Human papillomavirus nanoparticle formulations

Abstract

To provide virus-like particle drug conjugate formulations and use of the conjugates for treating ocular tumors or lesions.SOLUTION: The present disclosure provides, in some aspects, ophthalmic compositions comprising a virus-like particle (VLP) drug conjugate comprising photosensitive molecules conjugated to capsid proteins of a VLP and 2-(N-morpholino)ethanesulfonic acid (MES) or a pharmaceutically acceptable salt thereof. Surprisingly, the particular MES-based formulations provided herein eliminate visible aggregation and precipitation of the VLP drug conjugate, relative to other formulations tested, in some embodiments, without at a low salt concentration and / or with a pH that is suitable for ophthalmic administration (e.g., less than 7, such 6.5).SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 824,227, filed Mar. 26, 2019, under 35 U.S.C. § 119(e), which is incorporated herein by reference in its entirety.

Background Art

[0002] Background Fundamental concerns with biotherapeutic formulations, including ophthalmic formulations, are the stability and structural integrity of active molecules after multiple freeze - thaw cycles during transit and storage, the successful delivery of the drug to its site of action, as well as the rate and cost - effectiveness of development, and the end product. Many details are involved, including analytical methods and test protocols, containers and closures, delivery devices and dosage forms, excipients and stabilizers, and the compatibility of components.

Summary of the Invention

Means for Solving the Problems

[0003] Abstract The present disclosure provides, in some aspects, a VLP - drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of a virus - like particle (VLP) and an ophthalmic composition comprising 2 - (N - morpholino) ethanesulfonic acid (MES) or a pharmaceutically acceptable salt thereof. Surprisingly, certain MES - based formulations provided herein eliminate visible aggregation and precipitation of the VLP - drug conjugate in some embodiments without a low salt concentration and / or at a pH suitable for ocular administration (e.g., less than 7 (e.g., 6.5)).

[0004] Accordingly, in some aspects, there is provided an ophthalmic composition comprising a substantially isotonic solution (255 - 345 mOsm / L) of a VLP-drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of a VLP, wherein the VLP-drug conjugate is in suspension. As used herein, an isotonic solution has an isotonicity of approximately 290 mOsm / L, and a substantially isotonic solution has an isotonicity of 255 - 345 mOsm / L.

[0005] In some aspects, the ophthalmic composition comprises a VLP-drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of a VLP, wherein the VLP-drug conjugate does not aggregate to form visible microparticles.

[0006] In some embodiments, the ophthalmic composition further comprises at least one protecting reagent and at least one surfactant. In some embodiments, the ophthalmic composition further comprises at least one, at least two, or at least three reagents selected from trehalose dihydrate, magnesium chloride (MgCl2), sodium chloride (NaCl), and polysorbate 80 (PS80). In some embodiments, the ophthalmic composition further comprises trehalose dihydrate, MgCl2, NaCl, and PS80.

[0007] In some embodiments, the composition comprises 0.01% - 0.1% (w / v) VLP-drug conjugate. For example, the composition can comprise 0.04% (w / v) VLP-drug conjugate.

[0008] In some embodiments, the composition comprises 0.1% - 1.0% (w / v) MES or a pharmaceutically acceptable salt thereof (e.g., MES hemisodium salt). For example, the composition can comprise 0.4% (w / v) MES or a pharmaceutically acceptable salt thereof. In some embodiments, the composition comprises 5 mM - 50 mM MES or a pharmaceutically acceptable salt thereof. For example, the composition can comprise 20 mM MES or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the composition further comprises 1% to 10% (w / v) trehalose dihydrate. For example, the composition may further comprise 5% (w / v) trehalose dihydrate.

[0010] In some embodiments, the composition further comprises 0.1% to 1.0% (w / v) NaCl. For example, the composition may further comprise 0.37% (w / v) NaCl. In some embodiments, the composition further comprises 20 mM to 100 mM NaCl. For example, the composition may further comprise 63 mM NaCl.

[0011] In some embodiments, the composition further comprises 0.1% to 1.0% (w / v) MgCl2. For example, the composition may further comprise 0.2% (w / v) MgCl2. In some embodiments, the composition further comprises 5 mM to 25 mM MgCl2. For example, the composition may further comprise 10 mM MgCl2.

[0012] In some embodiments, the composition further comprises 0.01% to 0.1% (w / v) polysorbate 80. For example, the composition may further comprise 0.05% (w / v) polysorbate 80.

[0013] In some embodiments, the composition has a pH value of 5 to 8. For example, the composition may have a pH value of 6.5.

[0014] In some aspects, the ophthalmic composition comprises 0.43% (w / v) MES, 5% (w / v) trehalose dihydrate, 0.37% (w / v) NaCl, 0.2% MgCl2, 0.05% (w / v) polysorbate 80, and 0.04% (w / v) virus-like particle (VLP) - drug conjugate, wherein the VLP - drug conjugate comprises a photosensitive molecule conjugated to the capsid protein of the VLP. In some aspects, the ophthalmic composition comprises 20 mM MES, 5% (w / v) trehalose dihydrate, 63 mM NaCl, 10 mM MgCl2, 0.05% (w / v) polysorbate 80, and 0.04% (w / v) VLP - drug conjugate, wherein the VLP - drug conjugate comprises a photosensitive molecule conjugated to the capsid protein of the VLP.

[0015] In some embodiments, the photosensitive molecule comprises an infrared or near - infrared (e.g., phthalocyanine) dye molecule. For example, the photosensitive dye molecule may comprise an IRDye® 700DX molecule, an IRDye® 800CW molecule, or a mixture of IRDye® 700DX and IRDye® 800CW molecules. Other therapeutic and diagnostic photosensitive molecules are contemplated herein.

[0016] In some embodiments, the VLP comprises 10 - 1000 photosensitive molecules, 10 - 500 photosensitive molecules, 50 - 1000 photosensitive molecules, 50 - 500 photosensitive molecules, 100 - 1000 photosensitive molecules, or 100 - 500 photosensitive molecules. In some embodiments, the VLP comprises 300 photosensitive molecules.

[0017] In some embodiments, the VLP includes a papillomavirus capsid protein (e.g., a human papillomavirus capsid protein). For example, the papillomavirus capsid protein may include an L1 capsid protein, an L2 capsid protein, or a combination of L1 and L2 capsid proteins. In some embodiments, the L1 capsid protein is modified to reduce the immunogenicity of the VLP.

[0018] In some aspects, methods are also provided herein that include administering to the eye of a subject an ophthalmic composition of the present disclosure, where the subject has an eye cancer (e.g., ocular melanoma), and the ophthalmic solution is administered in an amount effective to treat the ocular melanoma. In some embodiments, the ophthalmic composition is administered by intravitreal injection. In some embodiments, the subject has uveal melanoma. In some embodiments, the subject has choroidal melanoma.

[0019] In some aspects, methods are further provided herein that include administering to the eye of a subject an ophthalmic composition of the present disclosure, where the subject has an indeterminate lesion, and the ophthalmic solution is administered in an amount effective to treat the indeterminate lesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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[0021] Detailed Description In some aspects, the present disclosure provides an ophthalmic composition comprising a VLP drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of a virus - like particle (VLP) and 2 - (N - morpholino)ethanesulfonic acid (MES). The chemical structure of MES contains a morpholine ring, has a molecular weight of 195.2, and its chemical formula is C6H 13It is NO4S. In some embodiments, the composition contains 0.1% - 1.0% (w / v) MES or a pharmaceutically acceptable salt thereof (e.g., MES hemisodium salt). For example, the composition may contain 0.1% - 0.9%, 0.1% - 0.8%, 0.1% - 0.7%, 0.1% - 0.6%, 0.1% - 0.5%, 0.1% - 4%, 0.2% - 1.0%, 0.2% - 0.9%, 0.2% - 0.8%, 0.2% - 0.7%, 0.2% - 0.6%, 0.2% - 0.5%, 0.2% - 0.4%, 0.3% - 1.0%, 0.3% - 0.9%, 0.3% - 0.8%, 0.3% - 0.7%, 0.3% - 0.6%, 0.3% - 0.5%, 0.3% - 0.4%, 0.4% - 1.0%, 0.4% - 0.9%, 0.4% - 0.8%, 0.4% - 0.7%, 0.4% - 0.6%, or 0.4% - 0.5% (w / v) MES. In some embodiments, the composition may contain 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) MES. In some embodiments, the composition may contain 0.41%, 0.42%, 0.43%, 0.44%, or 0.45% (w / v) MES. In some embodiments, the composition may contain 0.43% (w / v) MES. In some embodiments, the composition contains 5 mM - 50 mM MES. For example, the composition may contain 5 mM - 45 mM, 5 mM - 40 mM, 5 mM - 35 mM, 5 mM - 30 mM, 5 mM - 25 mM, 5 mM - 20 mM, 10 mM - 50 mM, 10 mM - 45 mM, 10 mM - 40 mM, 10 mM - 35 mM, 10 mM - 30 mM, 10 mM - 25 mM, 10 mM - 20 mM, 20 mM - 50 mM, 20 mM - 45 mM, 20 mM - 40 mM, 20 mM - 35 mM, 20 mM - 30 mM, or 20 mM - 25 mM MES. In some embodiments, the composition contains 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM MES. In some embodiments, the composition contains 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM MES.In some embodiments, the composition contains 20 mM MES.

