 to 36, wherein the first component comprises a therapeutic drug, an antibody, an antigen binding fragment, an enzyme, a growth factor, an oligopeptide, a cysteine-binding peptide, a growth factor, an antisense oligonucleotide, a locked nucleic acid, or an aptamer.
Item 38 is the therapeutic molecule of item 37, wherein the cysteine knot peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 92.
Item 39 is the therapeutic molecule of 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 of any one of items 1 to 39, wherein the first component binds VEGF.
Item 41 is the therapeutic molecule of item 40, wherein the first component that binds VEGF comprises ranibizumab, albesiezomib-dbll, and bevacizumab.
Item 42 is the therapeutic molecule of item 37, wherein the aptamer is pegylated.
Item 43 is the therapeutic molecule of item 37 or 42, wherein the aptamer is
Item 44 is the therapeutic molecule of any one of items 1 to 43, wherein the linker comprises GGGGS (SEQ ID NO: 27) or a multimer thereof, more particularly (GGGGS) 3 (SEQ ID NO: 28).
Item 45 is the therapeutic molecule of any one of items 1 to 42, wherein the linker comprises GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).
Item 46 is the therapeutic molecule of item 45, wherein the cysteine knot peptide is linked to the one or two second components via a linker comprising sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).
Item 47 is the therapeutic molecule of 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 medicament comprising a therapeutic molecule according to any one of items 1 to 47 and optionally a pharmaceutically acceptable excipient, diluent or carrier.
Item 49 is a composition for treating an eye disease or brain disease comprising the conjugate of any one of items 1 to 47 and optionally a pharmaceutically acceptable excipient, diluent or carrier.
Item 50 is the composition used in item 49, formulated for intraocular delivery, particularly for intravitreal injection.
Item 51 is a composition for use of any one of items 48 to 50, wherein (a) the composition is administered at most once every three months, particularly at most once every four months, more particularly at most once every six months; and/or (b) the elimination half-life of the first component in the conjugate is extended by at least 3-fold, at least 4-fold, or at least 5-fold as compared to the unbound first component.
Item 52 is the composition for use of 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 retinal Degeneration (DR), in particular proliferative DR or non-proliferative DR; retinal Vein Occlusion (RVO); or Geographic Atrophy (GA).
Item 53 is a method of treating an ocular disease in a subject, the method comprising administering to the subject a therapeutic molecule as defined in any one of items 1 to 47 or a composition as defined in any one of items 48 to 52.
Item 54 is a method of delivering a therapeutic molecule that targets a tissue of a patient, the method comprising administering the therapeutic molecule of any one of items 1-47 or the composition of any one of items 48-52 to the patient, and allowing the therapeutic molecule to provide long-lasting delivery of the first component to the target tissue.
Item 55 is the method of item 54, further comprising binding the therapeutic molecule to hyaluronic acid prior to the administering step.
Item 56 is the method of item 55, further comprising mixing a first solution comprising the therapeutic molecule with a second solution comprising the hyaluronic acid.
Item 57 is the method of item 56, wherein the mixing comprises a container.
Item 58 is the method of item 57, wherein the container is a two-compartment syringe.
Item 59 is the method of any one of items 56 to 58, wherein the mixing produces a therapeutic molecule that binds to hyaluronic acid, the therapeutic molecule being ready for administration to a subject.
Item 60 is the method of any one of items 54 to 59, wherein the administering step is a single injection.
Item 61 is the method of any one of items 54 to 60, wherein the target tissue comprises an eye or 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 the unmodified bioactive agent.
Aspect 63 is a conjugate comprising (a) a first component capable of binding to a therapeutic target in an eye; (b) One or more second components capable of binding to hyaluronic acid; and (c) one or more third components comprising hyaluronic acid, (d) wherein each second component is covalently bound to the first component and not to the third component.
Aspect 64 is the conjugate of aspect 63, wherein the first component is a protein, peptide, receptor or fragment thereof, ligand for a receptor, darpin, nucleic acid, RNA, DNA, or aptamer.
Aspect 65 is the conjugate of aspect 63 or 64, wherein the first component is an antibody or antigen-binding antibody fragment, particularly an antibody fragment, more particularly an antibody fragment lacking at least an Fc domain, particularly wherein the fragment is or comprises a (Fab ') 2 fragment, a Fab' fragment, or a Fab fragment, more particularly a Fab fragment.
Aspect 66 is the conjugate of any one of aspects 63 to 65, wherein the second component comprises a hyaluronic acid receptor CD44 (CD 44) domain, a brain-specific junction protein (BRAL 1) domain, a tumor necrosis factor stimulating gene 6 (TSG-6) domain, a lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1) domain, or a Hyaluronic Acid Binding Protein (HABP) domain, an aggrecan G1 (AG 1) domain, or a multifunctional proteoglycan G1 (VG 1) domain.
Aspect 67 is the conjugate of any one of aspects 63 to 66, wherein the conjugate comprises one or two second components, in particular 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 3kDa to 60kDa, specifically 4kDa to 30kDa, more specifically 5kDa to 20 kDa; and/or (b) has a molecular weight of at least 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa or 9 kDa; and/or (c) has a molecular weight of at most 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa or 15 kDa; and/or (d) has a modification that reduces degradation of hyaluronic acid in the eye.
Aspect 69 is the conjugate of any one of aspects 63 to 67, wherein (a) the first components and second components are comprised 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 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 bound directly to the first component or indirectly to the first component via a linker, particularly a linker of at least 4 amino acids and/or of at most 50 or at most 25 amino acids, more particularly the linker is (GxS) n or (GxS) nGm, wherein g=glycine, s=serine, (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3).
Aspect 70 is the conjugate of any one of aspects 63 to 69, wherein the therapeutic target is VEGF, C5, factor P, factor D, EPO, EPOR, IL-1β, IL-17A, IL-10, TNF, FGFR2, PDGF or ANG2, particularly VEGF.
Aspect 71 is the conjugate of any one of aspects 63 to 70, wherein (a) the first component is an antibody or antigen-binding antibody fragment against VEGF, in particular an anti-VEGF Fab; and/or (b) each of these one or two second components comprises a CD44 domain or a TSG-6 domain or a VG1 domain; and/or (c) the third component is hyaluronic acid having a molecular weight of 5kDa to 20kDa, (d) particularly wherein (e) the first component is an anti-VEGF Fab, and wherein each of the one or two second components comprises a CD44 domain, and wherein the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa; or (f) the first component is an anti-VEGF Fab, and wherein each of the one or two second components comprises a TSG-6 domain, and wherein the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa; or (g) the first component is an anti-VEGF Fab, and wherein each of the one or two second components comprises a VG1 domain, and wherein the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa.
Aspect 72 is the conjugate of any one of aspects 63 to 71, the first component being an antibody having a VH domain comprised in SEQ ID No. 5 and a VL domain comprised in SEQ ID No. 6, and the second component comprising or consisting of SEQ ID No. 4.
Aspect 73 is a composition comprising the conjugate of any one of aspects 63 to 72 and optionally a pharmaceutically acceptable excipient, diluent or carrier for use as a medicament.
Aspect 74 is a composition for treating an eye disease comprising the conjugate of any one of aspects 63 to 73 and optionally a pharmaceutically acceptable excipient, diluent or carrier.
Aspect 75 is the composition used in aspects 73 or 74 formulated for intraocular delivery, particularly for intravitreal injection.
Aspect 76 is the composition for use of any one of aspects 73 to 75, wherein (a) the composition is administered at most once every three months, particularly at most once every four months, more particularly at most once every six months; and/or (b) the elimination half-life of the first component in the conjugate is extended by at least 3-fold, at least 4-fold, or at least 5-fold as compared to the unbound first component.
Aspect 77 is the composition for use of 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 retinal Degeneration (DR), in particular proliferative DR or non-proliferative DR; retinal Vein Occlusion (RVO); or Geographic Atrophy (GA).
Aspect 78 is a method of treating an ocular disease in a subject, the method comprising administering to the subject a conjugate as defined in any one of aspects 63 to 72 or a composition as defined in any one of aspects 73 to 77.
Example 79 is a therapeutic molecule that targets tissue of a patient, the therapeutic molecule comprising a hyaluronic acid binding domain and a therapeutically active agent, wherein the hyaluronic acid binding domain comprises at least two linking domains of a multifunctional proteoglycan.
Embodiment 80 is a therapeutic molecule that targets tissue of a patient, the therapeutic molecule comprising a hyaluronic acid binding domain and a therapeutically active agent, wherein the hyaluronic acid binding domain comprises at least two linking domains of a multifunctional proteoglycan that binds to hyaluronic acid via an HA binding domain.
Embodiment 81 is the therapeutic molecule of embodiment 79 or 80, wherein the hyaluronic acid ranges from 400Da to 200kDa.
Embodiment 82 is the therapeutic molecule of embodiment 81, wherein the hyaluronic acid is at least 5kDa.
Embodiment 83 is the therapeutic molecule of embodiment 81 or 82, wherein the hyaluronic acid is 10kDa.
Embodiment 84 is the therapeutic molecule of any one of embodiments 79 to 83, wherein the hyaluronic acid provides a molar excess of binding equivalents to the linking domains of the multifunctional proteoglycans.
Embodiment 85 is the therapeutic molecule of any one of embodiments 79 to 84, wherein the hyaluronic acid allows a ratio of hyaluronic acid to therapeutic molecule ranging from 1.5:1 to 1:1.
Embodiment 86 is the therapeutic molecule of any one of embodiments 79 to 85, wherein the hyaluronic acid binding domain has a K of 10nM to 10 μm D 。
Embodiment 87 is the therapeutic molecule of any one of embodiments 79 to 86, wherein the hyaluronic acid binding domain has a K of 5nM to 8 μm D 。
Embodiment 88 is the therapeutic molecule of any one of embodiments 79 to 87, wherein the hyaluronic acid binding domain has a K of 100nM to 5 μm D 。
Embodiment 89 is the therapeutic molecule of any one of embodiments 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.
Embodiment 90 is the therapeutic molecule of any one of embodiments 79 to 89, wherein the hyaluronic acid binding domain is at least 95% identical to 86, 60, 32, or 29.
Embodiment 91 is the therapeutic molecule of any one of embodiments 79 to 90, wherein the hyaluronic acid binding domain comprises at least 1, at least 2, at least 3, at least 4, or at least 5 mutations.
Embodiment 92 is the therapeutic molecule of any one of embodiments 79 to 91, wherein the hyaluronic acid binding domain comprises 1 to 3 mutations, wherein the 1 to 3 mutations comprise a single amino acid substitution, a double amino acid substitution, and a truncation.
Embodiment 93 is the therapeutic molecule of any one of embodiments 79 to 92, wherein the hyaluronic acid binding domain comprises 1 to 5 mutations, wherein the 1 to 5 mutations comprise a single amino acid substitution, a double amino acid substitution, and a truncation.
Embodiment 94 is the therapeutic molecule of any one of embodiments 79 to 93, wherein the hyaluronic acid binding domain has a truncation mutation relative to SEQ ID No. 29.
Embodiment 95 is the therapeutic molecule of embodiment 94, wherein the truncation mutation comprises a truncation of 1 to 129 amino acids at the N-terminus.
Embodiment 96 is the therapeutic molecule of any one of embodiments 79 to 95, wherein the hyaluronic acid binding domain is a truncated sequence, wherein no Ig domain of a wild-type multifunctional proteoglycan is present.
Embodiment 97 is the therapeutic molecule of any one of embodiments 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.
Embodiment 98 is the therapeutic molecule of any one of embodiments 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.
Embodiment 99 is the therapeutic molecule of any one of embodiments 79 to 98, 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.
Embodiment 100 is the therapeutic molecule of any one of embodiments 79 to 99, wherein the hyaluronic acid binding domain comprises a mutation of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 in the following positions relative to SEQ ID No. 29: r160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.
Embodiment 101 is the therapeutic molecule of any one of embodiments 79 to 100, wherein the hyaluronic acid binding domain comprises 2, 3, 4, 5, or 6 mutations in the following positions relative to SEQ ID No. 29: r160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.
Embodiment 102 is the therapeutic molecule of any one of embodiments 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, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a and LYR325LFK.
Embodiment 103 is the therapeutic molecule of any one of embodiments 79 to 102, wherein the hyaluronic acid binding domain comprises at least one of Y208A and H306A.
Embodiment 104 is the therapeutic molecule of any one of embodiments 79 to 103, wherein the 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, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a and LYR325LFK.
Embodiment 105 is the therapeutic molecule of any one of embodiments 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, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a and LYR325LFK.
Embodiment 106 is the therapeutic molecule of any one of embodiments 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:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59.
Embodiment 107 is the therapeutic molecule of any one of embodiments 79 to 106, wherein the therapeutically active agent comprises an oligopeptide, protein, or nucleic acid.
Embodiment 108 is the therapeutic molecule of any one of embodiments 79-107, wherein the therapeutically active agent comprises an antibody, an antigen-binding fragment, a cysteine-binding peptide, a growth factor, or an aptamer.
Embodiment 109 is the therapeutic molecule of embodiment 108, wherein the therapeutically active agent is capable of binding an antigen.
Embodiment 110 is the therapeutic molecule of embodiment 109, wherein the therapeutically active agent is capable of binding to VEGF, htrA1, IL-33, C5, factor P, factor D, EPO, EPOR, IL-1 β, IL-17A, IL-10, tnfα, FGFR2, PDGF, or ANG2.
Embodiment 111 is the therapeutic molecule of any one of embodiments 109 or 110, wherein the therapeutically active agent is an antibody or antigen-binding fragment thereof (including but not limited to a Fab fragment, a F (ab ') 2 fragment, a Fab' fragment, a VhH fragment, a scFv-Fc fragment, or a minibody).
Embodiment 112 is the therapeutic molecule of any one of embodiments 109 or 110, wherein the therapeutically active agent is an oligopeptide or protein.
Embodiment 113 is the therapeutic molecule of embodiment 102, wherein the oligopeptide or protein is a cysteine-binding peptide or enzyme.
Embodiment 114 is the therapeutic molecule of embodiment 103, wherein the cysteine knot peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 92.
Embodiment 115 is the therapeutic molecule of any one of embodiments 79-108, wherein the therapeutically active agent is a growth factor comprising a fibroblast growth factor, a platelet-derived growth factor, a Nerve Growth Factor (NGF), VEGF, a Fibroblast Growth Factor (FGF), and an insulin-like growth factor I (IGF-I).
Embodiment 116 is the therapeutic molecule of embodiment 110, wherein the therapeutically active agent that binds VEGF comprises ranibizumab, aflibercept, ibuprofen, and bevacizumab.
Embodiment 117 is the therapeutic molecule of any one of embodiments 79 to 110, wherein the therapeutically active agent is a nucleic acid.
Embodiment 118 is the therapeutic molecule of embodiment 117, wherein the nucleic acid is an aptamer, an antisense oligonucleotide, and/or a locked nucleic acid.
Embodiment 119 is the therapeutic molecule of embodiment 118, wherein the aptamer binds to VEGF.
Embodiment 120 is the therapeutic molecule of any one of embodiments 108, 118 or 119, wherein the aptamer is pegylated.
Embodiment 121 is the therapeutic molecule of embodiment 108 or any one of embodiments 118-120, wherein the aptamer is
Embodiment 122 is the therapeutic molecule of any one of embodiments 79 to 121, wherein the therapeutically active agent and the hyaluronic acid binding domain are covalently linked via a linker.
Embodiment 123 is the therapeutic molecule of embodiment 122, wherein the linker is at least 4 amino acids.
Embodiment 124 is the therapeutic molecule of embodiment 122 or 123, wherein the linker is no more than 50 amino acids in length.
Embodiment 125 is the therapeutic molecule of any one of embodiments 122-124, wherein the linker is 4 to 25 amino acids.
Embodiment 126 is the therapeutic molecule of any one of embodiments 122-125, wherein the linker comprises (GxS) n or (GxS) nGm, wherein g=glycine, s=serine, and (x=3, n=3, 4, 5, or 6, and m=0, 1, 2, or 3) or (x=4, n=2, 3, 4, or 5, and m=0, 1, 2, or 3).
Embodiment 127 is the therapeutic molecule of any one of embodiments 122-126, wherein the linker comprises GGGS (SEQ ID NO: 84) or a multimer thereof, more particularly (GGGGS) 3 (SEQ ID NO: 85).
Embodiment 128 is the therapeutic molecule of any one of embodiments 122-125, wherein the linker comprises (GxS) n, wherein G = glycine, S = serine, and (n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
Embodiment 129 is the therapeutic molecule of any one of embodiments 122-125 or 128, wherein the linker comprises GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).
Embodiment 130 is the therapeutic molecule of any one of embodiments 79 to 107, wherein the therapeutically active agent comprises an anti-VEGF antigen-binding portion and a cysteine knot peptide.
Embodiment 131 is the therapeutic molecule of embodiment 130, wherein the cysteine knot peptide is linked to the hyaluronic acid binding domain via a linker comprising sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95).
Embodiment 132 is the therapeutic molecule of embodiment 130 or 131, wherein the sequence comprises (a) an anti-VEGF antigen-binding portion; and (b) 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.
Embodiment 133 is the therapeutic molecule of any one of embodiments 79-132, wherein the hyaluronic acid binding domain can non-covalently bind to hyaluronic acid.
Embodiment 134 is a method of delivering a therapeutic molecule that targets tissue of a patient comprising administering the therapeutic molecule of any one of embodiments 79 to 133 to the patient and allowing the therapeutic molecule to provide long-lasting delivery of the therapeutically active agent to the target tissue.
Embodiment 135 is the method of embodiment 134, further comprising binding the therapeutic molecule to hyaluronic acid prior to the administering step.
Embodiment 136 is the method of embodiment 135, further comprising mixing a first solution comprising the therapeutic molecule with a second solution comprising the hyaluronic acid.
Embodiment 137 is the method of embodiment 136, wherein the mixing comprises a vessel.
Embodiment 138 is the method of embodiment 137, wherein the container is a two-compartment syringe.
Embodiment 139 is the method of any one of embodiments 136-138, wherein the mixing produces a therapeutic molecule that binds to hyaluronic acid, the therapeutic molecule being ready for administration to a subject.
Embodiment 140 is the method of any one of embodiments 134-139, wherein the administering step is a single injection.
Embodiment 141 is the method of any one of embodiments 134-140, wherein the target tissue comprises an eye or brain.
Embodiment 142 is the method of any one of embodiments 134-141, 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 the unmodified therapeutically active agent.
Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. These objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiment(s) and together with the description, serve to explain certain principles described herein.
Drawings
FIG. 1 shows the size exclusion chromatography (SEC; TSKgel UP-SW3000,2 μm, 4.6X150 mm; running buffer 0.2MKPh,0.25M KCl,pH 6.2) analysis of the Fab-hyaluronan binding domain (Fab-HABD) fusion protein VPDF-2xCD44, pre-complexed or not with 10kDa Hyaluronan (HA). Fab-HABD was prepared as described in example 1 and tested as described in example 2.
Figures 2A to 2B show the vitronectinic Pharmacokinetics (PK) of rabbit anti-c-Met Fab (RabFab) and rabbit anti-c-Met Fab-VG1 Fab-HABD (RabFab-Fab-HABDs) following dose standardized Intravitreal (IVT) injection in new zealand white rabbits. Fig. 2A shows the change over time in the content of RabFab or RabFab-Fab-HABD present in the vitreous after IVT. Data showing RabFab fusions, i.e 125 I-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 125 I-LeizumabControl (0.5 mg/eye). Data points were dose normalized. FIG. 2B vitriokinetics of RabFab (0.15 mg/eye) or RabFab-2xTSG6 (0.026 mg/eye, 0.15 mg/eye or 2.5 mg/eye) monitored by fluorometry.
FIG. 3 shows histopathological images of OS rabbit eyes showing retinal degeneration 4 days after administration of TSG6 (SEQ ID NO: 32) via IVT.
FIGS. 4A-B show the IVT Pharmacokinetic (PK) profiles (mean drug concentration over time) of VPDF (unmodified; FIG. 4A) and VPDF-2xCD44+10kDa HA (FIG. 4B) in aqueous and vitreous humor.
Fig. 5A-C show different mixtures with porcine vitreous. Fig. 5A shows porcine vitreous mixed with unmodified anti-VEGF/anti-PDGF Fab fragment (VPDF), which is homogeneous (transparent). Fig. 5B shows a pig vitreous mixed with VPDF-2xCD44, which is heterogeneous (precipitated). FIG. 5C shows porcine vitreous mixed with 1% (w/v) HA 10kDa VPDF-2xCD44 pre-complex, which is homogeneous (transparent).
Fig. 6A to 6F show pig glass bodies mixed with different concentrations of VPDF-2xCD44. Fig. 6A:37.5mg/mL VPDF-2xCD44. Fig. 6B:9.4mg/mL VPDF-2xCD44. Fig. 6C:2.4mg/mL VPDF-2xCD44. Fig. 6D:0.6mg/mL VPDF-2xCD44. Fig. 6E:0.15mg/mL VPDF-2xCD44. Fig. 6F:0.04mg/mL VPDF-2xCD44.++ severe precipitation; ++ neutral precipitation; + mild precipitation; -a transparent glass body.
Figures 7A-C show the vitreous non-uniformity throughout the pig eye after injection of a designated VPDF-2xCD44 sample. Fig. 7A: buffer control. Fig. 7B: uncomplexed VPDF-2xCD44. Fig. 7C: VPDF-2xCD44 complexed with HA.
FIGS. 8A-B show the amino acid sequences of domain structures and linking domains of multifunctional proteoglycans. Multifunctional proteoglycans are endogenous substances to the vitreous humor. Fig. 8A shows the multifunctional proteoglycan domain: VG1 domain, GAG attachment domain and G3 domain. The VG1 domain (WT VG1; SEQ ID NO: 29) contains an Ig-like domain followed by two linking domains (i.e., link1 and Link 2) that are responsible for HA binding. FIG. 8B shows sequence alignment of the junction domains including TSG6 LD (SEQ ID NO: 4), VG1 Link1 (SEQ ID NO: 30), and VG1 Link2 (SEQ ID NO: 31).
FIGS. 9A-B show precipitation of TSG6 but not WT VG1 in porcine vitreous humor. Turbidity was observed after mixing TSG6 (but not WT VG 1) with 1:4 diluted (PBS) porcine vitreous. The final concentration of TSG6 and WT VG1 in the vitreous was about 1mg/mL. Figure 9A shows TSG6 vs. sediment observed after control-centrifugation. FIG. 9B shows that no precipitate was observed after WT VG1 vs. control-centrifugation.
FIGS. 10A-B show that RabFab-TSG6 precipitated in porcine vitreous, whereas RabFab-VG1 did not. Each of TSG6 or VG1 was recombinant attached to RabFab and chemically bound to Alexa488 via an N-hydroxysuccinimide (NHS) primary amine tag. FIG. 10A shows RabFab-TSG6. FIG. 10B shows RabFab-VG1.
FIGS. 11A-C show that VG1 and RabFab-VG1 did not precipitate in rabbit vitreous humor. FIG. 11A shows VG1 at about 40 g/L. FIG. 11B shows about 40g/L of RabFab-VG1. FIG. 11C shows about 17g/L of RabFab-VG1+10kDa HA. No precipitation was observed under any conditions.
