Human papillomavirus nanoparticle formulations

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. The present invention provides, for example, ophthalmic compositions comprising an approximately isotonic solution of a virus-like particle (VLP)-drug conjugate comprising a photosensitive molecule conjugated to a capsid protein of the VLP, wherein the VLP-drug conjugate is in suspension. The present invention further provides ophthalmic compositions 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 particulate matter.

Description

[Technical Field]

[0001] Related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 824,227, filed March 26, 2019, under Section 119(e) of the U.S. Patent Act, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background The fundamental concerns for biotherapeutic formulations, including ophthalmic formulations, are the stability and structural integrity of the active molecule during transfer and storage, after numerous freeze-thaw cycles, the successful delivery of the drug to its site of action, the speed and cost-effectiveness of development, and the final product. Many details are involved, including analytical methods and test protocols, containers and closures, delivery devices and dosage forms, excipients and stabilizers, and component compatibility. [Overview of the project] [Means for solving the problem]

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

[0004] Accordingly, in some aspects, ophthalmic compositions are provided herein that include a nearly isotonic solution (255-345 mOsm / L) of a VLP drug conjugate containing a photosensitive molecule conjugated to the capsid protein of a VLP, wherein the VLP drug conjugate is in a suspension. When used herein, the isotonic solution has an isotonicity of approximately 290 mOsm / L, and the nearly isotonic solution has an isotonicity of 255-345 mOsm / L.

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

[0006] In some embodiments, the ophthalmic composition further comprises at least one protective 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 contains 0.01% to 0.1% (w / v) VLP drug conjugates. For example, the composition may contain 0.04% (w / v) VLP drug conjugates.

[0008] In some embodiments, the composition contains 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 contain 0.4% (w / v) MES or a pharmaceutically acceptable salt thereof. In some embodiments, the composition contains 5 mM to 50 mM MES or a pharmaceutically acceptable salt thereof. For example, the composition may contain 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 above composition has a pH value of 5 to 8. For example, the above 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, where 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, where 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 IRDye® 700DX molecule, IRDye® 800CW molecule, or a mixture of IRDye® 700DX and IRDye® 800CW molecules. Other therapeutic and diagnostic photosensitive molecules are intended herein.

[0016] In some embodiments, the VLP contains 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 contains 300 photosensitive molecules.

[0017] In some embodiments, the VLP comprises a papillomavirus capsid protein (e.g., 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.

[0018] In some aspects, methods are also provided herein that include the step of administering the ophthalmic composition of the present disclosure to the eye of a subject, wherein 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 comprising the step of administering the ophthalmic composition of the present disclosure to the eye of a subject, wherein the subject has an unclassifiable lesion, and the ophthalmic solution is administered in an effective amount to treat the unclassifiable lesion. [Brief explanation of the drawing]

[0020] [Figure 1]Figure 1 illustrates the protein recovery per formulation when measured by Bradford total protein assay. The control formulation contains 20 mM potassium phosphate, 500 mM NaCl (pH 7.0). Formulation A contains 20 mM potassium phosphate, 5% (w / v) trehalose dihydrate, 10 mM MgCl2, 63 mM NaCl, 0.05% (w / v) PS80 (pH 7.0). Formulation B contains 50 mM HEPES, 5% (w / v) trehalose dihydrate, 10 mM MgCl2, 47 mM NaCl, 0.05% (w / v) PS80 (pH 7.5). Formulation C contains 20 mM MES, 5% (w / v) trehalose dihydrate, 10 mM MgCl2, 63 mM NaCl, 0.05% (w / v) PS80 (pH 6.5). The numbers for freeze-thaw (FT) are the number of times the sample was frozen and thawed. [Figure 2] Figure 2 illustrates the protein recovery per formulation when measured by UV-Vis. The formulation is as in Figure 1. The above UV-Vis measures the absorbance at 280 nm. The numbers for freeze-thaw (FT) are the number of times the sample was frozen and thawed. [Figure 3] Figure 3 shows a representative SDS-PAGE gel of the protein formulation after 5 freeze-thaw cycles. The formulation is as in Figure 1. The band at approximately 55 kDa is the L1 protein in the above virus-like particles (VLPs). [Figure 4] Figure 4 shows an image of a representative transmission electron microscopy (TEM) of virus-like particle drug conjugate (VLP drug conjugate). The formulation is as in Figure 1. [Figure 5] Figure 5 illustrates the particle size distribution of VLP drug conjugate when measured by transmission electron microscopy (TEM). The formulation is as in Figure 1. The numbers for freeze-thaw (FT) are the number of times the sample was frozen and thawed. [Figure 6]Figure 6 illustrates the average particle size of the VLP drug conjugate when measured by dynamic light scattering (DLS). The formulation is as shown in Figure 1. The average particle size is measured by diameter. The numbers for freeze-thaw (FT) are the number of times the sample was 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. [Figure 7] Figures 7A - 7D illustrate the in vitro EC50 kill curves. The formulation is as shown in Figure 1. Figure 7A shows the percent (%) dead cells using the unfrozen VLP drug conjugate (0 FT). Figure 7B shows the % dead cells using the VLP drug conjugate frozen once (1 FT). Figure 7C shows the % dead cells using the VLP drug conjugate frozen three times. Figure 7D shows the % dead cells using the VLP drug conjugate frozen five times. [Figure 8] Figure 8 illustrates a diagram of the conjugation-formulation workflow. The formulation is as shown in Figure 1. **Mode for Carrying Out the Invention**

[0021] **Detailed Description** 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). The chemical structure of MES contains a morpholine ring, has a molecular weight of 195.2, and its chemical formula is C6H The composition is NO4S. 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 comprises 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%, and 0.2% to 0.4%. The composition may contain 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 may contain 5 mM to 50 mM MES. For example, the above compositions may include 5mM-45mM, 5mM-40mM, 5mM-35mM, 5mM-30mM, 5mM-25mM, 5mM-20mM, 10mM-50mM, 10mM-45mM, 10mM-40mM, 10mM-35mM, 10mM-30mM, 10mM-25mM, 10mM-20mM, 20mM-50mM, 20mM-45mM, 20mM-40mM, 20mM-35mM, 20mM-30mM, or 20mM-25mM MES. In some embodiments, the above composition comprises 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 above composition comprises 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 above composition contains 20 mM MES.

