Virus-like particle conjugates for diagnosis and treatment of tumors

Abstract

The present application relates to virus-like particle conjugates for the diagnosis and treatment of tumors. The present disclosure relates to methods and compositions for diagnosing and / or treating tumors, such as ocular tumors, using virus-like particles conjugated to photosensitive molecules.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201480057289.9 and invention name “Virus-like particle conjugates for diagnosing and treating tumors”. The original application is the PCT international application PCT / US2014 / 056412 filed on September 18, 2014, which entered the Chinese national phase on April 18, 2016. Related applications

[0002] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 61 / 879,627, filed September 18, 2013, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of tumor diagnosis and treatment. Background Art

[0004] Although many treatments are available for cancer, many forms of cancer remain incurable, untreatable, or become resistant to standard treatments, and effective treatments for many cancers have undesirable side effects. Treatment of ocular cancers (e.g., ocular melanoma and retinoblastoma) is particularly challenging. Patients diagnosed with ocular melanoma have few treatment options depending on the size of the tumor, including: (1) surgery, such as resection, enucleation, or exenteration, all of which are highly invasive and primarily involve removing part of the eye and optic nerve (after surgery, the patient is typically fitted with an artificial eye); and (2) plaque brachytherapy, which is a type of radiation therapy in which a thin metal (e.g., gold) sheet covered on one side with radioactive seeds is sutured to the outer wall of the eye and the seeds are targeted to the tumor. At the end of treatment, the thin metal sheet is removed, and treatment typically lasts several days. Serious radiation-related complications include: most commonly cataract formation, followed by vitreous hemorrhage. Other complications include dry eyes, keratitis, radiation-induced iris neovascularization, neovascular glaucoma, radiation-induced retinopathy, radiation-induced optic neuropathy, episcleral deposits, scleral necrosis, and / or extraocular muscle changes. Radiation retinopathy has been reported to occur in 10% to 63% of patients treated with plaque brachytherapy, with an average time from treatment to the development of maculopathy of approximately 25.6 months. Summary of the Invention

[0005] The present disclosure provides, at least in part, methods and compositions for detecting and / or selectively targeting tumor cells (e.g., for diagnosing and / or treating cancer (e.g., eye cancer)). In some cases, the methods and compositions provided herein can be used to selectively kill cancerous tumor cells without damaging healthy cells. For example, virus-like nanoparticles comprising a photosensitive molecule (e.g., conjugated to a photosensitive molecule) can be selectively delivered to tumor cells and photoactivated by exposure to light. When photoactivated, the photosensitive molecule absorbs photons, and the absorbed energy causes molecular changes that cause toxicity (e.g., cytotoxicity). "Photosensitive virus-like nanoparticles" (also referred to herein as "photosensitive virus-like particles") refer to virus-like nanoparticles conjugated to a photosensitive molecule. Surprisingly, the conjugation of the photosensitive molecule to the virus-like nanoparticle does not interfere with the tissue / tumor tropism of the nanoparticle (e.g., the specificity of the virus-like nanoparticle for a particular host tumor tissue or tumor cell).

[0006] The virus-like nanoparticles (also referred to as virus-like particles (VLPs)) of the present disclosure are generally assembled by L1 capsid protein or a combination of L1 and L2 capsid proteins, and in some embodiments, the photosensitive molecule is conjugated to the capsid protein that forms the virus-like nanoparticle. Therefore, various aspects of the present disclosure provide tumor-targeting virus-like nanoparticles comprising a photosensitive molecule conjugated to the capsid protein.

[0007] Some aspects of the present disclosure also provide tumor-targeting virus-like particles comprising about 50 to about 500, about 50 to about 600, about 50 to about 700, about 50 to about 800, about 50 to about 900, or about 50 to about 1000 photosensitive molecules per particle. In some embodiments, the tumor-targeting virus-like particles comprise about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 photosensitive molecules per particle. In some embodiments, the tumor-targeting virus-like particles comprise 500 photosensitive molecules or 1000 photosensitive molecules per particle.

[0008] In some embodiments, the capsid protein is a papilloma virus capsid protein. For example, in some embodiments, the papilloma virus capsid protein is a non-human papilloma virus capsid protein, such as a bovine papilloma virus capsid protein. In some embodiments, the virus-like particle comprises a human papilloma virus capsid protein and does not cross-react with human papilloma virus (HPV) 16, HPV 18, or pre-existing antibodies specific for HPV.

[0009] In some embodiments, the virus-like particles comprise papillomavirus L1 or L1 / L2 proteins (e.g., human, bovine, or other species). In some embodiments, the L1 or L1 / L2 VLPs do not cross-react with neutralizing antibodies against human papillomavirus (HPV) 16, HPV18, or existing antibodies specific for other HPVs. However, in some embodiments, the virus-like particles comprise human papillomavirus capsid proteins of HPV16.

[0010] In some embodiments, the photosensitive molecule is conjugated to a surface-exposed peptide of the capsid protein.

[0011] In some embodiments, the virus-like particle comprises L1 capsid protein or a combination of L1 and L2 capsid proteins. In some embodiments, the virus-like particle consists of L1 capsid protein.

[0012] In some embodiments, the virus-like particle comprises a BPV L1 capsid protein (e.g., SEQ ID NO: 2), a combination of BPV L1 and BPV L2 capsid proteins. In some embodiments, the virus-like particle comprises an HPV L1 capsid protein or a combination of HPV L1 and HPV L2 capsid proteins. In some embodiments, the HPV L1 capsid protein is a variant HPV16 / 31 L1 protein (e.g., SEQ ID NO: 1) having modified immunogenicity and / or antigenicity. Thus, in some embodiments, the virus-like particle comprises or consists of a variant HPV16 / 31 L1 capsid protein or a combination of a variant HPV16 / 31 L1 capsid protein (e.g., SEQ ID NO: 1) and an HPV L2 capsid protein.

[0013] In some embodiments, the capsid protein of the virus-like particle has modified immunogenicity and / or antigenicity. A non-limiting example of such a capsid protein is the HPV16 / 31 L1 capsid protein (e.g., SEQ ID NO: 1). Compared to wild-type virus-like particles, virus-like particles comprising modified capsid proteins may be referred to herein as virus-like particles comprising modified immunogenicity and / or antigenicity.

[0014] In some embodiments, the photosensitive molecule is covalently conjugated to the capsid protein. In some embodiments, the photosensitive molecule is conjugated to an amino acid of the capsid protein. In some embodiments, the photosensitive molecule is conjugated to an amine group (e.g., aliphatic primary amine) of an amino acid of the capsid protein. In some embodiments, the photosensitive molecule is conjugated to an amine group (e.g., a side chain amine of lysine) of a capsid protein. In some embodiments, the photosensitive molecule is conjugated to an amine group of an arginine and / or histidine residue of a capsid protein. The present disclosure provides methods for conjugating photosensitive molecules to lysine and other amino acids containing amine groups.

[0015] In some embodiments, the photosensitive molecule does not compromise (e.g., prevent, interfere with, or inhibit) the binding of the virus-like particles to the surface of tumor cells. In some embodiments, the photosensitive molecule does not compromise (e.g., prevent, interfere with, or inhibit) the binding of the virus-like particles to heparan sulfate proteoglycan or other polysaccharides on the surface of tumor cells.

[0016] In some embodiments, the virus-like particle comprises from about 10 to about 1000 photosensitive molecules. In some embodiments, the virus-like particle comprises from about 50 to about 1000 photosensitive molecules. In some embodiments, the virus-like particle comprises from about 100 to about 1000 photosensitive molecules. In some embodiments, the virus-like particle comprises from about 100 to about 500 photosensitive molecules. In some embodiments, the virus-like particle comprises from about 500 to about 1000 or more photosensitive molecules.

[0017] In some embodiments, the virus-like particle comprises about 10 to about 1000 photosensitive molecules conjugated to lysine residues or other amino acid residues of the L1 capsid protein, the L2 capsid protein, or a combination of the L1 capsid protein and the L2 capsid protein.

[0018] In some embodiments, the photosensitive molecule is activated by infrared light, near infrared light, or ultraviolet light. A photosensitive molecule is considered to be "activated" when it absorbs a photon and the absorbed energy results in a molecular change that causes toxicity, as described elsewhere herein.

[0019] In some embodiments, the photosensitive molecule comprises a fluorescent dye, an infrared dye, a near-infrared dye, a porphyrin molecule, a chlorophyll molecule, or a combination of any two or more of the foregoing.

[0020] In some embodiments, the photosensitive molecule is a porphyrin molecule. Examples of porphyrin molecules used in accordance with the present disclosure include, but are not limited to, HpD (hematoporphyrin derivative), HpD-based molecules, BPD (benzoporphyrin derivative), ALA (5-aminolevulinic acid), and texaphyrin. In some embodiments, the porphyrin molecule is verteporfin ( ).

[0021] In some embodiments, the photosensitive molecule is a chlorophyll molecule. Examples of chlorophyll molecules for use in accordance with the present disclosure include, but are not limited to, chlorin, purpurin, and bacteriochlorin.

[0022] In some embodiments, the photosensitive molecule is a dye.Examples of dyes for use in accordance with the present disclosure include, but are not limited to, phthalocyanine and napthalocyanine.

[0023] In some embodiments, the phthalocyanine dye is both a fluorescent molecule and a near infrared molecule. For example, in some embodiments, the phthalocyanine dye is an IR700 dye (e.g., 700DX, IR700 dye is a fluorescent dye that absorbs and emits wavelengths in the near-infrared (NIR) spectrum, typically between 680 nm and 800 nm. Other fluorescent dyes that absorb and emit wavelengths in the NIR spectrum are also provided herein.