[0022] In some embodiments, the composition contains 0.01% - 0.1% (w / v) VLP-drug conjugate. For example, the composition may contain 0.01% - 0.09%, 0.01% - 0.08%, 0.01% - 0.07%, 0.01% - 0.06%, 0.01% - 0.05%, 0.01% - 0.04%, 0.02% - 0.1%, 0.02% - 0.09%, 0.02% - 0.08%, 0.02% - 0.07%, 0.02% - 0.06%, 0.02% - 0.05%, 0.02% - 0.04%, 0.03% - 0.1%, 0.03% - 0.09%, 0.03% - 0.08%, 0.03% - 0.07%, 0.03% - 0.06%, 0.03% - 0.05%, 0.03% - 0.04%, 0.04% - 0.1%, 0.04% - 0.09%, 0.04% - 0.08%, 0.04% - 0.07%, 0.04% - 0.06%, or 0.04% - 0.05% (w / v) VLP-drug conjugate. In some embodiments, the composition may contain 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% (w / v) VLP-drug conjugate. In some embodiments, the composition may contain 0.03%, 0.035%, 0.04%, 0.045%, or 0.05% (w / v) VLP-drug conjugate. In some embodiments, the composition contains 0.04% (w / v) VLP-drug conjugate.

[0023] In some embodiments, the composition contains 0.01% - 0.2% (w / v) VLP-drug conjugate. In some embodiments, the composition contains 0.01% - 0.3% (w / v) VLP-drug conjugate. In some embodiments, the composition contains 0.01% - 0.4% (w / v) VLP-drug conjugate. In some embodiments, the composition contains 0.01% - 0.5% (w / v) VLP-drug conjugate. In some embodiments, the composition may contain 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% (w / v) VLP-drug conjugate.

[0024] In some embodiments, the ophthalmic composition further comprises at least one, at least two, or at least three reagents selected from trehalose dihydrate, magnesium chloride (MgCl2), sodium chloride (NaCl), and polysorbate 80 (PS80). In some embodiments, the ophthalmic composition further comprises trehalose dihydrate and MgCl2. In some embodiments, the ophthalmic composition further comprises trehalose dehydrate and NaCl. In some embodiments, the ophthalmic composition further comprises trehalose dihydrate and PS80. In some embodiments, the ophthalmic composition further comprises MgCl2 and NaCl. In some embodiments, the ophthalmic composition further comprises MgCl2 and PS80. In some embodiments, the ophthalmic composition further comprises NaCl and PS80. In some embodiments, the ophthalmic composition further comprises trehalose dihydrate, MgCl2 and NaCl. In some embodiments, the ophthalmic composition further comprises trehalose dihydrate, MgCl2 and PS80. In some embodiments, the ophthalmic composition further comprises trehalose dihydrate, NaCl, and PS80. In some embodiments, the ophthalmic composition further comprises MgCl2, NaCl, and PS80. In some embodiments, the ophthalmic composition further comprises trehalose dihydrate, MgCl2, NaCl, and PS80.

[0025] Trehalose is a disaccharide formed by a 1,1-glycosidic bond between two α-glucose units. Trehalose forms rhombic crystals as a dihydrate, and the anhydrous form of trehalose readily re-acquires moisture to form the dihydrate. As shown herein, this excipient has a significant positive effect on the recovery and stability of VLP-drug conjugates in MES buffer containing NaCl and MgCl2. In some embodiments, the composition further comprises 1% to 10% (w / v) trehalose dihydrate. For example, the composition may comprise 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 2% to 10%, 2% to 9%, 2% to 8%, 2% to 7%, 2% to 6%, 2% to 5%, 3% to 10%, 3% to 9%, 3% to 8%, 3% to 7%, 3% to 6%, 3% to 5%, 3% to 4%, 4% to 10%, 4% to 9%, 4% to 8%, 4% to 7%, 4% to 6%, or 4% to 5%, 5% to 10%, 5% to 9%, 5% to 8%, 5% to 7%, or 5% to 6% (w / v) trehalose dihydrate. In some embodiments, the composition is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (w / v) and further comprises trehalose dihydrate. In some embodiments, the composition further comprises 4%, 4.5%, 5%, 5.5%, or 6% trehalose dihydrate. In some embodiments, the composition further comprises 5% trehalose dihydrate.

[0026] In some embodiments, for isotonicity, the composition further comprises a salt such as sodium chloride (NaCl). In some embodiments, the composition further comprises 0.1% - 1.0% NaCl. For example, the composition may comprise 0.1% - 0.9%, 0.1% - 0.8%, 0.1% - 0.7%, 0.1% - 0.6%, 0.1% - 0.5%, 0.1% - 4%, 0.2% - 1.0%, 0.2% - 0.9%, 0.2% - 0.8%, 0.2% - 0.7%, 0.2% - 0.6%, 0.2% - 0.5%, 0.2% - 0.4%, 0.3% - 1.0%, 0.3% - 0.9%, 0.3% - 0.8%, 0.3% - 0.7%, 0.3% - 0.6%, 0.3% - 0.5%, 0.3% - 0.4%, 0.4% - 1.0%, 0.4% - 0.9%, 0.4% - 0.8%, 0.4% - 0.7%, 0.4% - 0.6%, or 0.4% - 0.5% (w / v) NaCl. In some embodiments, the composition may comprise 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) NaCl. In some embodiments, the composition further comprises 0.35%, 0.35%, 0.37%, 0.38%, or 0.39% (w / v) NaCl. In some embodiments, the composition further comprises 0.37% (w / v) NaCl. In some embodiments, the composition further comprises 20 mM - 100 mM NaCl. For example, the composition may further comprise 20 mM - 90 mM, 20 mM - 80 mM, 20 mM - 70 mM, 20 mM - 60 mM, 20 mM - 50 mM, 30 mM - 100 mM, 30 mM - 90 mM, 30 mM - 80 mM, 30 mM - 70 mM, 30 mM - 60 mM, 30 mM - 50 mM, 40 mM - 100 mM, 40 mM - 90 mM, 40 mM - 80 mM, 40 mM - 70 mM, 40 mM - 60 mM, 40 mM - 50 mM, 50 mM - 100 mM, 50 mM - 90 mM, 50 mM - 80 mM, 50 mM - 70 mM, 50 mM - 60 mM, or 60 mM - 70 mM NaCl. In some embodiments, the composition further comprises 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, or 66 mM NaCl. In some embodiments, the composition further comprises 63 mM NaCl.

[0027] In some embodiments, the composition further comprises 0.1% to 1.0% (w / v) magnesium chloride (MgCl₂). For example, the composition may comprise 0.1% to 0.9%, 0.1% to 0.8%, 0.1% to 0.7%, 0.1% to 0.6%, 0.1% to 0.5%, 0.1% to 4%, 0.2% to 1.0%, 0.2% to 0.9%, 0.2% to 0.8%, 0.2% to 0.7%, 0.2% to 0.6%, 0.2% to 0.5%, 0.2% to 0.4%, 0.3% to 1.0%, 0.3% to 0.9%, 0.3% to 0.8%, 0.3% to 0.7%, 0.3% to 0.6%, 0.3% to 0.5%, 0.3% to 0.4%, 0.4% to 1.0%, 0.4% to 0.9%, 0.4% to 0.8%, 0.4% to 0.7%, 0.4% to 0.6%, or 0.4% to 0.5% (w / v) MgCl₂. In some embodiments, the composition may comprise 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) MgCl₂. In some embodiments, the composition further comprises 0.1%, 0.15%, 0.2%, 0.25%, or 3% (w / v) MgCl₂. In some embodiments, the composition further comprises 0.2% (w / v) MgCl₂. In some embodiments, the composition further comprises 5 mM to 25 mM MgCl₂. For example, the composition may comprise 5 mM to 20 mM, 5 mM to 15 mM, 5 mM to 10 mM, 10 mM to 25 mM, 10 mM to 20 mM, or 10 mM to 15 mM MgCl₂. In some embodiments, the composition further comprises 5 mM, 10 mM, 15 mM, 20 mM, or 25 mM MgCl₂. In some embodiments, the composition further comprises 8 mM, 9 mM, 10 mM, 11 mM, or 12 mM MgCl₂. In some embodiments, the composition further comprises 10 mM MgCl₂.