[0022] In some embodiments, the above composition contains 0.01% to 0.1% (w / v) VLP drug conjugates. For example, the above composition may contain 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%, and 0.02% to 0.0 It may contain 4%, 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 conjugates. 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 conjugates. In some embodiments, the composition may contain 0.03%, 0.035%, 0.04%, 0.045%, or 0.05% (w / v) VLP drug conjugates. In some embodiments, the composition may contain 0.04% (w / v) VLP drug conjugates.

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

[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 dissaccharide formed by a 1,1-glycosidic bond between two α-glucose units. Trehalose forms rhomboid crystals as a dihydrate, and the anhydrous form of trehalose readily regains water to form a 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 above composition may contain 1%~9%, 1%~8%, 1%~7%, 1%~6%, 1%~5%, 2%~10%, 2%~9%, 2%~8%, 2%~7%, 2%~6%, 2%~5%, 3%~10%, 3%~9%, 3%~8%, 3%~7%, 3%~6%, 3%~5%, 3%~4%, 4%~10%, 4%~9%, 4%~8%, 4%~7%, 4%~6%, or 4%~5%, 5%~10%, 5%~9%, 5%~8%, 5%~7%, or 5%~6% (w / v) trehalose dihydrate. In some embodiments, the composition further comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (w / v) 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, 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 contain 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%, and 0.2% to 0.4%. The composition may contain 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 contain 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 to 100 mM NaCl. For example, the above composition may further contain 20mM-90mM, 20mM-80mM, 20mM-70mM, 20mM-60mM, 20mM-50mM, 30mM-100mM, 30mM-90mM, 30mM-80mM, 30mM-70mM, 30mM-60mM, 30mM-50mM, 40mM-100mM, 40mM-90mM, 40mM-80mM, 40mM-70mM, 40mM-60mM, 40mM-50mM, 50mM-100mM, 50mM-90mM, 50mM-80mM, 50mM-70mM, 50mM-60mM, or 60mM-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.

[0027] In some embodiments, the above composition further comprises 0.1% to 1.0% (w / v) magnesium chloride (MgCl2). For example, the above composition may contain 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%, and 0.2% to 0.4%. The composition may contain 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) MgCl2. 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) MgCl2. In some embodiments, the above composition further comprises 0.1%, 0.15%, 0.2%, 0.25%, or 3% (w / v) MgCl2. In some embodiments, the above composition further comprises 0.2% (w / v) MgCl2. In some embodiments, the above composition further comprises 5mM to 25mM MgCl2. For example, the above 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 above composition further comprises 5mM, 10mM, 15mM, 20mM, or 25mM MgCl2. In some embodiments, the above composition further comprises 8mM, 9mM, 10mM, 11mM, or 12mM MgCl2. In some embodiments, the above composition further comprises 10mM 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 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 0.0 May contain 4%, 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) polysorbate 80. 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) polysorbate 80. In some embodiments, the composition may contain 0.04%, 0.045%, 0.05%, 0.055%, or 0.06% (w / v) polysorbate 80. In some embodiments, the composition may contain 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 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, where 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, where the VLP drug conjugate comprises a photosensitive molecule conjugated to the capsid protein of VLP.

[0031] In some embodiments, the formulation is visibly free of particulate matter. Visibly free of particulate matter means that a normal human being (e.g., without uncorrected visual defects and without using means to magnify the sample) does not perceive the presence of particulate matter (e.g., clumps, 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). A molecule or other substance / agent is considered "pharmaceutically acceptable" if it is approved or appropriable by a federal or state regulatory authority, or if it is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals (including humans). The excipient may be any inert (inactive) and 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 colorants.

[0033] The term “pharmaceutically acceptable salt” refers to a salt that, within the bounds of sound medical judgment, is appropriate for use in contact with human and lower animal tissues without excessive toxicity, irritation, or allergic reactions, and that balances the benefits / risks in a reasonable proportion. 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 (as incorporated herein by reference). Pharmaceutically acceptable salts of the compounds disclosed herein 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 organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or by other methods known in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include: adipines, alginates, ascorbic acid, aspartates, benzenesulfons, benzoates, bisulfates, borates, butyrates, camphorates, camphor sulfons, citrates, cyclopentanepropionates, diglucons, dodecyl sulfates, ethanesulfons, formates, fumarates, glucoheptons, glycerophosphates, glucons, hemisulfates, heptanoates, hexanoates, hydroiodides, and 2-hydroxyethansulfates. These include honates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, p-toluenesulfons, undecanoates, valersates, etc. Salts obtained from suitable bases include alkali metals, alkaline earth metals, ammonium, and N+(C1-4 alkyl) 4-salts.Typical alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions (e.g., halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates) where appropriate.

[0034] The above ophthalmic compositions are formulated as liquids in some embodiments. These liquids may be packaged, for example, in vials or pre-filled syringes. Other packaging and delivery forms are contemplated herein.

