Human papillomavirus nanoparticle preparation

Abstract

In some aspects, the present disclosure provides virus-like particle drug conjugate formulations and uses of the conjugates for treating ocular tumors or lesions.

Description

[0001] Related Applications

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 824,227, filed on March 26, 2019, which is incorporated herein by reference in its entirety. Background Art

[0003] Fundamental issues for biotherapeutic formulations, including ophthalmic formulations, are stability and structural integrity of the active molecule during shipping and storage, after multiple freeze-thaw cycles, successful delivery of the drug to its site of action, and speed and cost effectiveness of development and final product. Many details come into play, including analytical methods and testing protocols, containers and closures, delivery devices and dosage forms, excipients and stabilizers, and compatibility of ingredients. SUMMARY OF THE INVENTION

[0005] In some aspects, the present disclosure provides an ophthalmic composition comprising a VLP drug conjugate containing a photosensitive molecule conjugated to a capsid protein of a virus-like particle (VLP) and 2-(N-morpholino)ethanesulfonic acid (MES) or a pharmaceutically acceptable salt thereof. Surprisingly, in some embodiments, without having a low salt concentration and / or having a pH suitable for ophthalmic administration (e.g., less than 7, e.g., 6.5), relative to other tested formulations, a specific MES-based formulation provided herein eliminates visible VLP drug conjugate aggregation and precipitation.

[0006] Therefore, in some aspects, an ophthalmic composition is provided herein, comprising a near isotonic solution (255 to 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 a suspension. The isotonicity of the isotonic solution used herein is about 290 mOsm / L, and the isotonicity of the near isotonic solution is 255 to 345 mOsm / L.

[0007] In some aspects, an 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 particles.

[0008] In some embodiments, the ophthalmic composition further comprises at least one protective agent and at least one surfactant. In some embodiments, the ophthalmic composition further comprises at least one, at least two, or at least three agents selected from the group consisting of 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.

[0009] In some embodiments, the composition comprises 0.01% to 0.1% (w / v) VLP drug conjugate. For example, the composition may comprise 0.04% (w / v) VLP drug conjugate.

[0010] In some embodiments, the composition comprises 0.1% to 1.0% (w / v) MES or a pharmaceutically acceptable salt thereof (e.g., MES hemi-sodium salt). For example, the composition may comprise 0.4% (w / v) MES or a pharmaceutically acceptable salt thereof. In some embodiments, the composition comprises 5 mM to 50 mM MES or a pharmaceutically acceptable salt thereof. For example, the composition may comprise 20 mM MES or a pharmaceutically acceptable salt thereof.

[0011] 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.

[0012] 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 20mM to 100mM NaCl. For example, the composition may further comprise 63mM NaCl.

[0013] 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 5mM to 25mM MgCl2. For example, the composition may further comprise 10mM MgCl2.

[0014] 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.

[0015] In some embodiments, the pH of the composition is between 5 and 8. For example, the pH of the composition can be 6.5.

[0016] 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 20mM MES, 5% (w / v) trehalose dihydrate, 63mM NaCl, 10mM 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.

[0017] 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 700DX molecules, 800CW molecule, or 700DX and Mixture of 800CW molecules. Additional therapeutic and diagnostic photosensitive molecules are contemplated herein.

[0018] In some embodiments, the VLP comprises 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. In some embodiments, the VLP comprises 300 photosensitive molecules.

[0019] In some embodiments, the VLP comprises a papillomavirus capsid protein (e.g., a human papillomavirus capsid protein). For example, the papillomavirus capsid protein may comprise 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.

[0020] In some aspects, also provided herein are methods comprising administering an ophthalmic composition of the present disclosure to an eye of a subject, wherein the subject suffers from an eye cancer (e.g., an ophthalmic melanoma), and wherein the ophthalmic solution is administered in an amount effective to treat the ophthalmic melanoma. In some embodiments, the ophthalmic composition is administered by intravitreal injection. In some embodiments, the subject suffers from uveal melanoma. In some embodiments, the subject suffers from choroidal melanoma.

[0021] In some aspects, also provided herein are methods comprising administering an ophthalmic composition of the present disclosure to an eye of a subject, wherein the subject has an indeterminate lesion, and wherein the ophthalmic solution is administered in an amount effective to treat the indeterminate lesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The protein recovery of each formulation as measured by Bradford total protein assay is shown. The control formulation contains 20mM potassium phosphate, 500mM NaCl, pH 7.0. Formulation A contains 20mM potassium phosphate, 5% (w / v) trehalose dihydrate, 10mM MgCl2, 63mM NaCl, 0.05% (w / v) PS80, pH 7.0. Formulation B contains 50mM HEPES, 5% (w / v) trehalose dihydrate, 10mM MgCl2, 47mM NaCl, 0.05% (w / v) PS80, pH 7.5. Formulation C contains 20mM MES, 5% (w / v) trehalose dihydrate, 10mM MgCl2, 63mM NaCl, 0.05% (w / v) PS80, pH 6.5. The freeze-thaw (FT) number is the number of times a sample is frozen and thawed.

[0024] Figure 2 The protein recovery of each formulation as measured by UV-Vis is shown. Figure 1 The UV-Vis spectra were measured at 280 nm. The freeze-thaw (FT) number is the number of times a sample was frozen and thawed.

[0025] Figure 3 A representative SDS-PAGE gel of a protein preparation after 5 freeze-thaw cycles is shown. Figure 1 The band at about 55 kDa is the L1 protein in virus-like particles (VLPs).

[0026] Figure 4 Representative transmission electron micrograph (TEM) images of virus-like particle drug conjugates (VLP drug conjugates) are shown. Figure 1 as shown in .

[0027] Figure 5 Particle size distribution of VLP drug conjugates as measured by transmission electron microscopy (TEM) is shown. Figure 1 The freeze-thaw (FT) number is the number of times a sample is frozen and thawed.

[0028] Figure 6 The average particle size of the VLP drug conjugates as measured by dynamic light scattering (DLS) is shown. Figure 1 The average particle size is measured by diameter. The freeze-thaw (FT) number is the number of times a sample is frozen and thawed. N is the average diameter by number, V is the average diameter by volume, and I is the average diameter by intensity.

[0029] Figures 7A to 7D In vitro EC 50 Killing curve. Figure 1 as shown in . Fig. 7A Shown are the percentage (%) of dead cells using unfrozen (OFT) VLP drug conjugates. Figure 7B Shown are the % dead cells using VLP drug conjugates that had been frozen once (1 FT). Figure 7C Shown is the % dead cells using VLP drug conjugates that had been frozen three times. Fig.7D Shown is the % dead cells using VLP drug conjugates that had been frozen 5 times.