Fig. 12 shows Fluorescence Correlation Spectroscopy (FCS) measurements of VG1 interaction with ex vivo vitreous humor. Measurements showing slow diffusion indicate that proteins interact with the vitreous humor, while fast diffusion indicates no interaction. The dilution factor of the vitreous humor is shown on the top-leftmost column of the heat map, showing the undiluted control/sample; phosphate Buffered Saline (PBS), pH 7.4, is shown in the far right column; the middle column shows the dilution factor increasing from left to right. Measurement results of unbound control are shown in the top two rows. The measurement results of the following samples are shown in lines 3-8: free VG1, picFab-VG1+10 kDa HA (1:1), free VG1, rabFab-VG1 and RabFab-VG1+10kDa HA (1:1). The unbound control shows the fastest diffusion (fig. 12, lines 1 and 2). Although all samples showed significant delayed diffusion relative to the control, free VG1, pigFab-VG1 and RabFab-VG1 all showed delayed diffusion before the vitreous was diluted more than 6000-fold (FIG. 12, lines 3, 4, 6 and 7; undiluted to dilution factor 6,561). Slow diffusion was observed for samples co-formulated with 10kDa HA, but when the dilution factor was greater than 729 times, the effect disappeared (FIG. 12, line 5: picFab-VG1+10 kDa HA (1:1), and line 8: rabFab-VG1+10kDa HA (1:1); from 729 to PBS). These results indicate that VG1 can interact with endogenous HA.
FIG. 13 shows the results of thermal stress (i.e., protein stability) analysis against HtrA1-VG1 at 37 ℃. T0 = non-incubated control. T4wk=after 4 weeks of incubation.
FIG. 14 shows the average concentration of pigFab-VG1 in pig aqueous humor. The concentration was measured by mass spectrometry after injection of 1.8mg of pigFab-VG1 alone or precomplexed with an equal mass concentration of 10kDa HA via IVT. The average of several animals is shown, and the error bars represent standard deviation.
Figure 15 shows the percent inhibition of neovascularization by VPDF VG1 in rat laser-induced choroidal neovascularization (rat laser CNV).
Fig. 16A-C show histopathology of test article treated rabbit eyes 30 days after treatment. FIG. 16A shows WT VG1, FIG. 16B shows RabFab-VG1, and FIG. 16C shows RabFab-VG1 bound to HA.
Figures 17A-B show brain levels after mice received an intraventricular injection. Fig. 17A shows the amount of protein retained in the brain over time. Fig. 17B shows the level of exposure in the brain as measured by area under the curve (AUC). * P <0.01 and p <0.001 for comparison between groups. Anti gD = anti-herpes simplex virus-1 glycoprotein D. BRD = anti-gD Fab-VG1.
Fig. 18 shows the crystal structure of the WT VG1 and HA conjugates. Ig domains of VG1 are shown at the top of the figure, link1 structures are shown at the bottom right of the figure, and Link2 domains are shown at the bottom left of the figure. HA binding is indicated by the smaller HA molecule below and to the right of the VG1 molecule.
FIG. 19 shows an alignment of VG1 variants SEQ ID NO:29, SEQ ID NO: 33-59. The first 20 amino acids at the N-terminus are the signal sequences of the multifunctional proteoglycans (shown with). In-frame amino acids are conserved residues. All of these proteins bear a C-terminal His-tag for purification.
DESCRIPTION OF THE SEQUENCES
Table 1 provides a list of certain sequences cited herein. The amino acid sequence is provided from the N-terminus to the C-terminus.
Detailed Description
1. Definition
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention, the preferred methods and materials are now described.
The following terms and phrases used herein are intended to have the following meanings, unless otherwise indicated:
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 interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (CL). VH and VL regions can be further subdivided into regions of high variability termed Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved termed Framework Regions (FR). Each VH and VL is arranged from amino-terminus to carboxyl-terminus by three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (CIq).
As used herein, the term "antigen-binding fragment" or "antibody fragment (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 a desired antigen-binding activity. The antigen binding function of an antibody may be performed by fragments of the whole antibody. Antigens encompassed by the term "antibodiesExamples of binding fragments within the binding fragment "include, but are not limited to, examples of antibody fragments including, but not limited to: fab, fab '-SH, F (ab') 2 The method comprises the steps of carrying out a first treatment on the surface of the A diabody antibody; a linear antibody; single chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dabs); and multispecific antibodies formed from antibody fragments; fd fragment consisting of VH and CH1 domains; fv fragments consisting of the VL and VH domains of the antibody single arm; a single domain antibody (dAb) fragment consisting of a VH domain or a VL domain; and an isolated Complementarity Determining Region (CDR). For a review of certain antibody fragments, see Holliger and Hudson, nature Biotechnology 23:1126-1136 (2005). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker, enabling them to be a single protein chain, in which the VL and VH regions pair to form monovalent molecules, known as single chain Fv (scFv). These single chain antibodies may include one or more antigen binding fragments of the antibody. These antigen binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as whole antibodies. Antigen binding fragments may also incorporate single domain antibodies, large antibodies, minibodies, internal antibodies, diabodies, trisomy antibodies, tetrabodies, v-NAR and bis-scFv. The antigen binding fragments may be incorporated into a single chain molecule comprising a pair of tandem Fv fragments (VH-CH 1-VH-CH 1) together with a complementary light chain polypeptide to form a pair of antigen binding regions. The term "antibodies" includes polyclonal antibodies and monoclonal antibodies.
An aptamer is an oligonucleotide or peptide molecule that binds to a particular target molecule. Aptamers are typically created by selecting them from a large pool of random sequences, but natural aptamers are also found in riboswitches (riboswitches). The aptamer can be used as a macromolecular drug for basic research and clinical purposes. The aptamer may bind to a ribozyme and self-cleave in the presence of a target molecule. These compound molecules have additional research, industrial and clinical uses. More specifically, aptamers can be classified as DNA or RNA or XNA aptamers, which consist of (typically short) oligonucleotide chains, and peptide aptamers consisting of one (or more) short variable peptide domains are attached at both ends to a protein scaffold. Both DNA and RNA aptamers exhibit stable binding affinities to various targets. DNA and RNA aptamers have been selected for the same target. These targets include lysozyme, thrombin, interferon gamma, vascular Endothelial Growth Factor (VEGF), dopamine. In the case of VEGF, for example, the DNA aptamer is an analog of an RNA aptamer in which thymine replaces uracil.
"covalent bond" is also known as a molecular bond, and is a chemical bond that involves sharing electron pairs between atoms. These electron pairs are referred to as shared electron pairs or bonded electron pairs, and when they share electrons, a stable balance of interatomic attractive and repulsive forces is referred to as covalent bonds.
As used herein, the term "DARPin" (abbreviated as engineered ankyrin repeat protein (designed ankyrin repeat protein)) refers to an antibody mimetic protein that generally exhibits high specificity and high affinity target protein binding. They are typically genetically engineered from natural ankyrin and consist of at least three, typically four or five, repeat motifs of these proteins. For four or five repeat darpins, their molecular weights are about 14kDa or 18kDa, respectively. Examples of DARPin can be found, for example, in us patent 7,417,130.
A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective to achieve a desired therapeutic or prophylactic effect over a desired dosage and period of time.
The term "ocular disease" as used herein includes any ocular disease associated with pathological angiogenesis and/or atrophy. The term "eye disease" is synonymous with the terms "eye condition", "eye disorder", "ocular condition", "ocular disease", and "ocular disorder".
As used herein, "Fab-hyaluronic acid binding domain" and "Fab-HABD" refer to fusion proteins comprising Fab and hyaluronic acid binding domains. These terms are synonymous and may be used interchangeably throughout this disclosure.
As used herein, "hyaluronic acid (hyaluronic acid)", "hyaluronate (hyaluronate)", and "HA" refer to a compound having the formula (C) 14 H 21 NO 11 ) n Non-sulfated glycosaminoglycans and salts thereof.
As used herein, "hyaluronic acid binding domain (hyaluronic acid binding domain)", "hyaluronic acid binding moiety (hyaluronic acid binding moiety)", "HA binding domain (HA binding domain)", or "HABD" refers to any moiety capable of binding hyaluronic acid. In some cases, the HABD may be a domain of an HA-binding protein.
A ligand is a substance that forms a complex or conjugate with a biological molecule to achieve its biological purpose. In protein-ligand binding, a ligand is typically a molecule that generates a signal by binding to a site on a target protein. This binding typically results in a change in conformational isomerism (formation) of the target protein. In DNA-ligand binding studies, the ligand may be a small molecule, ion, or protein that binds to the DNA duplex. The relationship between the ligand and the binding partner is a function of charge, hydrophobicity, and molecular structure. The case of combining occurs in an infinitely small temporal and spatial range, so the rate constant is typically a very small number. The ligand may be a naturally occurring ligand or a non-naturally occurring ligand. Furthermore, it may be an agonist, partial agonist, antagonist or inverse agonist.
"non-covalent interactions (non-covalent interaction)" differ from covalent bonds in that it does not involve sharing of electrons, but rather involves more diffuse changes in intermolecular or intramolecular electromagnetic interactions. Non-covalent interactions can be classified into different categories such as static electricity, pi-effect, van der Waals force, and hydrophobic effect. Preferably, the conjugate is provided in an isolated form. The first component and the second component may be covalently bound to each other via a linker or directly.
Nucleic acids are biopolymers consisting of nucleotides, which are monomers formed by three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. The term "nucleic acid" is a generic term for DNA and RNA. If fructose is complex ribose, then the polymer is RNA (ribonucleic acid); if fructose is deoxyribose derived from ribose, then the polymer is DNA (deoxyribonucleic acid).
As used herein, the term "peptide linker" means a peptide comprising an amino acid sequence, preferably of synthetic origin.
Proteins are macromolecular biopolymers (polypeptides) consisting of long chains of one or more amino acid residues. Proteins perform a range of functions within an organism, including catalyzing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. The difference between proteins is mainly in their amino acid sequence, which is determined by the nucleotide sequence of their genes, and usually results in the folding of the protein into a specific three-dimensional structure, which determines its activity. Short polypeptides containing less than 20-30 residues are commonly referred to as peptides.
As used herein, "protein conjugate (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 be integrated into biological systems. These signals are typically chemical messengers (ligands) that bind to receptors, causing some form of cell/tissue reaction, such as a change in cellular activity. The mode of action of the receptor can be largely divided into three types: signal relay, amplification or integration. The relay forwards the signal, amplifies the effect of increasing the 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. Thus, in the context of the present invention, a receptor or fragment comprising a ligand binding site and its ligand are suitable binding counterparts (first component and therapeutic target).
As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "treatment") refers to a clinical intervention that attempts to alter the natural course of an individual to be treated, and may be performed for prophylaxis or in the course of clinical pathology. Desirable therapeutic effects include preventing the occurrence or recurrence of a disease or condition or symptom thereof, alleviating a condition or symptom of a disease, alleviating any direct or indirect pathological consequences of a disease, slowing the rate of disease progression, ameliorating or alleviating a disease state, and achieving the goal of alleviating or improving prognosis. In some aspects, the antibodies of the invention are used to delay the progression of a disease or to slow the progression of a disease.
The numerical range includes numbers defining the range. In view of the significant figures and errors associated with measurements, the measured and measurable values should be understood as approximations. Furthermore, the use of "include/comprise", "contain/contain" and "include/include" is not intended to be limiting. It is to be understood that both the foregoing general description and the detailed description are exemplary and explanatory only and are not restrictive of the teachings.
All numbers in the specification and claims are modified by the term "about". This means that each number includes minor variations defined as 10% of the stated value or range.
Unless specifically indicated in the specification, embodiments in the specification that reference "comprise various components" are also considered to be "consisting of" or "consisting essentially of" the referenced components; embodiments in the specification that reference "consisting of" various components are also considered to "comprise" or "consist essentially of" the recited components; and embodiments in the specification that reference "consisting essentially of the various components" are also considered to be "consisting of" or "comprising" the referenced components (this interchangeability is not applicable to the use of these terms in the claims). The term "or" is used in an inclusive sense, i.e., equivalent to "and/or (and/or)", unless the context clearly indicates otherwise.
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 examples (unless specifically indicated otherwise) are not intended to limit the scope of the invention to the particular methods, protocols, and reagents described herein as they may vary. The invention is intended to cover all alternatives, modifications and equivalents which may be included within the invention as defined by the appended claims and the included embodiments. Furthermore, the techniques used herein are for the purpose of disclosing specific embodiments only and are not intended to limit the scope of the disclosure. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "comprise," "include," and "include" are to be construed as inclusive rather than exclusive.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the required aim in any way. All publications (scientific and patent publications) cited herein are incorporated by reference. If any material incorporated by reference contradicts any term defined in the specification or any other explicit context of the specification, the specification controls. While the present teachings are described in connection with various embodiments, it is not intended to limit the present teachings to those embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
Therapeutic molecule comprising a therapeutically active agent (i.e., a first component) and a hyaluronic acid binding domain (HABD; i.e., a second component)
The present application provides therapeutic molecules that target tissue of a patient, the therapeutic molecules comprising a therapeutically 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 thus comprise the 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 component and the second component are covalently bound to each other, thereby forming a fusion protein. In some embodiments, the therapeutic molecule further comprises a peptide linker.
In some embodiments, the therapeutic molecule comprises a second component. In some embodiments, the therapeutic molecule comprises two or more second components. In particular, if an antibody or antigen-binding fragment thereof consisting of two proteins (i.e., one heavy chain or fragment thereof and one light chain or fragment thereof) is used, the therapeutic molecule may comprise two second components. In these embodiments, the first second component is attached to the heavy chain of the antibody or antigen-binding fragment and the second component is attached to the light chain of the antibody or antigen-binding fragment. In some embodiments, the first second component is attached to the C-terminus of the heavy chain of the Fab fragment and the second component is attached to the C-terminus of the light chain of the Fab fragment.
In some embodiments, the therapeutic molecule further comprises (in addition to the first component and the second component) one or more third components. The second component is covalently bound to the first component and the second component is non-covalently bound to the third component. In some embodiments, the third component is Hyaluronic Acid (HA). In some of these embodiments, the second component is capable of binding HA, and the therapeutic molecule protein (i.e., the first component covalently linked to the second component) can be pre-complexed with HA (i.e., the third component). In some of these embodiments, the first component, the second component, and the third component form a conjugate.
Non-limiting examples of the first component, the second component, and the third component are provided herein.
A. First component-therapeutically active agent
In many embodiments, the first component is capable of binding to a therapeutic target, making it a bioactive or therapeutically active agent. In some embodiments, the first component is capable of binding to a therapeutic target in the eye. As used herein, the term "capable of binding (capable of binding)" refers to a substance or agent or component that can specifically bind to a target and optionally modulate the activity of the target. In other words, the first component has therapeutic activity in the eye due to binding to a therapeutic target in the eye. In some embodiments, the first component can activate, deactivate, increase or decrease the activity of the therapeutic target after binding to the therapeutic target. In some embodiments, the therapeutic target is a suitable structure in the eye whose activity is associated with the ocular disease to be treated. In some embodiments, the first component binds to a component of the signal transduction cascade 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 member or binding domain has at least 10 for its corresponding target molecule 6 l/mol affinity. Preferably, the specific binding domain has 10 for its target molecule 7 Per mole, or even more preferably 10 8 Per mol or even most preferably 10 9 Affinity per mole. "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). As used herein, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibodies and antigens), unless otherwise indicated. The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K D ) To represent. Affinity can be measured by conventional methods known in the art. As the skilled person will appreciate, the term "specific" is used to indicate that the presence of other biomolecules does not significantly bind to the specific binding agent of the binding domain. Preferably, the level of binding to biomolecules other than the target molecule results in a binding affinity of only 10% or less, more preferably only 5% or less, of the affinity to the target molecule, respectively. Preferred specific binding agents will meet the minimum criteria for affinity and specificity described above.
In some embodiments, the first component comprises a protein, such as a receptor or fragment thereof, that binds to a therapeutic target, an antibody or fragment thereof, a growth factor, a cysteine knot 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 comprising 2 to 20 amino acids. In some embodiments, the protein is a polypeptide comprising 21 to 50 amino acids. In some embodiments, the protein is a polypeptide comprising more than 50 amino acids. In some embodiments, the protein is a protein complex comprising two or more chains or amino acids, wherein each amino acid chain may comprise any number of amino acids. In some embodiments, the first component is no greater than 80kDa. In some embodiments, the first component is greater than 80kDa.
In some embodiments, the first component comprises a nucleic acid that may be DNA or RNA. The nucleic acid can be complementary to a target-related nucleic acid (e.g., the nucleic acid is complementary to a 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. There may be many therapeutic targets in the eye. With the development of therapies that effectively target these molecules and pathways, there would be a need to provide improvement in visual outcome while reducing the therapeutic burden and risk associated with frequent IVT injections.
a) Pro-angiogenic, inflammatory and growth factor mediators
In some embodiments, the first component binds to a therapeutic target that is pro-angiogenic, inflammatory, and/or growth factor mediator. Pro-angiogenic, inflammatory and growth factor mediators are involved in retinal diseases such as neovascular age-related macular degeneration (AMD; wet AMD), diabetic retinal degeneration and retinal vein occlusion.
Examples of such pro-angiogenic, inflammatory or growth factor mediator molecules include, but are not limited to, platelet-derived growth factor (PDGF), angiogenin, S1P, integrin alpha v beta 3, integrin alpha v beta 5, integrin alpha 5 beta 1, beta-cytokine (betacelllin), apelin/APJ, erythropoietin, complement factor D and TNF alpha.
b) Proteins in age-related macular degeneration (AMD)
In some embodiments, the first component binds to a protein, which is typically associated with an increased risk of age-related macular degeneration (AMD). In some embodiments, the first component binds to a complement pathway component, such as C2, factor B, factor H, CFHR, C3B, 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 ocular diseases, such as diseases or disorders associated with diabetic retinal degeneration or macular edema. (see section III below)
The term "VEGF" refers to 165-amino acid vascular endothelial growth factors, related 121-, 189-and 206-amino acid vascular endothelial growth factors, and naturally occurring dual genes and processed forms of those growth factors. VEGF may refer to VEGF proteins from any species.
VEGF is essential in normal development and pathologic angiogenesis. Hypoxia-induced stellate cell secretion of VEGF is a key factor in directing retinal angiogenesis. Elevated VEGF levels also induce pathological growth of new blood vessels in the retina and choroid. Inhibition of angiogenic factors (e.g., VEGF) has become a major strategy for designing therapeutic approaches for the treatment of pathological ocular angiogenesis, including age-related macular degeneration, proliferative retinal degeneration, and retinal degeneration in premature infants.
The term "VEGF-mediated disorder (VEGF-mediated disorder)" refers to any disorder whose symptoms or disease states occur, progress, or persist in need of VEGF involvement. Exemplary VEGF-mediated disorders include, but are not limited to: age-related macular degeneration, neovascular glaucoma, diabetic retinal degeneration, macular edema, diabetic macular edema, pathologic myopia, retinal vein occlusion, retinal degeneration of premature infants, abnormal vascular proliferation associated with lens disease (phacomyces), edema (such as that 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 svgfr.
In some embodiments, the first component is an antibody or antigen binding fragment against VEGF, more particularly an anti-VEGF Fab. The present disclosure provides VEGF antibodies and antigen-binding fragments. Other anti-VEGF antibodies, VEGF antagonists, and VEGF receptor antagonists that may be used include, for example: ranibizumab, bevacizumab, albesipu, pipadatinib, CT-322 and anti-VEGF antibodies and fragments thereof, as described in US 2012/0014958, WO 1998/045331 and WO 2015/198243, which are incorporated herein by reference in their entirety. In some embodiments, the first component comprises a drug that targets 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 an erythropoietin receptor (EPOR). In many species, EPO refers to an erythropoietin protein. The protein sequences of human, cynomolgus monkey, mouse, rat, rabbit EPO are publicly available. Human EPO may also undergo hyperglycosylation. The term "EPO Receptor" or "EPOR" is used interchangeably to refer to the erythropoietin Receptor proteins in different species.
e) Angiogenin
In some embodiments, the first component binds to an angiopoietin, such as angiopoietin 2 (ANG 2). ANG2 is known to be a candidate therapeutic for wet AMD because it plays a role in both angiogenesis and immune activation, both of which involve pathological neovascularization of the eye. In the human eye, higher levels of ANG2 correlate with disease severity in wet AMD. Elevated intraocular ANG2 levels were also detected in diabetic retinal degeneration and retinal vein occlusion patients, suggesting that targeting intraocular ANG2 is of potential medical significance. ANG2 refers to a protein in a different species. Also, researchers have suggested the use of combined inhibition of VEGF-A/ANG2 to substantially reduce vascular leakage, immunoreactivity, and apoptosis.
f) Interleukin
In some embodiments, the first component binds to an interleukin, such as interleukin (IL-1β), IL-6, IL-10, IL-17A, and IL-19. Interleukins are associated with ocular diseases such as uveitis, an inflammatory disease that may be blinding. The interleukins may be derived from any species.
g) Platelet Derived Growth Factor (PDGF)
In some embodiments, the first component binds to a therapeutic target that is Platelet Derived Growth Factor (PDGF) or platelet derived growth factor subunit B (PDGF-BB). PDGF and PDGF-BB may be derived from any species. In some embodiments, the first component comprises a PDGF antagonist, such as those disclosed in section ii.a.2.e) below.
h)VPDF
In some embodiments, the first component binds to VEGF and PDGF. Various proteins, antibodies, antibody fragments, binding domains, agonists and antagonists may bind to VEGF and PDGF. As used herein, the term "anti-VP" refers to a bispecific antibody or fragment thereof that binds to VEGF and PDGF.
In some embodiments, the first component is a dual targeting Fab, i.e., a dutaFab. As used herein, "anti-VPDF (anti-VPDF)" refers to the dutab that binds to 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 comprising at least one C-terminal PDZ domain and ATP-independent chaperones associated with protein metabolism and cell fate. Clausen et al Molecular cell 10 (3): 443-445 (2002). Four HtrA proteins exist in humans: htrA1, htrA2, htrA3 and HtrA4. In humans, htrA1, htrA3 and HtrA4 share the same domain architecture: an N-terminal IGFBP-like module and a Kazal-like module, a protease domain comprising a trypsin-like fold, and a C-terminal PDZ domain. Human genetic studies have found that there is a strong correlation between progression of age-related macular degeneration (AMD) and Single Nucleotide Polymorphisms (SNPs) in the HtrA1 promoter region, leading to elevated levels of HtrA1 transcripts. Dewan et al, science 314:989-992 (2006); yang et al Science 314:992-933 (2006).
In some embodiments, the first component binds to HtrA1. In some embodiments, the first component binds to HtrA2. In some embodiments, the first component binds to HtrA3. In some embodiments, the first component binds to HtrA4.
j) Other therapeutic targets
In some embodiments, the first component binds to one of the following therapeutic targets: factor P, factor D, TNF alpha, FGFR, IL-6R, tie2, S1P, integrin αvβ3, integrin αvβ5, integrin α5β1, betacellulin (betacellin), apelin/APJ, complement factor D, TNF alpha, htrA1, ST-2 receptor, insulin, human growth factor, complement factor H, CD, CD46, CD55, CD59, complement receptor 1-associated (CRRY), nerve growth factor, pigment epithelium derived factor, endostatin, ciliary neurotrophic factor, complement factor 1 inhibitor, complement factor-like 1, complement factor I, and the like.