[0028] In some embodiments, the composition further comprises a surfactant, for example, to increase protein recovery and stability. In some embodiments, the composition further comprises 0.01% to 0.1% polysorbate 80. For example, the composition may further comprise 0.05% polysorbate 80. For example, the composition may further comprise 0.01% to 0.09%, 0.01% to 0.08%, 0.01% to 0.07%, 0.01% to 0.06%, 0.01% to 0.05%, 0.01% to 0.04%, 0.02% to 0.1%, 0.02% to 0.09%, 0.02% to 0.08%, 0.02% to 0.07%, 0.02% to 0.06%, 0.02% to 0.05%, 0.02% to 0.04%, 0.03% to 0.1%, 0.03% to 0.09%, 0.03% to 0.08%, 0.03% to 0.07%, 0.03% to 0.06%, 0.03% to 0.05%, 0.03% to 0.04%, 0.04% to 0.1%, 0.04% to 0.09%, 0.04% to 0.08%, 0.04% to 0.07%, 0.04% to 0.06%, or 0.04% to 0.05% (w / v) polysorbate 80. In some embodiments, the composition may comprise 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% (w / v) polysorbate 80. In some embodiments, the composition may comprise 0.04%, 0.045%, 0.05%, 0.055%, or 0.06% (w / v) polysorbate 80. In some embodiments, the composition may comprise 0.05% (w / v) polysorbate 80.

[0029] In some embodiments, the composition has a pH value of 5 to 8. For example, the composition may have a pH value of 5 to 7 or 6 to 8. In some embodiments, the composition has a pH value of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6., 6.7, 6.8, 6.9, or 7.0. In some embodiments, the composition has a pH value of 6.5.

[0030] In some embodiments, the ophthalmic composition comprises 0.43% (w / v) MES, 5% (w / v) trehalose dihydrate, 0.37% (w / v) NaCl, 0.2% (w / v) MgCl2, 0.05% (w / v) polysorbate 80, and 0.04% (w / v) virus-like particle (VLP) - drug conjugate, wherein the VLP - drug conjugate comprises a photosensitive molecule conjugated to the capsid protein of the VLP. In some embodiments, the ophthalmic composition comprises 20 mM MES, 5% (w / v) trehalose dihydrate, 63 mM NaCl, 10 mM MgCl2, 0.05% (w / v) polysorbate 80, and 0.04% (w / v) VLP - drug conjugate, wherein the VLP - drug conjugate comprises a photosensitive molecule conjugated to the capsid protein of the VLP.

[0031] In some embodiments, the formulation is free of visible particulates. Free of visible particulates means that a normal human (e.g., having no uncorrected visual defects and not using means to magnify the sample) does not observe the presence of particulates (e.g., lumps, aggregates) in the formulation.

[0032] The compositions provided herein, in some embodiments, comprise at least one pharmaceutically acceptable excipient (e.g., a carrier, buffer, and / or salt, etc.). A molecule or other substance / agent is considered "pharmaceutically acceptable" if it is approved or approvable by a regulatory agency of the federal or state government or is included in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals (including humans). An excipient can be any inert, non - toxic agent when administered in combination with the agents provided herein. Non - limiting examples of excipients include buffers (e.g., sterile saline), salts, carriers, preservatives, fillers, surfactants, and coloring agents.

[0033] The term "pharmaceutically acceptable salt" refers to salts that are suitable for use in contact with the tissues of humans and lower animals within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, etc., and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19 (incorporated herein by reference). Pharmaceutically acceptable salts of the compounds of the present disclosure include those obtained from appropriate inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or formed by using other methods known in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include the following: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pe Salts such as succinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Suitable salts obtained from appropriate bases include alkali metals, alkaline earth metals, ammonium, and N+(C1-4 alkyl)4-salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using appropriate counterions (e.g., halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate).

[0034] In some embodiments, the ophthalmic composition is formulated as a solution. The solution may be packaged, for example, in a vial or prefilled syringe. Other packaging and delivery forms are contemplated herein.

[0035] Virus-like particle conjugate Virus-like particle (VLP) In some embodiments, the VLP is a papillomavirus VLP. The VLP can be a human papillomavirus VLP (e.g., derived from a virus that can infect humans), while in other embodiments, the VLP is a non-human papillomavirus VLP. Examples of non-human VLPs include those derived from bovine papillomavirus, murine papillomavirus, cottontail rabbit papillomavirus, and macaque or rhesus papillomavirus, but are not limited thereto. In some embodiments, the VLP is a bovine papillomavirus VLP (e.g., assembled from BPV L1 capsid protein or a combination of BPV L1 and BPV L2 capsid proteins).

[0036] The capsid protein is a protein monomer, some of which form capsomere oligomers. A capsomere is the basic oligomeric structural unit of a viral capsid, which is the outer covering of proteins that protects the genetic material of viruses such as, for example, human papillomavirus (HPV). The capsid proteins of the present disclosure include papillomavirus L1 major capsid protein and papillomavirus L2 minor capsid protein. In some embodiments, the VLP of the present disclosure contains only the L1 capsid protein, while in other embodiments, the VLP contains a mixture (or combination) of L1 and L2 capsid proteins. In some embodiments, the VLP contains human papillomavirus capsid proteins. In some embodiments, the VLP contains non-human papillomavirus capsid proteins.

[0037] In some embodiments, the percentage of the L1 capsid protein in the VLP is higher than the percentage of the L2 capsid protein in the VLP. For example, in some embodiments, the percentage of the L1 capsid protein in the VLP is 80% - 100% (of the total number of capsid proteins in the virus-like particle). In some embodiments, the percentage of the L1 capsid protein in the VLP is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the percentage of the L2 capsid protein in the VLP is 1% - 25% (of the total number of capsid proteins in the VLP). For example, in some embodiments, the percentage of the L2 capsid protein in the VLP is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0038] In certain embodiments, the VLP comprises 12 to 72 L2 proteins. In certain embodiments, the VLP comprises 360 L1 proteins and 12 to 72 L2 proteins. In some embodiments, the capsid proteins assemble into VLPs having a diameter of 20 to 60 nm. For example, the capsid proteins can assemble into VLPs having a diameter of 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, or 60 nm.

[0039] VLPs according to the present disclosure can have modified immunogenicity and / or antigenicity with respect to wild-type papillomavirus VLPs. The VLPs can be assembled, for example, from capsomers having modified capsid proteins with modified immunogenicity and / or antigenicity. A modified capsid protein having "modified immunogenicity and / or antigenicity" is one that has been modified (e.g., mutated, substituted, deleted, pegylated, or inserted) either naturally or synthetically in amino acids to reduce or prevent recognition of the capsid protein by existing (e.g., endogenous) virus serotype-specific antibodies. The modified capsid protein can be a human papillomavirus (HPV) L1 variant, a non-human papillomavirus L1 variant, or a papillomavirus L1 variant based on a combination of amino acids from different HPV serotypes. For example, an L1 variant having modified immunogenicity and / or antigenicity can be a recombinant protein based on HPV serotype 16 and HPV serotype 31 (referred to herein as "modified HPV16 / 31 L1 protein"). This is described in International Publication Nos. WO 2010 / 120266, WO 2013 / 119877, and WO 2015 / 042325, each of which is incorporated herein by reference in its entirety.

[0040] Photosensitive molecule According to various aspects of the present disclosure, the photosensitive molecule can be conjugated to the capsid protein of the VLP (e.g., L1 and / or L2 capsid proteins). In some embodiments, the photosensitive molecule is covalently conjugated to the capsid protein of the VLP. In some embodiments, the photosensitive molecule is covalently conjugated to a lysine residue of the capsid protein of the VLP. The VLP conjugated to the photosensitive molecule can be referred to herein as a "VLP-drug conjugate". In some embodiments, the photosensitive molecule comprises an NHS (N-hydroxysuccinimide) ester group that reacts with an amine group of the capsid protein (e.g., the amine group of lysine or another amino acid) to form a covalent amide bond.