[0030] Figure 8 A conjugation-formulation workflow diagram is shown. Figure 1 as shown in . DETAILED DESCRIPTION OF THE INVENTION

[0032] In some aspects, the present disclosure provides an ophthalmic composition comprising a VLP drug conjugate containing 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 has a chemical formula of C6H 13NO4S. In some embodiments, the composition comprises 0.1% to 1.0% (w / v) MES or a pharmaceutically acceptable salt thereof (e.g., MES hemi-sodium salt). 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) MES. In some embodiments, the composition may comprise 0.1%, 0.2%, 0.3%, 04%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1% (w / v) MES. In some embodiments, the composition may comprise 0.41%, 0.42%, 0.43%, 0.44% or 0.45% (w / v) MES. In some embodiments, the composition may include 0.43% (w / v) MES. In some embodiments, the composition includes 5mM to 50mM MES. For example, the composition may include 5mM to 45mM, 5mM to 40mM, 5mM to 35mM, 5mM to 30mM, 5mM to 25mM, 5mM to 20mM, 10mM to 50mM, 10mM to 45mM, 10mM to 40mM, 10mM to 35mM, 10mM to 30mM, 10mM to 25mM, 10mM to 20mM, 20mM to 50mM, 20mM to 45mM, 20mM to 40mM, 20mM to 35mM, 20mM to 30mM, or 20mM to 25mM MES. In some embodiments, the composition comprises 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM or 50mM MES. In some embodiments, the composition comprises 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM or 25mM MES. In some embodiments, the composition comprises 20mM MES.

[0033] In some embodiments, the composition comprises 0.01% to 0.1% (w / v) VLP drug conjugate. For example, the composition may 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 ... 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) 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 comprise 0.03%, 0.035%, 0.04%, 0.045%, or 0.05% (w / v) VLP drug conjugate. In some embodiments, the composition may comprise 0.04% (w / v) VLP drug conjugate.

[0034] In some embodiments, the composition comprises 0.01% to 0.2% (w / v) VLP drug conjugate. In some embodiments, the composition comprises 0.01% to 0.3% (w / v) VLP drug conjugate. In some embodiments, the composition comprises 0.01% to 0.4% (w / v) VLP drug conjugate. In some embodiments, the composition comprises 0.01% to 0.5% (w / v) VLP drug conjugate. In some embodiments, the composition comprises 0.1%, 0.2%, 0.3%, 0.4% or 0.5% (w / v) VLP drug conjugate.

[0035] In some embodiments, the ophthalmic composition further comprises at least one, at least two or at least three agents selected from the following: 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 dihydrate 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.

[0036] Trehalose is a disaccharide formed by a 1,1-glycosidic bond between two α-glucose units. Trehalose forms rhombus crystals as a dihydrate, and the anhydrous form of trehalose easily regains water to form a dihydrate. As shown herein, the excipient has a significant positive effect on the recovery and stability of the VLP drug conjugate in a MES buffer containing NaCl and MgCl2. In some embodiments, the composition also 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 further comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% trehalose (w / v) 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.

[0037] In some embodiments, the composition further comprises a salt, such as sodium chloride (NaCl), for isotonicity. In some embodiments, the composition further comprises 0.1% to 1.0% NaCl. 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) NaCl. In some embodiments, the composition may include 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 also includes 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 to 100 mM NaCl. For example, the composition can further comprise 20mM to 90mM, 20mM to 80mM, 20mM to 70mM, 20mM to 60mM, 20mM to 50mM, 30mM to 100mM, 30mM to 90mM, 30mM to 80mM, 30mM to 70mM, 30mM to 60mM, 30mM to 50mM, 40mM to 100mM, 40mM to 90mM, 40mM to 80mM, 40mM to 70mM, 40mM to 60mM, 40mM to 50mM, 50mM to 100mM, 50mM to 90mM, 50mM to 80mM, 50mM to 70mM, 50mM to 60mM, or 60mM to 70mM 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.

[0038] 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 include 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 includes 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 5mM to 25mM MgCl2. For example, the composition may comprise 5mM to 20mM, 5mM to 15mM, 5mM to 10mM, 10mM to 25mM, 10mM to 20mM or 10mM to 15mM MgCl2. In some embodiments, the composition further comprises 5mM, 10mM, 15mM, 20mM or 25mM MgCl2. In some embodiments, the composition further comprises 8mM, 9mM, 10mM, 11mM or 12mM MgCl2. In some embodiments, the composition further comprises 10mM MgCl2.

[0039] In some embodiments, the composition further comprises a surfactant, for example to improve 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 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 include 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.

[0040] In some embodiments, the pH of the composition is 5 to 8. For example, the pH of the composition can be 5 to 7 or 6 to 8. In some embodiments, the pH of the composition is 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 pH of the composition is 6.5.

[0041] In some aspects, 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 aspects, the ophthalmic composition comprises 20mM MES, 5% (w / v) trehalose dihydrate, 63mM NaCl, 10mM 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.

[0042] In some embodiments, the formulation is substantially free of particles. Substantially free of particles means that a normal person (e.g., not having uncorrected visual impairment, not using means of magnifying the sample) would not perceive the presence of particles (e.g., clumps, aggregates) in the formulation.

[0043] In some embodiments, the compositions provided herein include at least one pharmaceutically acceptable excipient (e.g., carrier, buffer, and / or salt, etc.). A molecule or other substance / agent is considered "pharmaceutically acceptable" if it is approved or approved by a regulatory agency of the federal government or state government, or is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for animals (including humans). An excipient can be any inert (inactive), non-toxic agent administered in combination with an agent provided herein. Some non-limiting examples of excipients include buffers (e.g., sterile saline), salts, carriers, preservatives, fillers, surfactants, and colorants.

[0044] The term "pharmaceutically acceptable salt" refers to salts that are suitable for contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc. and commensurate with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. 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, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from suitable inorganic acids and bases and organic acids and bases. Some 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 formed 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 such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, 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, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N+(C1-4 alkyl)4-salts. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Additional pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counter ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates when appropriate.

[0045] In some embodiments, the ophthalmic composition is formulated as a solution. For example, the solution can be packaged in a vial or a pre-filled syringe. Other packaging and delivery forms are contemplated herein.

[0046] Virus-like particle conjugates

[0047] Virus-like particles (VLPs)

[0048] In some embodiments, the VLP is a papillomavirus VLP. The VLP may 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. Some examples of non-human VLPs include, but are not limited to, those derived from bovine papillomavirus, mouse papillomavirus, cotton rabbit papillomavirus, and macaque or rhesus monkey papillomavirus. In some embodiments, the VLP is a bovine papillomavirus VLP (e.g., assembled from a combination of BPV L1 capsid protein or BPVL1 and BPV L2 capsid protein).

[0049] Capsid protein is a protein monomer, and several of the monomers form capsomere oligomers. Capsomere is the basic oligomeric structural unit of the viral capsid, which is a protein shell that protects the genetic material of the virus (e.g., human papillomavirus (HPV)). The capsid protein of the present disclosure includes the major capsid protein of papillomavirus L1 and the minor capsid protein of papillomavirus L2. In some embodiments, the VLP of the present disclosure contains only 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 protein. In some embodiments, the VLP contains non-human papillomavirus capsid protein.