[0024] In some embodiments, the photosensitive molecule is selected from: phthalocyanine dyes (e.g., IR700 dyes, e.g. 700DX), porphyrin molecules (e.g., verteporfin, e.g. ) and combinations of phthalocyanine dyes and porphyrin molecules.

[0025] Some aspects of the present disclosure provide methods comprising administering any one of the virus-like particles or photosensitive virus-like particles provided herein to a subject having a tumor. In some embodiments, the method comprises activating the photosensitive molecule of the virus-like particle at a wavelength that allows visualization of the photosensitive molecule. Thus, in some embodiments, the photosensitive molecules of the present disclosure are used as imaging agents and / or diagnostic agents. In some embodiments, the method comprises activating the molecule at a wavelength of light that causes the photosensitive molecule to be cytotoxic. In some embodiments, the method comprises activating the photosensitive molecule at a wavelength of light that produces energy transfer within the tumor cell that causes direct and irreversible cell damage leading to necrosis. Thus, in some embodiments, the photosensitive molecules of the present disclosure are used as therapeutic and / or prophylactic agents.

[0026] Some aspects of the present disclosure provide methods comprising administering to a subject having a tumor a tumor-targeting virus-like particle comprising a photosensitive molecule conjugated to a capsid protein. In some embodiments, the method comprises activating the molecule of the virus-like particle at a wavelength that renders the photosensitive molecule visible. That is, the photosensitive molecule re-emitted light after photoexcitation. In some embodiments, the method comprises activating the molecule at a wavelength that renders the photosensitive molecule cytotoxic, thereby killing tumor cells. That is, the photosensitive molecule undergoes molecular changes after photoexcitation that cause the photosensitive molecule to become toxic to cells.

[0027] Some aspects of the present disclosure provide methods comprising administering to a subject having a tumor a tumor-targeting virus-like particle comprising about 50 to about 1000, about 50 to 500, or about 500 to 1000 photosensitive molecules. In some embodiments, the method comprises administering to a subject having a tumor a tumor-targeting virus-like particle comprising 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more photosensitive molecules. In some embodiments, the method comprises activating the molecule at a wavelength that makes the photosensitive molecule visible. In some embodiments, the method comprises activating the molecule at a wavelength that makes the photosensitive molecule cytotoxic, thereby killing tumor cells.

[0028] In some embodiments, the photosensitive molecule is laser activated. In some embodiments, the laser is an infrared laser, a near infrared laser, or an ultraviolet laser. In some embodiments, the infrared laser is 5 joules (J) to 100 J (or J / cm 2)(For example, 5J, 6J, 7J, 8J, 9J, 10J, 11J, 12J, 13J, 14J, 15J, 16J, 17J, 18J, 19J, 20J, 21J, 22J, 23J, 24J, 25J, 26J, 27J, 28J, 2 9J, 30J, 31J, 32J, 33J, 34J, 35J, 36J, 37J, 38J, 39J, 40J, 41J, 42J, 43J, 44J, 45J, 46J, 47J, 48J, 49J, 50J, 51J, 52J, 53J , 54J, 55J, 56J, 57J, 58J, 59J, 60J, 61J, 62J, 63J, 64J, 65J, 66J, 67J, 68J, 69J, 70J, 71J, 72J, 73J, 74J, 75J, 76J, 77J, 78J, 79J, 80J, 81J, 82J, 83J, 84J, 85J, 86J, 87J, 88J, 89J, 90J, 91J, 92J, 93J, 94J, 95J, 96J, 97J, 98J, 99J or 100J (or J / cm 2 )). In some embodiments, the laser is applied for about 5 seconds to about 5 minutes.

[0029] In some embodiments, the photosensitive molecule is activated about 30 minutes to about 48 hours after the virus-like particle is administered to the subject. For example, the photosensitive molecule can be activated 30 minutes after the virus-like particle is administered to the subject. In some embodiments, the photosensitive molecule is activated 1 hour, 2 hours (h), 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 or 24 hours after the virus-like particle is administered to the subject. In some embodiments, the photosensitive molecule is activated 1 day, 2 days or 3 days after the virus-like particle is administered to the subject.

[0030] In some embodiments, the tumor is an eye tumor or a tumor that has metastasized to the eye. For example, in some embodiments, the eye tumor is located in the vitreous, choroidal space, iris, ciliary body, sclera, fovea, retina, optic disc, or optic nerve.

[0031] In some embodiments, the tumor is located in the lung, pleura, liver, pancreas, stomach, esophagus, colon, breast, ovary, prostate, brain, meninges, testicles, gastrointestinal tract, kidney, or bladder.

[0032] In some embodiments, the tumor is accessible without surgical intervention.

[0033] In some embodiments, the tumor is located in the head, neck, cervix, throat, or skin.

[0034] In some embodiments, the tumor is an orphan disease or a rare disease.

[0035] In some embodiments, the tumor is cancerous. In some embodiments, the tumor is metastatic. In some embodiments, the tumor is precancerous or dysplastic.

[0036] In some embodiments, the virus-like particles are administered by injection. For example, the virus-like particles can be administered by intraocular injection into the vitreous or can be administered intravenously. In some embodiments, the virus-like particles are administered with a hollow needle or a coated needle, a mini-needle, or a micro-needle. In some embodiments, the virus-like particles are administered topically. In some embodiments, the virus-like particles are administered by implantation.

[0037] In some embodiments, the capsid protein is a papillomavirus capsid protein. For example, in some embodiments, the papillomavirus capsid protein is a non-human papillomavirus capsid protein, such as a bovine papillomavirus (BPV) capsid protein. In some embodiments, the virus-like particle comprises a human papillomavirus capsid protein and does not cross-react with human papillomavirus (HPV) 16, HPV 18, or existing antibodies specific for HPV. In some embodiments, the virus-like particle comprises a human papillomavirus type 16 capsid protein. In some embodiments, the VLP does not bind to antibodies specific for human papillomavirus (HPV) 16, HPV 18 VLPs, or existing antibodies specifically induced by HPV infection.

[0038] Some aspects of the present disclosure provide methods for monitoring tumors (e.g., eye tumors and malignant nevi) in a subject, the method comprising administering any one of the virus-like particles provided herein (e.g., a virus-like particle comprising a photosensitive molecule (e.g., a fluorescent dye or an infrared dye)) to the subject (e.g., to the eye of the subject) and detecting the location of the tumor. In some embodiments, the method comprises detecting the location of the tumor by irradiating the subject (e.g., the eye of the subject) with a laser (e.g., an ultraviolet laser or an infrared laser). In some embodiments, the method comprises identifying a subject suspected of having a tumor before administering the virus-like particles. In some embodiments, the method comprises diagnosing and / or treating a tumor by administering a photosensitive virus-like particle to a subject having or suspected of having a tumor or to a tumor of a subject.

[0039] Still other aspects of the present disclosure provide methods for selectively inhibiting cancer cell proliferation or killing cancerous cells without inhibiting the proliferation or viability of non-cancerous (e.g., normal, healthy) cells, the methods comprising administering any one of the tumor-targeting virus-like particles provided herein (e.g., a virus-like particle comprising a photosensitive molecule (e.g., an infrared dye)) to a tumor in a subject (e.g., to an ocular tumor in the subject), and effectively irradiating the cancerous cells of the tumor by subjecting the tumor to infrared laser light (e.g., at a wavelength of about 660 nm to about 740 nm and a dose of at least 8 joules).

[0040] In some embodiments, the present disclosure provides virus-like nanoparticles (also referred to as virus-like particles) comprising a photosensitive molecule conjugated to a papillomavirus L1 protein (e.g., a bovine papillomavirus L1 protein). In some embodiments, the virus-like nanoparticle has a diameter of 20 to 60 nanometers (e.g., 10, 25, 30, 35, 40, 45, 50, 55, or 60 nanometers). In some embodiments, the virus-like nanoparticle comprises 300 to 500 L1 (e.g., BPV L1) capsid proteins, for example, 360 L1 capsid proteins (e.g., based on icosahedral symmetry). It will be appreciated that in some embodiments, each of the virus-like nanoparticles comprises about 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 L1 (e.g., BPV L1) capsid proteins. However, in some embodiments, the virus-like nanoparticles comprise fewer than 300 L1 (e.g., BPV L1) capsid proteins.

[0041] In some embodiments, the present disclosure provides bovine papillomavirus virus-like nanoparticles covalently conjugated to 100 to 1000 photosensitive molecules (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 molecules). In some embodiments, the capsid protein of the bovine papillomavirus (BPV) virus-like nanoparticle comprises or consists of a BPV L1 capsid protein or a combination of BPV L1 and BPV L2 capsid proteins. In some embodiments, the photosensitive molecule is conjugated to the virus-like nanoparticle (or to the capsid protein of the virus-like nanoparticle) via a covalent bond formed by reacting an ester group in the photosensitive molecule with an amine group in the capsid protein to form an amide bond. Thus, in some embodiments, the capsid protein of the virus-like nanoparticle of the present disclosure is conjugated to the photosensitive molecule via an amide bond.

[0042] In some embodiments, the present disclosure provides virus-like nanoparticles comprising 300 to 500 BPV L1 capsid proteins and / or having a diameter of 20 to 60 nm, wherein at least some (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) are conjugated to 1 to 5 (e.g., 1, 2, 3, 4, or 5) photosensitive molecules (e.g., IR700 dye, e.g., 700DX) covalently conjugated (e.g., via an amide bond). The present disclosure also provides methods for producing virus-like nanoparticles and methods for administering virus-like nanoparticles to a subject as a diagnostic, therapeutic, or prophylactic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The mechanism of cell death induction using virus-like particles (VLPs) conjugated to photosensitive molecules is shown.