[0035] Virus-like particle conjugate Virus-like particles (VLPs) 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. Examples of non-human VLPs include, but are not limited to, those derived from bovine papillomavirus, mouse papillomavirus, cottontail rabbit papillomavirus, and macaque or rhesus monkey papillomavirus. In some embodiments, the VLP is a bovine papillomavirus VLP (e.g., assembled from the BPV L1 capsid protein or a combination of BPV L1 and BPV L2 capsid proteins).

[0036] Capsid proteins are protein monomers, some of which form capsomer oligomers. Capsomers are the basic oligomeric structural units of viral capsids, which are the outer coverings of proteins that protect the genetic material of viruses, such as human papillomavirus (HPV). The capsid proteins of this disclosure include papillomavirus L1 major capsid proteins and papillomavirus L2 minor capsid proteins. In some embodiments, the VLP of this disclosure comprises only L1 capsid proteins, while in other embodiments, the VLP comprises a mixture (or combination) of L1 and L2 capsid proteins. In some embodiments, the VLP comprises human papillomavirus capsid proteins. In some embodiments, the VLP comprises non-human papillomavirus capsid proteins.

[0037] In some embodiments, the percentage of L1 capsid protein in VLPs is higher than the percentage of L2 capsid protein in VLPs. For example, in some embodiments, the percentage of L1 capsid protein in VLPs is 80% to 100% (of the total number of capsid proteins in the virus-like particles). In some embodiments, the percentage of L1 capsid protein in VLPs 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 VLPs is 1% to 25% (of the total number of capsid proteins in VLPs). For example, in some embodiments, the percentage of 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 one embodiment, the VLP contains 12 to 72 L2 proteins. In another embodiment, the VLP contains 360 L1 proteins and 12 to 72 L2 proteins. In some embodiments, the capsid protein is assembled into a VLP having a diameter of 20 to 60 nm. For example, the capsid protein may be assembled into a VLP 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 this disclosure may have modified immunogenicity and / or antigenicity with respect to wild-type papillomavirus VLPs. Such VLPs may be assembled, for example, from capsomers having a modified immunogenicity and / or antigenicity variant capsid protein. The modified capsid protein having “modified immunogenicity and / or antigenicity” is naturally or synthetically modified (e.g., mutation, substitution, deletion, PEGylation, or insertion) in its amino acids to reduce or prevent recognition of the capsid protein by existing (e.g., endogenous) virus serotype-specific antibodies. The modified capsid protein may 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 derived from different HPV serotypes. For example, modified immunogenic and / or antigenic L1 variants may be recombinant proteins based on HPV serotypes 16 and 31 (referred herein to as “modified HPV16 / 31 L1 proteins”). These are described in International Publication Numbers WO 2010 / 120266, WO 2013 / 119877, and WO 2015 / 042325 (each of these in whole is incorporated herein by reference).

[0040] photosensitive molecules According to various aspects of this disclosure, a photosensitive molecule may be conjugated to the capsid protein of a VLP (e.g., L1 and / or L2 capsid protein). 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 a photosensitive molecule may be referred to herein as a “VLP drug conjugate”. In some embodiments, the photosensitive molecule contains an NHS (N-hydroxysuccinimide) ester group that reacts with an amine group of the capsid protein (e.g., an amine group of lysine or another amino acid) to form a covalent amide bond.

[0041] The ratio of photosensitive molecules (PMs) to VLPs can vary. In some embodiments, the VLP:PM ratio 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., a mixture of the same 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 VLPs are: 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 30-1000, 30-900, 30-800, 30 ~700 pieces, 30~600 pieces, 30~500 pieces, 30~400 pieces, 30~300 pieces, 30~200 pieces, 30~100 pieces, 40~1000 pieces, 40~900 pieces, 40~800 pieces, 40~700 pieces, 40~600 pieces, 40~500 pieces, 40 ~400 pieces, 40~300 pieces, 40~200 pieces, 40~100 pieces, 50~1000 pieces, 50~900 pieces, 50~800 pieces, 50~700 pieces, 50~600 pieces, 50~500 pieces, 50~400 pieces, 50~300 pieces, 50~200 pieces, 50~ 100 pieces, 60~1000 pieces, 60~900 pieces, 60~800 pieces, 60~700 pieces, 60~600 pieces, 60~500 pieces, 60~400 pieces, 60~300 pieces, 60~200 pieces, 60~100 pieces, 70~1000 pieces, 70~900 pieces, 70 ~800 pieces, 70~700 pieces, 70~600 pieces, 70~500 pieces, 70~400 pieces, 70~300 pieces, 70~200 pieces, 70~100 pieces, 80~1000 pieces, 80~900 pieces, 80~800 pieces, 80~700 pieces, 80~600 pieces, 80~ It may contain 500, 80-400, 80-300, 80-200, 80-100, 90-1000, 90-900, 90-800, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, or 100 or more photosensitive molecules.In some embodiments, the VLP may contain 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 contain more than 1000 (e.g., 1500, 2000, etc.) photosensitive molecules or fewer than 10 photosensitive molecules.

[0042] More than one photosensitive molecule can be conjugated to a single capsid protein. For example, one capsid protein (e.g., 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 in the capsid protein can be conjugated to photosensitive molecules. In some embodiments, one capsid protein can be conjugated to 1 to 2, 1 to 3, or 2 to 3 photosensitive molecules. Thus, the 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 one capsid protein.