[0050] In some embodiments, the percentage of L1 capsid protein in VLP is greater than the percentage of L2 capsid protein in VLP. For example, in some embodiments, the percentage of L1 capsid protein in VLP is 80% to 100% (of the total number of capsid proteins in virus-like particles). In some embodiments, the percentage of L1 capsid protein in VLP is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the percentage of L2 capsid protein in VLP is 1% to 25% (of the total number of capsid proteins in VLP). For example, in some embodiments, the percentage of L2 capsid protein in VLP is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0051] In some embodiments, the VLP contains 12 to 72 L2 proteins. In some embodiments, the VLP contains 360 L1 proteins and 12 to 72 L2 proteins. In some embodiments, the capsid proteins are assembled into VLPs with a diameter of 20 to 60 nm. For example, the capsid proteins can be assembled into VLPs with a diameter of 20, 25, 30, 35, 40, 45, 50, 55 or 60 nm.

[0052] VLPs according to the present disclosure may have modified immunogenicity and / or antigenicity relative to wild-type papillomavirus VLPs. VLPs may, for example, be assembled from capsomers having variant capsid proteins with modified immunogenicity and / or antigenicity. Variant capsid proteins with "modified immunogenicity and / or antigenicity" are variant capsid proteins that are naturally or synthetically modified (e.g., mutated, replaced, deleted, pegylated, or inserted) on amino acids to reduce or prevent recognition of capsid proteins by pre-existing (e.g., endogenous) viral serotype-specific antibodies. Variant capsid proteins may be human papillomavirus (HPV) L1 variants, non-human papillomavirus L1 variants, or papillomavirus L1 variants based on amino acid combinations from different HPV serotypes. For example, an L1 variant with modified immunogenicity and / or antigenicity can be a recombinant protein based on HPV serotype 16 and HPV serotype 31 (referred to herein as a "variant HPV16 / 31 L1 protein"), which is described in International Publication Nos. WO 2010 / 120266, WO 2013 / 119877, and WO 2015 / 042325, the entire contents of each of which are incorporated herein by reference.

[0053] Photosensitive molecules

[0054] According to various aspects of the present disclosure, the photosensitive molecule can be conjugated to the capsid protein (e.g., L1 and / or L2 capsid protein) of the VLP. 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 the lysine residue of the capsid protein of the VLP. The VLP conjugated with the photosensitive molecule may be referred to as a "VLP drug conjugate" herein. In some embodiments, the photosensitive molecule comprises an NHS (N-hydroxysuccinimide) ester group, which reacts with an amine group (e.g., an amine group of lysine or other amino acids) of the capsid protein to form a covalent amide bond.

[0055] The ratio of photosensitive molecules (PM) to VLPs can vary. In some embodiments, the ratio of VLP:PM is about 1:10 to about 1:1000, about 1:10 to about 1:500, about 1:50 to about 1:500, or about 1:50 to about 1:1000. That is, in some embodiments, the VLPs may contain about 10 to about 1000 photosensitive molecules (e.g., photosensitive molecules of the same type or a mixture of different types). In some embodiments, the ratio of VLP:PM 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 VLPs may comprise 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, 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 0 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 60 0, 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, 100 to 1000, 100 to 900, 100 to 800, 100 to 700, 100 to 600, 100 to 500, 100 to 400, 100 to 300, or 100 photosensitive molecules. In some embodiments, the VLP may comprise 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 light-sensitive molecules. In some embodiments, the VLP may comprise more than 1000 (e.g., 1500, 2000, etc.) light-sensitive molecules or less than 10 light-sensitive molecules.

[0056] More than one photosensitive molecule can be conjugated to a single capsid protein. For example, a single capsid protein (e.g., an L1 or L2 capsid protein) can be conjugated to 1 to 5 (e.g., 1, 2, 3, 4, or 5) photosensitive molecules. Thus, more than one amino acid of a capsid protein can be conjugated to a photosensitive molecule. In some embodiments, a single capsid protein can be conjugated to 1 to 2, 1 to 3, or 2 to 3 photosensitive molecules. Thus, a photosensitive molecule can be conjugated to 1, 2, 3, 4, or 5 different amino acids (e.g., lysine, arginine, and / or histidine or other amino acids) of a single capsid protein.

[0057] Some examples of photosensitive molecules used according to the present disclosure include, but are not limited to, fluorescent dyes, infrared dyes, near-infrared dyes, porphyrin molecules, and chlorophyll molecules. In some embodiments, VLPs include a combination of photosensitive molecules, such as therapeutic photosensitivity dye molecules and non-toxic imaging (e.g., fluorescent) dye molecules.

[0058] Some examples of photosensitizing dyes for use according to the present disclosure include, but are not limited to: 700DX, HpD, Porfimer sodium m-THPC, Temoporfin Verteporfin Palladium-bacterial-pheophorbide 5-ALA, 5-aminolevulinic acid 5-ALA methyl ester 5-ALA Benzyl Ester 5-ALA Hexyl Ester Lutetium(III)-texaphyrin or Motexafin-lutetium SnET2, Tin(IV)ethyletiopurpurin NPe6, mono-L-aspartate chloroe6, talaporfin sodium BOPP, Boronated Protoporphyrin Zinc Phthalocyanine Silicon Phthalocyanine Mixture of sulfonated aluminum phthalocyanine derivatives ATMPn, acetoxy-tetrakis(β-methoxyethyl-)porphin), TH9402 and dibromorhodamine methyl ester.

[0059] In some embodiments, the photosensitizing dye is a dye having the chemical formula C 74 H 96 N 12 Na4O 27S6Si3 and the following dye structures 700DX NHS Ester:

[0060]

[0061] Some examples of imaging dyes (e.g., fluorescent dyes) for use in accordance with the present disclosure include, but are not limited to: 800CW, acridine orange, acridine yellow, Alexa Fluor, 7-aminoactinomycin D, 8-anilinonaphthalene A-1-sulfonic acid, ATTO dye, auramine-rhodamine stain, benzanthrone, bimane, 9,10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)tetracene, bisbenzimide, black light 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-bisphenylanthracene, coumarin, DAPI, dark quencher, DiOC6, DyLight Fluor, Fluo-3, Fluo-4, FluoProbes, fluorescein, fluorescein isothiocyanate, fluorescence image-guided surgery, fluoro-jade staining, fura-2, fura-2-acetoxymethyl ester, GelGreen, GelRed, green fluorescent protein, heptamethine dye, Indian Yellow, Indo-1, firefly yellow, luciferin, MCherry, merocyanine, Nile Blue, Nile Red, fluorescent brightener, perylene, phloroglucin dye, 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, tetrasodium tris(bathophenanthroline disulfonate)ruthenium(II), Texas Red, Dadan Yellow, TSQ, umbelliferone, yellow fluorescent protein, and YOYO-1.