[0044] Figure 2 A comparison of bivalent targeting (eg, by antibodies) and multivalent targeting (eg, by VLPs) is shown.

[0045] Figure 3 A graph showing that the specificity of VLP binding to cells is mediated by heparan sulfate proteoglycan (HSPG) interactions and is inhibited by heparin is shown. It also shows that tumor cells are specifically killed only when photosensitive VLPs bind to cells and the cells are subjected to infrared irradiation.

[0046] Figure 4 Graphs showing the dependence of cell death on the dose of infrared radiation and the amount of delivered VLPs and photosensitizing molecules (eg, dyes) are shown.

[0047] Figure 5Shown are graphs showing in vitro ovarian cancer cell (SKOV-3) death following irradiation in the presence of VLPs conjugated to IR700 (designated PsV in the figure).

[0048] Figure 6A Shown is electrospray ionization-time-of-flight (ESI-TOF) analysis of control VLPs. Figure 6B Shown is an ESI-TOF analysis of VLPs (designated PsV in the figure) conjugated to 1000 molecules of IR700.

[0049] Figure 7 A to 7C show human epidermal growth factor receptor 2 negative (HER2 - ) graph of cell death in an ocular melanoma cell line (92.1) comparing the effectiveness of a bivalent agent (e.g., an antibody) and a multivalent agent (e.g., a photosensitive VLP, also referred to as a VLP conjugate, designated PsV in this figure).

[0050] Figure 8 A to 8C show human epidermal growth factor receptor 2 negative (HER2 + ) graph of cell death in an ovarian cancer cell line (SKOV-3) comparing the effectiveness of bivalent agents (e.g., antibodies) and multivalent agents (e.g., photosensitive VLPs, also known as VLP conjugates, designated PsV in this figure).

[0051] Figure 9 Shown is a graph showing that vaccine-induced anti-HPV16 neutralizing antibodies do not block BPV*IR700 VLP binding to the ocular melanoma cell line 92.1.

[0052] Figure 10A Shown Chemical structure of 700DX NHS ester. Figure 10B Shown Chemical structure of , where the reactive carboxyl group is circled.

[0053] Figure 11 The reaction scheme is shown, which involves (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC) and sulfo-N-hydroxysuccinimide (sulfo-NHS) mediated and VLP. In this scheme, ① represents And ② represents VLP. Note that there are two pathways to the desired end product. The presence of sulfo-NHS tends to stabilize the reaction and increase the production of the desired product.

[0054] Figure 12 Shown are histograms of representative samples of HSPG-dependent binding of virus-like nanoparticles comprising HPV16 capsid protein, variant HPV16 / 31 capsid protein, and BPV1 capsid protein (L1, or L1 and L2 proteins) to various types of cancer cells.

[0055] Figure 13A and 13B The resected tumor tissue is shown in the bright field ( Figure 13A ) and fluorescence ( Figure 13B ) of resected tumor tissues from PBS-injected negative control mice 12 hours after injection, photosensitive virus-like nanoparticle-injected mice (#3 and #4) 12 hours after injection, and photosensitive virus-like nanoparticle-injected mice (#1 and #2) 24 hours after injection.

[0056] Figure 14 Shown is the quantification of total tumor-associated virus-like nanoparticle-associated fluorescence in ex vivo TC-1 tumor samples excised 12 and 24 hours after intravenous injection of VLPs (same tumors as in FIG13 ).

[0057] Figure 15 A schematic diagram showing the experimental design of Example 14 is shown.

[0058] Figure 16A and 16B Shown is a graph of the percentage of cell death following in vivo administration of photosensitive virus-like nanoparticles (designated NP in this figure) and phototitration of subcutaneous 92.1 ocular melanoma (OM) cells (cell viability measured 24 hours after light treatment).

[0059] Figures 17A to 17C Shown Figure 16A and 16B The original histogram of the data is shown in .

[0060] Figure 18 (Upper panel) shows the results of subcutaneous inoculation of 2×10 5 Tissue samples obtained from animals that received TC-1 tumor cells and were administered: (1) no treatment; (2) 100 μg of virus-like nanoparticles (designated NP in the figure) assembled from variant HPV16 / 31 L1 and HPV L2 proteins and labeled with 700DX [no light]; (3) PBS and 50 J / cm 2 Light; (4) 200 μg virus-like nanoparticles and 50 J / cm 2 Light; (5) 100 μg virus-like nanoparticles and 50 J / cm 2 light; and (6) 50 μg virus-like nanoparticles with 50 J / cm 2 Light. Figure 18 (Lower panels) Shown are the percentage of dead cells for each of the six conditions tested.

[0061] Figure 19A A schematic diagram of the experiment described in Example 15 is shown. Figure 19B Shown is a graph of the percentage of survival in animals injected with virus-like nanoparticles (designated as nanoparticles in the figure) relative to controls (with light). Figure 19C Shown are the tumor volume (upper panel), "E7 tetramer" in each mouse + CD8 + T cells" and "IFN-γ secreting CD8 + cell".

[0062] Figure 20 Graphs showing results from a potency assay comparing the effects of photosensitive BPV virus-like nanoparticles and photosensitive HPV virus-like nanoparticles on cell viability.

[0063] Figure 21 Graphs showing results from binding assays comparing the binding of photosensitive BPV virus-like nanoparticles and photosensitive HPV virus-like nanoparticles to cells.

[0064] Figure 22 A graph showing tumor growth curves of head and neck cancer cells after treatment with photosensitive virus-like nanoparticles (designated PsV in the figure).

[0065] Figure 23A and 23B An example of a process for producing photosensitive virus-like nanoparticles of the present disclosure is depicted (eg, as described in Example 20). Specific implementation plan

[0066] Photodynamic therapy (PDT) is a form of light therapy that uses non-toxic photosensitive molecules that, when selectively exposed to light, become toxic and target and / or kill malignant cells and other diseased cells. The challenge faced by PDT in treating cancer is the delivery of high concentrations of photosensitive molecules exclusively to tumor cells. In order to achieve targeted delivery, antibodies can be used, but antibodies are limited by their ability to deliver 2 to 8 photosensitive molecules per antibody. In addition, there are important tumors that lack identified tumor receptor molecules and therefore cannot be targeted with antibodies. As a result, many tumors remain untreatable (e.g., ocular melanoma). In addition, many molecules targeted by antibody / dye conjugates (e.g., EGFR) are also found on the surface of non-tumor cells, resulting in unwanted off-target effects.

[0067] The present disclosure is based in part on the following surprising discovery: virus-like particles (VLPs) (e.g., papilloma VLPs) (also referred to herein as virus-like nanoparticles) can be chemically modified to carry many photosensitive molecules (e.g., IR700) without losing their ability to target tumors or structural stability. For example, in some embodiments, VLPs can be chemically modified to carry more than 50 molecules, more than 100 molecules, or more than 1000 molecules (or about 1000 photosensitive molecules). Virus-like particles assembled by L1, or L1 and L2 capsid proteins can selectively bind to and infect cancer cells without affecting non-cancerous cells, thereby minimizing the cytotoxicity of the treatment (see U.S. Patent Application Publication No. US20100135902A1, which is incorporated herein by reference in its entirety). In addition, in some embodiments, delivering high amounts of photosensitive molecules / particles can achieve selective killing of tumor cells with very small amounts of drugs (e.g., picomolar concentrations) after light irradiation.

[0068] The key cell binding feature of VLP is the presence of a large number of heparin binding sites on the capsid protein (e.g., L1). Surprisingly, conjugation of the photosensitive molecule to surface amino acids (e.g., conjugation via amide bonds to surface amino acids such as surface lysine residues, arginine residues, or histidine residues) does not disrupt the binding of VLP to heparan sulfate proteoglycans (HSPGs) on the surface of tumor cells. Although the present disclosure describes the conjugation of photosensitive molecules to surface exposed peptides of the capsid protein, it should be understood that the photosensitive molecule can be conjugated to any peptide of the capsid protein. That is, the photosensitive molecule can be conjugated only to the L1 protein or to a combination of L1 and L2 proteins. The protein and amino acid residue to which the photosensitive molecule is conjugated may depend on the composition of the virus-like particles.

[0069] The foregoing findings have important implications for the development of new targeted cancer therapies. For example, the photosensitive VLPs (also referred to as VLP conjugates) of the present disclosure offer certain advantages over other targeting molecules (e.g., antibodies) that have very limited delivery capabilities. In addition, the photosensitive VLPs of the present disclosure can be used to target a wide variety of tumors (e.g., eye tumors) that cannot be targeted by antibodies or other targeting molecules because suitable tumor surface-specific determinants have not yet been identified. In addition, the photosensitive VLPs can be used to treat distal metastases. In addition, the photosensitive molecules can be used to diagnose and treat early malignant or precancerous lesions (e.g., transforming, premalignant, or malignant eye moles).

[0070] As used herein, " virus-like particles " (VLP) refer to organized capsid-like structures (e.g., roughly spherical or cylindrical in shape) that include an ordered self-assembly of L1 or L1 and L2 capsomers and do not contain a viral genome. Virus-like particles are similar to true virions (virons) in morphology and antigenicity, but they lack viral genetic material (e.g., viral nucleic acids), thereby rendering the particles non-infectious. VLPs can be used to deliver agents (e.g., prophylactics, therapeutics, or diagnostics) or closed circular or linear DNA or RNA molecules to recipient cells. It should be understood that the terms "virus-like particles" or "VLPs" and "pseudoviruses" or "PsVs" are used interchangeably herein and can also be used interchangeably with the term "virus-like nanoparticles."