The term "factor D" refers to a factor D protein derived from any species.
The term "factor P" refers to a factor P protein derived from any species. Human factor P is available from complete Tech (Tyler, TX). Macaque factor P can be purified from macaque serum (protocol adapted from Nakano et al 1986,J Immunol Methods 90:77-83). Factor P is also known in the art as "Properdin".
The term "FGFR2" refers to fibroblast growth factor receptor 2 derived from any species.
2. Therapeutic agent
Any suitable therapeutic agent for treating an ocular disorder may be used as the first component (as described in section III below). In some embodiments, the first component comprises a putative therapeutic drug that binds to a target in the eye. In some embodiments, the first component binds to a humanized target. In some embodiments, the first component comprises a recognized therapeutic agent for treating an ocular disease.
a) VEGF targeting drugs
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 (original ESBA-1008, an anti-VEGF single chain antibody fragment; novartis) or bispecific anti-VEGF antibodies (e.g., anti-VEGF/anti-angiopoietin 2 bispecific antibodies such as RG-7716; roche)); (2) Soluble VEGF receptor fusion proteins (e.g.,>alopecuroide); (3) anti-VEGF(e.g., abicalcar pegol; molecular Partners AG/Allergan); or (4) an anti-VEGF aptamer (e.g., +.>Sodium pipadatinib).
In some embodiments, the first component comprises(ranibizumab), particularly for the treatment of eye diseases. In some cases, the ocular disease is age-related macular degeneration (AMD; e.g., wet AMD). In some cases, the ocular disease is Geographic Atrophy (GA). In some cases, the ocular disease is Diabetic Macular Edema (DME) and/or diabetic retinal degeneration (DR; e.g., non-proliferative DR (NPDR) or Proliferative DR (PDR)).
In some embodiments, the first component comprises RTH-258, which is particularly useful for treating eye diseases. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.
In some embodiments, the first component comprises(aflibercept), which is particularly useful for the treatment of ocular diseases. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA. In some cases, the ocular disease is DME and/or DR (e.g., NPDR or PDR).
In some embodiments, the first component comprises abacic pegol, which is particularly useful for treating eye diseases. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.
In some embodiments, the first component comprises(piperigatran sodium (pegaptanib sodium)), which is particularly useful for the treatment of eye diseases. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular 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 (original ESBA-1008, an anti-VEGF single chain antibody fragment; novartis), bispecific anti-VEGF antibodies (e.g., anti-VEGF/anti-angiopoietin 2 bispecific antibodies such as RG-7716; roche), soluble recombinant receptor fusion proteins (e.g., recombinant receptor fusion proteins>(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->(sodium pipadatinib; neXstar Pharmaceuticals/OSI Pharmaceuticals)), an aptamer capable of blocking VEGFR, an aptamer neutralizing anti-VEGFR antibodies, a small molecule inhibitor of VEGFR tyrosine kinase, an anti-VEGF->(e.g., abicap pegol; molecular Partners AG/Allergan), small interfering RNA that inhibits VEGF or VEGFR expression, VEGFR tyrosine kinase inhibitionAgents (e.g., 4- (4-bromo-2-fluoroanilino) -6-methoxy-7- (1-methylpiperidin) -4-ylmethoxy) quinazoline (ZD 6474), 4- (4-fluoro-2-methylindol-5-yloxy) -6-methoxy-7- (3-pyrrolidin-1-ylpropoxy) quinazoline (AZD 2171), wata-anib (vat anib) (PTK 787), chromatinib (semaxaminib) (SU 5416; SUGEN) and(sunitinib), and combinations thereof.
c) Anti-neoangiogenesis agents
In some embodiments, the first component comprises an agent having anti-neovascularization activity for treating an ocular disorder, the agent is, for example, an anti-inflammatory drug, a mammalian targeted rapamycin (mTOR) inhibitor (e.g., rapamycin,(everolimus) and +.>(temsirolimus), cyclosporine, tumor Necrosis Factor (TNF) antagonists (e.g., anti-tnfα antibodies or antigen-binding fragments thereof (e.g., infliximab, adalimumab, cetuzumab (certolizumab pegol), and golimumab)), or soluble receptor fusion proteins (e.g., etanercept), anticomplements, non-steroidal anti-inflammatory drugs (NSAIDs), or combinations thereof.
d) Neuroprotective agents
In some embodiments, the first component comprises an agent that has neuroprotective effects and can potentially reduce disease progression. For example, the agent may reduce the progression of dry AMD to wet AMD. Examples of neuroprotective agents include a class of drugs known as "neurotensins" which include drugs such as Dehydroepiandrosterone (DHEA) (PRASTERA TM And) Dehydroepiandrosterone sulfate and pregnenolone sulfide.
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., REGN 2176-3), (2) an anti-PDGF-BB pegylated aptamer (e.g.,e10030; ophthotech/Novartis), (3) soluble PDGFR receptor fusion proteins, (4) dual PDGF/VEGF antagonists/inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)), (5) dual-specific anti-PDGF/anti-VEGF antibodies, (6) anti-PDGFR antibodies, or (7) small molecule inhibitors (e.g., squalamine).
f) Complement system antagonists
In some embodiments, the first component comprises a complement system antagonist. Examples of complement system antagonists include: complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; opthotech)), anti-C5 antibodies (e.g., LFG-316; novartis), anti-properdin antibodies (e.g., anti-properdin antibodies; CLG-561; alcon), complement factor D antagonists (e.g., anti-complement factor D antibodies; lanpalizumab; roche), and C3 blocking peptides (e.g., APL-2; appelis).
g) Accepted medicaments for the treatment of ocular diseases
In some embodiments, the first component comprises a recognized therapeutic agent for treating an ocular disease. Treatment of ocular disorders is described in section III below. Examples of recognized drugs include: non-steroidal anti-inflammatory drugs (NSAIDs), steroids (e.g., for reducing inflammation and/or fibrosis), antibiotics, local ophthalmic anesthetics, ophthalmic adhesives (e.g., for post-operative wound closure), enzyme preparations (e.g., for vitrectomy), DNA or RNA (e.g., for gene therapy techniques), agents that mediate neuroprotection (such as providing neurotrophic factors, blocking excessive glutamate stimulation, stabilizing Ca) 2+ Constant, prevention of apoptosis, modulation of immune status via vaccination, induction of endogenous neuroprotective mechanisms, antioxidants, vitamins and mineral supplements).
In some embodiments, the first component comprises any suitable DME and/or DR therapeutic agent, particularly for use in therapyOcular diseases, including but not limited to VEGF antagonists (e.g.,or->) Corticosteroids (e.g., corticosteroid implant,)>Dexamethasone IVT implant; or->Fluocinolone acetonide IVT implant) or a corticosteroid (e.g., aneld) formulated for administration by IVT injection, or a combination thereof. In some cases, the ocular disease is DME and/or DR.
Further examples of recognized drugs for treating ocular disorders suitable for use as the first component condition include, but are not limited to:(verteporfin), a photoactivated drug, which is typically used in combination with photodynamic therapy using a non-thermal laser), PKC412, endoconn (NS 3728;NeuroSearch A/S), neurotrophic factors (e.g., glial-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide (dorzolamide), and combinations thereof >9-cis-retinaldehyde, ocular drugs (e.g., iodophor (phospholine iodide), diethylphosphoryl thiocholine (echo sulfate) or carbonic anhydrase inhibitors), vitamin Wo Si he (veovastat) (AE-941;AEterna Laboratories,Inc), sirna-027 (AGF-745;Sima Therapeutics,Inc), neurotrophins (including, by way of example only, NT-4/5, genentech), cand5 (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals), integrin antagonists (including those from Jerini AG and Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (such as, for example)EntreMed, inc.), cardiotrophin-1 (Genntech), 2-methoxyestradiol (Allergan/Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan), LYN-002 (Lynkeus Biotech), microalgae compounds (Aquasearch/Albany, mera Pharmaceuticals), D-9120 (Celltech Group plc), ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme/Celtrix), tyrosine kinase inhibitors (e.g., those described in U.S. Pat. No. 7,771,742, and VEGFR inhibitors SUGEN (SU 5416), or Pfizer' S Inlyta (dacmetinib)), (dacomitinib)) >(Lalatinib), NX-278-L (NeXstar Pharmaceuticals/Gilead Sciences), opt-24 (OPTIS France SA), retinal ganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A)&M University System), KP-102 (Krenitsky Pharmaceuticals), cyclosporin a, therapeutic agents for use in photodynamic therapy (e.g.,receptor-targeted PDT, bristol-Myers Squibb, co.; porphin sodium (porfimer sodium) for injection with PDT; verteporfin, QLT inc; rotapofungin (R), miravent Medical Technologies for use with PDT; sodium talaporfin (talaporfin sodium), nippon Petroleum, for use with PDT; and moxifloxacin lutetium (motexafin lutetium), pharmcycles, inc.), antisense oligonucleotides (including, for example, products tested by Novagali Pharma SA and ISIS-13650,Ionis Pharmaceuticals), and combinations thereof. />
In some embodiments, the first component comprises a tissue factor antagonist (e.g., hI-con1; iconic Therapeutics), an alpha-adrenergic receptor agonist (e.g., brimonidine tartrate (brimonidine tartrate); allergan), a peptide vaccine (e.g., S-646240; shonoogi), an amyloid beta antagonist (e.g., an anti-beta amyloid monoclonal antibody; GSK-933776), an S1P antagonist (e.g., an anti-S1P antibody; iSONEP) TM The method comprises the steps of carrying out a first treatment on the surface of the Lpath Inc.), ROBO4 antagonists, and anti-ROBO 4 antibodiesBody (e.g., DS-7080a;Daiichi Sankyo).
In some embodiments, the first component comprises a tryptophan-tRNA synthetase (TrpRS), squalamine,(anecortave acetate for long-acting suspensions; alcon, inc.), combretastatin A4 prodrug (CA 4P), and->(mifepristone-ru 486)), subconjunctival triamcinolone acetonide (subtenon triamcinolone acetonide), IVT crystalline triamcinolone acetonide, matrix metalloproteinase inhibitors (e.g., prinomastat); AG3340; pfizer), fluocinolone acetonide (including fluocinolone acetonide intraocular implants; bausch&Lomb/controlled delivery system), linezolid (linomide), inhibitors of integrin beta 3 function, angiostatin, and combinations thereof. These and other therapeutic agents are described, for example, in U.S. patent application No. US 2014/0017244, which is incorporated herein by reference in its entirety.
3. Antibodies and antigen binding fragments
In some embodiments, the first component comprises or is derived from an antibody or antigen binding fragment thereof capable of binding an antigen. The antibody or antigen binding fragment binds to an unrelated, non-target protein to a lesser extent than the antibody binds to the target by about 10%, as measured by, for example, surface Plasmon Resonance (SPR). In certain aspects, the dissociation constant (K) of an antibody or antigen-binding fragment that binds to the target D ) Is less than or equal to 1. Mu.M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM (e.g., 10) -8 M or less, e.g. 10 -8 M to 10 -13 M, e.g. 10 -9 To 10 -13 M). When antibody K D At 1 μm or less, the antibody or antigen binding fragment thereof is said to "specifically bind" to the target.
In some embodiments, the antibody or antigen binding fragment thereof comprises a bispecific antibody, an antibody lacking at least an Fc domain, a Fab fragment, (Fab') 2 Fragments, fab' fragments, vhH fragments, scFv-Fc fragments or microType antibody.
In some embodiments, the antibody or antigen binding fragment thereof binds to an antigen present in the eye. In some embodiments, the antibody or antigen binding fragment thereof may bind to VEGF, htrA1, IL-33, C5, factor P, factor D, EPO, EPOR, IL-1β, IL-17A, IL-10, TNF α, 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-ANG 2 antibody or antibody-binding fragment, or an anti-IL-1 β antibody or antibody-binding fragment. Examples of antibodies that bind VEGF include(Leizumab), ->(Abelmoschus),>(Bluoracelizumab-dbll) and (bevacizumab).
In some embodiments, the antibody comprises a bispecific antibody. In some embodiments, the bispecific antibody is an anti-VEGF/anti-Ang 2 bispecific antibody, such as RG-7716 or any of the bispecific anti-VEGF/anti-Ang 2 bispecific antibodies disclosed in WO 2010/069532 or WO 2016/073157, or variants thereof. In some embodiments, the bispecific antibody is an anti-VPDF antibody, i.e., an anti-VEGF and anti-PDGF dutaFab antibody.
In some embodiments, the first component is an anti-IL-6 antibody, such as EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890), cetuximab (siltuximab;) Olouzumab (olokizumab), clazakizumab (clazakizumab), cetuximab (sirukumab), ai Ximo mab (elsilimomab), OPR-003, MEDI5117, PF-04236921 or variants thereof. />
In some embodiments, the first component is an anti-IL-6R antibody, such as tobalizumab (tocilizumab;) (see, e.g., WO 1992/019579), sarilumab (Sarilumab), ALX-0061, SA237 or variants thereof.
In some embodiments, the first component is RabFab, which is an antigen-binding Fab fragment derived from a parent monoclonal antibody (G10) produced in rabbits, directed against a phosphorylated peptide derived from the intracellular domain of the human cMET receptor, and thus does not bind to extracellular targets in the eye. Shatz, W.et al mol.Pharm.,13 (9): 2996-3003 (2016).
In some embodiments, the antigen binding fragment comprises a peptide or polypeptide that is not an antibody or antigen binding fragment thereof.
4. Growth factors
In some embodiments, the first component comprises a growth factor. In some embodiments, the growth factor comprises fibroblast growth factor, platelet-derived growth factor, nerve Growth Factor (NGF), VEGF, fibroblast Growth Factor (FGF), and insulin-like growth factor-I (IGF-I).
5-hemi-absentamino acid peptide
In some embodiments, the first component comprises a cysteine knot peptide. In some embodiments, the cysteine-binding peptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 92 (cysteine-binding 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 section ii.a.2, supra, section ii.a.4, supra. In some embodiments, the first component comprises a cysteine knot peptide that is covalently linked to an anti-VEGF antigen binding fragment.
In some embodiments, the HABD (i.e., the second component) is covalently linked to the first component at a cysteine knot peptide. In some embodiments, the covalent linker comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 95. In some embodiments, the covalent linker comprises the sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 95). In some embodiments, the cysteine-binding peptide is covalently linked to VG1 with a C-terminal His tag (SEQ ID NO: 29). In some embodiments, the cysteine-binding peptide is covalently linked to VG1 (SEQ ID NO: 32) having an Ig domain deletion and a C-terminal His tag. In some embodiments, the cysteine knot peptide is covalently linked to VG1 with an N-terminal His tag. In some embodiments, the cysteine-binding peptide is covalently linked to VG1 with an Ig domain deletion and an N-terminal His tag.
B. Second component-Hyaluronic Acid Binding Domain (HABD)
In many embodiments, the second component comprises or is derived from an HA-binding protein (which comprises an HA-binding domain; HABD). In some embodiments, the second component comprises HABD. Examples of proteins comprising HABD include CD44, tumor necrosis factor-stimulated gene-6 (TSG 6), proteoglycans, brain-specific junction protein (BRAL 1), lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1), and aggrecan.
In some embodiments, the two second components may be different or the same. For example, the therapeutic molecule may comprise a second component comprising two CD44 domains, two TSG-6 domains, two VG1 domains, or any combination of the foregoing domains to form a pair of two different domains.
The eye is a complex tissue with several distinct compartments including cornea, aqueous humor, lens, vitreous humor, retina, retinal pigment epithelium and choroid. These compartments include extracellular macromolecules such as HA.
The term "hyaluronic acid binding protein" or "HA binding protein" refers to a protein or family of proteins that bind HA. Typically, these HA-binding proteins comprise HABD. A variety of HA-binding molecules are well known in the art and may be used as the second component (see, e.g., day et al, 2002,J Bio.Chem 277:4585; and Yang et al, 1994,EMBO J13:286-296). Exemplary HA-binding proteins include CD44, LYVE-1, aggrecan, multifunctional proteoglycans, brevican, neurocan, hyaluronan-binding protein 1 (HABP 1; also known as C1qBP/C1qR and p 32), HAPLN1 (also known as connexin and CRTL 1), hyaluronan and proteoglycan connexin 4 (HAPLN 4; also known as connexin 2), layilin, stabilin-1, stabilin-2, brain-specific connexin (BRAL 1) or tumor necrosis factor stimulating gene 6 (TSG-6), RHA M, bacterial HA synthase and type VI collagen.
Many HA-binding proteins and peptide fragments contain a common domain, which is about 100 amino acids in length, involved in HA binding; this Domain is called the "linking Domain" (Yang et al, EMBO J13:2, 286-296 (1994) and Mahoney et al, J Bio. Chem276:25,22764-22771 (2001)). Any of these proteins may be used in the present invention. Any HA binding protein, such as the HABD of the exemplary proteins described above, may be included in the second component to impart the ability to bind to HA. Preferably, the second component comprises a CD44 (CD 44) domain, a brain specific binding protein (BRAL 1) domain, a tumor necrosis factor stimulating gene 6 (TSG-6) domain, a lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1) domain, a Hyaluronic Acid Binding Protein (HABP) domain, an aggrecan G1 (AG 1) domain or a multifunctional proteoglycan G1 (VG 1) domain. Exemplary and suitable HA binding molecules (including peptide tags) for use in the eye are described in WO 2014/99997 and WO 2015/19824, the contents of which are incorporated herein by reference in their entirety. Any of the sequences described herein may be used in the present invention.
In some embodiments, the second component is covalently linked to the first component so as to reduce clearance of the first component from the eye, thereby extending its intraocular half-life. The first component may benefit from longer ocular retention time and/or longer time to act on ocular disease.
Furthermore, the second component may be non-covalently bound to a third component comprising HA to form a conjugate. In some embodiments, each second component in the conjugate may be bound 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 in several ranges; binding affinity may be modulated according to the mechanism of action of the therapeutically active agent. For example, if the site of action is in the vitreous humor, a high binding affinity may help retain the biological agent in the vitreous humor. Conversely, if the site of action is in the retina, lower binding affinity may aid the passage of the biological agent through the vitreous to reach the retina.
In many embodiments, the binding affinity of HABD to HA can be measured using methods including Surface Plasmon Resonance (SPR). Without being bound by theory, in some embodiments, the binding affinity of HABD to HA (K D ) The range comprises 10nM to 10. Mu.M, 5nM to 10nM and 100nM to 5. Mu.M.
In many embodiments, the interaction of HABD with HA can be observed. In some embodiments, interactions are observed using a method that includes Fluorescence Correlation Spectroscopy (FCS). In FCS, the diffusion of molecules can be determined by monitoring the fluorescence intensity of small volume portions in solution. The fluorescence intensity fluctuates due to the movement of the molecules, and quantitative analysis of these fluctuations can give the diffusion time of the molecules. By using fluorescent dyes with appropriate spectral properties, diffusion in biological matrices can be determined. In some embodiments, the observed value of FCS is related to the measured value of SPR.
In some embodiments, the HABD comprises a wild-type sequence compared to the protein from which it was derived. In some embodiments, the HABD may comprise one or more mutations in its protein sequence compared 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, the HABD comprises a single amino acid substitution or a double amino acid substitution. In many examples, substitutions may comprise conservative mutations, wherein an amino acid substitution changes the original amino acid to a different amino acid with similar biochemical properties. In other examples, substitutions may comprise non-conservative mutations, wherein an amino acid substitution changes the original amino acid to a different amino acid with different biochemical properties.
In some embodiments, the HABD comprises amino acids that 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 desired for long-acting treatment.
In some embodiments, the HABD comprises amino acids that contribute to the thermal stability of the HABD and/or therapeutic molecule. In some embodiments, these amino acids may remain 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 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to one of the reference sequences disclosed herein. In some embodiments, the HABD comprises 1 to 3 mutations, wherein the 1 to 3 mutations independently comprise a single amino acid substitution, a double amino acid substitution, an addition, a deletion, and a truncation. In some embodiments, the HABD comprises 1 to 5 mutations, wherein the 1 to 5 mutations independently comprise a single amino acid substitution, a double amino acid substitution, an addition, a deletion, and 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 comprises a linking domain, a GAG attachment domain, a transmembrane domain and a cytoplasmic domain. Several isoforms with different modular compositions treated by alternative splicing are described. In some embodiments, the second component is derived from or comprises a CD44 HA receptor domain. In some embodiments, the second component is derived from or comprises SEQ ID NO. 2.
2. Tumor necrosis factor stimulating gene 6 (TSG 6)
In some embodiments, the second component is derived from TSG6.TSG-6, also known as TNFAIP6, consists of an HA-binding attachment domain followed by a CUB domain. In some embodiments, the second component is derived from or comprises a TSG6 HA binding linking domain. In some embodiments, the second component is derived from or comprises SEQ ID NO. 4.
3. Multifunctional proteoglycan
In some embodiments, the second component is derived from a multifunctional proteoglycan. The multifunctional proteoglycan comprises the following domains: VG1, GAG attachment domain and G3 domain (figure 8A). The VG1 domain (SEQ ID NO: 29) contains the Ig domain, link1 and Link2 (FIG. 8A). In some embodiments, the second component comprises Link1 (SEQ ID NO: 30) and/or Link2 (SEQ ID NO: 31), wherein Link1 and/or Link2 is capable of binding HA.
a) Wild VG1
In some embodiments, the HABD comprises wild-type (WT) VG1, the amino acid sequence of which is shown in SEQ ID NO. 29. In some embodiments, the HABD comprises amino acid sequences as shown in Link1 (SEQ ID NO: 30) and/or Link2 (SEQ ID NO: 31).
b) Mutation VG1
In some embodiments, HABD comprises the mutation VG1. In many embodiments, VG1 mutations are relative to the amino acid sequences shown as SEQ ID NO. 29 (WT VG 1), 32 (VG1ΔIg), 60 (WT VG1 consensus sequence) or 86 (VG1ΔIg consensus sequence). In some embodiments, the HABD comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 29 (WT VG 1), 32 (VG1ΔIg), 60 (WT VG1 consensus sequence), or 86 (VG1ΔIg consensus sequence). In some embodiments, the HABD comprises a sequence that is at least 95% identical to SEQ ID NO. 29 (WT VG 1), 32 (VG1ΔIg), 60 (WT VG1 consensus sequence), or 86 (VG1ΔIg consensus sequence).
c) Truncated VG1
In some embodiments, the HABD comprises a truncation mutation relative to SEQ ID NO. 29 (WT VG 1) or 60 (WT VG1 consensus sequence). In some embodiments, the HABD comprises a truncation of 1 to 129 amino acids from the N-terminus of the multifunctional proteoglycan. In some embodiments, the HABD comprises a truncated sequence in which the Ig domain of the wild-type proteoglycan is absent. 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:32 (VG1ΔIg) or SEQ ID NO:86 (VG1ΔIg consensus sequence). In some embodiments, the HABD comprises a sequence that is at least 95% identical to SEQ ID NO. 32 (VG1ΔIg) or SEQ ID NO. 86 (VG1ΔIg consensus sequence). In some embodiments, the HABD comprises SEQ ID NO. 32 (VG1ΔIg).
d) Amino acid substitutions
In some embodiments, the HABD comprises at least one of the following amino acids relative to SEQ ID NO. 29: r160, Y161, E194, D197, Y208, R214, Y230, F261, D295, and R233. In some embodiments, the HABD comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the following amino acids relative to SEQ ID No. 29: r160, Y161, E194, D197, Y208, R214, Y230, F261, D295, and R233.