[0062] In some embodiments, the photosensitizing dye is a dye having the chemical formula C 50 H54 N3Na3O 17 S4 and the following dye structures 800CW NHS Ester:

[0063]

[0064] The photosensitive molecules of the present disclosure can be activated at a suitable wavelength. In some embodiments, the activation of the photosensitive molecules makes it cytotoxic or can produce cytotoxic molecules. Suitable wavelengths include but are not limited to ultraviolet wavelengths, visible light wavelengths, infrared wavelengths and near-infrared wavelengths. In some embodiments, the photosensitive molecules are activated and become cytotoxic at a wavelength of 600nm to 800nm ​​or 660nm to 740nm. In some embodiments, the photosensitive molecules are activated and become cytotoxic at a wavelength of about 600nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, 770nm, 780nm, 790nm or 800nm. In some embodiments, the photosensitive molecules are activated at a wavelength less than 600nm or greater than 800nm. The appropriate wavelength for activation of the photosensitive molecule will depend on the specific molecule used.

[0065] Production methods and drug conjugation

[0066] In order to produce the VLP drug conjugates of the present disclosure, mammalian cells, such as 293T cells (e.g., HEK293F cells) can be cultured (e.g., in suspension culture) and transiently transfected with nucleic acids (e.g., bicistronic plasmid DNA) encoding HPV L1 (or L1 and L2) capsid proteins. This induces protocapsid formation (e.g., as described in Bucket.al.Current Protocols in Cell Biology 26.1.1-26.1.19, December 2007). After cell mass (cell mass) is recovered and destroyed, protocapsid can be treated with benzonase to remove host DNA, and then in vitro maturation process is performed to form stable VLPs. After purification, VLP can be chemically conjugated with photosensitive molecules (e.g., IR700NHS esters) to produce VLP drug conjugates.

[0067] In some embodiments, the VLP drug conjugate is prepared by diluting 1:1 with 2X labeling buffer containing 100 mM HEPES pH 7.5, 20 mM MgCl2 and 10% (w / v) trehalose dihydrate. 700DXNHS ester is conjugated with a calculated molar excess of 300:1 (dye: VLP) for 2 hours. In some embodiments, the total protein concentration is determined by Bradford total protein assay (Pierce Bradford protein assay catalog #23200) using a micro-microplate procedure according to the manufacturer's instructions. In some embodiments, the VLP drug conjugate is then buffer exchanged into a specific formulation.

[0068] Thus, in some aspects, provided herein is a method for producing a photosensitive molecule, comprising (a) transiently transfecting cells with nucleic acids encoding one or more capsid proteins to form procapsids, (b) collecting procapsids and subjecting procapsids to an in vitro maturation process to form stable VLPs, and (c) chemically conjugating VLPs (capsids of VLPs) with 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, VLPs are conjugated to photosensitive molecules via amide bonds (e.g., by reacting an ester group of a photosensitive molecule with an amine group of an amino acid of a capsid protein of a virus-like nanoparticle).

[0069] Conjugation can be performed by any method known in the art. Some non-limiting examples of methods for conjugating photosensitive molecules to capsid proteins include reacting photosensitive molecules labeled with N-hydroxysuccinimide ester (NHS-ester) with amine groups on capsid proteins; reacting photosensitive molecules labeled with maleimide, iodoacetyl or pyridyl disulfide with sulfhydryl groups on capsid proteins; and reacting photosensitive molecules labeled with primary amines with carboxyl groups on capsid proteins.

[0070] In some embodiments, conjugation includes reacting a photosensitive molecule labeled with NHS-ester with an amine group on a capsid protein. Available amine groups are on the amino terminus of the capsid protein or on the ε-amino group on 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, 0.3M to 0.5M NaCl). In some embodiments, the photosensitive molecule and the capsid protein are mixed together in a ratio of 1: 200, 1: 300, 1: 400, 1: 500, 1: 600, 1: 700, 1: 800, 1: 900 or 1: 1000. In some embodiments, the conjugation reaction is carried out at room temperature.

[0071] The conjugated VLP and photosensitive molecule can be exposed to the first light and the second light during conjugation. In some embodiments, the first light is as described herein (e.g., having a wavelength of 470 to 610 nm and an intensity of at least 500 lux). In some embodiments, the second light is as described herein (e.g., white light with an intensity of at least 500 lux). Exposure to the first and / or second light during conjugation can be used to ensure that the photosensitive molecule is conjugated to the VLP, to collect samples from the conjugation reaction for quality control, or to check the uniformity of the conjugation reaction. Uniformity refers to a well-mixed solution that is clearly free of particles.

[0072] In some embodiments, the VLP and the photosensitive molecule are exposed to the first light for no more than 15 minutes 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.

[0073] In some embodiments, the VLP and the photosensitive molecule are exposed to the second light for no more than 15 minutes 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.

[0074] Treatment

[0075] Any type of tumor can be targeted according to the present disclosure. Some examples of tumors include, but are not limited to, those located in the eye, lung, pleura, liver, pancreas, stomach, esophagus, colon, breast, ovary, prostate, brain, meninges, testis, kidney, bladder, head, neck, cervix, larynx and / or skin. For example, the 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.

[0076] In some embodiments, the tumor is an eye tumor or lesion. An eye tumor or lesion may be located in the vitreous, choroidal cavity, suprachoroidal cavity, iris, ciliary body, sclera, fovea, retina, optic disc, or optic nerve. Therefore, in some embodiments, the object of the ophthalmic composition of the present disclosure is suffering from an eye tumor. The eye tumor may be, for example, uveal melanoma or choroidal melanoma. In some embodiments, the tumor is cancerous or malignant. In some embodiments, the tumor is metastatic. In some embodiments, the lesion is a precancerous lesion or an uncertain lesion. In some embodiments, the object of the ophthalmic composition of the present disclosure has an uncertain lesion.

[0077] In some embodiments, the subject to whom the ophthalmic composition of the present disclosure is administered has choroidal metastasis, which originates from elsewhere in the body and spreads to the eye. For example, choroidal metastasis may originate from lung cancer or breast cancer in men.

[0078] The ophthalmic compositions of the present disclosure are typically administered by intravitreal or suprachoroidal injection, although other routes of administration are contemplated herein.

[0079] 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.

[0080] Other implementation plans

[0081] 1. An ophthalmic composition comprising a near isotonic solution of a virus-like particle (VLP) drug conjugate, wherein the virus-like particle (VLP) drug conjugate comprises a photosensitive molecule conjugated to a capsid protein of the VLP, wherein the VLP drug conjugate is a suspension.