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

[0044] An example of a photosensitizing dye for use in accordance with this disclosure is IRDye. (登録商標)700DX, HpD, Porfimer sodium (Photofrin®, Photogem®, Photosan Hemporfin®), m-THPC, Temoporfin (Foscan®), Verteporfin (Visudyne®), HPPH (Photochlor®), Palladium-bacterial 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)-texaphyllin or Motexafin-lutetium (Lutex®, Lutrin®, Angrin®, Optrin®), SnET2, Ethyl etiopropyl princes(IV) (Tin (IV) Examples include, but are not limited to, ethyl etiopurpurin (Purlytin®, Photrex®), NPe6, mono-L-aspartylchloride e6, sodium talaporfin (Talporfin®, Laserphyrin®), BOPP, borated protoporphyrin (BOPP®), zinc phthalocyanine (CGP55847®), silicon phthalocyanine (Pc4®), mixture of sulfonated aluminum phthalocyanine derivatives (Photosens®), ATMPn, acetoxy-tetrakis(β-methoxyethyl-)porphycene), TH9402, and dibromolodamine methyl ester.

[0045] In some embodiments, the photosensitizing dye is of the chemical formula C 74 H 96 N 12 Na4O 27 S6Si3 and dye structure: [ka] IRDye (登録商標)It is 700DX NHS ester.

[0046] Examples of imaging dyes (e.g., fluorescent dyes) for use in accordance with this disclosure include, but are not limited to, IRDye® 800CW, acridine orange, acridine yellow, Alexa Fluor, 7-aminoactinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO dye, auramine-rhodamine dye, benzantrone, biman, 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 this disclosure can be activated at appropriate wavelengths. In some embodiments, the activation of the photosensitive molecules can make them cytotoxic or cause them to generate cytotoxic molecules. Appropriate wavelengths include, but are not limited to, ultraviolet, visible, infrared, 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 approximately 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 molecule is activated at wavelengths below 600 nm or above 800 nm. The appropriate wavelength for activating the photosensitive molecule depends on the specific molecule used.

[0049] Methods for production and drug conjugation To generate VLPs, the drug conjugates of this disclosure, mammalian cells (e.g., 293T cells (e.g., HEK293F cells)) may be grown (e.g., in a suspension culture) and transiently transfected with nucleic acids encoding HPV L1 (or L1 and L2) capsid proteins (e.g., bicistronic plasmid DNA). This induces the formation of a protocapsid (e.g., as described in Buck et al. Current Protocols in Cell Biology 26.1.1-26.1.19, December 2007). After cell aggregate recovery and disruption, the protocapsid can be subjected to benzoase treatment for host DNA removal and subsequent in vitro maturation processes to form stable VLPs. After purification, the VLPs can be chemically conjugated with a photosensitive molecule (e.g., IR700 NHS ester) to generate VLP drug conjugates.

[0050] In some embodiments, the VLP drug conjugate is prepared as follows: Conjugation is performed over 2 hours using IRDye® 700DX NHS Ester in a calculated molar excess of 300:1 (dye:VLP) after 1:1 dilution with 2× labeled 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 Bradford whole protein assay (Pierce Bradford protein assay catalog no. 23200) using a microplate procedure according to the manufacturer's instructions. The VLP drug conjugate is then buffer-exchanged to a specific formulation in some embodiments.

[0051] Accordingly, in some aspects, a method for generating photosensitive molecules is provided herein, comprising the steps of (a) transiently transfecting cells with nucleic acids encoding capsid proteins to form protocapsids, (b) collecting the protocapsids and subjecting them to an in vitro maturation process to form stable VLPs, and (c) chemically conjugating the VLPs (capsids of the VLPs) into 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 via an amide bond (for example, by reacting the ester group of the photosensitive molecule with the amine group of the amino acid of the capsid protein of the virus-like nanoparticle).

[0052] Conjugation may be carried out 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 (NHS-ester) labeled photosensitive molecule with an amine group on the capsid protein; reacting a maleimide, iodoacetyl group, or pyridyl disulfide 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 involves reacting an NHS-ester-labeled photosensitive molecule with an amine group on a capsid protein. The available amine groups are located at the amino terminus of the capsid protein or at an 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), 0.3 M to 0.5 M NaCl). In some embodiments, the photosensitive molecule and capsid protein are mixed together in ratios of 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, or 1:1000. The conjugation reaction is carried out at room temperature in some embodiments.

[0054] The combined VLP and photosensitive molecules may be exposed to a first and a second light during the conjugation process. 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 may be to ensure that the photosensitive molecules are conjugated to the VLP and to collect samples from the conjugation reaction for quality control or to check the conjugation reaction in terms of homogeneity. Homogeneity refers to a well-mixed solution in which no particulate matter is visibly present.

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

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

[0057] Treatment method Any type of tumor may be targeted in accordance with this 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, testes, kidneys, bladder, head, neck, cervix, larynx, and / or skin. For example, this application provides methods and compositions for targeting cervical cancer cells, ovarian cancer cells, melanoma cancer cells, lung cancer cells, head and / or cervical cancer cells, and bladder cancer cells. Other tumors may also be targeted.

[0058] In some embodiments, the tumor is a tumor or lesion of the eye. The tumor or lesion of the eye may be located in the vitreous humor, choroidal space, suprachoroidal space, iris, ciliary body, sclera, fovea, retina, optic disc, or optic nerve. Thus, in some embodiments, a subject administered with the ophthalmic composition of the Disclosure has a tumor of the eye. The tumor of the eye 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 unclassifiable lesion. In some embodiments, a subject administered with the ophthalmic composition of the Disclosure has an unclassifiable lesion.