[0041] The ratio of photosensitive molecules (PM) to VLP can vary. In some embodiments, the VLP:PM ratio is from about 1:10 to about 1:1000, from about 1:10 to about 1:500, from about 1:50 to about 1:500, or from about 1:50 to about 1:1000. That is, in some embodiments, a VLP can contain from about 10 to about 1000 photosensitive molecules (e.g., a mixture of the same type or different types). In some embodiments, the VLP:PM ratio is 1:10, 1:15, 1:20, 1:25, 1:50, 1:75, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950 or 1:1000.In some embodiments, the above VLP may contain 10 to 1000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 20 to 1000, 20 to 900, 20 to 800, 20 to 700, 20 to 600, 20 to 500, 20 to 400, 20 to 300, 20 to 200, 20 to 100, 30 to 1000, 30 to 900, 30 to 800, 30 to 700, 30 to 600, 30 to 500, 30 to 400, 30 to 300, 30 to 200, 30 to 100, 40 to 1000, 40 to 900, 40 to 800, 40 to 700, 40 to 600, 40 to 500, 40 to 400, 40 to 300, 40 to 200, 40 to 100, 50 to 1000, 50 to 900, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 60 to 1000, 60 to 900, 60 to 800, 60 to 700, 60 to 600, 60 to 500, 60 to 400, 60 to 300, 60 to 200, 60 to 100, 70 to 1000, 70 to 900, 70 to 800, 70 to 700, 70 to 600, 70 to 500, 70 to 400, 70 to 300, 70 to 200, 70 to 100, 80 to 1000, 80 to 900, 80 to 800, 80 to 700, 80 to 600, 80 to 500, 80 to 400, 80 to 300, 80 to 200, 80 to 100, 90 to 1000, 90 to 900, 90 to 800, 90 to 700, 90 to 600, 90 to 500, 90 to 400, 90 to 300, 90 to 200, 90 to 100, or 100 to 1000 photosensitive molecules.In some embodiments, the VLP may include 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 825, 950, 975, or 1000 photosensitive molecules. In some embodiments, the VLP may include more than 1000 (e.g., 1500, 2000, etc.) photosensitive molecules or less than 10 photosensitive molecules.

[0042] More than one photosensitive molecule may be conjugated to one capsid protein. For example, one capsid protein (e.g., L1 or L2 capsid protein) may be conjugated to 1-5 (e.g., 1, 2, 3, 4, or 5) photosensitive molecules. Thus, more than one amino acid in the capsid protein may be conjugated to the photosensitive molecule. In some embodiments, one capsid protein may be conjugated to 1-2, 1-3, or 2-3 photosensitive molecules. Thus, the photosensitive molecule may be conjugated to 1, 2, 3, 4, or 5 different amino acids (e.g., lysine, arginine and / or histidine, or other amino acids) of one capsid protein.

[0043] Examples of photosensitive molecules for use according to the present disclosure include, but are not limited to, fluorescent dyes, infrared dyes, near-infrared dyes, porphyrin molecules, and chlorophyll molecules. The VLP may, in some embodiments, include a combination of photosensitive molecules (e.g., a therapeutic photosensitizing dye molecule and a non-toxic imaging (e.g., fluorescent) dye molecule).

[0044] Examples of photosensitizing dyes for use according to the present disclosure include IRDye (登録商標)700DX, HpD, sodium porfimer (Photofrin®, Photogem®, Photosan Hemporfin®), m-THPC, temoporfin (Foscan®), verteporfin (Visudyne®), HPPH (Photochlor®), palladium-bacterio pheophorbide (Tookad®), 5-ALA, 5-aminolevulinic acid (Levulan®), 5-ALA methyl ester (Metvix®), 5-ALA benzyl ester (Benzvix®), 5-ALA hexyl ester (Hexvix®), lutetium(III)-texaphyrin or Motexafin-lutetium (Lutex®, Lutrin®, Angrin®, Optrin®), SnET2, tin(IV) ethyl etiopurpurin (Purlytin®, Photrex®), NPe6, mono-L-aspartyl chlorine e6, sodium talaporfin (Talporfin®, Laserphyrin®), BOPP, boronated protoporphyrin (BOPP®), zinc phthalocyanine (CGP55847®), silicon phthalocyanine (Pc4®), a mixture of sulfonated aluminum phthalocyanine derivatives (Photosens®), ATMPn, acetoxy-tetrakis(β-methoxyethyl-)porphycene, TH9402, and dibromorhodamine methyl ester, among others, but not limited to these.

[0045] In some embodiments, the photosensitizing dye has the chemical formula C 74 H 96 N 12 Na4O 27 S6Si3 and the dye structure:

Chemical formula

[0046] Examples of imaging dyes (e.g., fluorescent dyes) for use in accordance with the present disclosure include, but are not limited to: IRDye® 800CW, acridine orange, acridine yellow, Alexa Fluor, 7-aminoactinomycin D, 8-anilino-1-naphthalenesulfonic acid, ATTO dyes, auramine-rhodamine dyes, benzanthrone, bimane, 9,10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthacene, bisbenzimide, blacklight paint, calcein, carboxyfluorescein, carboxyfluorescein diacetate succinimidyl ester, carboxyfluorescein succinimidyl ester, 1-chloro-9,10-bis(phenylethynyl)anthracene, 2-chloro-9,10-bis(phenylethynyl)anthracene, 2-chloro-9,10-Diphenylanthracene, coumarin, DAPI, dark quencher, DiOC6, DyLight Fluor, Fluo-3, Fluo-4, FluoProbes, fluorescein, fluorescein isothiocyanate, fluorescence image-guided surgery, fluoro-jade stain, fura-2, fura-2-acetoxymethyl ester, GelGreen, GelRed, green fluorescent protein, heptamethine dye, Indian yellow, Indo-1, Lucifer yellow, luciferin, MCherry, Merocyanine, Nile blue, Nile red, optical brightener, perylene, phloxine, phycobilin, phycoerythrin, phycoerythrobilin, propidium iodide, pyranine, rhodamine, rhodamine 123, rhodamine 6G, RiboGreen, RoGFP, rubrene, (E)-stilbene, (Z)-stilbene, sulforhodamine 101, sulforhodamine B, SYBR Green I, synapto-pHluorin, tetraphenylbutadiene, tris(bathophenanthrolinedisulfonate)ruthenium(II) tetrasodium, Texas Red, Titan yellow, TSQ, umbelliferone, yellow fluorescent protein, and YOYO-1.,

[0047] In some embodiments, the photosensitizing dye has the chemical formula C 50 H 54 N3Na3O 17 S4 and the dye structure: [Chemical formula] and is IRDye (登録商標) 800CW NHS ester.

[0048] The photosensitive molecules of the present disclosure can be activated at appropriate wavelengths. In some embodiments, activation of the photosensitive molecules can render them cytotoxic or generate cytotoxic molecules. Suitable wavelengths include, but are not limited to, ultraviolet wavelengths, visible wavelengths, infrared wavelengths, and near-infrared wavelengths. In some embodiments, the photosensitive molecules are activated at wavelengths of 600 nm to 800 nm, or 660 nm to 740 nm, and become cytotoxic. In some embodiments, the photosensitive molecules are activated at wavelengths of about 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, or 800 nm and become cytotoxic. In some embodiments, the photosensitive molecules are activated at wavelengths less than 600 nm or greater than 800 nm. The appropriate wavelength for photosensitive molecule activation depends on the particular molecule being used.

[0049] Methods of production and drug conjugation To produce VLPs, the drug conjugates of the present disclosure, mammalian cells (e.g., 293T cells (e.g., HEK293F cells)) can be grown (e.g., in suspension culture) and transiently transfected with a nucleic acid encoding an HPV L1 (or L1 and L2) capsid protein (e.g., a bicistronic plasmid DNA). This induces the formation of protocapsids (e.g., as described in Buck et al., Current Protocols in Cell Biology 26.1.1-26.1.19, December 2007). After cell pellet harvest and disruption, the protocapsids can be subjected to host DNA elimination by benzonase treatment and subsequent in vitro maturation processes to form stable VLPs. After purification, the VLPs can be chemically conjugated to a photosensitive molecule (e.g., IR700 NHS ester) to produce VLP-drug conjugates.