In some embodiments, the HABD comprises a sequence in which amino acids may be mutated relative to wild-type to increase or decrease HA binding affinity. In some embodiments, the HABD 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. In some embodiments, the HABD 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. In some embodiments, the HABD comprises mutations in 2, 3, 4, 5, or 6 of the following positions relative to SEQ ID No. 29: r160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.
In some embodiments, the HABD comprises at least one of the following mutations relative to SEQ ID No. 29: R160A, Y161A, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a and LYR325LFK. In some embodiments, the HABD includes at least one of Y208A and H306A.
In some embodiments, the HABD comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the following mutations relative to SEQ ID No. 29: R160A, Y161A, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a and LYR325LFK. In some embodiments, the HABD comprises at least 2, 3, 4, 5, or 6 of the following mutations relative to SEQ ID No. 29: R160A, Y161A, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a and LYR325LFK.
In some embodiments, the HABD is SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ 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 junction protein (BRAL 1)
In some embodiments, the second component is derived from BRAL1.BRAL1 comprises an immunoglobulin domain, a junction domain module 1 and a junction domain module 2. The connection domain modules 1 and 2 are capable of binding HA. In some embodiments, the second component comprises a connection domain module 1 and/or a connection 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 that comprises a linking domain that binds to HA. In some embodiments, the second component comprises a linking domain from LYVE-1.
6. Aggrecan
In some embodiments, the second component is derived from aggrecan. Aggrecan comprises three globular domains: the G1 domain HAs the structural motif of the connexin and interacts with HA; the G2 domain is homologous to the G1 domain and is involved in product processing; the G3 domain constitutes the carboxy terminus of the core protein. In some embodiments, the second component comprises a G1 domain from aggrecan.
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 a first component and a second component) is pre-complexed with HA to form a conjugate. In some embodiments, the third component is HA having a molecular weight of 5kDa 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 of the first component is covalently linked to a K of less than or equal to 10.0. Mu.M D To a third component (i.e., hyaluronic acid). For example, the second component may be less than or equal to 9.0. Mu.M, 8.0. Mu.M, 7.0. Mu.M, 6.0. Mu.M, 5.0. Mu.M, 4.0. Mu.M, 3.0. Mu.M, 2.0. Mu.M, 1.5. Mu.M, 1.0. Mu.M, or 0.5. Mu.M K D Binds HA.
1. Hyaluronic Acid (HA)
Hyaluronic Acid (HA) is a linear glycosaminoglycan present in the extracellular matrix and cell surface. HA contains repeating disaccharide units of N-acetylglucosamine (GlcNac) and glucuronic acid (glcna) linked by alternating β1→3 glucuronic acid and β1→4 glucosamine (glucosaminodic) linkages to form a linear polymer. HA is further described in the following documents: necas et al, 2008,Veterinarni Medicina,53:397-411. Glycosaminoglycans are ubiquitous in the extracellular matrix of all vertebrates, as well as in the capsule of certain streptococcal strains. Functionally, HA molecules are important for maintaining highly hydrated extracellular matrix in tissues, which are involved in cell adhesion and support cell migration. The vitreous humor, which is mainly composed of HA in addition to water, is excellent in retaining moisture and structure in the central portion of the eye. It helps to keep the eyes lubricated and to replenish any water lost. HA also exhibits a variety of biological functions through interactions with a number of 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.
HA HAs a broad molecular weight range of 1000Da to 10000000 Da. The natural high molecular weight HA in tissues is degraded into small molecules in metabolic pathways through the lymphatic system, lymph nodes, liver and kidneys. Although HA is known to have a half-life in plasma of about 2.5 minutes to 5.5 minutes, it is reported to have a half-life in the vitreous of the eye of about 70 days. The unique physicochemical properties and various biological functions of HA have led to its widespread use in biomedical fields such as drug delivery, arthritis treatment, ophthalmic surgery and tissue engineering. In particular, HA HAs been widely studied for targeted specific and long-acting delivery of bio/drugs through various delivery routes. HA HAs been developed as an effective delivery vehicle for topical ophthalmic drugs, taking advantage of its viscoelastic and mucoadhesive properties.
HA having a defined size HAs been shown to be suitable for the present invention. Accordingly, HA may have a molecular weight of at least 2, 3, 4, 5, 6, 7, 8 or 9kDa and/or a molecular weight of at most 60, 50, 40, 30, 25, 20 or 15 kDa. In particular, suitable ranges for molecular weights are 3kDa to 60kDa, particularly 4kDa to 30kDa, more particularly 5kDa to 20kDa.
In some embodiments, unmodified naturally occurring HA is preferably used. In these embodiments, the use of unmodified naturally occurring HA reduces side effects. For example, pre-complexing HABD with 10kDa HA reduces in vitro precipitation in vitreous humor and reduces intraocular toxicity observed in pigs and rabbits. In other examples, in the case where the HABD is TSG-6 or CD44, intraocular toxicity such as inflammation and retina is observed when TSG-6 or CD44 is not pre-complexed with HA.
In some embodiments, HA is hyaluronate, which includes, but is not limited to, potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.
In some embodiments, HA may have minor chemical modifications. Chemical modifications can be used to reduce HA degradation, increase or decrease water solubility, 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, diepoxide, formaldehyde and dihalides are bifunctional reagents for crosslinking HA. Chemically modified HA formulations include, but are not limited to, aminoethyl methacrylate HA, adipic acid dihydrazide grafted HA, dimethyl ether complexed HA, HA-cysteine ethyl ester, urea crosslinked HA, and N-acetyl cysteine HA. Of particular interest are modifications that reduce HA degradation in the HA eye.
2. Pre-complexing therapeutic molecules 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, resulting in adverse effects, as described in example 5 below. In some cases, these effects may be caused by contact of free HABD with IVT HA at the injection site. Pre-compounding 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. When HABD is converted from interaction with pre-complexed HA to interaction with IVT HA, the diffusion time is slowed and the vitreous half-life is prolonged. Thus, in some embodiments, the therapeutic molecule is a conjugate, comprises the therapeutic molecule, and further comprises one or more third components comprising HA.
In some embodiments, the conjugate comprises a non-covalent interaction between the therapeutic molecule and HA. In some embodiments, the conjugate comprises a covalent interaction between the therapeutic molecule and HA.
In some embodiments, the conjugate may be an isolated conjugate, i.e., the conjugate is not within the individual to be treated. In some aspects, the conjugate is purified to a purity of greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods of assessing antibody purity, see, for example: flatman et al, J Chromatogr B848:79-87 (2007).
D. Fusion proteins
In some embodiments, the first component and the second component are proteins, more preferably comprised in a fusion protein; the first component and the second component are linked via a covalent linker.
Fusion proteins are proteins made up of two or more otherwise isolated proteins or peptides linked. The method results in a single polypeptide having the functional properties derived from each of the original, separate proteins. Proteins may be directly fused to each other. Proteins may also be fused via linkers that increase the likelihood that the proteins will fold independently of each other and behave as desired. Dimeric or multimeric fusion proteins can be produced by genetic engineering by fusion with the original protein of a peptide domain that induces protein complexation (such as with an antibody domain).
In some embodiments, the second component is directly bonded to the first component. This means that the second component immediately follows the first component (and vice versa), and that no other chemical element (atom or group) is present between these 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 last amino acid of the second component is immediately adjacent to the first amino acid of the first component.
In some embodiments, the second component is indirectly bound to the first component via a linker (particularly a peptide linker). In some embodiments, this means that the peptide linker is between the first component and the second component. In some embodiments, the peptide linker is 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 between the last amino acid of the second component and the first amino acid of the first component.
In some embodiments, these one or both second components are covalently bound to the N-terminus and/or the C-terminus of the first component. In some embodiments, the first component is an antibody or antigen binding fragment, and the one or two second components are covalently bound to the C-terminus of the first component (either 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 therapeutically active agent (i.e., the first component) and the HABD (i.e., the second component). In some embodiments, the linker comprises at least 4 amino acids. In some embodiments, the linker comprises 4 to 25 amino acids. In some embodiments, the linker comprises 5 to 100 amino acids. In some embodiments, the linker comprises 10 to 50 amino acids. In some embodiments, the linker is no more than 25 amino acids in length. In some embodiments, the linker is no more than 50 amino acids in length.
In some embodiments, the peptide linker comprises flexible residues (e.g., glycine and serine) such that adjacent protein domains can move freely relative to each other. Thus, in some embodiments, the peptide linker is a glycine-serine linker, i.e., a peptide linker consisting of a pattern of glycine and serine residues. In one embodiment, the peptide linker is (GxS) n Or (GxS) n G m Where g=glycine and s=serine. In these embodiments, x=3; n=3, 4, 5 or 6; and m=0, 1, 2 or 3. In other embodiments, x=4; n=2, 3, 4 or 5; and m=0, 1, 2 or 3. In some embodiments, x=4 and n=2 or 3. In some embodiments, x=4 and n=2.
In some embodiments, the peptide linker consists of GGGGS (SEQ ID NO: 27) or a multimer thereof, more particularly of (GGGGS) 3 (SEQ ID NO: 28).
In some embodiments, the peptide linker comprises (GS) n Wherein G is glycine and S is serine. In these embodiments, n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodimentsIn some embodiments, the linker is SEQ ID NO 95.
Certain examples of E.therapeutic molecules and bradycardias
1.VEGF
In some embodiments, the therapeutic molecule comprises (1) a first component comprising an anti-VEGF antibody, antibody fragment, antigen-binding fragment, or Fab; (2) One or two second components, wherein the second component comprises a CD44 HA receptor domain, a TSG6 domain and/or a VG1 domain.
In some embodiments, the conjugate comprises (1) a first component comprising an anti-VEGF antibody, antigen-binding fragment, antibody fragment, or Fab; (2) one or two second components; and (3) HA having a molecular weight in the range of 5kDa to 20 kDa.
a)G6.31
In some embodiments, the first component is an antibody comprising a G6.31 anti-VEGF Fab. In some embodiments, the first component is an antibody having the VH domain contained in SEQ ID No. 17. In some embodiments, the first component is an antibody having the VL domain contained in SEQ ID NO. 18. In some embodiments, the first component is an antibody comprising a VH domain as set forth in SEQ ID No. 105. In some embodiments, the first component is an antibody comprising the VL domain shown as SEQ ID NO. 106.
b)PigFab
In some embodiments, the first component is an antibody comprising a PigFab anti-VEGF Fab. In some embodiments, the first component is an antibody having the VH domain contained in SEQ ID No. 66. In some embodiments, the first component is an antibody having a VL domain comprised in SEQ ID NO. 65. In some embodiments, the first component is an antibody comprising a VH domain as set forth in SEQ ID No. 97. In some embodiments, the first component is an antibody comprising the VL domain shown as SEQ ID NO. 98.
c) Leizumab
In some embodiments, the first component is an antibody comprising ranibizumab. In some embodiments, the first component is an antibody having the VH domain contained in SEQ ID No. 77. In some embodiments, the first component is an antibody having the VL domain contained in SEQ ID NO. 76. In some embodiments, the first component is an antibody comprising a VH domain as shown in SEQ ID NO. 114. In some embodiments, the first component is an antibody comprising the VL domain shown as SEQ ID NO. 115.
d)CD44
In some embodiments, one or both of these second components comprises 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 comprises a TSG6 domain. In some embodiments, one or both of these second components comprises 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 comprises a VG1 domain. In some embodiments, these one or both second components comprise one or both of SEQ ID NOs 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.
In some embodiments, the therapeutic molecule comprises SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:76, and/or SEQ ID NO:77.
g) Cysteine Knot Peptide (CKP)
In some embodiments, the first component optionally further comprises a Cysteine Knot Peptide (CKP) in addition to the anti-VEGF antigen binding fragment. In some embodiments, CKP has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 92.
In some embodiments, the therapeutic molecule has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 93. In some embodiments, the anti-VEGF antigen binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 94.
In some embodiments, the therapeutic molecule comprises a first component comprising an anti-VEGF antigen-binding fragment, and the cysteine-binding peptide may further comprise a second component comprising HABD as described in section ii.b above.
In some embodiments, the conjugate comprises (1) a first component comprising an anti-VEGF antigen-binding fragment, (2) one or two second components comprising HABD, and (3) HA having a molecular weight in the range of 5kDa to 20 kDa.
2.NVS24
In some embodiments, the therapeutic molecule comprises (1) a first component comprising anti-VEGF antibody NVS24, and (2) a second component comprising a TSG6 (Lava 12) domain.
In some embodiments, the first component comprises an NVS24 antibody. In some embodiments, the first component is an antibody having the VH domain contained in SEQ ID No. 21. In some embodiments, the first component is an antibody having the VL domain contained in SEQ ID NO. 22. In some embodiments, the first component is an antibody comprising a VH domain as set forth in SEQ ID NO. 109. In some embodiments, the first component is an antibody comprising the VL domain shown as SEQ ID NO. 110.
a)TSG6(Lava12)
In some embodiments, the second component comprises a TSG6 (Lava 12) 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.
anti-VEGF and anti-PDGF dual targeting antibodies (anti-VP-dutaFab; anti-VPDF)
In some embodiments, the therapeutic molecule comprises (1) a first component capable of binding VEGF and PDGF (such as a bispecific antibody or a dual targeting antibody, dutaFab), as described in section ii.a.1.h above; and (2) one or two second components 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 contained in SEQ ID No. 5. In some embodiments, the first component is an antibody having a VL domain comprised in SEQ ID NO. 6. In some embodiments, the first component is an antibody comprising a VH domain as set forth in SEQ ID No. 99. In some embodiments, the first component is an antibody comprising the VL domain shown as SEQ ID NO. 100.
a)CD44
In some embodiments, one or both of these second components comprises a CD44 HA receptor domain. In some embodiments, one or both of these second components comprises 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 comprises a CD44-ko domain. In some embodiments, one or both of these second components comprises the 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 (Lava 12) 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 comprises a VG1 domain. In some embodiments, these one or both second components comprise one or both of SEQ ID NOs 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.
In some embodiments, the therapeutic molecule comprises SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 72, and/or SEQ ID NO 73.
4.RabFab
In some embodiments, the therapeutic molecule comprises (1) a first component comprising a RabFab antibody (as described in section ii.a.3 above); and (2) one or two second components comprising a TSG6 domain and/or a VG1 domain.
In some embodiments, the RabFab antibody comprises RabFab VH and VL domains. In some embodiments, the RabFab antibody comprises: a VH domain comprised in SEQ ID No. 13; and a VL domain comprised 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 comprises a TSG6 domain. In some embodiments, one or both of these second components comprises 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 comprises a VG1 domain. In some embodiments, these one or both second components comprise one or both of SEQ ID NOs 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.
In some embodiments, the therapeutic molecule comprises SEQ ID NO 63 and/or SEQ ID NO 64.
5.20D12v2.3
In some embodiments, the first component is an antibody comprising an anti-complement factor D antibody Fab (20d12v2.3). In some embodiments, the first component is an antibody having a VH domain contained in SEQ ID No. 75. In some embodiments, the first component is an antibody having the VL domain contained in SEQ ID NO. 74. In some embodiments, the first component is an antibody comprising a VH domain as set forth in SEQ ID No. 111. In some embodiments, the first component is an antibody comprising the VL domain shown as SEQ ID NO. 112.
a)VG1
In some embodiments, one or both of these second components comprises a VG1 domain. In some embodiments, these one or both second components comprise one or both of SEQ ID NOs 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.
In some embodiments, the therapeutic molecule comprises SEQ ID NO 74 and/or SEQ ID NO 75.
6.HtrA1
In some embodiments, the first component is an antibody comprising an antibody or antibody fragment capable of binding to human HtrA 1. In some embodiments, the first component is an antibody having the VH domain contained in SEQ ID No. 118. In some embodiments, the first component is an antibody having the VL domain contained in SEQ ID NO. 119. In some embodiments, the first component is an antibody comprising the VH domain shown as SEQ ID NO. 116. In some embodiments, the first component is an antibody comprising the VL domain shown as SEQ ID NO. 117.
a)VG1
In some embodiments, one or both of these second components comprises a VG1 domain. In some embodiments, these one or both second components comprise one or both of SEQ ID NOs 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 87.
In some embodiments, the therapeutic molecule comprises SEQ ID NO 118 and/or SEQ ID NO 119.
Treatment of eye diseases
Materials and methods are used in the treatment of ocular disorders. Ocular disorders may be characterized by the proliferation of new blood vessels, either altered or unregulated, and/or invasion of ocular tissues such as the retina or cornea into structures. Eye diseases may be characterized by a vision networkAtrophy of the membranous tissue (photoreceptors and underlying retinal pigment epithelial cells (RPE) and choroidal capillaries). Non-limiting eye diseases include, for example, age-related macular degeneration (AMD) (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and Geographic Atrophy (GA)), macular degeneration, macular edema, diabetic Macular Edema (DME) (e.g., focal, non-central DME, and diffuse, central-related DME), retinal degeneration, diabetic retinal Degeneration (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR) and high altitude DR), other ischemia-related retinal degenerations, ROP, retinal Vein Occlusion (RVO) (e.g., central (CRVO) and Branched (BRVO) forms), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal/choroidal neovascularization, central serous retinal degeneration (CSR), pathological myopia, von Hippel-Lindau, ocular histoplasmosis, FEVR, coats' disease, norrie disease, retinal abnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, redness, ocular vascular neovascularization, vascular neogenesis glaucoma, retinitis Pigmentosa (RP), hypertensive retinal degeneration, retinal hemangioma hyperplasia, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, macular edema (CME), vasculitis, optic papilla, retinitis, including but not limited to, retinal edema Ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g., allergic) conjunctivitis), leber congenital black Meng Zheng (also known as Leber's congenital amaurosis or LCA), uveitis (including infectious and non-infectious uveitis), and choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, ocular trauma, Diseases and other ophthalmic diseases, wherein the disease or condition is associated with angiogenesis, vascular leakage and/or retinal edema or retinal atrophy. Other exemplary ocular diseases include diseases caused by abnormal proliferation of fibrovascular or fibrous tissue, bagsIncluding all forms of proliferative vitreoretinal degeneration.
Exemplary diseases associated with corneal neovascularization (iris neovascularization, corner neovascularization, or cutaneous redness) include, but are not limited to, epidemic keratoconjunctivitis, vitamin a deficiency, excessive contact lens abrasion, atopic keratitis, upper limbic keratitis, pterygoid keratosis, sjogrens, rosacea, small vesicular disease (phlyctenosis), syphilis, mycobacterial infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infection, herpes zoster infection, protozoan infection, kaposi's sarcoma, predatory corneal ulcers, trie limbic keratosis (Terrien's marginal degeneration), limbic keratolytic, rheumatoid arthritis, systemic lupus erythematosus, multiple arteritis, trauma, wegener's sarcoidosis (Wegeners sarcoidosis), scleritis, steven's syndrome (sJohnson's disease), pemphigoid radial keratosis, and corneal transplantation.
Exemplary ocular diseases associated with choroidal neovascularization and retinal vascular defects including increased vascular leakage, aneurysms, and capillary drops include, but are not limited to, diabetic retinal degeneration, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, elastohydropseudoxanthoma, paget's disease, venous occlusion, arterial occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infection, lyme disease, systemic lupus erythematosus, premature retinal degeneration, retinal edema including macular edema, eilstonia (earles disease), bechets disease, infections that cause retinitis or choroiditis (e.g., multifocal choroids), postocular histoplasmosis, bests disease (vitelliform macular degeneration), myopia, visual fossa (optica), stargardt disease (Stargarts disease), ciliary body applanation, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma, and post-concurrence.
Exemplary ocular diseases associated with retinal tissue (photoreceptors and underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or advanced dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and/or geographic atrophy), diabetic retinal degeneration, stark's disease, skorsby fundus atrophy, retinal cleavage (abnormal division of the sensory layers of the retina), and retinitis pigmentosa.
In certain embodiments according to (or as applied to) any of the embodiments above, the ocular disease is an intraocular neovascular disease selected from the group consisting of: proliferative retinal degeneration, choroidal Neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemia-related retinal degenerations, diabetic macular edema, pathologic myopia, cheibia-lindau disease, ocular histoplasmosis, retinal Vein Occlusion (RVO) (including CRVO and BRVO), corneal neovascularization, retinal neovascularization, and premature retinal degeneration (ROP). In a preferred embodiment of the invention, the eye disease is age-related macular degeneration (AMD), in particular wet AMD or neovascular AMD; diabetic Macular Edema (DME); diabetic retinal Degeneration (DR), particularly proliferative DR or non-proliferative DR; retinal Vein Occlusion (RVO); or Geographic Atrophy (GA).
The therapeutic molecules, conjugates, and compositions disclosed herein are useful as medicaments for treating ocular diseases in mammalian subjects. Examples of mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human. In some embodiments, the therapeutic targets of the therapeutic molecules, conjugates, and compositions are targets in the human eye.
IV. method of treatment
Provided herein are methods of treating an ocular disorder comprising delivering a therapeutic molecule, conjugate, or composition to a tissue of a patient. In many embodiments, the methods include administering a therapeutic molecule such that the therapeutic molecule can provide long-lasting delivery of a therapeutically active agent to a target tissue. In many embodiments, the target tissue is in the eye.
A. Application method
The therapeutic molecule, conjugate or composition may be administered in any effective, convenient manner, including, for example, by topical, oral, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intratracheal, or intradermal routes, and the like. Preferably, the composition is suitable for administration to the eye, more particularly, the composition may be suitable for IVT administration. Thus, in a preferred embodiment, the composition is formulated for intraocular delivery, particularly for IVT injection. In treatment or as a prophylactic agent, the therapeutic molecule, conjugate, or composition may be administered to the individual as an injectable composition (e.g., as a sterile aqueous dispersion).
Without being bound by this theory, it is believed that injection of the conjugate may promote diffusion of HA from the pre-complexed HABD prior to interaction with the IVT HA. The concentration of free HABD in the vitreous is low due to slow dissociation. Lower intravitreal concentrations of free HABD may be less damaging to the eye than therapeutic molecules that are not pre-complexed with HA.
In some embodiments, the administering step is a single injection. In some embodiments, the administering step comprises more than one single injection.
B. Composition and method for producing the same
Provided herein are compositions for use as medicaments, particularly for the treatment of ocular diseases. The compositions may be referred to as pharmaceutical compositions, as they are intended for use in the pharmaceutical field or as medicaments, meaning formulations in a form that allows the biological activity of the active ingredient contained therein to be effective, and that are free of other components having unacceptable toxicity to the subject to whom the composition is to be administered.
In some embodiments, the composition comprises a therapeutic molecule. In some embodiments, the composition comprises a conjugate.
In some embodiments, the composition optionally comprises a pharmaceutically acceptable excipient, diluent or carrier, such as a buffer substance, stabilizer, preservative, or other ingredient, particularly commonly known ingredients associated with pharmaceutical compositions.