[0059] In some embodiments, subjects administered with the ophthalmic compositions of this disclosure have choroidal metastases originating somewhere in the body and propagating to the eyes. For example, such choroidal metastases may originate from breast cancer or, in men, from lung cancer.

[0060] The ophthalmic compositions of this disclosure are typically administered via intravitreous injection or suprachoroidal injection, but other routes of administration are contemplated herein.

[0061] In some embodiments, the ophthalmic composition (or any of its components) is formulated as a liquid. In some embodiments, the ophthalmic composition (or any of its components) is freeze-dried.

[0062] Further embodiments 1. An ophthalmic composition comprising a nearly isotonic solution of a VLP drug conjugate containing a photosensitive molecule conjugated to the capsid protein of a virus-like particle (VLP), wherein the VLP drug conjugate is present in a suspension. 2. An ophthalmic composition comprising a VLP drug conjugate containing a photosensitive molecule conjugated to the capsid protein of a virus-like particle (VLP), wherein the VLP drug conjugate does not aggregate to form visible particulate matter. 3. An ophthalmic composition according to paragraph 1 or 2, having a pH value of less than 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 cleansing agent. 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. The ophthalmic composition according to paragraph 6, further comprising at least two reagents selected from trehalose dihydrate, MgCl2, NaCl, and PS80. 8. The ophthalmic composition according to paragraph 7, further comprising at least three reagents selected from trehalose dihydrate, MgCl2, NaCl, and PS80. 9. The 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, comprising 0.1% to 1.0% (w / v) MES or a pharmaceutically acceptable salt thereof (e.g., MES hemi sodium salt). 11. The ophthalmic composition described in paragraph 10, comprising 0.4% (w / v) MES. 12. The ophthalmic composition according to any one of paragraphs 1 to 11, comprising 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, comprising 0.1% to 1.0% (w / v) NaCl. 15. The ophthalmic composition described in paragraph 14, comprising 0.4% (w / v) NaCl. 16. The ophthalmic composition according to any one of paragraphs 1 to 15, comprising 0.1% to 1.0% (w / v) MgCl2. 17. The ophthalmic composition described in paragraph 16, comprising 0.2% (w / v) MgCl2. 18. The ophthalmic composition according to any one of paragraphs 1 to 17, wherein the composition comprises 0.01% to 0.1% (w / v) PS80. 19. The ophthalmic composition described in paragraph 18, comprising 0.05% (w / v) PS80. 20. The ophthalmic composition according to any one of paragraphs 1 to 19, comprising 0.01% to 0.5% (w / v) VLP drug conjugate. 21. The ophthalmic composition according to paragraph 20, comprising 0.01% to 0.1% (w / v) VLP drug conjugate. 22. The ophthalmic composition according to paragraph 21, wherein the composition comprises a 0.04% VLP drug conjugate. 23. The ophthalmic composition according to any one of paragraphs 3 to 22, having a pH value of 6.5. 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 the capsid protein of the VLP. 25. The ophthalmic composition according to any one of paragraphs 1 to 24, wherein the photosensitive molecule comprises a dye molecule. 26. The ophthalmic composition according to paragraph 25, wherein the dye molecule comprises a phthalocyanine dye molecule. 27. The ophthalmic composition according to paragraph 26, wherein the phthalocyanine dye molecule comprises IRDye® 700DX. 28. The ophthalmic composition according to any one of paragraphs 1 to 27, wherein 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. 29. The ophthalmic composition according to any one of paragraphs 1 to 28, wherein the VLP comprises 50 to 500 photosensitive molecules. 30. The ophthalmic composition according to any one of paragraphs 1 to 29, wherein the VLP comprises a papillomavirus capsid protein. 31. The ophthalmic composition according to paragraph 30, wherein the papillomavirus capsid protein is human papillomavirus capsid protein. 32. The ophthalmic composition according to paragraph 31, wherein the papillomavirus capsid protein comprises an L1 capsid protein, an L2 capsid protein, or a combination of L1 and L2 capsid proteins. 33. The ophthalmic composition according to paragraph 32, wherein the L1 capsid protein is modified to reduce the immunogenicity of the VLP. 34. A method comprising the step of administering an ophthalmic solution described in any one of paragraphs 1 to 33 to the eye of a subject, wherein the subject has an intraocular melanoma, and the ophthalmic solution is administered in an amount effective for treating the intraocular melanoma. 35. The method according to paragraph 34, wherein the intraocular melanoma is a uveal melanoma or a choroidal melanoma. 36. A method comprising the step of administering an ophthalmic solution described in any one of paragraphs 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. 37. The ophthalmic composition is injected intravitreously according to any one of paragraphs 34 to 36. 37. A method comprising the step of administering an ophthalmic solution described in any one of paragraphs 1 to 33 to the eye of a subject, 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 intravitreously, according to any one of paragraphs 34 to 37. 39. The method according to any one of paragraphs 34 to 37, wherein the ophthalmic composition is injected into the suprachoroidal space of the eye. 40. The method according to paragraph 39, wherein the ophthalmic composition remains in the suprachoroidal space of the eye for at least one week. 41. The method according to paragraph 39 or 40, wherein no leukocyte infiltration is observed in the ciliary body and / or sclera at least 35 days after injection of the ophthalmic composition. 42. The method according to any one of paragraphs 34 to 41, wherein optical coherence tomography is normal in the eye of the subject after injection of the ophthalmic composition. 43. The method according to any one of paragraphs 34 to 42, wherein the intraocular pressure is normal in the eye of the subject after injection of the ophthalmic composition. 44. The ophthalmic composition described in any one of the preceding paragraphs, wherein the ophthalmic composition is free of DNase and / or RNase. 45. The ophthalmic composition described in any one of the preceding paragraphs, wherein the ophthalmic composition is sterile.