[0082] 2. An ophthalmic composition comprising a virus-like particle (VLP) drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of the VLP, wherein the VLP drug conjugate does not aggregate to form visible particles.

[0083] 3. The ophthalmic composition of paragraph 1 or 2, which has a pH value of less than 7.

[0084] 4. The ophthalmic composition of any of paragraphs 1 to 3, further comprising 2-(N-morpholino)ethanesulfonic acid (MES).

[0085] 5. The ophthalmic composition of any of paragraphs 1 to 4, further comprising at least one protective excipient and at least one detergent.

[0086] 6. The ophthalmic composition of any of paragraphs 1 to 4, further comprising at least one agent selected from the group consisting of trehalose dihydrate, magnesium chloride (MgCl2), sodium chloride (NaCl), and polysorbate 80 (PS80).

[0087] 7. The ophthalmic composition of paragraph 6, further comprising at least two agents selected from the group consisting of trehalose dihydrate, MgCl2, NaCl, and PS80.

[0088] 8. The ophthalmic composition of paragraph 7, further comprising at least three agents selected from the group consisting of trehalose dihydrate, MgCl2, NaCl, and PS80.

[0089] 9. The ophthalmic composition of paragraph 8, further comprising trehalose dihydrate, MgCl2, NaCl and PS80.

[0090] 10. The ophthalmic composition of any of paragraphs 1 to 9, wherein the composition comprises 0.1% to 1.0% (w / v) MES or a pharmaceutically acceptable salt thereof (eg, MES hemi-sodium salt).

[0091] 11. The ophthalmic composition of paragraph 10, wherein the composition comprises 0.4% (w / v) MES.

[0092] 12. The ophthalmic composition of any of paragraphs 1 to 11, wherein the composition comprises 1% to 10% (w / v) trehalose dihydrate.

[0093] 13. The ophthalmic composition of paragraph 12, wherein the composition comprises 5% (w / v) trehalose dihydrate.

[0094] 14. The ophthalmic composition of any of paragraphs 1 to 13, wherein the composition comprises 0.1% to 1.0% (w / v) NaCl.

[0095] 15. The ophthalmic composition of paragraph 14, wherein the composition comprises 0.4% (w / v) NaCl.

[0096] 16. The ophthalmic composition of any of paragraphs 1 to 15, wherein the composition comprises 0.1% to 1.0% (w / v) MgCl2.

[0097] 17. The ophthalmic composition of paragraph 16, wherein the composition comprises 0.2% (w / v) MgCl2.

[0098] 18. The ophthalmic composition of any of paragraphs 1 to 17, wherein the composition comprises 0.01% to 0.1% (w / v) PS80.

[0099] 19. The ophthalmic composition of paragraph 18, wherein the composition comprises 0.05% (w / v) PS80.

[0100] 20. The ophthalmic composition of any of paragraphs 1 to 19, wherein the composition comprises 0.01% to 0.5% (w / v) VLP drug conjugate.

[0101] 21. The ophthalmic composition of paragraph 20, wherein the composition comprises 0.01% to 0.1% (w / v) VLP drug conjugate.

[0102] 22. The ophthalmic composition of paragraph 21, wherein the composition comprises 0.04% VLP drug conjugate.

[0103] 23. The ophthalmic composition of any of paragraphs 3 to 22, wherein the pH of the composition is 6.5.

[0104] 24. An ophthalmic composition comprising 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 comprises a photosensitive molecule conjugated to a capsid protein of the VLP.

[0105] 25. The ophthalmic composition of any of paragraphs 1 to 24, wherein the photosensitive molecule comprises a dye molecule.

[0106] 26. The ophthalmic composition of paragraph 25, wherein the dye molecule comprises a phthalocyanine dye molecule.

[0107] 27. The ophthalmic composition of paragraph 26, wherein the phthalocyanine dye molecule comprises 700DX.

[0108] 28. The ophthalmic composition of any of paragraphs 1 to 27, wherein the VLP comprises 10 to 1000 photosensitizing molecules, 10 to 500 photosensitizing molecules, 50 to 1000 photosensitizing molecules, 50 to 500 photosensitizing molecules, 100 to 1000 photosensitizing molecules, or 100 to 500 photosensitizing molecules.

[0109] 29. The ophthalmic composition of any of paragraphs 1 to 28, wherein the VLP comprises 50 to 500 photosensitive molecules.

[0110] 30. The ophthalmic composition of any of paragraphs 1 to 29, wherein the VLP comprises a papillomavirus capsid protein.

[0111] 31. The ophthalmic composition of paragraph 30, wherein the papillomavirus capsid protein is a human papillomavirus capsid protein.

[0112] 32. The ophthalmic composition of paragraph 31, wherein the papillomavirus capsid protein comprises L1 capsid protein, L2 capsid protein, or a combination of L1 and L2 capsid proteins.

[0113] 33. The ophthalmic composition of paragraph 32, wherein the L1 capsid protein is modified to reduce the immunogenicity of the VLP.

[0114] 34. A method comprising administering the ophthalmic solution of any of paragraphs 1 to 33 to an eye of a subject, wherein the subject has ocular melanoma, and wherein the ophthalmic solution is administered in an amount effective to treat the ocular melanoma.

[0115] 35. The method of paragraph 34, wherein the ocular melanoma is uveal melanoma or choroidal melanoma.

[0116] 36. A method comprising administering the ophthalmic solution of any of paragraphs 1 to 33 to an eye of a subject, wherein the subject has an indeterminate lesion, and wherein the ophthalmic solution is administered in an amount effective to treat the indeterminate lesion.

[0117] 37. The method of any of paragraphs 34 to 36, wherein the ophthalmic composition is injected intravitreally.

[0118] 37. A method comprising administering the ophthalmic solution of any of paragraphs 1 to 33 to an eye of a subject, wherein the subject has choroidal metastases, and wherein the ophthalmic solution is administered in an amount effective to treat the indeterminate lesion.

[0119] 38. The method of any of paragraphs 34 to 37, wherein the ophthalmic composition is injected intravitreally.

[0120] 39. The method of any of paragraphs 34 to 37, wherein the ophthalmic composition is injected into the suprachoroidal space of the eye.

[0121] 40. The method of paragraph 39, wherein the ophthalmic composition remains in the suprachoroidal space of the eye for at least 1 week.

[0122] 41. The method of paragraph 39 or 40, wherein no leukocyte infiltration is observed in the ciliary body and / or sclera after at least 35 days after injection of the ophthalmic composition.

[0123] 42. The method of any of paragraphs 34 to 41, wherein optical coherence tomography is normal in the subject's eye following injection of the ophthalmic composition.

[0124] 43. The method of any of paragraphs 34 to 42, wherein intraocular pressure is normal in the eye of the subject following injection of the ophthalmic composition.

[0125] 44. The ophthalmic composition of any of the preceding paragraphs, wherein the ophthalmic composition is free of DNase and / or RNase.