In general, the nature of the optional ingredients or additional ingredients will depend on the particular form of the composition and the mode of administration employed. The pharmaceutically acceptable carrier may enhance or stabilize the composition or may be used to facilitate the preparation of the composition. Such carriers can include, but are not limited to, physiologically compatible saline, buffered saline, dextrose, water, glycerol, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, and combinations thereof. The composition may additionally comprise one or more other therapeutic agents, particularly those suitable for treating or preventing diseases or disorders associated with, for example, eye diseases such as retinal vascular diseases. The formulation should be adapted to the mode of administration. For example, parenteral formulations typically comprise injectable fluids which include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol and the like, as carriers. In addition to the biologically neutral carrier, the pharmaceutical composition to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, pH buffering agents and the like.
The composition may comprise a stabilizer. The term "stabilizer" refers to a substance that protects the composition from adverse conditions, such as those that occur during heating or freezing, and/or extends the stability or shelf life of the conjugates of the invention under certain conditions or states. Examples of stabilizers include, but are not limited to: sugars such as sucrose, lactose, and mannose; sugar alcohols such as mannitol; amino acids such as glycine or glutamic acid; and proteins such as human serum albumin or gelatin.
C. Effective dose
Typically, a therapeutically effective amount or effective dose of a therapeutic molecule or conjugate is employed in the pharmaceutical compositions of the present disclosure. The therapeutic molecules and conjugates are formulated into pharmaceutical dosage forms by 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 may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form that facilitate administration and ensure uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect associated with the desired pharmaceutical carrier.
The actual dosage level of the active ingredient (i.e., therapeutic molecule and conjugate) in the pharmaceutical composition can be varied to achieve amounts of the active ingredient effective to achieve the desired therapeutic response for the particular patient, the composition, and the mode of administration that is non-toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors including: activity of the particular compositions of the invention employed; route of administration; the time of application; the rate of excretion of the particular compound employed; duration of treatment; other drugs, compounds and/or materials used in combination with the particular composition employed; age, sex, weight, disease, general health and prior history of the patient receiving the treatment; and the like. Dosage levels may be selected and/or adjusted to achieve a therapeutic response determined using one or more of the ocular/visual assessments described herein. The physician or veterinarian can begin administering the therapeutic molecule of the conjugate employed in the pharmaceutical composition at a dosage below the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, the effective dosage of the compositions described herein for treating ocular disorders depends on a number of different factors including the mode of administration, the target site, the physiological state of the patient, whether the patient is a human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic.
Titration of therapeutic doses is required to optimize safety and efficacy. The doses administered for IVT using the conjugates of the invention may range from 0.1 mg/eye to 10 mg/eye per injection. A single dose per eye may be administered by injecting each eye 1 or more times. For example, a single dose of 20 mg/eye may be delivered in 2 doses, 10mg per injection, giving a total dose of 20mg. The volume per injection may be between 10 microliters and 50 microliters, while the volume per dose may be between 10 microliters and 100 microliters. Consider the dosage and regimen approved by the U.S. Food and Drug Administration (FDA) for Lucentis. Other dosages and protocols suitable for anti-VEGF antibodies or antigen-binding fragments are described in US 2012/0014958, and are incorporated herein by reference in their entirety.
The composition may be administered multiple times. The interval between single doses may be weekly, monthly or yearly. The intervals may also be irregular, as determined by the desired retreatment of the patient, based on, for example, visual acuity or macular edema. Furthermore, alternative dosing intervals may be determined by a physician and administered once a month or as necessary to exert utility. The effectiveness is based on the condition of the eye and the kind and severity of eye diseases, e.g. characterized by pathologic growth, anti-VEGF treatment rate, retinal thickness as determined by Optical Coherence Tomography (OCT), and visual acuity. Dosage and frequency can vary depending on the half-life of the conjugate of the invention in the patient and the level of the therapeutic target (e.g., VEGF, C5, EPO, factor P, etc.). However, in a preferred embodiment of the invention, the composition is administered at most once every three months, in particular at most once every four months, more in particular once every six months. This reflects the extended half-life (and thus the duration of effectiveness) of the first component in the conjugate as compared to the corresponding unbound (free) first component. Accordingly, the elimination half-life of the first component in the conjugate is extended by at least 3-fold, at least 4-fold, or at least 5-fold as compared to the unbound first component. The relative increase in elimination half-life of the first component in the conjugate compared to the free first component can be determined by administering these molecules by IVT injection and measuring the residual concentration at different time points using analytical methods known in the art, such as ELISA, mass spectrometry, western blot, radioimmunoassay, or fluorescent labeling. Blood concentration may also be measured and used to calculate the rate of clearance from the eye (Xu L et al, invest Ophthalmol Vis ScL,54 (3): 1816-24 (2013)). In general, molecules (e.g., antibodies or fragments) that are part of the conjugate exhibit longer intraocular half-lives than the free molecules. The half-life of the conjugate in the eye may 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 aspects it is contemplated that the half-life of the conjugate may 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 co-administration (wherein two or more therapeutic agents are contained in the same or separate formulations) as well as separate administration, in which case administration of the therapeutic molecule and conjugate may occur before, simultaneously with, and/or after administration of the additional therapeutic agent(s). In certain embodiments, the therapeutic molecule, conjugate, or composition is administered simultaneously with the additional compound. In certain embodiments, the therapeutic molecule, conjugate, or composition is administered before or after the additional compound. In some embodiments, administration of the therapeutic molecule, conjugate, or composition and administration of the additional therapeutic agent are within about one, two, three, four, or five months of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days.
Any suitable therapeutic agent for treating an ocular disorder may be used as the additional compound, particularly as an agent for treating an ocular disorder. Eye diseases are described in section III above. Furthermore, any of the molecules described in section ii.a above as components of therapeutic molecules may also be used as additional compounds used in combination therapies.
In some embodiments, the additional compound is an anti-angiogenic agent as described in section II.A.1.h) and Carmeliet et al, nature 407:249-257 (2000) above. Other suitable anti-angiogenic agents include corticosteroids, angiogenesis inhibiting steroids, anecortave acetate, angiostatin, endostatin, tyrosine kinase inhibitors, matrix Metalloproteinase (MMP) inhibitors, insulin-like growth factor binding protein 3 (IGFBP 3), stromal cell derived factor (SDF-1) antagonists (e.g., anti-SDF-1 antibodies), pigment Epithelium Derived Factor (PEDF), gamma secretase delta-like ligand 4, integrin antagonists, hypoxia-inducible factor (HIF) -1 alpha antagonists, protein kinase CK2 antagonists, drugs (e.g., anti-vascular endothelial cadherin (CD-144) antibodies and/or anti-SDF-1 antibodies) that inhibit stem cells (e.g., endothelial progenitor cells) located at a neovascular site, and combinations thereof.
The therapeutic molecule, conjugate or composition may also be administered in combination with a therapeutic or surgical method for treating an ocular disease (e.g., AMD, DME, DR, RVO or GA), including, for example: laser photocoagulation (e.g., total retinal photocoagulation (PRP)), latent laser action, macular hole surgery, macular displacement surgery, implantable micro telescope, PHI-motion angiography (also known as micro laser therapy and branch vessel therapy (feeder vessel treatment)), photon beam therapy, microstimulation therapy, retinal detachment and vitrectomy, scleral buckle, subretinal surgery, pupil hyperthermia, optical system I therapy, use of RNA interference (RNAi), in vitro rheological processes (also known as membrane filtration and rheo-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 implantable retinal epithelial cells, diacrino, inc.; retinal cell grafts, e.g., astellas Pharma US, inc, neuron, CHA Biotech), acupuncture, and combinations thereof.
The therapeutic molecule, conjugate, or composition may also be combined with a vision cycle modulator (e.g., imistat hydrochloride (emixustat hydrochloride)); squalamine (e.g., OHR-102;Ohr Pharmaceutical); vitamin and mineral supplements (e.g., those disclosed in Age-Related Eye Disease Study 1 (AREDS 1; zinc and/or antioxidants) and Studiy 2 (AREDS 2; zinc, antioxidants, lutein, zeaxanthin, and/or omega-3 fatty acids)); cell-based therapies, e.g., NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; janssen), opregen (suspension of RPE cells; cell Cure Neurosciences) or MA09-hRPE cell transplantation (Ocata Therapeutics) were administered in combination.
In some embodiments, the additional therapeutic agent is an AMD therapeutic agent. For example, the anti-PDGFR antibody REGN2176-3 may be used in combination with Abelmoschus (aflibercept)And (5) blending together. In some cases, these co-formulations may be administered in combination with a therapeutic molecule, conjugate, or composition.
In some embodiments, the additional compound comprises a lentiviral vector (e.g., retinoStat) that expresses endostatin and angiostatin.
In certain embodiments, the additional compound binds to a second biomolecule selected from the group consisting of: IL-1 beta; IL-6; IL-6R; IL-13; IL-13R; PDGF; angiogenin; ang2; tie2; S1P; integrins αvβ3, αvβ5 and α5β1; beta-cytokine (betacelllin); apelin/APJ; erythropoietin; complement factor D; tnfα; htrA1; a VEGF receptor; ST-2 receptor; and proteins genetically linked to the risk of AMD, such as complement pathway components C2, factor B, factor H, CFHR3, C3B, C5a and C3a; htrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; TNFRSF10A. In certain embodiments, the additional compound is an antibody or 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 comprises an eye, brain, bone, and/or tumor. In some embodiments, the tissue comprises a retina. In some embodiments, the therapeutic molecule, conjugate, or composition is injected into the eye, brain, bone, or tumor. In some embodiments, the therapeutic molecule, conjugate, or composition is injected into the vitreous, cerebrospinal fluid, or synovial fluid. In some embodiments, the therapeutic molecule, conjugate, or composition is injected subcutaneously.
In some embodiments, the therapeutic molecule, conjugate, or composition provides improved compatibility, longer residence time, and/or longer half-life relative to the injection site as compared to the unmodified therapeutically active agent. In some embodiments, the therapeutic molecule, conjugate, or composition may further provide a longer duration of pharmacological action in the target tissue than the unmodified therapeutically active agent.
In some embodiments, the therapeutic molecule, conjugate, or composition provides improved vitreous compatibility, longer vitreous residence time, longer vitreous half-life, and/or improved duration of pharmacological action as compared to the unmodified therapeutically active agent. In some embodiments, the therapeutic molecule, conjugate, or composition provides improved compatibility, longer residence time, longer half-life, and/or longer duration of pharmacological action in the brain as compared to the unmodified therapeutically active agent.
F. Binding therapeutic molecules to HA
In some embodiments, the method comprises binding the therapeutic molecule to HA (i.e., pre-complexing the therapeutic molecule with HA to form a conjugate) prior to the administering step. In these embodiments, pre-complexing allows the therapeutic molecule to bind to HA. In some of these embodiments, HA is bound to the HABD of the therapeutic molecule. Examples of HABDs are described above in section ii.b.
In some embodiments, the method comprises mixing a first solution comprising the therapeutic molecule with a second solution comprising HA. In some embodiments, the mixing comprises a container. Examples of containers include vials, single-chamber syringes, and two-chamber syringes. In some embodiments, the mixing produces a therapeutic molecule that binds to HA, ready for administration to a subject.
In some embodiments, the HA ranges in size from 400Da to 200kDa. In some embodiments, HA is at least 5kDa. In some embodiments, the HA is 10kDa. In some embodiments, the HA size/content allows for a molar excess of HA relative to the HA binding sites to be present in the binding or pre-complexing mixture. In some embodiments, the HA size/content provides a molar excess of binding equivalents for HABD. In some embodiments, the HA size/number allows a ratio of HA to therapeutic molecule of 1.5:1 to 1: 1.
Examples
The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be implemented in view of the general description given above.
The following example discusses a fusion protein comprising a Fab fragment or peptide and a hyaluronic acid binding domain, i.e. Fab-HABD. Examples 1 to 7 relate to CD44 and/or TSG6 HABD. Examples 8 to 18 relate to VG1HABD.
Example 1 production of Fab-hyaluronan binding domain fusion protein (Fab-HABD) and complexing with HA
A fusion protein of the Fab fragment and the hyaluronic acid binding domain (hereinafter referred to as Fab-HABD) was generated (table 2). Fab-HABD (referred to herein as "version 1X") is formed by recombinant fusion of HABD to the C-terminus of the heavy chain of a Fab fragment via a Gly-Ser containing linker sequence. In some cases, an additional HABD is fused to the C-terminus in the light chain of the Fab fragment (referred to herein as "version 2").
E Fab-HABD was generated using Fab fragments that specifically bind to VEGF and PDGF (referred to as "VPDF"), digoxigenin (referred to as "Dig"), and VEGF (clone "G6.31").
HABD is derived from CD44 (SEQ ID NO: 2) or TSG6 (SEQ ID NO: 4).
Dig antibodies were covalently linked to one or both CD44 HA receptor domains and used as non-binding control molecules (SEQ ID NOS: 9 to 12).
A. Materials and methods
1. Protein expression
Expression plasmids for the various Fab-HABD were generated by either restricted cloning or gene synthesis using standard molecular biology techniques. Separate expression vectors are generated for each polypeptide chain. Expression was performed in HEK293 cells (ThermoFisher) and expressed as 1:1, and mixing the expression plasmids in a ratio.
In some cases, TSG6 is expressed in e.
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 4000rpm at 4℃for 20 minutes. Thereafter, the cell-free supernatant was filtered through a 0.22 μm vial top filter and stored in a refrigerator (-20 ℃).
Fab-HABD was purified from cell culture supernatants by affinity chromatography using anti-Ckappa and anti-CH 1 resins and Size Exclusion Chromatography (SEC).
Briefly, the cells were buffered in 1 XPBS (10 mM Na 2 HPO 4 、1mM KH 2 PO 4 Cell culture supernatants after sterile filtration were captured on kappa select resin (GE Healthcare), equilibrated with 137mM NaCl and 2.7mM KCl, pH 7.4, washed with equilibration buffer, and eluted with 100mM sodium citrate (pH 2.8). The eluted antibody fractions were pooled and the pH was adjusted to 7.5. Then after passing through 1 XPBS buffer (10 mM Na 2 HPO 4 、1mM KH 2 PO 4 Proteins were captured on CaptureSelect IgG-CH1 resin (Life Technologies), equilibrated with 137mM NaCl and 2.7mM KCl, pH 7.4), washed with elution buffer, and eluted with 100mM sodium citrate (pH 2.8). The concentration of the protein samples was determined on a Nanodrop 800 spectrophotometer (Thermo Scientific) at 280 nm.
Analytical SEC was performed via HiLoad 16/60Superdex 200 preparative column (GE Healthcare) using 20mM histidine, 140mM NaCl (pH 6.0) buffer at a flow rate of 1.5 mL/min.
The combined fractions containing the antibodies from size exclusion chromatography were frozen at-80 ℃ and stored for further use.
In some cases, TSG6 is purified from e. Briefly, use was made of a solution consisting of 7M guanidine hydrochloride, 50mMColi cells were extracted with a buffer consisting of Tris-HCl, 100mM sodium tetrasulphonate and 20mM sodium sulphite. UsingAfter homogenization, centrifugation and filtration of the supernatant, his-tagged proteins were captured on a Ni-NTA column (GE Healthcare) equilibrated with 6M guanidine hydrochloride, 25mM Tris-HCl (pH 8.6). The column was washed with 25mM Tris-HCl (pH 8.6), 0.1% Triton X-114 and eluted with a buffer containing 250mM imidazole. TSG6 eluted from the column was refolded by the following method: it was diluted to 1.5mg/mL and then dialyzed overnight at 4℃with a solution of 0.5M guanidine hydrochloride, 0.5M L-arginine, 1mM reduced Glutathione (GSH) and 1mM oxidized glutathione (GSSG). After buffer exchange to 25mM sodium acetate (pH 5.0), the mixture was passed through SP-Sepharose TM The refolded material was purified by cation exchange chromatography on (GE Healthcare).
In some cases, rabFab-1xTSG6 and RabFab-2xTSG6 are secreted by stably transfected Chinese Hamster Ovary (CHO) cells and purified from the cell culture medium. These proteins do not need to refold. RabFab-1xTSG6 has this Fab fused to TSG via a gly-gly-gly-gly-ser linker; HABD is at the C-terminus of HC. RabFab-2xTSG6 has this Fab fused to TSG6 via a gly-gly-gly-gly-ser linker; one HABD is at the C-terminus of HC and the other is at the C-terminus of LC. Both proteins had His-tags at the C-terminus of the heavy chain, which were used for purification. These Fab-HABDs were purified from CHO supernatants using 3 column chromatography steps, including: (1) Capture on antigen affinity column, e.g., shatz, w. et al, mol.pharm.,13 (9): 2996-3003 (2016), (2) separation of His-tagged material on a Nickel-NTA column, followed by (3) cation exchange chromatography on SP-Sepharose.
3. And Hyaluronic Acid (HA) and complexes
Fab-HABD was combined with sodium hyaluronate (Lifecore, biomedical) at 1:1 (w/w) to form a Fab-HABD-HA conjugate (hereinafter referred to as Fab-HABD-HAs). After mixing, the conjugate was concentrated and rebuffered with an Amicon Ultra10kDa cut-off concentrator (Millipore). The final formulation was 20mM histidine pH 6.0, 260mM sucrose, 140mM NaCl, 0.02% Tween 20. Finally, the conjugate was filtered through a 0.22 μm filter (Ultrafree-MC, centri nine gal Units 0.22 μm, GV Durapore). The formation of protein-HA conjugates was monitored by shifting towards shorter retention times in SEC compared to the corresponding Fab-HABD (see figure 1).
EXAMPLE 2 molecular Properties of Fab-HABD
EXAMPLE 2.1 interaction with HA
A. Materials and methods
The ability of HABD of Fab-HABD to bind HA was examined. Binding of Fab-CD44 and Fab-TSG6 Fab-HABD to HA was tested by SPR using a Biacore T200 instrument (GE Healthcare) (Table 3). Briefly, fab-CD44 Fab-HABD was injected over an HA-coated chip (SCBS HY, xantect Bioanalytics GmbH, germany) in a concentration range of 3.7nM to 300nM each in 80 or 120 seconds. In some experiments, HA-coated chips were prepared by indirectly coupling biotin-HA (Sigma-Aldrich, st.louis, missouri U.S.) to a streptavidin (GEHealthcare) -coated S-series sensing chip SA. The dissociation phase was monitored for 600 seconds. Subsequently, by injection of 10mM glycine (pH 1.5) for 60 seconds or injection of 3M MgCl 2 And last for 30 seconds to regenerate the surface. The bulk refractive index difference was corrected by subtracting the response obtained from the buffer injection. All experiments were performed at 25℃using PBS-T (10mM Na2HPO4,1mM KH2PO4, 137mM NaCl,2.7mM KCl pH 7.4,0.05%Tween-20). Using BIAevaluation software, the resulting curve was fitted to 1:1Langmuir binding model. All experiments were performed using PBS-T (10 mM Na at 25℃ 2 HPO 4 ，1mM KH 2 PO 4 137mM NaCl,2.7mM KCl pH 7.4,0.05%Tween-20).
Furthermore, the interaction of VDPF-2xCD44 with HA was tested by Isothermal Titration Calorimetry (ITC). Briefly, fab-CD44 fusion was treated with PBS (10 mM Na 2 HPO 4 ，1mM KH 2 PO 4 137mM NaCl,2.7mM KCl pH 7.4) dialysis. After dialysis, the HA is dissolved with the remaining buffer amount so that all molecules are in exactly the same buffer condition to avoid any phase with the bufferA mismatch of off. HA molecules were loaded into the sample cell at a concentration 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 Fab-CD44 fusion protein at a concentration of 150. Mu.M. Titration experiments were performed at 25 ℃. The affinity constant K and the stoichiometric ratio N were calculated using a set of site models in Origin 7.0 (OriginLab Corporation).
Similarly, the interaction of TSG6 with 10kDa HA was measured using ITC (table 4), except that in these experiments TSG6 (20 μm) was placed in the calorimeter cell and titrated with HA in a syringe (50 μm). As described above, a solution containing PBS was prepared, and the measurement temperature was 25℃or 37 ℃. These measurements were performed on an Auto PEAQ ITC instrument (Malvern Instruments). Analysis of the data as described in the previous paragraph, except that N is fixed at 1.0 and HA concentration and affinity constant K are variable parameters.
B. Results
K binding of Fab-CD44s and Fab-TSG6s to HA as measured by SPR D As shown in table 3.
The strength of the interaction is determined by the HABD sequence and the binding nature of the interaction (i.e., version 2 exhibits higher functional affinity via close binding to HA).
ITC analysis (table 4) gave the following HA affinities calculated to have binding site concentrations of 400-745 μm, indicating an estimated stoichiometric ratio of 8-15 TSG6 molecules per 10kDa HA chain. Similar experiments using 50. Mu.M VPDF-2xCD44 in the pool and 150. Mu.M 10kDa HA in the syringe gave K D 25. Mu.M, and an apparent stoichiometric ratio of 4.5 VPDF-2xCD44 per 10kDa HA chain. The weaker HA binding affinity of CD44 requires higher concentrations to be used in ITC experiments. From the divalent HA binding properties of the 2xCD44 fusion and the higher molecular weight of CD44 compared to TSG6, it is expected that the binding stoichiometry of 10kDa is 2-3 times greater for 1 xCD 6 compared to 2xCD44. The strength of the interaction is determined by the sequence of the HABD and the binding of the interaction (i.e., version 2xExhibit higher functional affinity via close binding to HA).
In terms of stoichiometry of interaction, it was found that on average 1.5 VPDF-2xCD44 could be bound per 10kDa HA molecule, while 5 VPDF-2xCD44 could be bound per 50kDa HA molecule.
Based on SPR measurements, VPDF-2xCD44 is able to bind both VEGF and PDGF ligands. Binding of VEGF and PDGF to VPDF-2xCD44 was compared to their binding to unmodified VPDF. Briefly, PDGF was coupled to S-series sensor chip CM5 (GE Healthcare) using standard coupling chemistry to give a surface density of about 4000 Resonance Units (RU). After injection of 3. Mu.g/mL of VPDF-2xCD44 fusion and unmodified VPDF control, respectively, 5. Mu.g/mL of VEGF was injected to demonstrate that Fab binds to ligand PDGF and VEGF simultaneously. Subsequently, the surface was regenerated by injection of 10mM glycine pH 2.0 for 60 seconds. SPR measurements confirm that fusion of HABD to the C-terminus of the VPDF Fab fragment heavy chain does not interfere with ligand interaction with the target protein.
EXAMPLE 2.2 stability of Fab-HABD
The use of Fab-HABD for long-lasting delivery in the eye requires that the protein remain stable at body temperature over a range of up to several months. A prerequisite for this is that the thermal stability of the Fab-HABD is above 37 ℃.
A. Materials and methods
The thermal stability of VPDF-2xCD44 and TSG6 was tested by static light scattering and protein autofluorescence. The samples were diluted to about 1mg/mL and the temperature was raised from 25 ℃ to 80 ℃ using an Optim instrument (Avacta inc.) at a heating rate of 0.1 ℃/min. During this process, light scattering and fluorescence data after irradiation with 266nm laser light were recorded. The onset of aggregation temperature was measured to be about 75 deg.c, which is defined as the temperature at which the scattering intensity increases. At the same time, the fluorescence emission spectrum was recorded.
For VPDF-CD44, two transitions were measured at about 56℃and 79℃when plotting the center of gravity average of the fluorescence spectrum versus temperature. These transitions indicate protein (possibly Fab and CD44 domains) denaturation. Thus, any scattering or spectral changes associated with thermal unfolding were much higher than 37 ℃, indicating good stability of the Fab-HABD.