[0063] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter they refer to, and in some cases may encompass the entire document.

[0064] The indefinite articles "a" (one, one) and "an" (one, one), as used herein and in the claims, should be understood to mean "at least one" unless the opposite is explicitly stated.

[0065] Unless the opposite is clearly indicated, it should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of that method is not necessarily limited to the order in which the steps or actions of that method are described.

[0066] In the claims and the above specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and “composed of” should be understood to mean unrestrictive, that is, including but not limited to these. Only the transitional phrases “consisting of” and “consisting essentially of” are considered restrictive or semi-restrictive transitional phrases, respectively, as set forth in Section 2111.03 of the U.S. Patent and Trademark Office's Examination Manual.

[0067] The terms “approximately” and “substantially” preceding a number mean ±10% of the stated number. Where a range of values ​​is provided, each value between the upper and lower limits of that range is specifically intended and described herein. [Examples]

[0068] In the initial evaluation of VLP drug conjugate formulations, we tested historical phosphate-based high-salt formulations and three alternative formulations to assess the parameters of the formulation material in relation to in-process recovery and repeated freeze-thaw cycles during bulk VLP drug conjugate formulation. The collected data included protein recovery by Bradford whole protein assay, A280, SDS-PAGE band formation patterns, VLP morphology by transmission electron microscopy, VLP size distribution by dynamic light scattering, VLP drug conjugate efficacy, ocular distribution of VLP conjugates after suprachoroidal injection, and in vivo safety of VLP drug conjugates.

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

[0070] Example 1: Process Recovery Bulk VLP drug conjugates were prepared by buffer exchange and concentration using tangential flow filtration (TFF) into four formulations (A, B, C, D). The control included a previously used phosphate / high-salt based formulation. Three further trehalose-containing formulations were evaluated with the intention that the combination of buffer pH and trehalose excipient would protect the bioconjugate. Findings gathered during the TFF procedure included visible aggregation formation in the PBS / high-salt control formulation and phosphate-based trehalose / PS80-containing formulation A. No evidence of aggregation was observed for HEPES or MES-based formulations containing trehalose and PS80 (formulations B and C, respectively). Process recovery was determined using the Bradford whole protein assay. Two phosphate-based formulations (in which evidence of agglutination was observed) resulted in approximately 48–58% process recoveries, which was significantly lower than the recoveries for the HEPES-based formulation (69.7%) and the MES-based formulation (79.3%) (Table 1). The concentrations of the final formulations ranged from 0.25 to 0.66 mg / mL. To avoid further processing and sample loss, it was decided to characterize these samples without further processing. [Table 1]

[0071] Example 2: Recovery after freezing and thawing The samples were filled into 2.0 mL CZ resin vials. Many vials from each formulation were divided into 2-8 oWhile samples were stored at 1C, additional vials were frozen. Samples were subjected to one, three, or five freeze-thaw cycles, and total protein recovery was determined using the Bradford assay or UV-VIS. This data is shown in Table 2 below and as graphs in Figures 1 and 2. [Table 2]

[0072] Within the error margin of the analytical assay used (estimated at approximately 20%), no apparent protein loss was observed, regardless of final formulation conditions or the number of freeze-thaw cycles. Consistent recovery was also obtained from each sample when total protein recovery was tracked by analysis 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 also imaged by standard Coomassie staining and fluorescence scanning using an Odyssey scanner to detect the fluorescence of the DX700 dye. Newly prepared samples and samples subjected to one, three, or five 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 that underwent five 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, representing the L1 protein, may be observed, moving at approximately 55 kDa. A faint band may also be observed above the 62 kDa marker, representing the L2 protein. Above the 98 kDa marker, a species ladder is observed, indicating the presence of a certain form of the oligomeric material. The properties of this substance are the subject of other technical reports, and LC-MS analysis of gel-digested samples performed by SGS reveals that this substance is a protein containing L1 and L2 sequences. Visually, its band formation pattern is consistent across all samples, and the band intensity and fluorescence signal can be observed to correspond to Bradford and absorbance datasets.

[0074] Example 4: Morphology and particle size distribution of virus-like particles by transmission electron microscopy. Particle size was estimated by measuring the diameter of approximately 60–90 particles, and the capsomer material was assembled using AMT software. The majority of VLPs appeared to assemble completely and were observed to have a certain size range, with shapes being primarily spherical or ellipsoidal (Figure 4). A similar range in apparent shape was 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 graphed in Figure 5. During the freeze-thaw stability studies, no significant differences in VLP drug-conjugate particle size were observed between the three buffer conditions evaluated—control and between formulations B and C. Formulation A was not evaluated using TEM. No significant changes in VLP drug-conjugate size range distribution or macroscopic morphological characteristics were observed throughout the numerous freeze-thaw cycles during the study. [Table 3]

[0075] Example 5: Particle size analysis using dynamic light scattering To complement TEM morphological and size distribution analysis, 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 the motion and structural changes of nanoparticles in solution (i.e., degradation or VLP oligomerization), providing information on the average particle size and frequency distribution. DLS data are summarized in Table 4 and shown as a graph in Figure 6. Both show average diameter values ​​by number (N), volume (V), and intensity (I). Each shows a snapshot of the average particle size distribution and reports the average value of the overall distribution. No significant changes were observed in the average particle size distribution, regardless of formulation or the number of freeze-thaw cycles. In summary, the DLS and TEM measurements of VLPs are in agreement, indicating that VLPs are of comparable size and morphology, regardless of formulation or the number of freeze-thaw cycles. [Table 4]

[0076] Example 6: Efficacy Evaluation The functional properties of VLP drug conjugates were evaluated using an in vitro cell death assay. Data generated for each of the four tested formulations—freshly prepared material and material stored in a final sealed container and subjected to one, three, or five freeze-thaw cycles—are shown in Figures 7A–7D. Due to limitations in personnel capacity and the assay itself, data points were limited to one of the predicted EC50s. Regardless of formulation or the number of freeze-thaw cycles, the VLP drug conjugates remained potent.