[0071] As used herein, "tumor-targeted virus-like particles" refer to VLPs that target tumor (eg, cancerous) cells but not non-tumor (eg, non-cancerous, or normal, healthy) cells (eg, in intact tissue).

[0072] VLPs according to the present disclosure may have modified immunogenicity and / or antigenicity relative to wild-type papillomavirus VLPs. The VLPs may, for example, be assembled from capsomers of variant capsid proteins having modified immunogenicity and / or antigenicity. A variant capsid protein having "modified immunogenicity and / or antigenicity" is a capsid protein whose amino acids have been naturally or synthetically modified (e.g., mutated, replaced, deleted, pegylated, or inserted) to reduce or prevent recognition of the capsid protein by existing (e.g., endogenous) viral serotype-specific antibodies. The variant 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 from different HPV serotypes. For example, the 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 "variant HPV16 / 31 L1 protein" - SEQ ID NO: 1), which is described in International Publication No. WO / 2010 / 120266 (which is incorporated herein by reference in its entirety).

[0073] In some embodiments, the VLP is a papillomavirus VLP. The VLP can be a human papillomavirus VLP (e.g., 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, murine papillomavirus, cotton-rabbit papillomavirus, and macaque or rhesus macaque papillomavirus particles. In some embodiments, the VLP is a bovine papillomavirus virus-like nanoparticle (e.g., type 1 virus-like nanoparticle) (e.g., assembled from a combination of BPV L1 capsid protein or BPV L1 and BPV L2 capsid proteins).

[0074] As used herein, "capsid protein" refers to protein monomers, some of which form capsomere oligomers. As used herein, "capsomere" refers to the basic oligomeric structural unit of the viral capsid, which is the outer protein covering that protects the genetic material of a virus (e.g., human papillomavirus (HPV)). The capsid proteins of the present disclosure include the papillomavirus L1 major capsid protein and the papillomavirus L2 minor capsid protein. In some embodiments, the VLPs of the present disclosure comprise only the L1 capsid protein, while in other embodiments, the VLPs comprise a mixture (or combination) of the L1 and L2 capsid proteins.

[0075] In some embodiments, the percentage of L1 capsid protein in the virus-like particle is greater than the percentage of L2 capsid protein in the virus-like particle. For example, in some embodiments, the percentage of L1 capsid protein in the virus-like particle is 80% to 100% (of the total number of capsid proteins in the virus-like particle). In some embodiments, the percentage of L1 capsid protein in the virus-like particle 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 the virus-like particle is 1% to 25% (of the total number of capsid proteins in the virus-like particle). For example, in some embodiments, the percentage of L2 capsid protein in the virus-like particle is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0076] In some embodiments, the virus-like particle comprises 12 to 72 L2 proteins. In some embodiments, the virus-like particle comprises 360 L1 proteins and 12 to 72 L2 proteins. In some embodiments, the capsid proteins assemble into virus-like nanoparticles having a diameter of 20 to 60 nm. For example, the capsid proteins can assemble into virus-like nanoparticles having a diameter of 20, 25, 30, 35, 40, 45, 50, 55, or 60 nm.

[0077] As used herein, "outer capsid protein" refers to the capsid protein exposed on the surface of the VLP. In some embodiments, the outer capsid protein (eg, L1 protein) is conjugated to (eg, at least one) photosensitive molecule.

[0078] As used herein, a "photosensitive molecule" refers to a non-toxic molecule that becomes "activated" (also referred to as "photoactivated") when selectively exposed to light. In some embodiments, the activated photosensitive molecule re-emits light after photoexcitation (e.g., a fluorophore). In some embodiments, the activated photosensitive molecule may become toxic or may produce a toxic molecule after photoexcitation. For example, a class of photosensitive molecules known as photosensitizers may transition to an excited state after absorbing light and undergo intersystem crossing of oxygen to produce singlet oxygen. This singlet oxygen rapidly attacks any organic compound it encounters and is therefore highly cytotoxic.

[0079] According to various aspects of the present disclosure, the photosensitive molecule can be conjugated to the capsid protein of the VLP (e.g., L1 and / or L2 capsid 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 the photosensitive molecule may be referred to herein as a "VLP conjugate" or "photosensitive VLP". In some embodiments, the photosensitive molecule comprises an NHS (N-hydroxysuccinimide) ester group, which reacts with an amine group of the capsid protein (e.g., an amine group of lysine or other amino acids) to form a covalent amide bond.

[0080] The ratio of photosensitive molecules (PM) to VLPs can vary. In some embodiments, the ratio of VLP:PM is from about 1:10 to about 1:1000, from about 1:10 to about 1:500, from about 1:50 to about 1:500, or from about 1:50 to about 1:1000. That is, in some embodiments, the VLPs may contain from about 10 to about 1000 photosensitive molecules. 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 VLP may comprise 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 light-sensitive molecules. In some embodiments, the VLP may comprise more than 1000 light-sensitive molecules or less than 10 light-sensitive molecules.

[0081] More than one photosensitive molecule can be conjugated to a single capsid protein. For example, a single 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 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.

[0082] Examples of photosensitive molecules used in accordance with the present disclosure include, but are not limited to, fluorescent dyes, infrared dyes, near-infrared dyes, porphyrin molecules, and chlorophyll molecules.

[0083] Examples of fluorescent dyes used in accordance with the present disclosure include, but are not limited to, acridine orange, acridine yellow, Alexa Fluor, 7-aminoactinomycin D, 8-anilinonaphthalene-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-diphenylanthracene, coumarin, DAPI, dark quencher, quencher), DiOC6, DyLightFluor, Fluo-3, Fluo-4, FluoProbe, fluorescein, fluorescein isothiocyanate, fluorescein image-guided surgery, fluoro-jade dye, fura-2, fura-2-acetoxymethyl ester, GelGreen, GelRed, green fluorescent protein, heptamethine dye), Indian Yellow, Indo-1, Lucifer Yellow, Luciferin, M Cherry, Merocyanine, Nile Blue, Nile Red, optical brightener, perylene, phloxine, phycobilin, phycoerythrin, phycoerythrin, 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(barophenanthroline disulfonate)ruthenium(II), Texas Red, titanium yellow, TSQ, umbelliferone, yellow fluorescent protein, and YOYO-1.

[0084] Examples of photosensitizing dyes used in accordance with the present disclosure include, but are not limited to, HpD, porfimer sodium m-THPC, Temoporfin Verteporfin HPPH Palladium-bacterial-pheophorbide 5-ALA, 5-aminolevulinic acid 5-ALA methyl ester 5-ALA benzyl ester 5-ALA hexyl ester Lutetium(III)-porphyrin or motexafin-lutetium SnET2, tin(IV) ethoxylate NPe6, mono-L-aspartyl dihydrochlorin e6, talaporfin sodium BOPP, boronated protoporphyrin Zinc phthalocyanine Silicon phthalocyanine Mixture of sulfonated aluminum phthalocyanine derivatives ATMPn, acetoxy-tetra(β-methoxyethyl-)porphyrinene), TH9402, and dibromorhodamine methyl ester.

[0085] Examples of photosensitizing dyes used in accordance with the present disclosure include those useful for fluorescence imaging (e.g., near-infrared (NIR) fluorescent dyes), such as La Jolla and 700DX.

[0086] The present disclosure also provides a method of administering to a subject having a tumor a tumor-targeting virus-like particle comprising a photosensitive molecule conjugated to a capsid protein, or a method of administering to a subject having a tumor a tumor-targeting virus-like particle comprising about 50 to about 1000 (e.g., 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000) photosensitive molecules.

[0087] In some embodiments, the subject is a mammal, such as a human.

[0088] The mode of administration can be by injection, infusion, implantation, topical administration, or by any other means commonly used to deliver virus-like particles. In some embodiments, a hollow needle, coated needle, microneedle, or micromanipulator needle is used, depending on the area of ​​injection. In some embodiments, the mode of administration is by injection into the intraocular space or into the vitreous body of the eye (e.g., to target an ocular tumor or a tumor that has metastasized to the eye).

[0089] Agents that can be used to deliver the virus-like particles of the present disclosure include, but are not limited to, saline, MgCl2, trehalose, sodium hyaluronate, polysorbate 20, polysorbate 80, or any combination of two or more of the foregoing agents.

[0090] The photosensitive molecules of the present disclosure can be activated at a suitable wavelength. In some embodiments, the activation of the photosensitive molecules makes them cytotoxic or can produce cytotoxic molecules. Suitable wavelengths include but are not limited to: ultraviolet wavelengths, visible wavelengths, infrared wavelengths or 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 the following wavelengths: 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.

[0091] The photosensitive molecules of the present disclosure can be activated by infrared light, near infrared light, or ultraviolet light, depending on the type of molecule. For example, in some embodiments, an infrared laser, a near infrared laser, or an ultraviolet laser can be used to activate the photosensitive molecules of the VLP conjugate. The energy delivered by the laser can range from about 5J to about 100J, from about 5 joules (J) to about 50J, or from about 8J to about 36J. In some embodiments, the energy delivered by the laser is 8J, 9J, 10J, 11J, 12J, 13J, 14J, 15J, 16J, 17J, 18J, 19J, 20J, 21J, 22J, 23J, 24J, 25J, 26J, 27J, 28J, 29J, 30J, 31J, 32J, 33J, 34J, 35J, 36J, 37J, 38J, 39 J, 40J, 41J, 42J, 43J, 44J, 45J, 46J, 47J, 48J, 49J, 50J, 51J, 52J, 53J, 54J, 55J, 56J, 57J, 58J, 59J, 60J, 61J, 62J, 63J, 64J, 65J, 66J, 67J, 68J, 69J, 70J, 71J, 72J, 73J, 74J, or 75 J. In some embodiments, the energy delivered by the laser is 10J, 20J, 30J, 40J, 50J, 60J, 70J, 80J, 90J, or 100J.