[0050] In some embodiments, the VLP-drug conjugate is prepared as follows: Conjugation is carried out for 2 hours using IRDye® 700DX NHS Ester at a calculated molar excess of 300:1 (dye:VLP) after 1:1 dilution in 2× labeling buffer containing 100 mM HEPES pH 7.5, 20 mM MgCl2, and 10% (w / v) trehalose dihydrate. The total protein concentration is determined in some embodiments by the Bradford total protein assay (Pierce Bradford Protein Assay catalog number 23200) using a microplate procedure according to the manufacturer's instructions. The VLP-drug conjugate is then buffer-exchanged into a specific formulation in some embodiments.

[0051] Accordingly, provided herein is a method of generating a photosensitive molecule, comprising: (a) transiently transfecting a cell with a nucleic acid encoding one or more capsid proteins, thereby forming a procapsid; (b) collecting the procapsid and subjecting the procapsid to a maturation process in vitro, thereby forming a stable VLP; and (c) chemically conjugating the VLP (the capsid of the VLP) to 10 to 1000 (e.g., 10 to 500, 50 to 1000, 50 to 500, 100 to 1000, 100 to 500, 100, 200, 300, 400, or 500) photosensitive molecules. In some embodiments, the VLP is conjugated to the photosensitive molecule through an amide bond (e.g., by reacting an ester group of the photosensitive molecule with an amine group of an amino acid of the capsid protein of the virus-like nanoparticle).

[0052] Conjugation can be by any method known in the art. Non-limiting examples of methods for conjugating a photosensitive molecule to a capsid protein include reacting an N-hydroxysuccinimide ester (NHS-ester) labeled photosensitive molecule with an amine group on the capsid protein; reacting a maleimide, iodoacetyl group, or pyridyldisulfide labeled photosensitive molecule with a sulfhydryl group on the capsid protein; and reacting a primary amine labeled photosensitive molecule with a carboxyl group on the capsid protein.

[0053] In some embodiments, conjugation includes reacting an NHS-ester labeled photosensitive molecule with an amine group on the capsid protein. Available amine groups are on the amino terminus of the capsid protein or on the ε-amino group of any lysine amino acid. In some embodiments, the photosensitive molecule is conjugated to the capsid protein in a suitable buffer (e.g., PBS (pH 7.2), 0.3 M to 0.5 M NaCl). In some embodiments, the photosensitive molecule and the capsid protein are mixed together at a ratio of 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, or 1:1000. The conjugation reaction is, in some embodiments, carried out at room temperature.

[0054] The conjugated VLP and photosensitive molecule can be exposed to a first light and a second light during conjugation. In some embodiments, the first light is as described herein (e.g., having a wavelength of 470 - 610 nm and an intensity of at least 500 lux). In some embodiments, the second light is as described herein (e.g., white light having an intensity of at least 500 lux). Exposure to the first and / or second light during conjugation can ensure that the photosensitive molecule is conjugated to the VLP and to collect samples from the conjugation reaction for quality control or to check the conjugation reaction with respect to homogeneity. Homogeneity refers to a well-mixed solution in which particulate matter is not visibly present.

[0055] In some embodiments, the VLP and the photosensitive molecule are exposed to the first light for 15 minutes or less during conjugation. In some embodiments, the VLP and the photosensitive molecule are exposed to the first light for 2 to 8 minutes during conjugation. In some embodiments, the VLP and the photosensitive molecule are exposed to the first light for 3 to 10 minutes during conjugation.

[0056] In some embodiments, the VLP and the photosensitive molecule are exposed to the second light for 15 minutes or less during conjugation. In some embodiments, the VLP and the photosensitive molecule are exposed to the second light for 1 to 5 minutes during conjugation. In some embodiments, the VLP and the photosensitive molecule are exposed to the second light for 3 to 10 minutes during conjugation.

[0057] Method of treatment Any type of tumor can be targeted according to the present disclosure. Examples of tumors include, but are not limited to, tumors located in the eye, lung, pleura, liver, pancreas, stomach, esophagus, colon, chest, ovary, prostate, brain, meninges, testis, kidney, bladder, head, neck, cervix, larynx and / or skin. For example, the present application provides methods and compositions for targeting cervical cancer cells, ovarian cancer cells, melanoma cancer cells, lung cancer cells, head and / or neck cancer cells, and bladder cancer cells. Other tumors can also be targeted.

[0058] In some embodiments, the tumor is an eye tumor or lesion. The eye tumor or lesion can be located in the vitreous body, choroidal space, suprachoroidal space, iris, ciliary body, sclera, fovea, retina, optic disc, or optic nerve. Thus, in some embodiments, the subject to which the ophthalmic composition of the present disclosure is administered has an eye tumor. The eye tumor can be, for example, a uveal melanoma or a choroidal melanoma. The tumor is, in some embodiments, cancerous or malignant. In some embodiments, the tumor is metastatic. In some embodiments, the lesion is a pre-cancerous lesion or an unclassifiable lesion. In some embodiments, the subject to which the ophthalmic composition of the present disclosure is administered has an unclassifiable lesion.

[0059] In some embodiments, the subject to which the ophthalmic composition of the present disclosure is administered has a choroidal metastasis that originated somewhere in the body and spread to the eye. For example, the choroidal metastasis can originate from breast cancer or lung cancer in men.

[0060] The ophthalmic composition of the present disclosure is typically administered via intravitreal injection or suprachoroidal injection, although other routes of administration are contemplated herein.

[0061] In some embodiments, the ophthalmic composition (or any component thereof) is formulated as a solution. In some embodiments, the ophthalmic composition (or any component thereof) is lyophilized.