[0126] 45. The ophthalmic composition of any of the preceding paragraphs, wherein the ophthalmic composition is sterile.

[0127] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which they are individually cited, which in some cases may be incorporated in their entirety.

[0128] Unless explicitly stated to the contrary, as used herein in the specification and claims, nouns modified by an unquantified word should be understood to mean "at least one".

[0129] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are listed, unless explicitly stated to the contrary.

[0130] In the claims and the above specification, all transitional phrases such as "comprising", "including", "with", "having", "containing", "involving", "having", "comprising", etc. shall be understood as open-ended, i.e., meaning including but not limited to. As provided in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0131] The terms "about" and "substantially" preceding a numerical value mean ±10% of the recited numerical value.

[0132] Where a range of values ​​is provided, every value between the upper and lower limit of that range is specifically contemplated and described herein. Example

[0133] In the preliminary evaluation of VLP drug conjugate formulations, a historical phosphate-based high-salt formulation and three alternative formulations were tested to evaluate the parameters of in-process recovery after bulk VLP drug conjugate formulation and the response of formulated material to repeated freeze-thaw cycles. The collected data included protein recovery obtained by Bradford total protein assay, A280, SDS-PAGE band pattern, VLP morphology obtained by transmission electron microscopy, VLP size distribution obtained by dynamic light scattering, VLP drug conjugate efficacy, eye distribution of VLP conjugates after suprachoroidal injection, and safety of VLP drug conjugates in vivo.

[0134] The data below unexpectedly show that recovery of the VLP drug conjugate was improved when the product formulation was changed to a MES-based formulation containing 5% trehalose dihydrate, with no visible signs of aggregation during or after removal of excess dye using tangential flow filtration (TFF).

[0135] Example 1: Process Step Recovery

[0136] Raw material VLP drug conjugates are prepared into each of four preparations (A, B, C, D) by buffer exchange and concentration using tangential flow filtration (TFF). The control contains historical phosphate / high salt-based preparations. Three other trehalose-containing preparations were evaluated, and the purpose is that the combination of buffer pH and trehalose excipient will protect the bioconjugate. The observations collected during the TFF operation include the formation of visible aggregation in the PBS / high salt control preparation and the phosphate-based and trehalose / PS80-containing preparation A. For the preparations based on HEPES or MES containing trehalose and PS80 (formulations B and C, respectively), no signs of aggregation are seen. Step recovery is determined using Bradford total protein assay. Two phosphate-based preparations (wherein signs of aggregation are seen) obtain about 48 to 58% of step recovery, which is significantly lower than the recovery (table 1) in the preparations based on HEPES (69.7%) and based on MES (79.3%). The concentration of the final prepared sample is 0.25 to 0.66 mg / mL. In order to avoid further handling operations and sample loss, it was decided to evaluate the properties of these samples without further processing.

[0137] Table 1: Step recovery by Bradford total protein assay

[0138]

[0139] Example 2: Recovery after freeze-thaw

[0140] Samples were filled into 2.0 mL CZ resin vials. Multiple vials from each formulation were stored at 2 to 8°C while 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. The data are shown in Table 2 below and in graphical form. Figures 1 to 2 middle.

[0141] Table 2: Recovery by Bradford total protein assay

[0142]

[0143] Within the error of the analytical determination used (expected to be approximately 20%), there was no apparent loss of protein regardless of final formulation conditions or number of freeze-thaw cycles. Consistent recoveries were also obtained from each sample when analyzed by UV-VIS at 280 nm to follow total protein recovery.

[0144] Example 3: Evaluation by SDS-PAGE

[0145] Samples from each formulation were analyzed by SDS-PAGE and the gels were imaged by standard Coomassie staining and also by fluorescence, using an Odyssey scanner to detect the fluorescence of the DX700 dye. Freshly prepared samples and those 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 of samples subjected to 5 freeze-thaw cycles are shown in Figure 3 In. Visual evaluation of SDS-PAGE gel reveals the complex band pattern characteristics of VLP drug conjugates. It can be seen that the main band as L1 protein migrates at about 55kDa. Fuzzy bands can also be seen above the 62kDa marker, and it represents L2 protein. Above the 98kDa marker, a ladder of material is seen, indicating that there is a certain form of oligomeric material. The nature of this material is the theme of other technical reports, and the LC-MS analysis of the in-gel digestion samples carried out by SGS reveals that this material is a protein comprising L1 and L2 sequences. Visually, the band pattern is consistent in all samples, and it can be seen that the band intensity and fluorescence signal are parallel to Bradford and absorbance data sets.

[0146] Example 4: Morphology and size distribution of virus-like particles obtained by transmission electron microscopy

[0147] The size of the particles was estimated by measuring the diameter of approximately 60 to 90 particles and assembled capsole material using the AMT software. Most VLPs appeared fully assembled and were observed to have a variety of sizes and were primarily spherical or ellipsoidal in shape. Figure 4 A similar range of apparent morphologies was observed in EM studies of HPV16 L1 and HPV11 L1 based VLPs and in unfractionated preparations of rabbit papillomavirus. TEM data for samples subjected to 1, 3 and 5 freeze-thaw cycles are shown in Table 3 and in graphical form. Figure 5 During the freeze-thaw stability study, no significant differences in particle size of the VLP drug conjugates were observed between the three buffer conditions evaluated - control, formulation B and formulation C. Formulation A was not evaluated using TEM. No significant changes in the size range distribution or overall morphological properties of the VLP drug conjugates were observed through multiple freeze-thaw cycles during the study.

[0148] Table 3: Particle size data obtained by TEM

[0149]

[0150] Example 5: Particle size analysis using dynamic light scattering

[0151] To complement the morphology and size distribution analysis by TEM, the average size of the VLPs in each formulation was estimated by DLS measurements before freezing and after 1, 3, or 5 freeze-thaw cycles. Laser-based DLS can monitor the movement and structural changes of nanoparticles in solution (i.e., degradation or VLP oligomerization) and provides information related to the average size and frequency distribution of the particles. The DLS data are summarized in Table 4 and presented in Figure 4. Figure 6 In both cases, the average diameter values ​​expressed by number (N), volume (V) and intensity (I) are shown. Each snapshot of the average particle size distribution is given, and the average value of the overall distribution is reported. Regardless of the formulation and the number of freeze-thaw cycles, the average particle size distribution does not show significant changes. In general, the DLS and TEM measurements of VLPs are consistent and show that VLPs have considerable size and morphology, regardless of the formulation and the number of freeze-thaw cycles.

[0152] Table 4: Average particle size obtained by DLS

[0153]

[0154] Example 6: Efficacy Evaluation

[0155] The functional properties of the VLP drug conjugates were evaluated using an in vitro killing assay. Data generated in each of the four tested formulations of freshly prepared material and material stored in final container closures and subjected to 1, 3, or 5 freeze-thaw cycles are shown in Figures 7A to 7D Limited staffing capacity and the assay itself limited the data points to either side of the expected EC50. The VLP drug conjugate remained potent regardless of formulation and number of freeze-thaw cycles.