[0092] Light or laser light can be applied to the photosensitive molecules (or photosensitive VLPs) for about 5 seconds to about 5 minutes. For example, in some embodiments, light or laser light is applied to the photosensitive molecules for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 seconds to activate the molecules. In some embodiments, laser light is applied to the photosensitive molecules for 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 minutes, or longer. It should be understood that the length of time light or laser light is applied to the photosensitive molecules can vary depending on, for example, the energy (e.g., wattage) of the laser light. For example, a laser light with a lower wattage can be applied to the photosensitive molecules for a longer time to activate the molecules.

[0093] Light or laser can be applied to the photosensitive molecule (or VLP conjugate) from about 30 minutes to about 48 hours after administering the VLP conjugate. For example, in some embodiments, light or laser is applied to the photosensitive molecule 30, 35, 40, 45, 50 or 55 minutes after administering the VLP conjugate. For example, in some embodiments, light or laser is applied to the photosensitive molecule 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after administering the VLP conjugate. In some embodiments, light or laser is applied to the photosensitive molecule 36 or 48 hours after administering the VLP conjugate.

[0094] Light or laser light can be applied directly to the tumor site. For example, a VLP conjugate targeted to an ocular tumor can be activated by irradiating the eye.

[0095] According to the present disclosure, any type of tumor can be a target. 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, testicles, kidney, bladder, head, neck, cervix, and / or skin.

[0096] In some embodiments, the tumor is an ocular tumor. The ocular tumor can be located in the vitreous body, choroidal cavity, iris, ciliary body, sclera, fovea, retina, optic disc, or optic nerve.

[0097] In some embodiments, the tumor is cancerous or malignant. In some embodiments, the tumor is metastatic. Other tumors can also be targeted. For example, the present application provides methods and compositions for targeting cervical cancer cells, ovarian cancer cells, melanoma cancer cells, lung cancer cells, head and / or neck cancer cells, and bladder cancer cells. Composition

[0098] In some embodiments, the virus-like particles (virus-like nanoparticles) of the present disclosure are virus-like nanoparticles conjugated with photosensitive molecules. The virus-like nanoparticles contain one or two types of capsid proteins from papillomaviruses. In some embodiments, the capsid proteins are modified. The capsid proteins typically self-assemble into "empty" proto-capsids (e.g., spherical particles containing an empty core) with a diameter of about 55 nm. After the proto-capsids mature to form virus-like nanoparticles (virus-like particles), the virus-like nanoparticles are then conjugated with photosensitive molecules (e.g., IR700 dye, for example). 700DX, by Manufactured infrared dyes) are chemically conjugated.

[0099] In some embodiments, the photosensitive virus-like nanoparticles are provided in a sterile solution (e.g., 1 or 2 ml) in a disposable vial (e.g., a borosilicate glass vial). In some embodiments, the photosensitive virus-like nanoparticles are provided in a sterile aqueous solution, which optionally includes NaCl, KCl, Na2HPO4.2H2O, KH2PO4, or any combination of two or more of the foregoing. In some embodiments, NaCl may be present in the solution at a concentration of 400 to 600 mMol (e.g., 500 mMol). In some embodiments, KCl may be present in the solution at a concentration of 2 to 6 mMol (e.g., 2.7 mMol). In some embodiments, Na2HPO4.2H2O may be present in the solution at a concentration of 5 to 15 mMol (e.g., 10 mMol). In some embodiments, KH2PO4 may be present in the solution at a concentration of 1 to 3 mMol (e.g., 2 mMol).

[0100] In some embodiments, the photosensitive virus-like nanoparticles are diluted and administered intraocularly using a sterile syringe or needle commonly used for ophthalmic procedures. The present disclosure also provides other routes of administration and administration to other tumors and / or metastases, as described elsewhere herein.

[0101] In some embodiments, each virus-like nanoparticle comprises 12 to 72 capsomeres, wherein each capsomere comprises 5 molecules of L1 capsid protein (e.g., 55 to 56 kD per molecule) and 1 molecule of L2 capsid protein (e.g., 52 kD per molecule). In some embodiments, each virus-like nanoparticle comprises 12 to 72 capsomeres, wherein each capsomere comprises only L1 capsid protein (e.g., 5 molecules of L1 protein per capsomere).

[0102] In some embodiments, each virus-like nanoparticle has 10 to 1000 molecules (e.g., 500 molecules) of a photosensitive molecule (IR700 dye, e.g. 700DX) is chemically conjugated (e.g., via an amide bond) to at least one amino acid (e.g., a lysine amino acid) of a protein. Methods for producing virus-like particles

[0103] To produce the photosensitive virus-like nanoparticles of the present disclosure, mammalian cells (e.g., 293T cells (e.g., HEK293F cells)) can be cultured (e.g., in suspension culture) and transiently transfected with nucleic acids encoding BPV or HPV L1 (or L1 and L2) capsid proteins. This induces the formation of protocapsids (e.g., as described in Buck et al. Current Protocols in Cell Biology 26.1.1-26.1.19, December 2007). After cell mass recovery and disruption, the protocapsids can be treated with benzonase to clear host DNA and subjected to a subsequent in vitro maturation process to form stable virus-like nanoparticles. After purification, the virus-like nanoparticles can be chemically conjugated with a photosensitive molecule (e.g., IR700 NHS ester) to produce photosensitive virus-like nanoparticles. Figure 23 shows a schematic representation of an example of the production process provided herein.

[0104] Thus, in some aspects, provided herein are methods for producing photosensitive molecules comprising (a) transiently transfecting cells with nucleic acids encoding one or more capsid proteins, thereby forming procapsids, (b) collecting the procapsids and subjecting the procapsids to an in vitro maturation process, thereby forming stable virus-like nanoparticles, and (c) conjugating the virus-like nanoparticles with 50 to 1000 photosensitive molecules. In some embodiments, the virus-like nanoparticles are conjugated with 500 photosensitive molecules. In some embodiments, the virus-like nanoparticles are conjugated to the photosensitive molecules via an amide bond (e.g., by reacting an ester group of the photosensitive molecule with an amine group of an amino acid of a virus-like nanoparticle capsid protein). Example Example 1- Conjugation of 700DX

[0105] VLPs (e.g., virus-like nanoparticles comprising a combination of variant HPV16 / 31 L1 protein and HPV L2 protein) and photosensitive molecules (e.g., Chemical conjugation of IR700 (e.g., IR700DX) is performed as follows. Generally, a solution of VLPs is maintained at a concentration of 1 mg / ml in PBS, pH 7.2 and 0.3 to 0.5 M NaCl. 700DX) molecules as dry NHS (N-hydroxysuccinimide) esters (NHS-esters react with amine groups on proteins to form covalent amide bonds) Figure 10A) are supplied by the manufacturer. Available amine groups on proteins are the amino termini of proteins or the epsilon-amino groups on amino acids (e.g., lysine). Dry solid IR700-NHS ester was dissolved in DMSO at a concentration of 5 mg / ml and stored frozen. Generally, different VLP:dye ratios were achieved by mixing different amounts of IR700-NHS with a fixed amount of VLP (usually a 1 mg / ml solution in 1 ml of PBS). Typical ratios and amounts of IR700-NHS are listed in the table below: Table 1

[0106] To achieve a 200:1 ratio, 1 mg / ml VLPs in 1 ml of PBS were mixed with 3.2 μl of IR700-NHS ester solution. These reactions were allowed to proceed for 2 to 4 hours at room temperature. After the reaction was complete, the VLPs were purified by heparin affinity column chromatography to separate unbound IR700-NHS from the newly formed VLP-IR700 conjugates (also known as photosensitive VLPs). Example 2- Conjugation

[0107] Conjugation to VLPs followed a slightly different protocol than IR700-NHS. The molecules need to be functionalized with NHS before conjugation to VLPs. This functionalization is achieved by using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride). EDC is used to functionalize molecules with free carboxylic acid molecules, such as (See Figure 10B , circled), and the NHS moiety is efficiently transferred to the reagent in the presence of sulfo-NHS. This reaction scheme is summarized in Figure 11 Briefly, approximately 2 mM EDC and 2× molar excess of sulfo-NHS were mixed with different amounts of After 15 minutes at room temperature, the reaction was stopped by adding 2-mercaptoethanol to a final concentration of 20 mM. The reaction mixture was added to 1 mg of VLP at a concentration of 1 mg / ml in PBS, pH 7.2 + 0.3-0.5 M NaCl and incubated at room temperature for 2 to 4 hours. Finally, unreacted components were separated from the VLP conjugate by heparin affinity column chromatography. Example 3 - VLP binding specificity is mediated by HSPG and inhibited by heparin.