[0062] Further embodiments 1. An ophthalmic composition comprising a substantially isotonic solution of a VLP-drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of a virus-like particle (VLP), wherein the VLP-drug conjugate is in suspension. 2. An ophthalmic composition comprising a VLP-drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of a virus-like particle (VLP), wherein the VLP-drug conjugate does not aggregate to form visible microparticles. 3. An ophthalmic composition according to paragraph 1 or 2, having a pH value of less than 3.7. 4. An ophthalmic composition according to any one of paragraphs 1 to 3, further comprising 2-(N-morpholino)ethanesulfonic acid (MES). 5. An ophthalmic composition according to any one of paragraphs 1 to 4, further comprising at least one protective excipient and at least one detergent. 6. An ophthalmic composition according to any one of paragraphs 1 to 4, further comprising at least one reagent selected from trehalose dihydrate, magnesium chloride (MgCl2), sodium chloride (NaCl), and polysorbate 80 (PS80). 7. An ophthalmic composition according to paragraph 6, further comprising at least two reagents selected from trehalose dihydrate, MgCl2, NaCl, and PS80. 8. An ophthalmic composition according to paragraph 7, further comprising at least three reagents selected from trehalose dihydrate, MgCl2, NaCl, and PS80. 9. An ophthalmic composition according to paragraph 8, further comprising trehalose dihydrate, MgCl2, NaCl, and PS80. 10. The ophthalmic composition according to any one of paragraphs 1 to 9, wherein the composition comprises 0.1% to 1.0% (w / v) MES or a pharmaceutically acceptable salt thereof (for example, MES hemisodium salt). 11. The ophthalmic composition according to paragraph 10, wherein the composition comprises 0.4% (w / v) MES. 12. The ophthalmic composition according to any one of paragraphs 1 to 11, wherein the composition comprises 1% to 10% (w / v) trehalose dihydrate. 13. The ophthalmic composition according to paragraph 12, wherein the composition comprises 5% (w / v) trehalose dihydrate. 14. The ophthalmic composition according to any one of paragraphs 1 to 13, wherein the composition comprises 0.1% to 1.0% (w / v) NaCl. 15. The ophthalmic composition according to paragraph 14, wherein the composition comprises 0.4% (w / v) NaCl. 16. The ophthalmic composition according to any one of paragraphs 1 to 15, wherein the composition contains 0.1% to 1.0% (w / v) MgCl2. 17. The ophthalmic composition according to paragraph 16, wherein the composition contains 0.2% (w / v) MgCl2. 18. The ophthalmic composition according to any one of paragraphs 1 to 17, wherein the composition contains 0.01% to 0.1% (w / v) PS80. 19. The ophthalmic composition according to paragraph 18, wherein the composition contains 0.05% (w / v) PS80. 20. The ophthalmic composition according to any one of paragraphs 1 to 19, wherein the composition contains 0.01% to 0.5% (w / v) VLP-drug conjugate. 21. The ophthalmic composition according to paragraph 20, wherein the composition contains 0.01% to 0.1% (w / v) VLP-drug conjugate. 22. The ophthalmic composition according to paragraph 21, wherein the composition contains 0.04% VLP-drug conjugate. 23. The ophthalmic composition according to any one of paragraphs 3 to 22, wherein the composition has a pH value of 6.5. 24. An ophthalmic composition containing 0.43% (w / v) 2-(N-morpholino)ethanesulfonic acid (MES), 5% (w / v) trehalose dihydrate, 0.37% (w / v) sodium chloride, 0.2% (w / v) magnesium chloride, 0.05% (w / v) polysorbate 80, and 0.04% (w / v) virus-like particle (VLP)-drug conjugate, wherein the VLP-drug conjugate contains a photosensitive molecule conjugated to the capsid protein of VLP. 25. The ophthalmic composition according to any one of paragraphs 1 to 24, wherein the photosensitive molecule contains a dye molecule. 26. The ophthalmic composition according to paragraph 25, wherein the dye molecule contains a phthalocyanine dye molecule. 27. The ophthalmic composition according to paragraph 26, wherein the phthalocyanine dye molecule contains IRDye® 700DX. 28. The VLP is an ophthalmic composition according to any one of paragraphs 1 to 27, comprising 10 to 1000 photosensitive molecules, 10 to 500 photosensitive molecules, 50 to 1000 photosensitive molecules, 50 to 500 photosensitive molecules, 100 to 1000 photosensitive molecules, or 100 to 500 photosensitive molecules. 29. The VLP is an ophthalmic composition according to any one of paragraphs 1 to 28, comprising 50 to 500 photosensitive molecules. 30. The VLP is an ophthalmic composition according to any one of paragraphs 1 to 29, comprising a papillomavirus capsid protein. 31. The papillomavirus capsid protein is a human papillomavirus capsid protein, and the ophthalmic composition is as described in paragraph 30. 32. The papillomavirus capsid protein is an ophthalmic composition according to paragraph 31, comprising an L1 capsid protein, an L2 capsid protein, or a combination of L1 and L2 capsid proteins. 33. The L1 capsid protein is modified to reduce the immunogenicity of the VLP, and the ophthalmic composition is as described in paragraph 32. 34. A method comprising the step of administering to the eye of a subject an ophthalmic solution according to any one of paragraphs 1 to 33, wherein the subject has intraocular melanoma, and the ophthalmic solution is administered in an amount effective to treat the intraocular melanoma. 35. The intraocular melanoma is uveal melanoma or choroidal melanoma, and the method is as described in paragraph 34. 36. A method comprising the step of administering to the eye of a subject an ophthalmic solution according to any one of paragraphs 1 to 33, wherein the subject has an unclassifiable lesion, and the ophthalmic solution is administered in an amount effective to treat the unclassifiable lesion. 37. The ophthalmic composition is injected into the vitreous body, and the method is as described in any one of paragraphs 34 to 36. 37. A method comprising the step of administering to the eye of a subject an ophthalmic solution according to any one of paragraphs 1 to 33, wherein the subject has choroidal metastasis, and the ophthalmic solution is administered in an amount effective to treat the unclassifiable lesion. 38. The ophthalmic composition is injected into the vitreous body by the method according to any one of paragraphs 34 to 37. 39. The ophthalmic composition is injected into the suprachoroidal space of the eye by the method according to any one of paragraphs 34 to 37. 40. The ophthalmic composition remains in the suprachoroidal space of the eye for at least one week by the method according to paragraph 39. 41. No leukocyte infiltrates are observed in the ciliary body and / or sclera at least 35 days after injection of the ophthalmic composition by the method according to paragraph 39 or 40. 42. Optical coherence tomography is normal in the eye of the subject after injection of the ophthalmic composition by the method according to any one of paragraphs 34 to 41. 43. Intraocular pressure is normal in the eye of the subject after injection of the ophthalmic composition by the method according to any one of paragraphs 34 to 42. 44. The ophthalmic composition does not contain DNase and / or RNase, and is the ophthalmic composition according to any one of the preceding paragraphs. 45. The ophthalmic composition is sterilized, and is the ophthalmic composition according to any one of the preceding paragraphs.

[0063] All references, patents, and patent applications disclosed herein are incorporated by reference for the subject matter to which each is cited, and in some cases may include the entire document.

[0064] It should be understood that, as used herein in the specification and claims, the indefinite articles "a" and "an" mean "at least one" unless the contrary is clearly indicated.

[0065] It should also be understood that, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited, unless the contrary is clearly indicated.

[0066] In the claims, as well as in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," etc. should be understood to mean that they are unrestricted, i.e., include, but are not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are to be considered restrictive or semi-restrictive transitional phrases, respectively, as shown in Section 2111.03 of the USPTO Patent Examination Handbook.

[0067] The terms "about" and "substantially" preceding a numerical value mean ±10% of the recited numerical value. When a range of values is provided, each value between the upper and lower ends of that range is specifically contemplated and described herein.

Examples

[0068] In the initial evaluation of the VLP-drug conjugate formulation, past phosphate-based high-salt formulations and three alternative formulations were tested to evaluate the in-process recovery during the formulation of the bulk VLP-drug conjugate and the parameters of the formulated material in response to repeated freeze-thaw cycles. The data collected included protein recovery by Bradford total protein assay, A280, SDS-PAGE banding pattern, VLP morphology by transmission electron microscopy, VLP size distribution by dynamic light scattering, VLP-drug conjugate potency, ocular distribution of the VLP conjugate after suprachoroidal injection, and in vivo VLP-drug conjugate safety.

[0069] The following data unexpectedly shows that VLP drug conjugate recovery increased when the product formulation was changed to an MES-based formulation containing 5% trehalose dihydrate without evidence of visible aggregation during or after excess dye removal using tangential flow filtration (TFF).

[0070] Example 1: Process Step Recovery Bulk VLP drug conjugates were prepared by buffer exchange into each of four formulations (A, B, C, D) and concentration using tangential flow filtration (TFF). Controls included past phosphate / high salt-based formulations. Three additional trehalose-containing formulations were evaluated with combinations of buffer pH and trehalose excipient with the intent of protecting the bioconjugate. Findings collected during the TFF procedure included the formation of visible aggregates in the PBS / high salt control formulation and the phosphate-based trehalose / PS80-containing formulation A. No evidence of aggregation was noted for HEPES- or MES-based formulations containing trehalose and PS80 (formulations B and C, respectively). Process recovery was determined using the Bradford total protein assay. Two phosphate-based formulations (where evidence of aggregation was noted) yielded approximately 48 - 58% process recovery, significantly lower than the recovery in the HEPES-based formulation (69.7%) and the MES-based formulation (79.3%) (Table 1). The concentration of the final formulated samples was between 0.25 - 0.66 mg / mL. To avoid further processing steps and sample loss, it was determined to evaluate the characteristics of these samples without further processing steps.

Table 1

[0071] Example 2: Recovery after Freeze-Thaw Samples were filled into 2.0 mL CZ resin vials. Multiple vials from each formulation were at 2 - 8 oWhile storing at C, additional vials were frozen. Samples were subjected to 1, 3, or 5 freeze - thaw cycles and total protein recovery was determined using Bradford assay or UV - VIS. This data is shown in Table 2 below and graphed in Figures 1 - 2.

Table 2

[0072] Within the error of the analytical assay used (predicted to be about 20%), there was no apparent loss of protein, independent of the final formulation conditions and the number of freeze - thaw cycles. Consistent recoveries were also obtained from each sample when total protein recovery was tracked by UV - VIS at 280 nm.

[0073] Example 3: Evaluation by SDS - PAGE Samples from each formulation were analyzed by SDS-PAGE and the gels were imaged both by standard Coomassie staining and by fluorescence using an Odyssey scanner to detect the fluorescence of the DX700 dye. Freshly prepared samples and samples subjected to 1, 3, or 5 freeze-thaw cycles were analyzed. Samples were loaded by volume. Representative gel images showing Coomassie-stained gels and fluorescence scans of the same gels for samples subjected to 5 freeze-thaw cycles are shown in Figure 3. Visual evaluation of the SDS-PAGE gels reveals the characteristic complex banding pattern of the VLP-drug conjugate. A major band corresponding to the L1 protein can be observed, migrating at approximately 55 kDa. Faint bands can also be observed above the 62 kDa marker, representing the L2 protein. Above the 98 kDa marker, a species ladder indicating the presence of some form of oligomeric material is observed. The nature of this material is the subject of other technical reports, and LC-MS analysis of in-gel digested samples performed by SGS reveals that this material is a protein containing L1 and L2 sequences. Visually, the banding pattern is consistent across all samples, and it can be observed that the band intensities and fluorescence signals correspond to the Bradford and absorbance datasets.