[0156] Example 7: Ocular distribution of VLP drug conjugates following suprachoroidal injection in New Zealand white rabbits

[0157] In New Zealand White (NZW) rabbits, AlexaFluor488*VLP was injected into the suprachoroidal space (SCS) to evaluate the in vivo ocular distribution of VLP conjugates (study PK-RPE-003). AlexaFluor488*VLP was used instead of VLP drug conjugates because it has similar physiochemical properties and is formulated in the same MES buffer as VLP drug conjugates, but is more suitable for in vivo imaging. Optical coherence tomography (OCT) and fundus autofluorescence (FAF) were used to evaluate the ocular distribution over time. The data showed that at ≤0.5 hours after administration, the distribution after 100 μl was injected into the SCS was about 75% of the posterior part of the eyeball and remained relatively constant throughout the study. Fluorescence was very strong within the 168 hour interval after administration and began to weaken at 240 hours after administration. At 504 hours after administration, no significant fluorescence above the baseline could be detected. The data demonstrate that VLP drug conjugates formulated in MES buffer are well distributed into the SCS lumen and persist for at least 168 hours (1 week).

[0158] Example 8: In vivo safety evaluation of VLP drug conjugates (non-clinical)

[0159] In order to evaluate the non-clinical safety of VLP drug conjugates, VLP drug conjugates were used in dog studies. The VLP drug conjugates were administered at a single dose level by suprachoroidal space (SCS) 100 μl injection at a dose of 20 μg / eye, followed by laser photoactivation 6 to 8 hours after injection into the dog. Dogs were treated once a week for 3 weeks (3 injections in total, followed by 50 J / cm 2Laser treatment). After the 3rd week treatment, the animals were observed for 7 days (terminal sacrifice) or 35 days (recovery period) to evaluate the reversibility, persistence or delayed onset of the effect. There were only minor microscopic ocular findings related to the VLP drug conjugate. Histopathology showed minimal / mild leukocyte infiltration into the choroidal cavity, and 50% of the animals had minimal leukocyte infiltration into the ciliary body and sclera. The minimal / mild leukocyte infiltration subsided between the first observation time point (animals terminally sacrificed at 7 days) and the second observation time point (animals in the recovery period at 35 days), and was not observed at the second observation time point. In all animals, optical coherence tomography (OCT) was normal (i.e., no retinal pigment epithelium (RPE) / retinal changes or retinal thinning were detected), and all studied eyes maintained normal retinal structure. The intraocular pressure (IOP) of the subject animals was evaluated during the study, and the intraocular pressure of all animals was normal. No systemic clinical findings, weight changes and clinical pathological findings were reported. In short, the data show that the VLP drug conjugates prepared in MES buffer delivered as multiple SCS injections are safe in vivo.

[0160] Materials and methods

[0161] sample

[0162] Four different formulations as outlined in Table 5 were used to formulate the VLP drug conjugates.

[0163] Table 5: Sample preparation

[0164]

[0165] equipment

[0166] The equipment required to perform the formulation steps described herein is provided in Table 6.

[0167] Table 6: Equipment

[0168]

[0169]

[0170] Reagents and solvents

[0171] Alcian Blue, 1.5% Uranyl Acetate, RO-DI water, Carbon film 300 Mesh Copper Grid, Fine-tip tweezers, Pipette, Petri Dish, Parafilm, and filter paper.

[0172] Manufacturing and formulation of VLP drug conjugates

[0173] VLP drug conjugates were prepared from stock VLP preparations. After 1:1 dilution with 2X labeling buffer containing 100mM HEPES pH 7.5, 20mM MgCl2 and 10% trehalose dihydrate, bioconjugation was performed with DX700-NHS ester at a calculated molar excess of 300:1 (dye: VLP) for 2 hours. The total protein concentration was determined using a microplate procedure by Bradford total protein assay (Pierce Bradford Protein Assay Catalog #23200) according to the manufacturer's instructions. The stock VLP drug conjugate buffer was then exchanged into a control formulation (phosphate / high salt) and three candidate formulations, each containing trehalose and PS80, with three different pHs (A, B and C). The entire experimental process is shown in Figure 8 middle.

[0174] Product Strength

[0175] VLP drug conjugate product strength was determined by Bradford total protein assay (Pierce Bradford protein assay catalog #23200, according to manufacturer's instructions, for microplate format). A280nm was measured on undiluted samples using a nanodrop UV-VIS spectrophotometer blanked with sample-matched buffer.

[0176] SDS-PAGE analysis

[0177] SDS-PAGE analysis of VLP drug conjugate products was performed using TGX standards, Any kD gel (Bio-Rad #5671125). Standard gel images were captured using an imaging camera, and fluorescence images were captured by scanning the gel on an Odyssey CLx. Fluorescence analysis was performed on an Odyssey CLx using Imagestudio software.

[0178] Dynamic Light Scattering

[0179] 50 μL of the tested 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 5 times. The diluted sample was then pipetted into a plastic cuvette (cuvette) (FisherCat#14-955-125), and the cuvette was placed in a DLS sample holder. Samples were analyzed using 90Plus particle size determination (sizing) software (Brookhaven Instruments). In brief, the diluted sample was run 3 times at 25 ° C and 90 ° detector angle, each run for 2 minutes. Four independent distributions were recorded: number, volume, intensity and surface area. The polydispersity index for each sample run was also recorded to determine the uniformity of the sample run.

[0180] Transmission electron microscopy

[0181] Aliquots of VLP drug conjugate samples prepared in different buffers were prepared and provided to the Electron Microscopy Center of Northeastern University without freezing (2 to 8°C) or frozen storage at -80°C and subsequently thawed 1, 3 or 5 times, and then analyzed. Samples were analyzed by TEM as follows: a carbon-coated EM grid (carbon film 300 mesh copper grid) was placed on a drop of Alcian blue dye (placed on a paraffin film portion) for 30 seconds, and the carbon side was facing up. Excess dye was removed by gently blotting with filter paper. The EM grid was washed by gently wiping the grid surface for 1 to 2 seconds on a drop of water (placed on a paraffin film portion) with deionized sterile water. For each wash, fresh water drops were used to repeat the wash twice. After washing, the grid was placed on a 10 μl sample suspension drop for about 1 minute. The loaded grid was gently blotted to remove excess sample, and then washed 3 times with deionized water using the same technique as described in step 2. The loaded grid was then stained by gently wiping it on the surface of a drop of 1.5% uranyl acetate stain for 1 to 2 seconds and blotted dry with filter paper. The staining step was repeated 3 more times on the same drop of uranyl acetate. For TEM imaging, a Jeol 1010AMT camera (located at the Center for Electron Microscopy, Northeastern University, Boston) was used. After drying for several minutes, the prepared grid was placed in a TEM. Images were captured at magnifications of ×15000, ×25000, ×30000, ×40000, ×50000, and ×200000. The diameters of the particles and assembled shell particles within the target area were measured at ×40000 magnification using AMT software.