[0108] Suspended SK-OV-3 cells were treated under the following conditions: without VLPs (e.g., virus-like nanoparticles comprising a combination of variant HPV16 / 31 L1 protein and HPV L2 protein), with 488( Figure 3 , "AF488*PsV") or IR700( Figure 3 , "IR700*PsV")-conjugated VLPs, or the same VLP conjugates incubated in the presence of HSPG. Following incubation, these cultures were subjected to 4 joules of 690 nm near-infrared light. Parallel groups of unirradiated cells served as controls. Following irradiation, the cultures were assessed for the extent of cell death. Figure 3 The only condition that showed significant cell killing was when cells were exposed to IR700*PsV and 4 joules of light. Similar cell death was not observed under exposure to AF488*PsV, indicating that cell death is specific to the IR700 dye conjugate. In addition, cell death was almost completely abolished in the presence of HSPG, indicating that VLP binding to cells is crucial for IR700-mediated cell death. Example 4 - Cell death depends on infrared radiation and the amount of VLPs and IR700.

[0109] Suspended SK-OV-3 cells were treated with different concentrations of VLPs (e.g., virus-like nanoparticles comprising a combination of variant HPV16 / 31 L1 protein and HPV L2 protein) that had been conjugated with different amounts of IR700 dye (e.g., 700DX). VLPs not conjugated with IR700 dye were used as controls. After incubation, these cultures were subjected to 0 or 16 joules of 690 nm near-infrared light. Following light treatment, the extent of cell death was assessed. Figure 4 Cell death was shown to be dependent on both the presence of IR700 dye and light treatment. This was supported by the observation that cell death was dependent on both VLP concentration and IR700 dye conjugation ratio. Example 5 - In vitro cell death of SKOV-3 cells following treatment with IR700-conjugated VLPs following irradiation.

[0110] SKOV-3 ovarian cancer cells were plated in 24-well plates and treated with two different concentrations of photosensitive VLP particles (e.g., virus-like nanoparticles containing a combination of variant HPV16 / 31 L1 and HPV L2 proteins conjugated to IR700 dye) (2.5 μg (red) and 0.25 μg (blue)) for 1 hour at 37°C. After binding, the cells were washed and then treated with 4J of light. Cell death was determined based on enzymatic assessment of LDH release (determined by measuring absorbance at 490 nm). Three different molar ratios of VLP:IR700 conjugation were tested: 1:500, 1:1000, and 1:2000. Figure 5It is shown that for both concentrations tested, maximal cell death efficacy was observed at a VLP:IR700 ("PsV:IR70") ratio of 1:1000. Detergent-mediated cell lysis was used as a positive control. Example 6 - Structural evaluation of the IR700-PsV complex

[0111] FIG6 shows the comparison of control VLP (PsV) (A) and IR700 conjugated VLP (PsV) (B) ESI-TOF analysis. Figure 6B In the , the reaction was set up to achieve conjugation of 1000 IR700 molecules per VLP (PsV) molecule. The signal peak in the ESI-TOF scan corresponds to the VLPL1 protein. Relative to the control sample, a transfer of 5517 amu was observed in the conjugated sample, which corresponds to an average of 3 conjugated IR700 molecules (1840 amu) per L1 protein or approximately 1000 IR700 molecules per VLP (generally, each VLP has 360 L1s). Example 7 - Combination of Agents Determines the Extent of Cell Death in Ocular Melanoma Cell Lines.

[0112] Suspended ocular melanoma cell line (92.1; HER2 - ) were exposed to different dilutions of IR700 dye (e.g. 700DX) conjugated Antibodies or VLPs conjugated to IR700 dye (e.g., virus-like nanoparticles comprising a combination of variant HPV16 / 31 L1 protein and HPV L2 protein). Parallel cultures were then evaluated for agent binding ( Figure 7 C) or in the absence of ( Figure 7 B) or exists ( Figure 7 A) Cell death under 16 joules of 690 nm near-infrared light. Figure 7 C shows that the binding of VLP to 92.1 ocular melanoma cells is concentration-dependent, while there is basically no Antibody binding. Figure 7 B shows that in the absence of light, there is no cell death. Figure 7 A shows concentration-dependent cell death only in cells treated with photosensitive VLPs. Example 8 - Drug combination determines the extent of cell death in ovarian cancer cell lines.

[0113] Suspended SK-OV-3 cells (HER2 - ) were exposed to different dilutions of IR700 dye (e.g. 700DX) conjugated Antibodies or VLP particles (e.g., virus-like nanoparticles comprising a combination of variant HPV16 / 31 L1 protein and HPV L2 protein). Parallel cultures were then evaluated for or VLP binding ( Figure 8 C) or in the absence of ( Figure 8 B) or exists ( Figure 8 A) Cell death under 16 joules of 690 nm near-infrared light. Figure 8 C shows that VLP binding reached saturation in SK-OV-3 cells. Binding was also concentration dependent, but to a reduced extent relative to VLPs. Figure 8 B shows that in the absence of light, there is no cell death. Figure 8 A shows concentration-dependent cell death under both conditions, but similar to binding, the response is saturated under VLPs, whereas under These data indicate that VLPs conjugated with IR700 (PsV-IR700) are more effective than those conjugated with IR700. (Herceptin-IR700) is more effective. Example 9 - Vaccine-induced anti-HPV16 neutralizing antibodies do not block the binding of BPV*IR700 VLPs to the ocular melanoma cell line 92.1.

[0114] Serum samples containing different antibodies were tested for their ability to inhibit the binding of photosensitive VLP particles (eg, HPV16 VLPs or BPV VLPs) to the 92.1 ocular melanoma cell line. Figure 9 Display "No serum" or "Naive serum" ( The results showed that the presence of IR700 dye in the presence of serum (serum) did not neutralize VLP binding activity. Furthermore, the observed blocking activity was specific for the viral serotype. Specifically, only human papillomavirus-like particles conjugated to IR700 dye (HPV16-IR700) were neutralized by serum containing HPV16 antibodies. Bovine papillomavirus-like particles conjugated to IR700 (BPV-IR700) were not neutralized by serum containing HPV16-specific antibodies. Example 10 - Immunogenicity Evaluation

[0115] In studies similar to those described in Example 9, neutralization titers were determined by serially diluting sera containing antibodies against HPV16 or BPV. The results, shown in Table 2, show that antibodies against HPV16 neutralize only HPV16. Furthermore, antibodies against BPV neutralize only BPV. Therefore, there is no cross-reactivity between HVP16 antibodies and BPV, and vice versa. Table 2 Example 11 - Binding Studies

[0116] The purpose of this example was to evaluate the binding of virus-like nanoparticles to various types of cancer cells. The virus-like particles contained human papillomavirus 16 (HPV16) capsid protein, variant HPV16 / 31 L1 capsid protein, and bovine papillomavirus (BPV) capsid protein. In addition, virus-like nanoparticles containing both L1 and L2 capsid proteins, or only L1 capsid protein, were tested to determine whether the binding of virus-like nanoparticles to cancer cells was dependent on L2. The results of this study showed that BPV virus-like nanoparticles and HPV virus-like nanoparticles had comparable binding.

[0117] A large panel of cell lines was screened, including: heterozygous cell lines (e.g., 293TT, HaCaT, PAM-212, and TC-1), cervical cell lines (e.g., HeLa, SiHa, CaSki, and C-33A), ovarian cell lines (e.g., MOSEC, SHIN-3, SK-OV-3, WF-3, ES-2, A2780, OVCAR-3, and OVCAR-4), melanoma cell lines (e.g., B16F10, SKMEL-2, SKMEL-5, SKMEL-28, and UACC), ocular melanoma cell lines (e.g., 92.1, MKT-BR, OCM-1, and UW-1), lung cell lines (e.g., NCI-H23, NCI-H322M, NCI-H460, and NCI-H522), head and neck cell lines (e.g., CAL-33 (HPV-), FaDu (HPV-), HSC-3 (HPV-), SNU-1076 (HPV-), UM-SCC-47 (HPV+), UPCI-SSC-90 (HPV+), and UPCI-SCC-154 (HPV+)), and bladder cell lines (e.g., 5637, J82, RT112, SCaBER, SVHUC, T24, UMUC-3, UMUC-5).

[0118] Prior to the experiment, virus-like nanoparticles were conjugated to AlexaFluor 488 to allow for easy and direct analysis of the binding of virus-like nanoparticles to the cell surface. AlexaFluor 488 was linked to virus-like nanoparticles using N-hydroxysuccinimide (NHS)-ester chemistry, which does not interfere with binding. Each virus-like nanoparticle was tested at concentrations of 10 μg / ml, 1 μg / ml, and 0.1 μ / ml.

[0119] The cells were trypsinized to remove them from the plastic surface of the tissue culture plates, washed, and allowed to recover in growth medium on a rocking platform at 37°C for 4 hours. The cells were then washed, counted, and plated at 1 × 10 5Cells / well were placed in phosphate buffered saline (PBS) / 2% fetal bovine serum (FBS) in a 96-well round-bottom plate. Virus-like nanoparticles were added to the cells in a final volume of 100 μl PBS / 2% FBS. Virus-like nanoparticles pre-incubated with heparin (1 mg / ml, 1 hour, 4°C) were also added to the wells as a control. The cells and virus-like nanoparticles were then incubated at 4°C for 1 hour (in the dark), washed twice with PBS / 2% FBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Finally, the cells were washed again, resuspended in 200 μl PBS / 2% FBS and analyzed using BD FACSDIVA TM (BD Biosciences, San Jose, CA) and FlowJo software in BD FACSCANTO TM Analyses were performed on a BD Biosciences II (BD Biosciences, San Jose, CA).