[0074] Example 4: Viral-like particle morphology and particle size distribution by transmission electron microscopy The particle size was estimated by measuring the diameters of approximately 60 - 90 particles, and the capsomer material was assembled using AMT software. Most of the VLPs appeared to be fully assembled, were observed to have a certain size range, and had shapes mainly spherical or ellipsoidal (Figure 4). Similar ranges in apparent shape were observed in EM studies of HPV16 L1 - and HPV11 L1 - based VLPs, as well as in unfractionated preparations of rabbit papillomavirus. TEM data for samples subjected to 1, 3, and 5 freeze - thaw cycles are presented in Table 3 and graphically in Figure 5. No significant differences in the particle size of the VLP - drug conjugate were observed among the three buffer conditions - control, formulation B, and formulation C evaluated during the freeze - thaw stability study. Formulation A was not evaluated using TEM. No significant changes in the VLP - drug conjugate size range distribution or macroscopic morphological characteristics were observed through multiple freeze - thaw cycles during the study.

Table 3

[0075] Example 5: Particle Size Analysis Using Dynamic Light Scattering To complement the morphological and size distribution analysis by TEM, the average size of VLPs in each formulation was estimated by DLS assay before freezing and after 1, 3, or 5 freeze - thaw cycles. Laser - based DLS can monitor changes in the motion and structure of nanoparticles in solution (i.e., degradation or VLP - oligomerization) and provides information on the average size and frequency distribution of the particles. The DLS data are summarized in Table 4 and shown graphically in Figure 6. In both, the values of the average diameter by number (N), volume (V), and intensity (I) are shown. Each shows a snapshot of the average particle size distribution and reports the average value for the entire distribution. No obvious changes were observed in the average particle size distribution, regardless of the formulation and the number of freeze - thaw cycles. In summary, the DLS and TEM measurements of VLPs are consistent, indicating that VLPs are of comparable size and morphology, regardless of the formulation and the number of freeze - thaw cycles.

Table 4

[0076] Example 6: Efficacy Evaluation The functional properties of the VLP-drug conjugate were evaluated using an in vitro killing assay. Data generated in each of the four test formulations of freshly prepared material and material stored in the final container closure and subjected to 1, 3, or 5 freeze-thaw cycles are shown in Figures 7A - 7D. Due to limitations in personnel capabilities and the assay itself, the data points were restricted to either side of the predicted EC50. Irrespective of the formulation and the number of freeze-thaw cycles, the VLP-drug conjugate remained potent.

[0077] Example 7: Ocular Distribution of VLP-Drug Conjugate after Supraciliary Injection in New Zealand White Rabbits The in vivo ocular distribution of the VLP conjugate was evaluated by injecting AlexaFluor488*VLP into the supraciliary cavity (SCS) of New Zealand White (NZW) rabbits (Study PK-RPE-003). AlexaFluor488*VLP was used in place of the VLP-drug conjugate because it has similar physicochemical characteristics, is formulated in the same MES buffer as the VLP-drug conjugate, and is more suitable for in vivo imaging. Ocular distribution was evaluated over time by optical coherence tomography (OCT) and fundus autofluorescence (FAF). From the data, it was shown that the distribution after a 100 μl injection into the SCS was approximately 75% in the posterior eye at ≤ 0.5 hours post-administration and remained relatively constant over the duration of the study. Fluorescence was strong over the interval of 168 hours post-administration and began to weaken at the 240-hour interval post-administration. No significant fluorescence above baseline could be detected at the 504-hour interval post-administration. This data suggests that the VLP-drug conjugate formulated in MES buffer is well-distributed in the SCS cavity and its duration is at least 168 hours (1 week).

[0078] Example 8: In Vivo Safety Evaluation (Non-Clinical) of VLP-Drug Conjugates To evaluate the non-clinical safety of the VLP-drug conjugate, the VLP-drug conjugate was administered in a canine study. The VLP-drug conjugate was administered at a single dose level via a 100 μl injection into the suprachoroidal space (SCS) at a dose of 20 μg / eye, followed by laser photoactivation 6 - 8 hours after injection into the dogs. The dogs were treated once a week for 3 weeks (a total of 3 injections, followed by laser treatment at 50 J / cm 2 at 6 - 8 hours after injection each week). After the third once-a-week treatment, the animals were observed for 7 days (eventually sacrificed) or 35 days (recovery phase) to evaluate reversibility, persistence, or delay in onset of effects. There were minor VLP-drug conjugate-related microscopic eye findings. Histopathology showed minimal / slight leukocyte infiltration into the choroidal space, and 50% of the animals had minimal leukocyte infiltration into the ciliary body and sclera. The minimal / slight leukocyte infiltration dissipated between the first observation time point (animals sacrificed at 7 days) and the second observation time point (animals in the recovery phase at 35 days) when it was not observed. Optical coherence tomography (OCT) was normal in all animals (i.e., no retinal pigment epithelium (RPE) / retinal changes or retinal thinning were detected), and all tested eyes retained normal retinal structure. Intraocular pressure (IOP) of the subject animals was evaluated during the study and was normal for all animals. No systemic clinical findings, weight changes, or clinicopathological findings were reported. In summary, the data suggest that the VLP-drug conjugate formulated in MES buffer delivered as multiple SCS injections is safe in vivo.

[0079] Materials and Methods Samples The VLP-drug conjugate was formulated using four different formulations as outlined in Table 5.

Table 5

[0080] Equipment The devices necessary for performing the formulation process described in this specification are provided in Table 6.

Table 6

[0081] Reagents and Solvents Alcian Blue, 1.5% uranyl acetate, RO-DI water, carbon film 300 mesh copper grid, precision tweezers, pipette, Petri dish, paraffin, filter paper.

[0082] VLP Drug Conjugate Production and Formulation The VLP drug conjugate was prepared from the bulk VLP preparation. Bioconjugation was performed with DX700-NHS Ester for 2 hours at a calculated molar excess of 300:1 (dye:VLP) after dilution 1:1 with 2× labeling buffer containing 100 mM HEPES (pH 7.5), 20 mM MgCl2, and 10% trehalose dihydrate. The total protein concentration was determined according to the manufacturer's instructions using the Bradford total protein assay (Pierce Bradford protein assay Catalog number 23200) using the micro-microplate procedure. The stock VLP drug conjugate was then buffer-exchanged into a control formulation (phosphate / high salt) and three candidate formulations (each containing trehalose and PS80 at three different pHs (A, B, and C)). The overall flow of the experiment is shown in Figure 8.

[0083] Product Strength The product strength of the VLP drug conjugate was determined according to the manufacturer's instructions regarding the microplate format using the Bradford total protein assay (Pierce Bradford protein assay Catalog number 23200). A280nm was performed on the undiluted sample using a NanoDrop UV-VIS spectrophotometer blanked with the buffer matched to the sample.

[0084] SDS-PAGE Analysis SDS-PAGE analysis of the VLP-drug conjugate product was performed using a TGX criterion, Any kD gel (Bio-Rad #5671125). Standard gel images were captured using an imaging camera, and fluorescence images were captured by scanning the gel with an Odyssey CLx. Fluorescence analysis was performed using the Imagestudio software of the Odyssey CLx.

[0085] Dynamic light scattering Fifty microliters of each test sample was added to 150 μL of filtered PBS (phosphate buffered saline, Boston Bioproducts catalog number BM220-S) and gently mixed by pipetting up and down five times. The diluted sample was then pipetted into a plastic cuvette (Fisher catalog number 14-955-125), and the cuvette was placed in a DLS sample holder. The sample was analyzed using 90 Plus particle sizing software (Brookhaven Instruments). Briefly, the diluted sample was run three times at a detector angle of 90° at 25 °C for 2 minutes per run. Four separate distributions were recorded: number, volume, intensity, and surface area. The polydispersity index for each sample run was also recorded to determine the homogeneity of the sample run.