B. Results
The two transitions of VPDF-2xCD44 were measured at 56℃and 79℃respectively. These two T' s m Indicating a shift in the denaturation of VPDF-2xCD44, wherein the CD44 domain is denatured at 56℃and the Fab is denatured at 79 ℃.
For TSG6, the observed T was measured monset At 35 ℃, actually measure T m 43 ℃.
Example 3 in vivo efficacy in rat laser Choroidal Neovascularization (CNV)
A. Materials and methods
Fab-HABD was studied in an in vivo rat model of laser-induced choroidal neovascularization (rat laser CNV) to test the following hypothesis: (1) Fab-HABD, while binding to IVT HA, is effective in vivo (i.e., fab-HABD can inhibit neovascularization); and (2) Fab-HABD has a longer in vivo efficacy compared to the corresponding unmodified Fab fragment.
To this end, rats received IVT injections of protein formulation 1 or 3 weeks before receiving laser injury (6 laser burns per eye). One week after setting up the laser lesions, the lesions of the vessel growth were analyzed using Fluorescence Angiography (FA) imaging.
Fab-HABD was compared to the corresponding unmodified Fab fragment. To test the sustained efficacy of Fab-HABD, the dose of unmodified Fab was titrated to the "lowest effector dose" (i.e., there was only a low degree of detectable inhibition of neovascularization compared to the vehicle over the duration of the rat model), indicating that Fab-HABD had more sustained efficacy over the same dose and duration of the model.
B. Results
All tested Fab-HABD showed inhibition of neovascularization in vivo (Table 5). This result suggests that Fab-HABD, although bound to HA in the vitreous, reaches the relevant tissues to exert pharmacological effects.
All tested Fab-HABDs showed longer duration pharmacological effects compared to the unmodified Fab fragments within the same model set-up and at the same dose (table 5). This suggests that the ability to bind to IVT HA may prolong pharmacological effects in vivo.
Despite the significant differences in affinity for HA, the resolution of in vivo models is insufficient to distinguish between persistence of efficacy of different molecules. At the low non-therapeutic doses applied in the model, tolerability problems were not detected. All eyes of rats receiving Fab-HABD doses were completely normal, without any disturbing signs, comparable to eyes receiving buffer only during the in vivo phase.
EXAMPLE 4 Rabbit Pharmacokinetic (PK) studies with RabFab, rabFab-1xTSG6 and RabFab-2xTSG6
A. Materials and methods
Proteins for animal studies were formulated via dialysis in 20mM histidine-acetate, 150mM NaCl (pH 5.5) or Phosphate Buffered Saline (PBS) (pH 7.4). The formulation is isotonic and has an osmotic pressure of between 300mOsm/kg and 340mOsm/kg as measured by freezing point method. Size Exclusion Chromatography (SEC) analysis showed that the monomer fraction of all proteins in these formulations was > 95%. At the final dosing concentration, endotoxin levels were assessed as less than 0.1EU per eye.
In the vitreous half-life study, an additional pharmacokinetic study was performed in which control (PBS, n=1), 0.15 mg/eye AlexaFluor-488-labeled RabFab (n=2), or AlexaFluor-488 (AF-488) -labeled RabFab-2xTSG6 at doses of 0.05 mg/eye (n=2), 0.15 mg/eye (n=2), and 2.5 mg/eye (n=4) were administered to both eyes of female rabbits by 1TV injection, in a total volume of 50 μl. The concentrations of the test article in the vitreous and aqueous humor at the indicated time points were measured using a fluorometry as previously described. Dickmann, l.j. Et al, invest. Ophthalmol. Vis. Sci.,56 (11): 6991-6999 (2015). Pharmacokinetic parameters were estimated by non-atrioventricular analysis using a concentration-time curve of Phoenix WinNonlin (Certara inc., mountain View, CA). For the concentration-time morphology generated using the fluorescence brightness method, sampling results within 48 hours prior to dosing were excluded from PK analysis due to high variability, which may be attributed to individual differences at the site of administration and subsequent diffusion of the test article through the vitreous. Dickmann, l.j. Et al, invest. Ophthalmol. Vis. Sci.,56 (11): 6991-6999 (2015). PK analysis was performed using a non-atrioventricular analysis method, with Clearance (CL) calculated as CL = dose/AUC, where dose is known and AUC is measured using a linear trapezoidal method. The steady state volume of distribution was calculated as v=cl/kel using the clearance values and the elimination rate constants obtained from the end stage slope. The elimination half-life is calculated as t 1/2=ln (2)/kel.
B. Results
The ability of HA binding to affect intraocular residence time was initially examined using Pharmacokinetic (PK) experiments in new zealand white rabbits. Rabbits are often used for early PK studies following administration of test IVT, although the HA concentration of rabbit vitreous (about 65 μg/mL) is much lower than that of human vitreous (100-400 μg/mL) or other preclinical species such as pigs (with a vitreous HA of about 180 μg/mL) or cynomolgus monkeys (with a vitreous HA of about 150 μg/mL). The study was designed to inject 0.3 mg/eye of RabFab, 0.3 mg/eye of RabFab-1xTSG6 or 0.5 mg/eye of RabFab-2xTSG6 with IVT. Recovery experiments using proteins in ex vivo added vitreous humor showed that RabFab and rabfab=1xtsg 6 could be quantified using ELISA with the anti-idiotypic detection antibody previously described (Shatz et al, 2016Molecular Pharmaceutics). However, the recovery obtained by ELISA using RabFab-2xTSG6 was poor, allowing radiochemical determination of the vitreous concentration of the material. In PK studies, rabFab-2xTSG6 was used 125 Iodine is radiolabeled.
As shown in fig. 2A, PK parameters are summarized in table 6, with RabFab-1xTSG6 and RabFab-2xTSG6 both exhibiting longer vitreous residence times compared to free RabFab. The half-life of RabFab-1xTSG6 is 1.4 times longer than that of RabFab, while the half-life of RabFab-2xTSG6 is increased by 2.2 times. These results indicate that fusion of Fab to HABD can increase the retention time of these molecules in the eye chamber. Given the higher vitreous HA concentration of other species, it is expected that the half-life of Fab-HABD will be more greatly extended in those animals.
Furthermore, the vitreous half-life study showed an increase of approximately 3 to 4-fold compared to the vitreous half-life of RabFab-2xTSG6 observed by the fluorometry, no apparent dependence on dose within the range evaluated (fig. 2B); however, the duration of the study for 21 days was not sufficient to reliably determine pharmacokinetic parameters, and it was estimated that approximately 40% of the RabFab-2xTSG6 remained intravitreally at the end of the study.
EXAMPLE 5 RabFab-1xTSG6 and Rabbit eye tolerance to TSG6
A. Materials and methods
Toxicity of free TSG6 and RabFab-1xTSG6 single ITV administration was evaluated in New Zealand white rabbits. A single IVT dosing study (table 7) was designed and performed for 4 weeks. Anti-drug antibodies (ADA) against RabFab-1xTSG6 or free TSG6 were measured in serum by ELISA. Plates were coated with RabFab-1xTSG-6 or free TSG6, incubated with serum collected from study animals, and anti-drug antibodies were detected with HRP-conjugated goat anti-rabbit Fc antibodies.
B. Results
In general, animals receiving RabFab-1xTSG-6 had less severe results than animals receiving free TSG6 administration. Animals receiving free TSG6 administration had significant clinical observations. Although 4 animals received necropsy on day 4 as planned, the other 4 animals were sacrificed in advance on day 12 or 17 instead of day 30 due to significant clinical observations and animal welfare issues. These clinical observations included eyelid and conjunctival swelling and redness, eye closure of the animal as the staff approaches, and ocular inflammation and irritation. Animals receiving free TSG6 administration exhibited significant posterior incipient cataract and variable retinal vascular weakening by 3 days post-dose, and these results correlated with microscopic results of lens and external to complete retinal degeneration. Similar but less severe results were also present in animals receiving RabFab-1xTSG6 administration. From day 7 after dosing, all animals developed significant inflammation, predominantly monocytes. Inflammation and retinal degeneration are multifocal with evidence of retinal detachment, hypertrophy and vimentin in the outer Zhou Qianyi, glial Fibrillary Acidic Protein (GFAP) and glutamine synthetase positive Muller cells. Histopathological images showing retinal degeneration of TSG6 4 days after administration via IVT are shown in fig. 3.
Evidence of anti-drug antibodies (ADA) was present in the serum of both animals receiving RabFab-1xTSG6 administration and necropsy on day 4. However, one animal received ADA pre-dosing, while the remaining 3 animals in the treatment group did not. Animals in this group that received post necropsy were negative for serum ADA on days 4 and 8, but became ADA positive on day 15. Because of the poor sensitivity of this assay, analysis of serum ADA responses in animals treated with free TSG6 is not yet known.
In general, animals receiving RabFab-1xTSG6 had lower severity results than animals receiving free TSG6 administration (Table 8). Each animal had a cataract, but the cataract was punctiform in nature and no correlation was found in the microtome. There was no clinical evidence of retinal degeneration, but microscopic evidence of very slight to mild outer retinal degeneration was present in a single eye. Similar moderate to severe vitreous and aqueous humor cells appeared from day 8. Animals were euthanized on days 4 and 17.
Evaluation of the anti-RabFab response is complicated by the fact that 3/8 animals had values above the threshold prior to dosing (2 animals received free TSG6 administration, 1 animal received rabfab=1 xTSG6 administration). However, after administration of the test article, 3/4 animals receiving administration of RabFab-1xTSG6 exhibited new ADA titers or increased ADA titers: of which 1 animal was euthanized on day 4 and the other two animals were euthanized on day 17. In contrast, animal 1, which received administration of free TSG6 alone (necropsy on day 4), had elevated ADA titers compared to prior to dosing.
Early onset of clinical signs and microscopic lesions indicate a direct role for TSG6 in retinal and lens degeneration; however, the findings at later time points are confused with unexpectedly strong ADA responses. The peripheral migration of Muller cells was judged to be a nonspecific response following damage to the rabbit retina or detachment of the retina.
EXAMPLE 6 Pharmacokinetic (PK) of therapeutic dose in minipigs
The aim of this study was to determine intraocular and systemic PK parameters of Fab-HABD and Fab-HABD-HAs and the resulting intraocular half-life (t 1/2 ) Prolonged, wherein the minipig is administered once by IVT Injection (IVT). In addition, studies were performed on anti-drug antibodies (ADA), intraocular tolerance, and ophthalmic pathology (in some study subjects).
A. Materials and methods
14 pieces ofThe following test (50 μl/eye) was received by both eyes of the SPF minipigs at therapeutic doses (table 9).
Blood and aqueous samples of animals receiving test article dosing were collected periodically throughout the study period (up to 9 weeks) following IVT dosing, and vitreous humor was collected shortly after planned euthanasia to track the systemic and intraocular PK of the test article. The plasma, aqueous and vitreous samples were further analyzed for the presence of ADA by analyzing the concentrations of the test samples in the plasma, aqueous and vitreous.
B. Results: in the in vivo phase of the study, visual observations of the eyes of 2 out of 5 animals receiving VPDF-2xCD44 indicated that porcine eyes were unable to tolerate IVT injections of the trial, resulting in premature sacrifice of these animals. One eye of each animal was provided for histopathological evaluation. Briefly, these visual observations were: vitreous opacity, lower than normal vitreous viscosity, is ultimately a sign of vision loss. Histopathological findings in the eye include perivascular/vascular moderate mixed cell inflammation, mainly mononuclear cell infiltration of iris, ciliary body, trabecular meshwork and retina. Retinal degeneration includes ganglion cell degeneration, cell loss in INL, photoreceptor aggregation, and nuclear translocation of the PR layer. In addition, eosinophilic proteins and mixed cell infiltration and fibrils were observed in the vitreous. No abnormalities were found in the optic nerve.
Visual observations of eyes of at least 1 of 5 animals receiving VPDF-2xCD44+10kDa HA were significantly less severe than those of VPDF-2xCD 44. The reddish/white films in the anterior chamber of the eyes were briefly noted to accumulate behind the cornea, but were not considered to require premature termination.
In summary, complexation of VPDF-2xCD44 with HA (i.e., occupying the CD44 HA binding site with HA prior to IVT injection) does improve intraocular tolerability of VPDF-2xCD 44.
No visual observations or tolerance problems were found in the group of animals receiving unmodified VPDF.
PK results for the test samples VPDF-2xCD44 and VPDF-2xCD44+10kda HA were derived from aqueous humor and vitreous humor, calculated from individual concentration time data by non-aqueous humor analysis, and are graphically presented in fig. 4A-4B.
Although IVT t of unmodified VPDF for 5.8 days 1/2 IVT t of VPDF-2xCD44+10kDa HA located within the expected range of such molecules, but for 48 days 1/2 Corresponding to an approximately 8-fold increase in intraocular residence time compared to unmodified VPDF. In summary, VPDF-2xcd44+10kda showed significantly improved tolerability compared to VPDF-2xCD44 not complexed with HA, and significantly improved half-life in the eye compared to unmodified VPDF.
EXAMPLE 7 Pre-compounding with HA to improve vitreous compatibility
Macroscopic observations from in vivo mini-pig studies (i.e., vitreous turbidity) indicate that VPDF-2xCD44 incompatibility with pig vitreous (i.e., precipitate formation) can be alleviated 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 be detected for other Fab-HABDs as well, we developed an ex vivo test system to detect vitreous degeneration.
An in vitro "drop" test was developed to assess the vitreous compatibility of several Fab-HABDs when pre-compounded with HA. This example illustrates that Fab-HABD (i.e., conjugate) pre-complexed with HA is vitreally compatible in an in vitro experiment. The vitreous incompatibility observed previously may be caused by free HABD, which is alleviated by HA pre-compounding. The results show that the incompatibility of Fab-HABD with free HABD is concentration dependent. Furthermore, CD44ko (a Fab-HABD mutant comprising point mutations that disable HA binding) is compatible with the vitreous both in pre-complexed and isolated forms.
EXAMPLE 7.1 Pre-complexing of VPDF-2xCD44 with 10kDa HA improved Intravitreal (IVT) tolerance
A. Materials and methods
In the first test, pig vitreous was homogenized 10x in a Dounce homogenizer and cleared of debris by centrifugation at 10000g for 2 minutes. A drop of 2 μl of homogenized vitreous was then applied to a glass microscope slide. In addition, 2. Mu.l of the sample (i.e., fab-HABD or Fab-HABD-HA at the indicated concentrations) was added to the top of the vitreous drops without further mixing. The samples were checked for non-uniformity and sedimentation in bright field mode by light microscopy at 40 x magnification 1 minute after droplet merger.
B. Results
Pig glass mixed with unmodified VPDF at a concentration of 200mg/mL in 20mM histidine, 140mM NaCl (pH 6.0) was homogeneous and clear (FIG. 5A), while pig glass mixed with VPDF-2xCD44 at a concentration of 20mg/mL in 20mM histidine, 140mM NaCl (pH 6.0) was heterogeneous and exhibited clear precipitate signs (FIG. 5B).
The results indicate that VPDF-2xCD44 is also incompatible with porcine vitreous after IVT injection in vivo. Thus, vitreous incompatibility may be one root cause of tolerance problems in VPDF-2xCD 44.
Pre-complexation of VPDF-2xCD44 at a concentration of 20mg/mL with 1% (w/v) HA (10 kDa, lifecore, biomedica) in 20mM histidine, 140mM NaCl (pH 6.0) resulted in vitreous compatibility (FIG. 5C). This result reflects the results of the above-described in vivo study of minipigs, which showed that pre-complexing of VPDF-2xCD44 with 10kDa HA improved IVT tolerance.
EXAMPLE 7.2 vitreous incompatibility of VPDF-2xCD44 depending on concentration
A. Materials and methods
To test the concentration dependence of the vitreous incompatibility of VPDF-2xCD44, 2. Mu.l of porcine vitreous was mixed with a 1:4 dilution of VPDF-2xCD44 in 20mM histidine, 140mM NaCl (pH 6.0) at an initial concentration of 37.5 mg/mL. The mixture of vitreous and protein was examined for vitreous inhomogeneities by optical microscopy.
B. Results
The detected non-uniformity was dependent on the protein concentration (Table 10; FIGS. 6A-6F). The vitreous compatibility of VPDF-2xCD44 reaches between 0.6 and 0.15 mg/mL. Correlating these results with the results of the in vivo minipig study described above (VPDF-2 xCD44 concentration=17.4 mg/mL) suggests that similar non-uniformities that may be caused by IVT injection of a VPDF-2xCD44 solution at a concentration of 17.4mg/mL may be the root cause of the tolerability problem observed.
EXAMPLE 7.3 vitreous incompatibility of VPDF-2xCD44 related to its interaction with Intravitreal (IVT) HA
A. Materials and methods
To test whether the vitreous incompatibility of VPDF-2xCD44 is caused by the interaction of VPDF-2xCD44 with IVT HA, we designed variants of this molecule (VPDF 2xCD 44-ko) that contain a point mutation in the HA binding site of CD44 that eliminates binding to HA, while leaving the remainder of the protein bound intact (referred to herein as "ko variants").
B. Results
CD44ko variants show the same behavior in terms of transient expression, purification and biophysical properties (analytical grade particle size screening, denaturing SDS capillary electrophoresis) and identity is confirmed by mass spectrometry. The introduction of the HA binding site mutation resulted in complete loss of affinity as shown by the SPR results (tested using the same method as example 2).
When the vitreous compatibility of this VPDF-2xCD44-ko variant was tested at the same concentration as the 2x VPDF as described in example 7.2 above, no vitreous inhomogeneities were detected, indicating vitreous compatibility (table 11).
EXAMPLE 7.4 VPDF-2xCD44 vitreous compatibility after hyaluronidase pretreatment
A. Materials and methods
Furthermore, we tested the vitreous compatibility of VPDF-2xCD44 in porcine vitreous pretreated with hyaluronidase to degrade HA. For this purpose, hyaluronidase (Sigma) from porcine testis was dissolved in PBS at a concentration of 2mg/mL (> 1.5U/. Mu.L). mu.L of hyaluronidase solution was added to 50. Mu.L of pig vitreous and incubated at 37℃for 2 hours. Control samples were treated with PBS buffer only.
B. Results
As a result, VPDF-2xCD44 did not exhibit non-uniformity when mixed with hyaluronidase-pretreated vitreous at a concentration of 20mg/mL, possibly due to high molecular weight HA degradation.
Taken together, these results suggest that vitreous incompatibility may be the root cause of the problem of tolerance in VPDF-2xCD44 and the interaction of CD44-HABD with high molecular weight IVT HA.
EXAMPLE 7.5 vitreous incompatibility of Fab-HABD was related to interaction of HABD with vitreous HA at specific concentrations
A. Materials and methods
To test whether vitreous incompatibility is merely a characteristic of VPDF-2xCD44, we tested for vitreous non-uniformities of other Fab-HABD or HABD alone as described in examples 7.2 and 7.3 above. The proteins tested are as described in example 1.
B. Results
VPDF-1xCD44 exhibited comparable vitreous non-uniformities to VPDF-2xCD 44. The results indicate that the increase in binding and potential crosslinking of HA polymers version 2 is not related to vitreous incompatibility. The results indicate that the interaction between CD44 HABD and IVT HA is related to vitreous incompatibility (table 12).
Fab-HABD comprising the TSG6 domain showed comparable vitronectin unevenness to Fab-HABD comprising CD 44. Fab component G6.31 showed no vitronelation at the same concentration, whereas isolated TSG6 domains showed vitronelation. This result again supports the notion that vitreous incompatibility relates to the interaction of HABD with vitreous HA at specific concentrations.
Example 7.6 vitreous incompatibility can be addressed by pre-compounding with HA
A. Materials and methods
To test whether the vitreous incompatibility performance of the detected VPDF-1xCD44 and TSG6 variants was resolved by pre-complexing with HA, we generated conjugates shown in Table 13 containing 1% (w/v) HA (10 kDa, lifecore, biomedica).
B. Results
All Fab-HABD tested were resolved for vitronectin heterogeneity by pre-complexing with 10kDa HA (Table 13). These results indicate that pre-complexing of HA binding proteins with pure HA can be used as a method to improve vitreous compatibility and thus the potential tolerability problem of these molecules.
EXAMPLE 7.7 vitreous incompatibility of Fab-HABD is not characteristic of porcine vitreous
A. Materials and methods
To test whether the detected Fab-HABD vitreous incompatibility containing CD44 and TSG6 is an effect that occurs only in porcine vitreous, we used rabbit vitreous instead of porcine vitreous, and conducted compatibility tests as described in examples 7.1 to 7.6 above.
B. Results
For all Fab-HABD tested, the same vitreous incompatibility as porcine vitreous was detected. Furthermore, all vitreous incompatibilities detected in rabbit vitreous can be resolved by pre-complexing of Fab-HABD with 10kDa HA.
These results indicate that vitreous incompatibility is not characteristic of porcine vitreous.
EXAMPLE 7.8 in vivo injection induces vitreous heterogeneity
A. Materials and methods
To obtain a link between the results of the ex vivo vitreous compatibility test and the tolerability results of the in vivo mini-pig study, we tested whether vitreous inhomogeneities could be detected in the whole pig eye and solved by HA pre-compounding.
To this end, whole pig eyes were harvested immediately after slaughter and 50. Mu.l of a solution of VPDF-2xCD44 in 20mM histidine, 140mM NaCl (pH 6.0) at a concentration of 17.4mg/mL was injected therein +/-1% (w/v) HA 10kDa. Eyes (same as in vivo mini-pig study above). The eyes were then transferred to HBSS (Lonza, biowhisttaker) and stored at 37 ℃ for 4 hours. After incubation, the eyes were opened and the vitreous was removed for non-uniformity.
B. Results
As shown in fig. 7A-7C, vitreous incompatibility at the time of injection can be addressed by pre-compounding Fab-HABD with HA. Injecting buffer did not result in IVT non-uniformity, resulting in a transparent vitreous (fig. 7A). Injection of VPDF-2xCD44 resulted in dense white non-uniformities (precipitations) in the vitreous surrounding the injection side (fig. 7B). The vitreous of eyes injected with VPDF pre-compounded with pure HA showed significant differences (fig. 7C): although a non-uniformity was detected, the density and thickness of the non-uniformity throughout the glass body was significantly reduced.
These results indicate that the VPDF-2xCD44 induced non-uniformity also occurred near the injection site in the whole pig eye. Without being bound by this theory, we believe that the same non-uniformities are induced upon in vivo injection and may be the root cause of the tolerance problem observed.
In addition, pre-complexing of VPDF-2xCD44 with HA reduces the non-uniformities observed around the injection site. We believe that the same effect results in the in vivo observation of improved tolerability of VPDF-2xCD 44-HA. In combination with the observation that vitreous incompatibilities in examples 7.5 and 7.6 also occur with another TSG6 HABD and are equally solvable by preconditioning with pure HA, we consider this approach as a general principle of improving vitreous compatibility and thus IVT tolerance of HABD-containing proteins.
EXAMPLE 8 multifunctional proteoglycans VG1 and VG1ΔIg HABD capable of binding HA
HABDs of multifunctional proteoglycans were studied to determine whether they could be used as HABDs that provided better intraocular tolerability and intraocular residence time than TSG6 and CD44 HABDs.