[0120] The results using TC-1, HeLa, SK-OV-3, SKMEL-28, 92.1, NCI-H322M, HSC-3, UPCI-SCC-154, and T24 cell lines are shown in histograms. Figure 12 In. By Figure 12 It is clear that in the binding assay, all virus-like nanoparticles, regardless of their serotype or composition (L1 versus L1 / L2), bound to cancer cells. Furthermore, heparin competed for binding, indicating that virus-like nanoparticle binding was specific and HSPG-dependent. Example 12 - Biodistribution Time Course

[0121] The purpose of this example was to evaluate the prolonged localization and clearance of virus-like particles from tumors following intravenous injection into tumor-bearing animals.

[0122] Pure virus-like nanoparticles were visualized by staining with IR700 dye (e.g. 700DX) were prepared by labeling virus-like nanoparticles (e.g., virus-like nanoparticles comprising a combination of variant HPV16 / 31 L1 protein and HPV L2) at a virus-like nanoparticle:dye ratio of 1:500. TM The density titer medium was purified by density gradient ultracentrifugation.

[0123] In C57Bl / 6 mice, 2 × 10 5Tumors were generated using TC-1 cancer cells. Approximately two weeks later, the animals were randomly divided into treatment groups. Tumor-bearing animals received either PBS or 200 μg of photosensitive virus-like nanoparticles (100 μl volume) by intravenous injection. Twelve or 24 hours after injection, the animals were euthanized. After euthanasia, tumor tissue was harvested and stained with IR700 dye (e.g., 700DX) fluorescence was imaged, which indicated the presence of photosensitive virus-like nanoparticles.

[0124] Figure 13B Tumor tissue obtained from both 12 and 24 hour time points showed detectable IR700 dye (e.g., 700DX) fluorescence, while no fluorescence was detected in the PBS control (12 hour time point). Quantitative total fluorescence in tumor tissues was plotted on Figure 14 As depicted in the figure. Example 13 - Biodistribution Time Course

[0125] The purpose of this example was to evaluate the time course of tumor localization and clearance of virus-like particles following intravenous injection into tumor-bearing animals.

[0126] The purified virus-like nanoparticles were labeled with Alexa Fluor 488 in the lysate and stained using OPTIPREP. TM Density titer media are purified by density gradient ultracentrifugation.

[0127] Albino C57Bl / 6 mice were injected subcutaneously with 2 × 10 5 TC-1 cancer cells were used to generate tumors. Approximately 2 weeks later, 200 μg of photosensitive virus-like nanoparticles were delivered in a volume of 100 μl by intravenous injection. Tumors were harvested at the following time points after injection of photosensitive virus-like nanoparticles: T=1, 2, 4, 8, 12, 24, 48, and 72 hours. After harvesting, tumor fragments were frozen for microscopic evaluation. For this microscopic evaluation, tissue sections were further stained. Rabbit polyclonal serum against HPV16 was used in combination with AlexaFluor-488 secondary antibody. Blood vessels were co-stained with rat anti-CD31 antibody and anti-rat AlexaFluor-594 secondary antibody. Cell nuclei were highlighted with DAPI.

[0128] The data (in situ images not shown) demonstrate the presence of photosensitive virus-like nanoparticles at the 1-hour time point. The localization of the signal appears to be associated with blood vessels. The highest staining level appears to occur at the 8-hour time point, and at the 8-hour time point, the photosensitive virus-like nanoparticles appear to be spreading from the blood vessels to the tumor cells. Finally, at the 24-hour and 48-hour time points, very low virus-like nanoparticle signals were shown in the tumor. Example 14 - In vivo efficacy following systemic administration

[0129] The study shown in this example was designed to measure tumor viability 24 hours after a single treatment. This study established a guiding protocol for long-term in vivo studies.

[0130] The complete study design is shown in Figure 15 Animals were randomly assigned based on the range of tumor sizes so that large and small tumors were found in each group (n=3, saline-treated group; n=5, IR700 (eg The virus-like nanoparticles were administered intravenously 12 hours before light treatment. 100 micrograms (100 μg) and 200 μg doses were tested. Light treatment consisted of 25 J (62.3 seconds at 400 mW) or 50 J (125 seconds at 400 mW). After 24 hours, the tumors were harvested and treated with collagenase and DNase to produce a single cell suspension. BD F-1 was then administered. Yellow stain was used and cells were placed on FACSCANTO TM II. Data are reported as the percentage of dead cells, as indicated by the fluorescence shift in the Pacific orange channel ( Figure 16A ).

[0131] A single dose of 200 μg of IR700 (e.g. 700DX) photosensitive virus-like nanoparticles (NPs) can kill most tumor cells after being treated with 50J light ( Figure 16B and Figure 17C When the tumors were treated with 25J of light, the killing level at 200μg NPs was reduced by nearly half ( Figure 16B and Figure 17B 100 μg of NPs were insufficient to induce killing at 25 J of light ( Figure 16B and Figure 17B ); however, some degree of tumor death was observed at a dose of 50J ( Figure 16B and Figure 17C This study provides the necessary IR700 for in vivo studies (e.g., 700DX) Photosensitive virus-like nanoparticles and light dosage information. Example 15 - Immune System Activation Studies

[0132] The TC-1 tumor model enables the detection of anti-tumor immunity induction after treatment with virus-like nanoparticles in immunocompetent animals. The TC-1 tumor line develops from C57Bl / 6 lung epithelial cells immortalized with the HPV16 oncogenes E6 and E7 and a mutant gene expressing c-Ha-Ras (Lin KY, et al., Cancer Research. 56(1):21-6, 1996). These cells can be implanted subcutaneously or, for studying metastasis models, can be injected intravenously to inoculate the cells in the lungs. For nearly two decades, these cells have been used to test the efficacy of E6 and E7 therapeutic vaccines. E7 has a unique MHC class I epitope in the C57Bl / 6 background that has been shown to be protective if a CD8 T-cell response can be elicited against it (H-2D b , aa 49-57RAHYNIVTF) (Feltkamp MC, et al. European Journal of Immunology. 23(9): 2242-9, 1993). These responses can be detected by staining cells for tetramers and restimulating the cells with the peptides followed by intracellular cytokine staining.

[0133] Dose response study: Animals were subcutaneously inoculated with 2×10 5 About two weeks after inoculation, the animals were randomly divided into six groups: (1) untreated control; (2) 100 μg virus-like nanoparticles (labeled with 700DX and a combination of variant HPV16 / 31 L1 protein and HPV L2 protein) control; (3) PBS with 50 J / cm 2 Light control; (4) 200 μg virus-like nanoparticles and 50 J / cm 2 Light; (5) 100 μg virus-like nanoparticles and 50 J / cm 2 light; and (6) 50 μg virus-like nanoparticles with 50 J / cm 2 Light. Mice received PBS or virus-like nanoparticles in a 100 μl volume by intravenous injection, and 12 hours later, light was applied to the tumor using a 690 nm laser. 24 hours later, the tumors were harvested, digested to produce a single-cell suspension, and stained with a viability stain to measure the percentage of dead cells ( Figure 18 , above).

[0134] Several animals in the high-dose group experienced symptoms associated with tumor lysis syndrome, which may be attributed to the massive and rapid tumor necrosis and release of intracellular components into the animals' systems. 2There were no deaths in the "light" group, but mice in this group did show some signs of disease ( Figure 18 , above). “100 μg nanoparticles without light” and “PBS with 50 J / cm 2 The "light" group showed no signs of disease, indicating that the observed response was due to the combination of virus-like nanoparticles and light. Overall, there was significant necrosis in all groups receiving virus-like nanoparticles and light. Maximal killing occurred in all groups and no dose response was observed ( Figure 18 , below).

[0135] Survival study: Animals were subcutaneously inoculated with 2 × 10 5 TC-1 cells. Approximately 2 to 3 weeks after inoculation, animals were randomly divided into treatment groups (25 μg virus-like nanoparticles) and placebo groups (PBS only). The mice received two rounds of treatment, three days apart. The following was considered as one treatment: a single intravenous injection of 100 μl of 25 μg virus-like nanoparticles or sterile PBS, followed 12 hours later by a 690 nm laser at 50 J / cm 2 Tumor size was measured every 3 to 4 days and animals were treated with light when their tumors reached >1500 mm 3 hour( Figure 19A ) and euthanized him.

[0136] When the tumor is less than 500 mm 3 In animals treated with virus-like nanoparticles, the growth of tumors was delayed or irradiated ( Figure 19B and 19C In the placebo group, there was no effect on tumor growth kinetics. The two animals with the smallest tumors at the start showed no signs of tumors within 7 days of the first treatment, and three animals showed signs of tumor reduction ( Figure 19C ).

[0137] Immunological studies: For immunological readouts, blood was collected on day 0 (before the first treatment), day 10, and day 17. Red blood cells were lysed and the remaining cells were split in half, with one half stained for cell surface markers (CD62L, CD127, CD103, CD69, CD4, CD8, CD3, H2-D b The other half was restimulated with HPV16 E7 peptide 49-57 for 4.5 hours and then stained with antibodies against CD4, CD8 and IFN-γ and a viability dye to distinguish live cells.

[0138] In the blood of two animals whose tumor growth was controlled, "E7 tetramers" were detected. + CD8 + T-cells" and "IFN-γ-secreting CD8+ cells" (after restimulation with E7 peptide), indicating that a potential anti-tumor response had been elicited ( FIG. 19 ). Example 16 - Histological Analysis

[0139] The effect of photosensitive virus-like nanoparticles (e.g., virus-like nanoparticles containing a combination of variant HPV16 / 31 L1 protein and HPV L2 protein conjugated to IR700 dye) at the histological level was evaluated using a mouse xenograft model. 6 92.1 uveal melanoma cells were implanted into the subcutaneous space of the hind flank of nu / nu mice. Tumors were allowed to reach approximately 200 mm. 3 At this time, animals were treated by intravenous injection of 200 μg of photosensitive virus-like nanoparticles. 12 hours after injection of photosensitive virus-like nanoparticles, 50 J / cm 2 The tumor site was irradiated with 690 nm near-infrared light. After another 24 hours, the animals were euthanized and the tumor tissues were excised, fixed in formalin, embedded in paraffin, and processed for standard histological examination.