[0077] Example 7: Distribution of VLP drug conjugates in the eyes after suprachoroidal injection in New Zealand White rabbits. The in vivo ocular distribution of VLP conjugates was evaluated by injecting AlexaFluor488*VLP into the superior choroidal space (SCS) of New Zealand White (NZW) rabbits (Study PK-RPE-003). AlexaFluor488*VLP was used instead of VLP drug conjugates because it has similar physiological and chemical characteristics, is formulated in the same MES buffer as VLP drug conjugates, but is more suitable for in vivo imaging. Ocular distribution was evaluated over time by optical coherence tomography (OCT) and fundus autofluorescence (FAF). Data showed that after 100 μl injection into the SCS, approximately 75% were located posteriorly within the eyeball at ≤0.5 hours post-administration and remained relatively constant throughout the duration of the study. Fluorescence was strong throughout the 168-hour post-administration interval and began to weaken at the 240-hour post-administration interval. No significant fluorescence above baseline was detected at the 504-hour post-administration interval. This data suggests that the VLP drug conjugate formulated in MES buffer is adequately distributed in the SCS lumen, and that this distribution lasts for at least 168 hours (1 week).

[0078] Example 8: In vivo safety evaluation of VLP drug conjugates (non-clinical) To evaluate the non-clinical safety of VLP drug conjugates, they were administered in a canine study. The VLP conjugates were administered at a single dose level of 20 μg / eye via 100 μl injection into the superior choroidal space (SCS), followed by laser photoactivation 6–8 hours after injection. Dogs were treated once a week for three weeks (a total of three injections, followed by 50 J / cm² of laser treatment 6–8 hours after injection each week). 2 (Laser treatment). After the third weekly treatment, animals were observed for 7 days (ultimately sacrificed) or 35 days (recovery phase) to assess the reversibility, duration, or delayed onset of the effect. There were very small microscopic ocular findings associated with VLP drug conjugates. Histopathology showed minimal / slight leukocyte infiltration into the choroidal space, with 50% of animals having minimal leukocyte infiltration into the ciliary body and sclera. Minimal / slight leukocyte infiltration dissipated between the first observation time point (animals ultimately sacrificed at 7 days) and the second observation time point (animals in the recovery phase at 35 days) where it was not observed. Optical coherence tomography (OCT) was normal in all animals (i.e., no retinal pigment epithelial (RPE) / retinal changes or retinal thinning were detected), and all eyes examined retained normal retinal structure. Intraocular pressure (IOP) in all test animals was assessed during the study and was normal in all animals. No systemic clinical findings, changes in body weight, or clinicopathological findings were reported. In summary, the data suggest that the VLP drug conjugate formulated in MES buffer and delivered as multiple SCS injections is safe in vivo.

[0079] material and method sample The VLP drug conjugates were formulated using four different combinations, as outlined in Table 5. [Table 5]

[0080] Device The apparatus necessary to carry out the formulation process described herein is provided in Table 6. [Table 6]

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

[0082] VLP drug conjugate manufacturing and formulation VLP drug conjugates were prepared from bulk VLP preparations. Bioconjugation was performed by diluting 1:1 with 2× labeled buffer containing 100 mM HEPES (pH 7.5), 20 mM MgCl2, and 10% trehalose dihydrate, followed by incubation in a calculated molar excess of 300:1 (dye:VLP) on a DX700-NHS Ester for 2 hours. Total protein concentration was determined by the Bradford whole protein assay (Pierce Bradford protein assay catalog no. 23200) using a micro-microplate procedure according to the manufacturer's instructions. The stock VLP drug conjugates were then buffer-exchanged to a control formulation (phosphate / high salt) and three candidate formulations (each containing trehalose and PS80 at three different pH levels (A, B, and C)). The overall experimental flow is shown in Figure 8.

[0083] product strength The product strength of VLP drug conjugates was determined by the Bradford whole protein assay (Pierce Bradford Protein Assay Catalog No. 23200) according to the manufacturer's instructions for the microplate format. Undiluted samples were subjected to A280nm using a nanodrop UV-VIS spectrophotometer with a sample-specific buffer blank.

[0084] SDS-PAGE analysis SDS-PAGE analysis of VLP drug conjugate products was performed using 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 on the Odyssey CLx.

[0085] Dynamic light scattering 50 μL of the test sample was added to 150 μL of filtered PBS (phosphate-buffered saline, Boston Bioproducts catalog no. BM220-S) and gently mixed by pipetting five times. The diluted sample was then pipetted into a plastic cuvette (Fisher catalog no. 14-955-125), which 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 25°C for 2 minutes per run with a 90° detector angle. Four distinct 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 runs.