The multifunctional proteoglycans were identified as having tandem repeat linking modules. As shown in fig. 8A, the amino acid sequence of the multifunctional proteoglycan encodes an Ig-like domain that is followed by two linking modules such that the N-terminal fragment of the multifunctional proteoglycan (designated herein as WT VG 1) comprises an N-terminal Ig-like domain and 2 linking domains. In this example we obtained WT VG1 and truncated variants v1Δig without Ig domains and tested whether they bind to HA. Furthermore, in the examples below, in vitro vitreous compatibility of WT VG1 and Fab-HABD consisting of Fab and WT VG1 and tolerability of IVT when injected into rabbits and minipigs were tested.
A. Materials and methods
Expression plasmids for various proteins were generated by restriction cloning and/or gene synthesis using standard molecular biology techniques. Expression was performed in CHO or HEK293 cells.
The supernatant was collected by centrifugation at 4000rpm at 4℃for 20 minutes. Thereafter, the cell-free supernatant was filtered through a 0.22 μm vial top filter and stored in a refrigerator (-20 ℃).
The His-tagged mutants of WT VG1 and VG1ΔIg were purified from the cell culture supernatant by affinity chromatography using Ni-NTA resin and SEC. Briefly, cell culture supernatants, sterile filtered, were captured on HisTrap resin, washed and eluted using buffer containing high concentration imidazole. The eluted protein fractions were pooled and concentrated, and then SEC analyzed using 20mM histidine-acetate, 150mM NaCl (pH 5.5) as running buffer.
WT VG1 and VG1 Δig were tested for binding to HA by SPR using Biacore T200 instrument (GE Healthcare). Briefly, WT VG1 and VG1 Δig were injected over an S-series CM5 chip (GE Healthcare Life Science Solutions) indirectly coated with biotin-HA by immobilized streptavidin (Creative PEGWorks, north Carolina) in 80 seconds or 120 seconds. The injection concentration ranged from 0.5nM to 1. Mu.M. The dissociation phase was monitored for 300 seconds to 600 seconds. Subsequently, by injection of 1M MgCl 2 And last for 15 seconds to regenerate the surface. All experiments were performed using PBS (10 mM Na at 25℃ 2 HPO 4 ，1mM KH 2 PO 4 137mM NaCl,2.7mM KCl,pH 7.4). Using BIAevaluation software, the resulting HA binding curve was fitted to 1:1Langmuir binding model.
B. Results
Multifunctional proteoglycan HABD capable of bindingHA. HA-binding K of various proteins D As shown in table 14.
EXAMPLE 9 glycosaminoglycan-binding morphology of WT VG1 and TSG6 proteins
A. Materials and methods
Glycosaminoglycan (GAG) binding morphology of WT VG1 and TSG6 was determined by SPR measurement of binding to heparin sulfate and chondroitin sulfate using Biacore T200 instrument (GE Healthcare). Briefly, proteins were injected over 180 seconds onto S-series CM5 chips (GE Healthcare Life Science Solutions) indirectly coated with biotin-heparin sulfate or biotin-chondroitin sulfate via streptavidin. The injection concentration ranges from about 5nM to 1000nM, respectively. The dissociation phase was monitored for 120 seconds. Subsequently, by injection of 1M MgCl 2 And last for 30 seconds to regenerate the surface.
B. Results
The results indicated that WT VG1 was more selective in binding than TSG6 (table 15). No binding of WT VG1 to heparin sulfate or chondroitin sulfate was observed, whereas TSG6 was tightly bound to both heparin and chondroitin sulfate.
EXAMPLE 10 Fab-HABD comprising VG1 HABD capable of binding antigen and HAA. Materials and methods
A.1. Construct design
Fab-HABD is produced by or by recombinant 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-VG 1 fusions, a peptide (EETI) is produced or can be produced that is attached to both the N-terminus of WT VG1 (EETI-VG 1) and the C-terminus of WT VG1 (VG 1-EETI). Two additional constructs were also generated in which a TEV cleavage site was introduced between EETI and WT VG1 (EETI-TEV-VG 1 and VG 1-TEV-EETI). The joints used or possible to use are shown in table 16.
A.2. Generation of species matched surrogate porcine anti-VEGF Fab
Since searches of the abYsis database did not result in a known example of pairing of heavy and light chains of porcine (susscrofa) IgG, we attempted to generate antibodies that bind actively to known antigens by CDR grafting from anti-VEGF Fab (g6.31.aarr). Searching in NCBI-Expressing Sequence Tag (EST) database for a G6.31 backbone (V H 4/V L K2 Pig mRNA EST with high sequence identity. Several sequences were selected and CDRs from g6.31.aarr were grafted into the appropriate framework regions to generate "porcine-derived" g6.31.aarr. The heavy and light chain sequences were randomly paired and expressed in 293Expi or CHO cells in 30mL cultures. Purification was performed on Capto L resin, followed by size exclusion chromatography analysis, and the purified cells were examined by SDS-PAGE, mass spectrometry, and assessed for binding to human and porcine VEGF. A sequence with good affinity for VEGF was selected for amplification and subsequent tox/PK analysis and recombined with VG1 to generate PigFab-VG1.
A.3. Protein expression and purification
Protein expression was performed by transfection of the cationic lipids of the DNA constructs into CHO or 293Expi cells. The culture volume was 30mL to 35L. For some constructs, a rapidly stable cell line is generated to increase protein production per unit culture volume.
Purification was performed by affinity chromatography, wherein Ni-NTA resin was used for the 6 x-histidine tag molecule, or Gamma bind Plus resin was used for Fab fusion. In some cases, a secondary ion exchange step is performed before the final particle size screening step is performed on the Sephadex resin.
Ha binding
To confirm that VG1 retains its HA binding properties as Fab-HABD, SPR was used as previously described in example 2.1. Experiments were performed using single cycle kinetics and dissociation was monitored for up to 600 seconds. The protein concentration tested varied between different proteins, but ranged between 500nM and 6.25 nM.
A.5. Antigen binding
Antigen binding was tested by direct immobilization of the corresponding antigen on a S-series CM5 chip (GE Healthcare) and binding was measured by SPR as described in example 2.1. Different protein concentrations were used based on known interaction affinities.
B. Results
B.1. Hyaluronic Acid (HA) binding
All HA binding data were fit to 1 using BIAevaluation software: 1Langmuir binding model. K of various proteins D As shown in table 17.
B.2. Antigen binding
Table 18 shows the proteins and the K measured analyzed for their antigen binding capacity D . The C-terminal fusion of VG1 to the heavy chain of the various Fab did not affect antigen binding. For EETI-VG1 fusion, the flexibility of the linker and attachment site affects antigen binding. More flexible linker and C-terminal fusions are preferred.
EXAMPLE 11 ex vivo vitreous compatibility of HABD
A. Materials and methods
This example describes the measurement of the solubility of the VG1 domain in the vitreous humor. The vitreous humor was prepared using a Dounce homogenizer and then centrifuged at 10000x g for 2 minutes to remove debris, which was used for these studies.
Additional experiments utilized Alexa 488-labeled proteins, allowing both bright field and fluorescence microscopy to be used to monitor precipitation in ex vivo vitreous humor. Equal volumes of test article were mixed with the vitreous humor by sequential injection 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 by microscopy.
B. Results
After mixing TSG6 with porcine vitreous humor previously diluted 1:4 with PBS (pH 7.4), the solution became turbid (FIG. 9A) and precipitation was observed after centrifugation of the mixture. In contrast, after mixing VG1 with pig glass bodies in a ratio of 1:4 and 1:1, the solution remained clear (FIG. 9B), and no precipitation was observed after centrifugation.
Further, precipitation of RabFab-TSG6 was observed in ex vivo pig glass (FIG. 10A), whereas no precipitation was observed for RabFab-VG1 (FIG. 10B). Similarly, no precipitation was observed in the ex vivo rabbit glass bodies as compared to VG1 (FIG. 11A), rabFab-VG1 (FIG. 11B) or formulations containing equal concentrations (on a mass basis) of RabFab-VG1 and 10kDa HA (FIG. 11C).
In contrast to TSG6, a pre-formulation of VG1 with 10kDa HA is not always desirable to prevent precipitation in the vitreous humor in vitro.
EXAMPLE 12 off-body interaction of VG1 with vitreous humor
A. Materials and methods
The interaction of isolated VG1 and Fab-VG1 Fab-HABD with ex vivo vitreous humor was examined using Fluorescence Correlation Spectroscopy (FCS). VG1 and Fab-VG1 were covalently labeled on lysine residues using PEG4-DY 647-N-hydroxysuccinimide ester. The fluorescence emission of DY647 can be excited by a laser of 594nm or 633nm and detected at a longer wavelength. The reaction chemistry is controlled such that the label level per molecule is no greater than 1 fluorescent dye. Porcine vitreous humor was collected from the eyes of freshly slaughtered animals and homogenized using a Dounce homogenizer. This material was treated with Phosphate Buffered Saline (PBS) pH 7.4 at 1:3 serial dilution. Labeled test pieces were added to each diluted aliquot to a final concentration of 20nM. The test pieces were (1) free VG1, (2) pI gFab-VG1, (3) pigFab-VG1 mixed with 10kDa HA in a 1:1 equiweight ratio, (4) RabFab-VG1 and (5) RabFab-VG1 mixed with 10kDa HA in a 1:1 equiweight ratio. After incubation for 2 hours at room temperature, FCS was performed.
B. Results
The FCS measurement results are shown in fig. 12. All samples showed significantly delayed diffusion when incubated with undiluted or slightly diluted glass bodies compared to incubation in buffer (PBS) alone. For free VG1, pigFab-VG1 and RabFab-VG1, this delayed diffusion continued until the vitreous body was diluted more than 6000-fold (FIG. 12, lines 3, 4, 6 and 7; from undiluted to dilution factor reaching 6561). For samples co-formulated with 10kDa HA, slow diffusion was also observed, but this effect disappeared when the dilution factor of the vitreous humor was > 729 times (FIG. 12, line 5: picFab-VG1+10 kDa HA (1:1), and line 8: rabFab-VG1+10kDa HA (1:1), from the dilution factor 729 to PBS). These results indicate a strong interaction between the vitreous component (most likely the endogenous high molecular weight HA of the vitreous humor) and the test article containing VG 1. VG1 can interact with endogenous HA even in the presence of low molecular weight HA at small dilutions of the vitreous humor (FIG. 12, line 5: picFab-VG1+10 kDa HA (1:1), and line 8: rabFab-VG1+10kDa HA (1:1)). This suggests that VG1 and Fab-VG1 can dissociate from the 10kDa HA and bind to the HA present in the vitreous humor. However, once the vitreous humor is greatly diluted, there will be no sufficiently high concentration of high molecular weight HA competing for VG1 binding to low molecular weight HA (FIG. 12, line 5: picFab-VG1+10 kDa HA (1:1), and line 8: rabFab-VG1+10kDa HA (1:1), from a dilution factor of 729 to PBS). VG1 bound to the low MW HA gave little or negligible diffusion slowing compared to unbound material (FIG. 12, PBS control was used for row 5: picFab-VG1+10 kDa HA (1:1), and row 8: rabFab-VG1+10kDa HA (1:1), these samples had 10kDa HA without added vitreous).
EXAMPLE 13 Effect of Pre-complexing with 10kDa HA on the thermal stress stability of Fab-VG1
A. Materials and methods
The effect of pre-complexing of Fab-VG1 with 10kDa HA on thermal stress stability was tested using anti-HtrA 1-VG1 protein. In these experiments, anti-HtrA 1-VG1 was formulated at a concentration of 3mg/mL with Phosphate Buffered Saline (PBS) pH 7.4, with or without 1.8mg/mL of 10kDa HA added thereto. The concentration of 1.8mg/mL (180. Mu.M) of 10kDa HA was 5-fold molar excess over the anti-HtrA 1-VG1 concentration (35. Mu.M). These formulations were incubated at 37℃for 4 weeks and then analyzed by non-reducing capillary electrophoresis sodium dodecyl sulfate (NR CE-SDS), as described in Michels et al 2007 (Anal. Chem.79, 5963). Aggregates resistant to SDS denaturation can be detected by NR CE-SDS, in addition to the monomer species and fragments.
B. Results
As shown in FIG. 13 and summarized in Table 19, pre-complexing with 10kDa HA inhibited SDS-stabilized aggregate formation in anti-HtrA 1-VG 1. The formation rate of the High Molecular Weight Form (HMWF) was reduced from 1.2% per week to 0.1% per week. The presence of 10kDa HA also appears to affect fragmentation, although less than the effect on aggregation, the rate of formation of the low molecular weight form (LWMF) is reduced by a factor of about 2 when complexed with HA. These results indicate that at neutral pH, the inclusion of 10kDa HA in the formulation stabilizes the anti-HtrA 1-VG1 under thermal stress conditions.
Example 14.Intraocular tolerance to VG1 and VG1 Fab-HABD
A. Materials and methods
A.1. Intravitreal (IVT) injection and endpoint assessment
UsingThe tolerance of IVT injections of VG1 and Fab-VG1 Fab-HABD was assessed. The design of this study is shown in table 20.
Both eyes of each minipig received 50 μl of single injection via IVT. Based on historical data, this volume was well tolerated in small pigs. IVT injection procedures are performed by a certified veterinary ophthalmic doctor. Group 1 minipigs received vehicle control injections. Group 2 minipigs received isolated WT VG1 (produced as described in example 8 above) treatment. Group 3 minipigs received pigFab-VG1 (produced as described in example 10 above) and group 4 minipigs received pigFab-VG1 pre-formulated with an equal weight of 10kDa HA. All assays were formulated with 20mM histidine-acetate, 150mM NaCl (pH 5.5) to the indicated protein concentration. The doses of pigFab-VG1 in groups 3 and 4 represent the maximum feasible dose to maintain total endotoxin levels of less than 0.05 Endotoxin Units (EU) per eye. This level of endotoxin has been previously found to be tolerated in small pig intraocular studies. Given the molecular weight difference between WT VG1 (about 30 kDa) and pigFab-VG1 (about 80 kDa), the dose level in group 2 represents 1.6HA binding molar equivalents per dose compared to groups 3 and 4.
The following parameters and endpoints were evaluated in this study: mortality, clinical signs, body weight, ophthalmology (examination, intraocular pressure measurement, wide-field color fundus imaging, OCT imaging and electroretinogram [ ERG ]), bioassays, toxicological parameters, anti-drug antibody assessment, total autopsy results, and histopathological examination.
Ophthalmoscopes were performed on both eyes of all surviving animals via indirect ophthalmoscopes and slit lamp biomicroscopes by a certified veterinary ophthalmologist. All animals were subjected to ophthalmic examination prior to treatment and on day 1 (post-dosing), 3, 5, 8, 15, 17, 22 and 29.
Intraocular pressure (IOP) is measured by a professionally authenticated ophthalmologist on both eyes of all surviving animals while performing an ophthalmic examination by applanation tonometry. Intraocular pressure was measured for all animals before treatment and on day 1 (post-dose), 3, 5, 8, 15, 17, 22 and 29 days.
On day 29, all surviving animals were imaged using Clarity RetCam Shuttle on a wide-field color fundus. If visible, an attempt is made to take a photograph of the applied test article.
On day 29, optical coherence tomography was performed using a Heidelberg Spectralis HRA/OCT system; a single, vertical, high resolution line scan is performed through the optical nerve.
All surviving animals were evaluated for ERG on day 29. Animals were acclimatized in the dark for a minimum of 1 hour prior to ERG. Full field flash ERG with Ganzfeld dome stimulation (flash intensity meets ISCEV standard parameters and photopic time is 5 minutes (report Gamma); amplitude and delay values are measured from the trace.
Blood samples (about 0.5 mL) were taken from all surviving animals via the anterior vena cava through the thoracic access for determination of serum concentration of the test article. Animals were not fasted prior to blood collection except for time intervals consistent with other program forbids. Blood was collected once before treatment, on day 1 (6 hours and 12 hours after administration) and on days 2, 3, 5, 8, 12, 15, 22 and 29, respectively.
Blood samples were collected into serum separation tubes and allowed to form clots at controlled room temperature until centrifugation at 1300g for 10 minutes at controlled room temperature within 60 minutes after collection. Within 30 minutes after centrifugation began, the resulting serum was made into 1 aliquot and placed into a pre-labeled 0.50mL two-dimensional bar code Matrix tube (Thermo Cat 3744). All aliquots were flash frozen on dry ice and stored frozen at-60 to-90 ℃.
The presence of anti-drug antibodies (ADA) in serum samples was detected in ELISA assays. The test article was immobilized on an assay plate, incubated with serum and washed, and then immunocomplexes were detected with an anti-pig IgG reagent having an Fc portion bound to horseradish peroxidase for a glycolytic assay.
On day 15, aqueous humor was collected from all animals by a certified veterinary ophthalmologist. The eye position was fixed using conjunctival forceps for aqueous humor collection while the tip of a 31 gauge needle was inserted obliquely at an angle of approximately 90 degrees into the sclera after the limbus. The angle of the needle is then made shallow before the needle enters the anterior chamber between the iris and the cornea. The syringe plunger is slowly withdrawn to aspirate a maximum available volume of aqueous humor, which can be up to 50 μl at maximum. The needle was removed and the scleral tissue was approximated to the puncture site and grasped with conjunctival forceps. The same sample collection procedure was performed on the lateral eye. The collected samples were stored in 1.0mL glass matrix trakmates 2D barcode storage tubes, then capped with TPE caps. Samples were frozen in liquid nitrogen and stored frozen at-60 ℃ to-90 ℃. The assay content in aqueous humor was determined using mass spectrometry-based assays.
A.2. Sample preparation
The PigFab standard calibration curve was performed by adding different amounts of PigFab to a porcine aqueous base diluted with 25mM ammonium bicarbonate. The standards/samples were then treated as follows: disulfide bonds from cysteinyl residues were reduced with 10mM DTT at 60℃for 1 hour, and then thiol groups were alkylated with 55mM iodoacetamide at room temperature in the dark for 45 minutes. The standard/sample was then digested with 36 μg/mL trypsin (sequencing grade trypsin, V5111, promega) and incubated overnight at 37 ℃. After digestion, heavy peptides are added to the standard and sample solutions. A linear calibration curve was obtained over a concentration range of 0.5-12. Mu.g/mL.
A.3. Labeled peptides
Peptide standards (New England Peptide, gardner, MA, USA) containing heavy isotope labeling in R (LLIYSASFLYSGVPSR m/z: 891.98+2) amino acids are commercially available. Characterization and concentration data are provided by the manufacturer. The labeled peptide was stored in 1mL of water at-80 ℃.
A.4. Analysis by Mass Spectrometry (MS)
Special column (130) for peptide analysis using ACQUITY UPLC CSH C on an acquisition UPLC (Waters Corporation, milford, mass.)1.7 μm,1mm×100 mm) digests from PigFab were separated under gradient elution conditions. The column was kept at 50 ℃ and the autosampler sample tray was kept at 8 ℃. The mobile phase was water (A) containing 0.1% FA and acetonitrile (B) containing 0.1% FA at a flow rate of 0.04mL/min. Samples were eluted with the following gradient: b increased from 2% to 90% in 2 minutes and then decreased to 2% B in 2 minutes to rebalance the column. The sample volume was 10. Mu.L.
Triple Quad 6500 Mass Spectrometry (Ab Sciex, framington, mass.) operates in positive ion Multiplex Reaction Monitoring (MRM) mode, equipped withTurbo V ion source. The PigFab precursor (Q1) ion was monitored as LLIYSASFLYSGVPSR (m/z: 886.98+2), a declustering potential of 90V was used, and the product (Q3) ion was monitored as 359.20m/z, and the collision energy as 29eV. Two other product ions (765.39 m/z and 602.33 m/z) were also monitored as qualitative ions with collision energies of 37eV and 30eV, respectively. The MS/MS setup parameters are as follows: ion spray voltage, 4500V; air curtain, 30psi; atomizer gas (GS 1), 25psi; the temperature is 300 ℃; dwell time, 50ms. Heavy peptides (891.97 m/z) of PigFab were also generated and quantified using a 369.204m/z transition with a collision energy of 29eV.
Data acquisition was performed using Sciex analysis software version 1.7.1 (TripleTOF). Raw data is displayed with PeakView 2.2.
B. Results
All animals survived to the planned expiration date. Thus, unscheduled euthanasia need not be implemented. Test article-related ophthalmic test results include vitreous opacity and very slight posterior uveitis in the area of test article injection.
The ophthalmic examination results were as follows:
(1) Very slight turbidity was observed in the temporal vitreous in 2 out of 6 animals on groups 2 (WT VG 1) -1. The result resolved on day 3 and was not present until termination of the study. 1 of the 4 animals developed very mild post-uveitis on day 15, which resolved on day 17. Very mild posterior uveitis is considered clinically insignificant.
(2) Group 3 (pigFab-VG 1) -day 1, all 6 animals showed vitreous opacity in the temporal vitreous of the test article injection area. On day 3, the results persisted in all 6 animals, and on day 5, 4/4 animals exhibited signs of diffusion involving the central vitreous. From day 8 to day 22 we observed a decrease in the affected vitreous area, whereas on day 29 only 2 of the 4 animals developed slight opacities in the temporal vitreous. Regional vitreous opacity does not appear to be inflammatory in nature, but appears to have a local effect on vitreous consistency.
(3) Group 4 (pigFab-vg1+10 kDa HA) -ophthalmic examination results related to the test article were not obtained for the duration of the study.
At all time points throughout the study, the intraocular pressure values for all animals were within the normal range.
At any time point, all animals did not show anti-drug antibodies (ADA) against the test article.
The color fundus photographs and optical coherence tomography images taken on day 29 showed that all animals of groups 2 and 4 had normal vitreous and retinal morphology. All animals of group 3 had very slight local vitreous opacities on fundus photographs and very slight post-vitreous sensitization on OCT, consistent with ophthalmic examination results.
Electroretinogram analysis was performed on all animals on day 29. At a Scotopic 0.01 light intensity, the b-wave amplitude was moderately reduced for both eyes of animal No. 3006. All other light intensities were within the normal range, indicating that the animal had background abnormalities affecting the function of the dim retina. Animal numbers 2005 and 4003 exhibited some asymmetry between eyes with slightly reduced amplitude OD compared to OS. This result is suspected to be related to recording conditions, including non-central eye positioning OD. No results indicating the effect of the test article were found in any animals.
In the eyes or optic nerve, no visual inspection was found in connection with the test article.
All macroscopic observations of animals treated with the test article were background findings of the species or were considered to be accidental findings and independent of the test article. These observations occur at low rates, lack of a clear dose relationship in terms of incidence or severity, and/or there are no relevant microscopic findings associated with the test article.
In the eye or optic nerve, there was no microscopic findings associated with the test article.
All microscopic observations were considered to be accidental findings and independent of the test article. These observations are known background findings of the species and/or have similar incidence and severity for animals treated with control and test.
The level of PigFab-VG1 in aqueous samples was determined by mass spectrometry. High aqueous levels were obtained and maintained for 30 days after IVT injection of 1.8 mg/eye PigFab-VG1 or pre-complexed PigFab-VG1 with an equal mass of 10kDa HA in small pig eyes (FIG. 14). These results indicate that there are measurable levels of test product in the small pig eyes during the 4 week study period. The concentration at 30 days was at least an order of magnitude higher than the observed value of unmodified Fab (figure 4A).