[0086] Transmission electron microscopy Aliquots of VLP-drug conjugate samples prepared in different buffers were provided either unfrozen (2 - 8°C) or frozen and stored at -80°C once, three times, or five times before being thawed and then analyzed at the Electron Microscopy Center of Northeastern University. The samples were analyzed by TEM as follows: A carbon-coated EM grid (300 mesh copper grid with carbon film) was placed on top of a droplet of alcian blue dye (placed on top of a parafilm section) with the carbon side up for 30 seconds. Excess dye was removed by gently blotting with filter paper. The EM grid was washed with sterile deionized water by gently touching its grid surface to the surface of a water droplet (placed on top of a parafilm section) for 1 - 2 seconds. This washing was repeated two more times, using a fresh water droplet for each wash. During washing, the grid was placed on top of a 10 μl droplet of the sample suspension for approximately 1 minute. The loaded grid was gently blotted to remove excess sample and then washed three times with deionized water using the same technique as described in step 2. The loaded grid was then stained by gently touching its surface to the surface of a droplet of 1.5% uranyl acetate dye for 1 - 2 seconds and blotted with filter paper. This staining step was repeated three more times with the same droplet of uranyl acetate. For TEM imaging, a Jeol 1010 AMT camera (located at the Electron Microscopy Center at Northeastern University (Boston)) was used. The prepared grid was placed into the TEM after drying for several minutes. Images were captured at magnifications of ×15000, ×25000, ×30000, ×40000, ×50000, and ×200000. The diameter of the particles and the capsomers assembled within the region of interest were measured at a magnification of ×40000 using AMT software.

[0087] Potency The in vitro potency of the VLP-drug conjugate was determined by performing a cell death assay (Aura-SOP-008) against the tumor cell line OCM-1. Due to sample and capacity limitations, only three product levels were evaluated near the predicted EC50 of the product. Tumor cells were incubated with the VLP-drug conjugate at various concentrations on ice for 1.5 hours. After this incubation, the cells were washed to remove unbound VLP-drug conjugate and then resuspended in sample diluent. Half of the cells to which the VLP-drug conjugate had bound for each VLP-drug conjugate dilution were dispensed into three wells of a 96-well plate (1 / 2 well, black, clear bottom) and irradiated with light at a wavelength of 690 nm at 25 J / cm2. The non-irradiated cells were used as controls. In certain embodiments, for example, the following items are provided. (Item 1) An ophthalmic composition comprising an almost isotonic solution of a VLP-drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of a virus-like particle (VLP), wherein the VLP-drug conjugate is in suspension, the ophthalmic composition. (Item 2) An ophthalmic composition comprising a VLP-drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of a virus-like particle (VLP), wherein the VLP-drug conjugate does not aggregate to form visible microparticles, the ophthalmic composition. (Item 3) The ophthalmic composition according to item 1 or 2, having a pH value of less than 7. (Item 4) The ophthalmic composition according to any one of items 1 to 3, further comprising 2-(N-morpholino)ethanesulfonic acid (MES). (Item 5) The ophthalmic composition according to any one of items 1 to 4, further comprising at least one protective excipient and at least one detergent. (Item 6) The ophthalmic composition according to any one of items 1 to 4, further comprising at least one reagent selected from trehalose dihydrate, magnesium chloride (MgCl2), sodium chloride (NaCl), and polysorbate 80 (PS80). (Item 7) The ophthalmic composition according to item 6, further comprising at least two reagents selected from trehalose dihydrate, MgCl2, NaCl, and PS80. (Item 8) The ophthalmic composition according to item 7, further comprising at least three reagents selected from trehalose dihydrate, MgCl2, NaCl, and PS80. (Item 9) The ophthalmic composition according to item 8, further comprising trehalose dihydrate, MgCl2, NaCl, and PS80. (Item 10) The ophthalmic composition according to any one of items 1 to 9, wherein the composition contains 0.1% to 1.0% (w / v) MES. (Item 11) The ophthalmic composition according to item 10, wherein the composition contains 0.4% (w / v) MES. (Item 12) The ophthalmic composition according to any one of items 1 to 11, wherein the composition contains 1% to 10% (w / v) trehalose dihydrate. (Item 13) The ophthalmic composition according to item 12, wherein the composition contains 5% (w / v) trehalose dihydrate. (Item 14) The ophthalmic composition according to any one of items 1 to 13, wherein the composition contains 0.1% to 1.0% (w / v) NaCl. (Item 15) The ophthalmic composition according to item 14, wherein the composition contains 0.4% (w / v) NaCl. (Item 16) The ophthalmic composition according to any one of items 1 to 15, wherein the composition contains 0.1% to 1.0% (w / v) MgCl2. (Item 17) The composition is the ophthalmic composition according to item 16, containing 0.2% (w / v) MgCl2. (Item 18) The composition is the ophthalmic composition according to any one of items 1 to 17, containing 0.01% to 0.1% (w / v) PS80. (Item 19) The composition is the ophthalmic composition according to item 18, containing 0.05% (w / v) PS80. (Item 20) The composition is the ophthalmic composition according to any one of items 1 to 19, containing 0.01% to 0.5% (w / v) VLP-drug conjugate. (Item 21) The composition is the ophthalmic composition according to item 20, containing 0.01% to 0.1% (w / v) VLP-drug conjugate. (Item 22) The composition is the ophthalmic composition according to item 21, containing 0.04% (w / v) VLP-drug conjugate. (Item 23) The composition is the ophthalmic composition according to any one of items 3 to 22, having a pH value of 6.5. (Item 24) An ophthalmic composition containing 0.43% (w / v) 2-(N-morpholino)ethanesulfonic acid (MES), 5% (w / v) trehalose dihydrate, 0.37% (w / v) sodium chloride, 0.2% (w / v) magnesium chloride, 0.05% (w / v) polysorbate 80, and 0.04% (w / v) virus-like particle (VLP)-drug conjugate, wherein the VLP-drug conjugate contains a photosensitive molecule conjugated to the capsid protein of VLP The ophthalmic composition containing the same. (Item 25) The photosensitive molecule is contained in the ophthalmic composition according to any one of items 1 to 24. (Item 26) The pigment molecule is contained in the ophthalmic composition according to item 25. (Item 27) The phthalocyanine pigment molecule is contained in the ophthalmic composition according to item 26, including IRDye® 700DX. (Item 28) The VLP is the ophthalmic composition according to any one of items 1 to 27, comprising 10 to 1000 photosensitive molecules, 10 to 500 photosensitive molecules, 50 to 1000 photosensitive molecules, 50 to 500 photosensitive molecules, 100 to 1000 photosensitive molecules, or 100 to 500 photosensitive molecules. (Item 29) The VLP is the ophthalmic composition according to any one of items 1 to 28, comprising 50 to 500 photosensitive molecules. (Item 30) The VLP is the ophthalmic composition according to any one of items 1 to 29, comprising a papillomavirus capsid protein. (Item 31) The papillomavirus capsid protein is a human papillomavirus capsid protein, and the ophthalmic composition is as described in item 30. (Item 32) The papillomavirus capsid protein is the ophthalmic composition according to item 31, comprising an L1 capsid protein, an L2 capsid protein, or a combination of L1 and L2 capsid proteins. (Item 33) The L1 capsid protein is the ophthalmic composition according to item 32, which is modified to reduce the immunogenicity of the VLP. (Item 34) A method comprising the step of administering the ophthalmic solution according to any one of items 1 to 33 to the eye of a subject, wherein the subject has intraocular melanoma, and the ophthalmic solution is administered in an amount effective to treat the intraocular melanoma. (Item 35) The intraocular melanoma is uveal melanoma or choroidal melanoma, and the method is as described in item 34. (Item 36) A method comprising the step of administering the ophthalmic solution according to any one of items 1 to 33 to the eye of a subject, wherein the subject has an unclassifiable lesion, and the ophthalmic solution is administered in an amount effective to treat the unclassifiable lesion. (Item 37) A method comprising the step of administering to the eye of a subject an ophthalmic solution according to any one of items 1 to 33, wherein the subject has choroidal metastasis and the ophthalmic solution is administered in an amount effective to treat the unclassifiable lesion. (Item 38) The method according to any one of items 34 to 37, wherein the ophthalmic composition is injected into the vitreous body. (Item 39) The method according to any one of items 34 to 37, wherein the ophthalmic composition is injected into the suprachoroidal cavity of the eye. (Item 40) The method according to item 39, wherein the ophthalmic composition remains in the suprachoroidal cavity of the eye for at least one week. (Item 41) The method according to item 39 or 40, wherein no leukocyte infiltrate is observed in the ciliary body and / or sclera at least 35 days after injection of the ophthalmic composition. (Item 42) The method according to any one of items 34 to 41, wherein optical coherence tomography is normal in the eye of the subject after injection of the ophthalmic composition. (Item 43) The method according to any one of items 34 to 42, wherein the intraocular pressure is normal in the eye of the subject after injection of the ophthalmic composition.

Claims

[Claim 1] The invention described in the specification.

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