[0182] Effectiveness

[0183] The in vitro efficacy of VLP drug conjugates was determined by carrying out cell killing assay (Aura-SOP-008) to tumor cell line OCM-1. Due to sample and capacity limitations, only three product levels close to the expected EC50 of the product were evaluated. Tumor cells were incubated on ice for 1.5 hours with VLP drug conjugates of varying concentrations. After this incubation, cells were washed to remove unbound VLP drug conjugates, and subsequently resuspended in sample diluent. Half of the cells bound by the VLP drug conjugates for each VLP drug conjugate diluent were distributed to 3 holes of 96-well plates (1 / 2 hole, black, transparent bottom) and irradiated with 25J / cm2 of 690nm wavelength light. Unirradiated cells were used as controls.

Claims

1. An ophthalmic composition comprising a near-isotonic solution of a virus-like particle (VLP) drug conjugate, wherein the virus-like particle (VLP) drug conjugate comprises a photosensitive molecule conjugated to a capsid protein of the VLP, wherein the VLP drug conjugate is a suspension, and wherein the near-isotonic solution comprises 2-(N-morpholino)ethanesulfonic acid (MES) and has an isotonicity of 255 to 345 mOsm / L, wherein the VLP comprises a papillomavirus capsid protein.

2. An ophthalmic composition comprising a near-isotonic solution of a virus-like particle (VLP) drug conjugate, wherein the virus-like particle (VLP) drug conjugate comprises a photosensitive molecule conjugated to a capsid protein of the VLP, wherein the VLP drug conjugate does not aggregate to form visible particles, and wherein the near-isotonic solution comprises 2-(N-morpholino)ethanesulfonic acid (MES) and has an isotonicity of 255 to 345 mOsm / L, wherein the VLP comprises a papillomavirus capsid protein.

3. The ophthalmic composition according to claim 1 or 2, which has a pH value of less than 7.

4. The ophthalmic composition of claim 1 or 2, further comprising at least one protective excipient and at least one detergent.

5. The ophthalmic composition of claim 1 or 2, further comprising at least one agent selected from the group consisting of trehalose dihydrate, magnesium chloride MgCl2, sodium chloride NaCl, and polysorbate 80 PS80.

6. The ophthalmic composition of claim 5, further comprising at least two agents selected from the group consisting of trehalose dihydrate, MgCl2, NaCl, and PS80.

7. The ophthalmic composition of claim 6, further comprising at least three agents selected from the group consisting of trehalose dihydrate, MgCl2, NaCl, and PS80.

8. The ophthalmic composition of claim 7, further comprising trehalose dihydrate, MgCl2, NaCl and PS80.

9. The ophthalmic composition of claim 1 or 2, wherein the composition comprises 0.1% to 1.0% w / v MES.

10. The ophthalmic composition of claim 9, wherein the composition comprises 0.4% w / v MES.

11. The ophthalmic composition of claim 1 or 2, wherein the composition comprises 1% to 10% w / v trehalose dihydrate.

12. The ophthalmic composition of claim 11, wherein the composition comprises 5% w / v trehalose dihydrate.

13. The ophthalmic composition of claim 1 or 2, wherein the composition comprises 0.1% to 1.0% w / v NaCl.

14. The ophthalmic composition of claim 13, wherein the composition comprises 0.4% w / v NaCl.

15. The ophthalmic composition of claim 1 or 2, wherein the composition comprises 0.1% to 1.0% w / v MgCl2.

16. The ophthalmic composition of claim 15, wherein the composition comprises 0.2% w / v MgCl2.

17. The ophthalmic composition of claim 1 or 2, wherein the composition comprises 0.01% to 0.1% w / v PS80.

18. The ophthalmic composition of claim 17, wherein the composition comprises 0.05% w / v PS80.

19. The ophthalmic composition of claim 1 or 2, wherein the composition comprises 0.01% to 0.5% w / v VLP drug conjugate.

20. The ophthalmic composition of claim 19, wherein the composition comprises 0.01% to 0.1% w / v VLP drug conjugate.

21. The ophthalmic composition of claim 20, wherein the composition comprises 0.04% w / v VLP drug conjugate.

22. The ophthalmic composition of claim 3, wherein the pH of the composition is 6.

5.

23. An ophthalmic composition comprising 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 comprises a photosensitive molecule conjugated to a capsid protein of the VLP, wherein the VLP comprises a papillomavirus capsid protein.

24. The ophthalmic composition of claim 1 or 2, wherein the photosensitive molecule comprises a dye molecule.

25. The ophthalmic composition of claim 24, wherein the dye molecules comprise phthalocyanine dye molecules.

26. The ophthalmic composition of claim 25, wherein the phthalocyanine dye molecule comprises:

27. The ophthalmic composition of claim 1 or 2, wherein the VLP comprises 10 to 1000 photosensitive molecules.

28. The ophthalmic composition of claim 27, wherein the VLP comprises 50 to 500 photosensitive molecules.

29. The ophthalmic composition of claim 1 or 2, wherein the papillomavirus capsid protein is a human papillomavirus capsid protein.

30. The ophthalmic composition of claim 29, wherein the papillomavirus capsid protein comprises an L1 capsid protein, an L2 capsid protein, or a combination of L1 and L2 capsid proteins.

31. The ophthalmic composition of claim 30, wherein the L1 capsid protein is modified to reduce the immunogenicity of the VLP.

32. Use of the ophthalmic composition of any one of claims 1 to 31 for the preparation of a medicament for use in a method comprising administering the ophthalmic composition to an eye of a subject, wherein the subject has ocular melanoma, and wherein the ophthalmic composition is formulated for administration in an amount effective to treat the ocular melanoma.

33. The use of claim 32, wherein the ocular melanoma is uveal melanoma or choroidal melanoma.

34. The use of claim 32 or 33, wherein the ophthalmic composition is formulated for intravitreal injection.

35. The use of claim 32 or 33, wherein the ophthalmic composition is formulated for injection into the suprachoroidal space of the eye.

36. The use of claim 35, wherein the ophthalmic composition remains in the suprachoroidal space of the eye for at least 1 week.

37. The use of claim 35, wherein no leukocyte infiltration is observed in the ciliary body and / or sclera after at least 35 days after injection of the ophthalmic composition.

38. The use of claim 32 or 33, wherein optical coherence tomography is normal in the subject's eye following injection of the ophthalmic composition.

39. The use of claim 32 or 33, wherein intraocular pressure is normal in the eye of the subject following injection of the ophthalmic composition.

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