In Gottingen minipigs, WT VG1 (1.13 mg/eye), pigFab-VG1 (1.8 mg/eye) or pigFab-VG1+10kDa HA (1.8 mg/eye) was well tolerated by individual dose levels administered by a single IVT. All animals survived to the planned termination time, no abnormal clinical observations, and body weight was not affected. The lack of a detectable immune response to the test article upon a single injection during the study, thereby allowing for assessment of the direct effect of the test article. Ophthalmic examination results are limited to: a short-lived very slight vitreous turbidity occurred at the test article injection site, which resolved on day 3 (WT VG-1); and vitreous opacities developed near the test injection site that did not completely regress at study termination (pigFab-VG 1). The absence of ophthalmoscopes with the pigFab-VG1+10kDa HA found that the IOP, OCT and ERG results were normal for all animals. In the eye or optic nerve, there is no macroscopic or microscopic effect associated with the test article.
EXAMPLE 15 efficacy of VPDF-VG1 in rat laser-induced choroidal neovascularization (rat laser CNV)
Fab-HABD was studied in an in vivo rat model of laser-induced choroidal neovascularization (rat laser CNV) to test the following hypothesis: (1) Fab-HABD is effective in vivo (i.e., fab-HABD inhibits neovascularization); and (2) the duration of in vivo efficacy of the Fab-HABD is comparable to or better than that of the unmodified Fab fragment.
A. Materials and methods
Rats received IVT injections of protein formulation 1 week or 3 weeks before receiving laser injury (6 laser burns per eye). One week after setting up the laser lesions, the lesions of the vessel growth were analyzed using Fluorescence Angiography (FA) imaging.
Fab-HABD was compared to the corresponding unmodified Fab fragment. To test the sustained efficacy of Fab-HABD, the dose of unmodified Fab was titrated to the "lowest effector dose" (i.e., there was only a low degree of detectable inhibition of neovascularization compared to the vehicle over the duration of the rat model), indicating that Fab-HABD had more sustained efficacy over the same dose and duration of the model.
B. Results
As shown in FIG. 15, administration of VPDF-VG1 7 or 21 days prior to laser treatment effectively inhibited CNV lesions. In this study, the persistence 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 purpose of this study was to determine intraocular tolerance of the test articles WT VG1, rabFab-VG1 and RabFab-VG1 pre-formulated with 1:1 (w/w) 10kDa HA over a 30 day observation period after a single bilateral IVT injection into male New Zealand white rabbits. The study design is shown in table 21.
The following parameters and endpoints were evaluated in this study: mortality, clinical signs, body weight, food consumption, ophthalmology (i.e., examination, intraocular pressure measurement, wide-field color fundus imaging, OCT and ERG), bioassays, toxicological kinetic parameters, anti-drug antibody assessment, total autopsy results, and histopathological examination.
B. Results
Based on the assessment of body weight and food consumption, no systemic trial related effects were present. There was no macroscopic necropsy findings with naked eyes, and all tissues were considered to be within normal range.
The presence of anti-drug antibodies (ADA) in rabbit serum was determined before dosing and on days 8, 15, 22 and 29 of the study. 3 of 6 animals receiving WT VG1 administration were assigned measurable serum ADA prior to administration. Similarly, 1/6 and 0/6 animals receiving RabFab-VG1 or RabFab-VG1+10kDa HA treatment were assigned with pre-existing serum ADA for the test article, respectively. On day 8, all animals in the WT VG1 and RabFab-VG1 groups showed ADA in serum against the test article and remained positive for the duration of the study, whereas in the RabFab-VG1+10kDa HA group, 2/3 of the animals had serum ADA. On day 15, all animals were positive for serum ADA and continued until the end of the study.
In animals treated with WT VG1, rabFab-VG1 and RabFab-VG1+10kDa HA, clinical ophthalmoscope results were consistent with the development of anterior and posterior uveitis, but with varying severity between treatment groups. For example, WT VG1 causes moderate anterior and posterior uveitis to occur on day 22, including some ocular subcapsular cataracts. In contrast, most animals treated with RabFab-VG1 exhibited mild uveitis at the same time point. Furthermore, animals treated with RabFab-vg1+10kDa HA showed only minimal to mild anterior and posterior uveitis. In each treatment group, improvement in uveitis signs occurred at day 29 after initiation of systemic anti-inflammatory treatment (which also includes topical ocular treatment of animals receiving WT VG1 administration) at day 18. The drop in intraocular pressure was consistent with active uveitis, while the degree of change in vitreous opacity also corresponded to the severity of uveitis in the different groups. In these cases, the vitreous opacity was limited to only weak vitreous opacity in animals treated with RabFab-VG1+10kDa HA, whereas WT VG1 animals showed moderate opacity and posterior cataract. Treatment-dependent effect severity was also observed by OCT and ERG, where reduction in scotopic and photopic amplitude indicated severe changes and degeneration in retinal function in eyes treated with WT VG 1.
The endoscopic effect was also most pronounced in eyes receiving WT VG1 treatment (fig. 16A) compared to other trials, where the RabFab-VG1 effect (fig. 16B) was less severe and the RabFab-vg1+10kDa HA effect (fig. 16C) was limited to only the vitreous and severity was only very mild to mild. The WT VG 1-associated vitreous inflammation includes areas near the optic nerve head, retinal detachment and varying degrees of necrosis, and the inflammatory cell layer adjacent to the posterior capsule of the lens. In addition, the anterior chamber contains homogeneous eosinophilic material, which is consistent with serum proteins. In the retina, WT VG 1-related inflammation is characterized by the extension of mixed cell types to the retinal parenchyma, with minimal to significant necrosis and vascular and perivascular inflammation. Reactive Muller cells were observed in the center of the retina.
Similar to WT VG1, eyes treated with RabFab-VG1 showed very slight to moderate diffuse photoreceptor lesions, which are typically associated with reactive Muller cells. However, the RabFab-VG1 photoreceptor layer lesion is different from WT VG 1-related retinal necrosis, in that the lesion is selective for the photoreceptor layer only, whereas retinal necrosis involves multiple retinal layers. In addition, eyes treated with both WT VG1 and RabFab-VG1 had fibrovascular membrane characteristics in the vitreous. In these cases, the membrane consists of fibroblasts, a large number of new blood vessels and early collagen deposition. Traction bands separated from the membrane were also observed. In contrast to WT VG1 and RabFab-VG1, the RabFab-VG1+10kDa HA-related effects are limited to very mild to mild mononuclear inflammation, which is limited to the vitreous and inner limiting membrane.
In summary, a single IVT administration of WT VG1, rabFab-VG1 or RabFab-VG1+10kDa HA to New Zealand white rabbits resulted in the development of pre-and post-uveitis, most severe in the animals administered WT VG1, moderate in the animals administered RabFab-VG1 and mild to moderate in the animals administered RabFab-VG1+10kDa HA. In addition to inflammation, WT VG1 and RabFab-VG1 present significant retinal necrosis and retinal degeneration, respectively, which correlate with reduced ERG amplitude in microscopy. At the end of the study, no sign of active anterior uveitis was present in the RabFab-vg1+10kDa HA eye, and only very slight chronic posterior uveitis was observed. Although the interpretation of these results is confused with the ADA observations on day 15 for all the test articles, pre-complexing or binding of RabFab-VG1 with 10kDa HA does appear to improve the tolerance of the Fab-HABD in rabbit eyes.
EXAMPLE 17 brain Retention of Fab-VG1
HA binding by VG1 was tested by mouse intraventricular injection to achieve the ability to remain in the brain. For these purposes, the non-target binding antibody anti-herpes simplex virus-1 glycoprotein D (anti-gD) is used as a Fab fragment (anti-gD Fab), intact IgG (anti-gD IgG) or as a fusion protein with VG1 (anti-gD Fab-VG1; BRD).
A. Materials and methods
A.1. Animals
Wild type C57BL/6 mice used in these studies were obtained from the Kansas university breeding group. The Institutional Animal Care and Use Committee (IACUC) approval of kansase university was obtained using a living animal protocol (AUS 75-15; approval date: 25 of 1 st year 2021). 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 unrestricted access to feed and water by the animals.
A.2. Binding of antibodies to IRDye800CW NHS esters
Antibodies were conjugated to IRDye800 according to the manufacturer's instructions. Briefly, antibodies were reacted with IRDye800 in PBS containing 10% potassium phosphate buffer pH 9 (v/v) at 25℃for 2 hours. Excess dye was removed using a Zeba Spin desalting column (Fisher Scientific) with a molecular weight cut-off of 7 kDa. Bound antibody purity was assessed using SDS-PAGE. SDS-PAGE gels were scanned at 800nm using an Odyssey CLx NIR scanner to confirm that all excess dye had been removed.
A.3. Intra-cerebral injection
Healthy C57BL/6 mice of 5-10 weeks old were anesthetized with 1.5-2% isoflurane and placed in a stereotactic instrument (Stoelting Co.). A midline sagittal incision was made to expose the mouse skull and identify the bregma. A small hole is opened at the position of 1.0mm on the right side of the skull and 0.3mm before bregma. A10. Mu.L Hamilton syringe (No. 7762-06) with a 33 gauge removable needle was fitted to the stereotactic apparatus and used to inject 5. Mu.L of 1mg/mL antibody solution into the lateral ventricle of the mouse to a depth of 2.25 mm. The antibody was infused at a rate of 1. Mu.L/min. Blood samples (about 100 μl) from the mandibular vein were collected into frozen plasma collection tubes containing heparin lithium as an anticoagulant prior to euthanasia. Samples were kept on ice until centrifugation at 10000 Xg for 3 minutes and plasma was stored at-80℃until analysis. After various time points, mice were sacrificed by transcardiac infusion of HBSS ice-cold solution containing 0.1% tween-20 while deeply anesthetizing the mice with 4-5% isoflurane. Brain, heart, lung, liver, spleen and kidney were collected and kept on ice until analysis.
A.4. Antibody organ quantification
The isolated organs were weighed and mechanically homogenized in 1mL PBS. Standard near infrared fluorescence (NIRF) antibody solutions were prepared by diluting stock solutions with varying amounts of PBS. A calibration curve was then generated for each organ in a 96-well plate by adding 10 μl of standard solution to 100 μl of homogeneous blank organ and scanning each well using OdysseyClx 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 receiving intraventricular injections were compared to a calibration curve to determine antibody deposition. Similarly, plasma analysis was performed with blank plasma diluted 5-fold first. Then, 10 μl of antibody standard was added to 100 μl aliquots of diluted plasma to generate a standard curve, which was compared to plasma samples of mice receiving intraventricular injections.
B. Results
As shown in FIG. 17, anti-gDFab-VG 1 (BRD; SEQ ID NO:121 and SEQ ID NO: 124) persists longer in the brain and provides greater exposure levels (expressed as area under the curve (AUC)) than equivalent doses of anti-gD Fab (SEQ ID NO:120 and SEQ ID NO: 121) or anti-gD IgG (SEQ ID NO:121 and SEQ ID NO: 122). These differences in exposure levels were statistically significant, with the p-value of anti-gD Fab-VG1 being less than 0.01 compared to anti-gD Fab; the p-value of anti-gD Fab-VG1 is less than 0.001 compared to anti-gD IgG.
EXAMPLE 18 Generation of VG1 affinity variants
A. Materials and methods
Crystallization of WT VG1 and identification of HA-binding residues
Crystallization conditions of HA-bound WT VG1 were determined using commercial crystallization screens (Hampton Research and Qiagen). The structure of HA-bound VCAN is obtained by soaking crystals with HA 6-mers. Crystals were harvested and flash frozen in liquid nitrogen without cryoprotectant. Diffraction data were collected on either a Pilatus 6M or Eiger 16M detector (dectrics), respectively, using a Stanford synchrotron radiation light source (SSRL) beam line 12-2 or 14-1. The structure is iteratively refined by modeling in COOT and then refined with REFMAC5, BUSTER or Phenix-Refine. Adams, p.d. et al Acta crystal r.d biol. Crystal r.,66 (Pt 2): 213-221 (2010); blanc, e et al Acta crystal grogr.d biol. 2210-2221 (2004); emsley, P. et al, acta crystal r.D biol. Crystal r.,66:486-501 (2010); emsley and Cowtan, acta crystal r.D biol. Crystal, 66:2126-2132 (2004); murshudov, G.N. et al Acta crystal r.D biol. Crystal r.,67:355-367, (2011).
The WT VG1-HA conjugate structure was analyzed using PyMol and/or chirea (fig. 18) and residues that interacted with HA were identified based on hydrogen bonding, electrostatic and hydrophobic interaction potentials.
A.2. Differential scanning fluorescence method (DSF)
Thermal stability of WT VG1 and single amino acid variants was measured using Differential Scanning Fluorescence (DSF). Briefly, 0.1mg/mL of purified protein was mixed with Sypro Orange dye in PBS. Each sample was treated in 0.05 °/second increments over a temperature gradient range of 25 ℃ to 95 ℃ and the increase in fluorescence was monitored at 585 nm. The original fluorescent units were plotted as negative derivatives using custom excel macros and Tm was calculated.
A.3. VG1 variants based on WT VG1-HA conjugate crystal structure design
Based on the WT VG1-HA conjugate crystal structure (FIG. 18), HA was found to bind only to link1 domain. Thus modeling was used to predict link 2 residues that are likely to be involved in HA binding. To verify the crystal structure and identify mutants that attenuate the HA binding affinity of WT VG1, the residues that the crystal contacts are mutated to alanine or, in some cases, to a surrogate amino acid. Furthermore, some WT VG1 residues do not make crystal contact with HA but are important for HA binding in TSG6 (based on sequence alignment between VG1 domain structure and TSG-6 domain; FIG. 8B), they also mutate to alanine. To examine the combined effect of mutations, several double site mutants were formed, such as Lys260 and Phe261 changed to Arg and Tyr (KF 260 RY), respectively, and tested for HA binding capacity. Table 22 lists VG1 variants produced by the method as described in example 10. The amino acid sequence alignment of the VG1 variants generated and tested is shown in FIG. 19.
A.4. Molecular characterization
HA binding was measured by SPR as described in example 10. Mutants were injected for 120 seconds and dissociation was monitored for 180 seconds.
B. Results
B.1.VG1 variants have reduced HA binding levels
Mutants R160A, Y161A and D197A showed reduced HA binding in the range of 2 to 7 μm. Table 23 shows the measured k of each VG1 variant measured by SPR a (M -1 s -1 )、k d (s -1 ) And K D (M)。
Stability of VG1 variants
Table 24 shows the measured Tm (melting temperature;. Degree. C.) of the VG1 mutants produced and of each mutant. While most mutations had little or no effect on thermal stability compared to WT VG1, tm of Y208A and H306A showed improvements of 2.16 ℃ and 2.81 ℃, respectively.
Equal form
The previous written description is believed to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and describe the best mode contemplated by the inventors. It should be understood, however, that the embodiments may be practiced in many ways, regardless of the level of detail in the foregoing description, and should be interpreted in accordance with the claims and any equivalents thereof.
As used herein, the term "about" refers to a value (including, for example, integers, fractions and percentages) whether or not explicitly indicated. The term "about" generally refers to a range of values (e.g., +/-5-10% of the range) that one of ordinary skill in the art would consider equivalent to the value (e.g., having the same function or result). When terms such as "at least" and "about" precede the list of values or ranges, these terms will modify all values or ranges provided in the list. In some cases, the term "about" may include numerical values rounded to the nearest significant figure.

Claims (31)

1. A therapeutic molecule comprising:
a. a first component capable of binding to a therapeutic target in the eye,
b. one or more second components capable of binding to hyaluronic acid, wherein the one or more second components are covalently bound to the first component, and
c. optionally, one or more third components comprising hyaluronic acid,
wherein the one or more third components, if present, are non-covalently bound to the one or more second components.
2. The therapeutic molecule of claim 1, wherein the first component is a protein, peptide, receptor or fragment thereof, ligand for a receptor, darpin, nucleic acid, RNA, DNA, or aptamer.
3. The therapeutic molecule according to claim 1 or 2, wherein the first component is selected from an antibody, an antigen binding fragment, in particular an antibody fragment, more in particular an antibody fragment at least lacking an Fc domain, in particular wherein the fragment is or comprises (Fab') 2 Fragments, fab' fragments, fab fragments, vhH fragments, scFv-Fc fragments and miniantibodies, more particularly Fab fragments.
4. The therapeutic molecule of any one of claims 1-3, wherein the second component comprises a hyaluronic acid receptor CD44 (CD 44) domain, a brain-specific junction protein (BRAL 1) domain, a tumor necrosis factor stimulating gene 6 (TSG-6) domain, a lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1) domain, or a Hyaluronic Acid Binding Protein (HABP) domain, an aggrecan G1 (AG 1) domain, or a multifunctional proteoglycan G1 (VG 1) domain.
5. The therapeutic molecule according to any one of claims 1 to 4, wherein the conjugate comprises one second component or two second components that are identical to each other.
6. The therapeutic molecule of any one of claims 1-4, wherein the third component is hyaluronic acid, wherein the hyaluronic acid
a. Has the following characteristics of
i. A molecular weight selected from 3kDa to 60kDa, 4kDa to 30kDa, 5kDa to 20kDa or 400Da to 200 kDa;
molecular weight of at least 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa or 9 kDa; or (b)
Molecular weights of up to 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa or 15 kDa;
b. providing a molar excess of binding equivalents to the one or both second components; and
c. has a modification that reduces degradation of hyaluronic acid in the eye.
7. The therapeutic molecule of any one of claims 1-6, wherein the second component is capable of K at 10nM to 10 μΜ, 5nM to 8 μΜ, or 100nM to 5 μΜ D Binds to hyaluronic acid.
8. The therapeutic molecule of any one of claims 1-7, wherein
a. The first component and the second component are comprised in a fusion protein, in particular wherein one or both of the second components are covalently bound to the N-terminus and/or the C-terminus of the first component, more in particular wherein the first component is an antibody or antigen binding fragment, and wherein the one or both 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, in particular a linker of at least 4 amino acids and/or of at most 50 or at most 25 amino acids, more in particular the linker is (GxS) n Or (GxS) n G m 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).
9. The therapeutic molecule of any one of claims 1-8, wherein the therapeutic target is VEGF, C2, C3a, C3b, C5a, htrA1, IL-33 factor P, factor D, EPO, EPOR, IL-1 beta, IL-17A, IL-10, tnfa, FGFR2, PDGF, or ANG2.
10. The therapeutic molecule according to any one of claims 1 to 9, wherein
a. The first component is an antibody or antigen binding fragment directed against VEGF; and/or
b. Each of the one or two second components comprises a CD44 domain or a TSG-6 domain or a VG1 domain; and/or
c. The third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa.
11. The therapeutic molecule according to any one of claims 1 to 10, wherein
a. The first component is an anti-VEGF antibody or antigen-binding fragment, the one or two second components comprise a CD44 domain, and the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa;
b. the first component is an anti-VEGF antibody or antigen-binding fragment, the one or two second components comprise a TSG-6 domain, and the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa; or (b)
c. The first component is an anti-VEGF antibody or antigen-binding fragment, the one or two second components comprise VG1 domains, and the third component is hyaluronic acid having a molecular weight of 5kDa to 20 kDa.
12. The therapeutic molecule of any one of claims 1-11, wherein
a. The first component comprises
A VH domain of seq ID NO 97, 99, 105, 109 or 114; and
a VL domain of SEQ ID NO 98, 100, 106, 110 or 115; and is also provided with
b. The second component comprises SEQ ID NO. 2.
13. The therapeutic molecule of any one of claims 1-11, wherein
a. The first component comprises
A VH domain of seq ID NO 97, 99, 105, 109 or 114; and
a VL domain of SEQ ID NO 98, 100, 106, 110 or 115; and is also provided with
b. The second component comprises SEQ ID NO. 4.
14. The therapeutic molecule of any one of claims 1-11, wherein
a. The first component comprises
A VH domain of seq ID NO 97, 99, 105, 109 or 114; and
a VL domain of SEQ ID NO 98, 100, 106, 110 or 115; and is also provided with
b. The second component comprises SEQ ID NO 86, 60, 32 or 29.
15. The therapeutic molecule of claim 14, wherein the second component is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 86, 60, 32 or 29.
16. The therapeutic molecule of claim 14 or 15, wherein the second component comprises 1 to 5 mutations, wherein the 1 to 5 mutations comprise a single amino acid substitution, a double amino acid substitution, and/or a truncation.
17. The therapeutic molecule according to any one of claims 14 to 16, wherein the second component has a truncation mutation relative to SEQ ID No. 29.
18. The therapeutic molecule of claim 17, wherein the truncation mutation comprises a truncation of 1 to 129 amino acids at the N-terminus.
19. The therapeutic molecule of any one of claims 14-18, wherein the second component is a truncated sequence, wherein no Ig domain of a wild-type multifunctional proteoglycan is present.
20. The therapeutic molecule of any one of claims 14-19, wherein the second component comprises a mutation in 1, 2, 3, 4, 5, or 6 of the following positions relative to SEQ ID No. 29: r160, Y161, E194, D197, Y208, R214, M222, Y230, R233, K260, F261, D295, Y296, H306, R312, L325, Y326 and R327.
21. The therapeutic molecule of any one of claims 14 to 20, 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, D A, D197S, Y208A, Y208F, R K, M4232 42230A, Y4815A, K260A, K260R, F A, D233A, K260R, F Y, KF RY, D295A, D295S, Y296A, Y296F, DY295SF, H306A, R312A, L A, Y52326A, R326A, R a and LYR325LFK.
22. The therapeutic molecule of any one of claims 14-21, wherein the second component comprises at least one of Y208A and H306A.
23. The therapeutic molecule according to claim 14 or 15, wherein the second component is SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58 or SEQ ID No. 59.
24. The therapeutic molecule according to any one of claims 1-23, wherein the first component further comprises a cysteine knot peptide.
25. The therapeutic molecule according to claim 24, wherein the cysteine knot peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to seq id No. 92.
26. The therapeutic molecule of claim 24 or 25, wherein the amino acid sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 93 or SEQ ID No. 94.
27. A composition for use as a medicament, the composition comprising a therapeutic molecule according to any one of claims 1 to 26 and optionally a pharmaceutically acceptable excipient, diluent or carrier.
28. A composition for use in the treatment of an eye disease or brain disease, the composition comprising a therapeutic molecule according to any one of claims 1 to 26 and optionally a pharmaceutically acceptable excipient, diluent or carrier.
29. The composition for use according to claim 28, formulated for intraocular delivery, in particular for intravitreal injection.
30. The composition for use according to claim 28 or 29, wherein the eye disease is age-related macular degeneration (AMD), in particular wet AMD or neovascular AMD, diabetic Macular Edema (DME), diabetic Retinopathy (DR), in particular proliferative DR or non-proliferative DR, retinal Vein Occlusion (RVO) or Geographic Atrophy (GA).
31. A method of delivering a therapeutic molecule that targets a tissue of a patient, comprising administering to the patient the therapeutic molecule of any one of claims 1-26 or the composition of any one of claims 27-30, and allowing the therapeutic molecule to provide a long-lasting delivery of the first component to a target tissue.
CN202180070826.3A 2020-10-15 2021-10-14 Non-covalent protein-hyaluronic acid conjugates for long-acting ocular delivery Pending CN116761634A (en)

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