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

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

1. An ophthalmic composition comprising a nearly isotonic solution of a VLP drug conjugate containing a photosensitive molecule conjugated to the capsid protein of a virus-like particle (VLP), wherein (i) the VLP drug conjugate is in a suspended state and does not aggregate to form visible particulate matter, (ii) the nearly isotonic solution comprises 2-(N-morpholino)ethanesulfonic acid (MES), trehalose dihydrate, polysorbate 80, and a 50 mM to 100 mM salt, and (iii) the nearly isotonic solution has an isotonicity of 255 to 345 mOsm / L.

2. The ophthalmic composition according to claim 1, having a pH value of less than 7.

3. The ophthalmic composition according to claim 1 or 2, comprising at least one pharmaceutically acceptable excipient.

4. The salt is magnesium chloride (MgCl ２ An ophthalmic composition according to any one of claims 1 to 3, comprising ) and sodium chloride (NaCl).

5. The ophthalmic composition according to any one of claims 1 to 4, comprising 0.1% to 1.0% (w / v) MES.

6. The ophthalmic composition according to claim 5, wherein the composition comprises 0.4% (w / v) MES.

7. The ophthalmic composition according to any one of claims 1 to 6, comprising 1% to 10% (w / v) trehalose dihydrate.

8. The ophthalmic composition according to claim 7, wherein the composition comprises 5% (w / v) trehalose dihydrate.

9. The composition is an ophthalmic composition according to any one of claims 1 to 8, comprising 0.2% to 0.6% (w / v) NaCl.

10. The ophthalmic composition according to claim 9, wherein the composition comprises 0.4% (w / v) NaCl.

11. The composition comprises 0.1% to 1.0% (w / v) MgCl ２ An ophthalmic composition according to any one of claims 1 to 10, comprising:

12. The composition comprises 0.2% (w / v) MgCl ２ The ophthalmic composition according to claim 11, comprising:

13. The ophthalmic composition according to any one of claims 1 to 12, comprising 0.01% to 0.1% (w / v) polysorbate 80.

14. The ophthalmic composition according to claim 13, wherein the composition comprises 0.05% (w / v) polysorbate 80.

15. The ophthalmic composition according to any one of claims 1 to 14, comprising 0.01% to 0.5% (w / v) VLP drug conjugate.

16. The ophthalmic composition according to claim 15, wherein the composition comprises 0.01% to 0.1% (w / v) of a VLP drug conjugate.

17. The ophthalmic composition according to claim 16, wherein the composition comprises 0.04% (w / v) VLP drug conjugate.

18. The composition is an ophthalmic composition according to any one of claims 2 to 17, having a pH value of 6.

5.

19. 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 the capsid protein of the VLP.

20. The ophthalmic composition according to any one of claims 1 to 19, wherein the photosensitive molecule comprises a dye molecule.

21. The ophthalmic composition according to claim 20, wherein the aforementioned pigment molecule comprises a phthalocyanine pigment molecule.

22. The ophthalmic composition according to claim 21, wherein the phthalocyanine dye molecule comprises IRDye® 700DX.

23. The ophthalmic composition according to any one of claims 1 to 22, wherein the VLP comprises 10 to 1,000 photosensitive molecules, 10 to 500 photosensitive molecules, 50 to 1,000 photosensitive molecules, 50 to 500 photosensitive molecules, 100 to 1,000 photosensitive molecules, or 100 to 500 photosensitive molecules.

24. The ophthalmic composition according to any one of claims 1 to 23, wherein the VLP comprises 50 to 500 photosensitive molecules.

25. The ophthalmic composition according to any one of claims 1 to 24, wherein the VLP comprises a papillomavirus capsid protein.

26. The ophthalmic composition according to claim 25, wherein the papillomavirus capsid protein is human papillomavirus capsid protein.

27. The ophthalmic composition according to claim 26, wherein the papillomavirus capsid protein comprises an L1 capsid protein, an L2 capsid protein, or a combination of L1 and L2 capsid proteins.

28. The ophthalmic composition according to claim 27, wherein the L1 capsid protein is modified to reduce the immunogenicity of the VLP.

29. An ophthalmic composition according to any one of claims 1 to 28, wherein the ophthalmic composition is administered to the eye of a subject, wherein the subject has an intraocular melanoma, and the ophthalmic composition is administered in an amount effective for treating the intraocular melanoma.

30. The ophthalmic composition according to claim 29, wherein the intraocular melanoma is a uveal melanoma or a choroidal melanoma.

31. An ophthalmic composition according to any one of claims 1 to 28, wherein the ophthalmic composition is administered to the eye of a subject, wherein the subject has an unclassifiable lesion, and the ophthalmic composition is administered in an amount effective to treat the unclassifiable lesion.

32. An ophthalmic composition according to any one of claims 1 to 28, wherein the ophthalmic composition is administered to the eye of a subject, wherein the subject has choroidal metastasis, and the ophthalmic composition is administered in an amount effective to treat the choroidal metastasis.

33. The ophthalmic composition described above is injected into the vitreous humor, as described in any one of claims 29 to 32.

34. The ophthalmic composition according to any one of claims 29 to 32, wherein the ophthalmic composition is injected into the suprachoroidal space of the eye.

35. The ophthalmic composition according to claim 34, wherein the ophthalmic composition remains in the suprachoroidal space of the eye for at least one week.

36. The ophthalmic composition according to claim 34 or 35, wherein no leukocyte infiltration is observed in the ciliary body and / or sclera at least 35 days after injection of the ophthalmic composition.

37. The ophthalmic composition according to any one of claims 29 to 36, wherein optical coherence tomography is normal in the eye of the subject after injection of the ophthalmic composition.

38. The ophthalmic composition according to any one of claims 29 to 37, wherein the intraocular pressure is normal in the eye of the subject after injection of the ophthalmic composition.

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