[0140] Hematoxylin and eosin (H&E) images revealed a high degree of necrosis compared to the untreated group (image not shown). Tumors treated with photosensitive virus-like nanoparticles and laser had a pale appearance when compared to control tumors. Examination at higher magnification revealed that cells in tumors treated with photosensitive virus-like nanoparticles exhibited significant cytoplasm loss compared to control-treated tumors. Furthermore, the extent of necrosis covered the entire tumor, leading to the conclusion that NIR light penetrated the entire depth of the tumor tissue. Example 17 - Virus-like nanoparticle activity in an orthotopic xenograft model of uveal melanoma

[0141] The most common primary malignant tumor of the eye is uveal melanoma (UM). Approximately 2,000 patients are diagnosed annually in the United States, with a higher incidence in Europe. Although several treatment options exist for UM, none reliably controls tumor growth, preserves vision, and minimizes radiation-related side effects.

[0142] Virus-like nanoparticle phototherapy (PT) is a new molecular targeted cancer treatment that involves a two-stage process requiring both drug administration and photoactivation. The researchers used a novel photosensitive virus-like nanoparticle (NP) conjugated to 700DX, a near-infrared (NIR) phthalocyanine dye used as a photosensitizer, followed by application of non-thermal NIR light designed to treat adults with primary uveal melanoma.

[0143] In this study, the anticancer activity of photosensitive virus-like nanoparticles was evaluated in an orthotopic xenograft model of uveal melanoma. In this model, human uveal melanoma cells were implanted into the choroidal cavity of immunosuppressed rabbits and allowed to grow. When tumors were observed by fundoscopy, animals were assigned to treatment or control groups. In both cases, the animals were tracked for progressive tumor growth or response to treatment by fundoscopy and ultrasound. After the study was completed, the tumor-bearing eyes were also examined by gross pathology and histopathology.

[0144] This study was conducted using a total of 20 rabbits implanted with the 92.1 uveal melanoma cell line. In total, 11 of the 20 animals developed tumors. Two animals died unexpectedly during the follow-up period prior to treatment; these animals served as untreated controls. Several animals that were not laser-treated developed additional ocular tumors; these animals served as internal controls. Animals with tumors in the anterior chamber were excluded from the study.

[0145] All treated tumors showed major tumor responses compared to control animals, as evidenced by fundus examination, gross pathology, and histopathological evaluation. Retinal tissue adjacent to the tumors was unaffected by the treatments.

[0146] In summary, based on the extent of tumor response and necrosis observed following administration of photosensitive virus-like nanoparticles and laser light, the treatment methods provided herein may be used to treat uveal melanoma.

[0147] Study schedule: Two treatment groups: 1) whole tumor treatment; 2) no treatment. Table 3 Animals arrive April 22 / 23, 2014 Tumor cell implantation April 29 / 30, 2014 Start processing May 20, May 27, and June 3, 2014 End of study June 24, 2014 Draft report July 25, 2014 Methods and experimental design: Testing system Table 4 Species: rabbit strain: New Zealand White Rabbit Number and gender Total reservations: 20 Total number of studies proposed: 20 gender: F Age at receipt: 6 months source: Charles River Logo: RFID and ear tags Model Cell culture

[0148] Human uveal melanoma cell line 92.1 (kindly provided by Dr. Jerry Y. Niederkorn, University of Texas Southwestern Medical Center, Dallas, TX) was cultured in complete medium (RPMI-1640 with 10% fetal bovine serum, 100 U / mL penicillin G, 250 ng / mL amphotericin B, and 100 μg / mL streptomycin solution) at 37° C. in 5% CO . Animals and induction of immunosuppression

[0149] New Zealand white rabbits with an average initial weight of approximately 3 kg were used in this study. Rabbits were immunosuppressed with daily subcutaneous injections of cyclosporine A (CsA; santobumin 50 mg / mL; Novartis Pharmaceuticals, Cambridge, MA, USA). CsA administration was maintained throughout the experiment to prevent spontaneous tumor regression. The dosage regimen was 15 mg / kg daily for 3 days prior to cell inoculation, then for 4 weeks, followed by 10 mg / kg daily until the end of the experiment. Further dose reductions were made at the discretion of the veterinarian. The CsA dose was adjusted daily based on the body weight of each animal. Body weights were measured daily and posted in the room where the rabbits were housed.

[0150] During the follow-up period, the animals were monitored daily for signs of CsA toxicity, such as gum hypertrophy, salivation, diarrhea, and weight loss. If the animal showed early signs of CsA toxicity (e.g., loss of appetite), veterinary staff were consulted immediately for supportive management, such as appetite stimulants and GI motility enhancers. Adjustments to the injection dose may also be considered based on the veterinarian's advice. Cell implantation

[0151] On day 3 after CsA treatment, animals were anesthetized by intramuscular injection of ketamine (40 mg / kg) and xylazine (6 mg / kg). After anesthesia, 1 to 3 drops of 0.5% proparacaine hydrochloride were applied to the right eye, and a 1.0 × 10 6 92.1 human uveal melanoma cells were injected into the suprachoroidal space of the right eye of the rabbit in a volume of 100 μl suspension. Briefly, a sterile drape was placed on the eye to avoid any hair or eyelash contamination, and the conjunctiva was cleaned with 10% betadine solution. Next, a suture was used under the eye muscle to turn the eye forward, and after the conjunctiva was incised, a sclerotomy was performed approximately 10 mm from the limbus. Then, a cannula was inserted into the slerotomy (1 / 3 to 1 / 2 of its length) and the cells (100 μL, containing 1.0×10^6 cells) were injected into the suprachoroidal space. The needle was slowly withdrawn, and the sutures closing the scleral incision were tightened to ensure minimal reflux at the injection site. A drop of antibiotic eye solution (erythromycin ointment) was applied to the surgical wound to prevent infection. Housing, feed, water and environmental conditions, acclimatization

[0152] In group housing, animals were housed in groups of 6 and fed with fresh, palatable and nutritious food at will. Clean, drinkable and uncontaminated water was provided at will. Environmental control was set to maintain a temperature of 22 ± 4 ° C (68 ± 5 ° F) and a relative humidity of 50% ± 20%. A 12-hour light / dark cycle was maintained. After the animals arrived at the facility, they were allowed to acclimate for at least 5 days before baseline evaluation. After baseline fundus examination evaluation, the animals were assigned to the test group. Test article and control article Table 5: Vehicles for test articles identity: PBS Storage conditions: 4℃, up to 3 months, protected from light Operation precautions: Standard PPE Table 6: Test Articles Table 7: Laser Energy settings: 600mW Duration: 83 seconds Energy density: <![CDATA[50J / cm 2 ]]> Spot size: 5.0mm wavelength: 690nm Preparation of dosage forms

[0153] The test article was diluted 1:1 in sterile water for injection. Administration of test / control articles

[0154] Administration: Photosensitive virus-like nanoparticles or saline were administered via intraocular injection into the vitreous.

[0155] Laser Application: Laser treatment uses Coherent Opal The laser was applied using a slit lamp system that delivered 690 nm light at a power of 600 mW for a duration of 83 seconds with a total fluence of 50 J / cm 2 The laser spot size was set to 5 mm in diameter and thus, tumors larger than this size were lasered with overlapping spots. In cases where a clear distinction of tumor boundaries could not be made due to ocular complications (e.g., vitritis, retinal detachment), the entire suspected area could be lasered.

[0156] Mortality / Morbidity Assessment: With the exception of two animals that died from complications of CsA, all animals remained in good health throughout the experiment (see below).

[0157] Clinical Observations: All animals were observed daily by animal facility personnel; observations were recorded. Most animals experienced some degree of weight loss and loss of appetite, which was attributed to CsA. Ophthalmology

[0158] Frequency: Ophthalmological examination with fundus examination and ultrasound is performed weekly.

[0159] Procedure: The animals were sedated and their right eyes were dilated using ophthalmic phenylephrine hydrochloride and tropicamide drops. Next, the eyes were examined fundus-wise using an indirect binocular ophthalmoscope. Any ocular complications were recorded by the ophthalmologist. When a tumor was identified, the size was estimated by comparing it to the optic disc (optic disc diameter [DD]; 1 DD = approximately 1.75 mm). For ultrasound readings, an ultrasound probe was applied to the eye immediately after the fundus examination to visualize the location of the tumor determined by fundus examination. Ultrasound measurements proved technically difficult, primarily because some tumors were located too peripherally to be properly visualized. As a result, the maximum tumor size could not always be measured; and in most cases only the height could be quantified. Final Operation and Anatomic Pathology

[0160] Unplanned Deaths: Per protocol guidelines, one of the 20 animals used in this study was euthanized due to weight loss (>20% of arrival body weight).One animal died unexpectedly from gastrointestinal stasis caused by CaA toxicity.

[0161] Planned Euthanasia: At the end of the study, animals were euthanized according to the recognized American Veterinary Medical Association (AVMA) guidelines. Animals were bled under anesthesia using a combination of ketamine-xylazine-acepromazine (0.75 mg / kg, 5 mg / kg, and 20 to 35 mg / kg, respectively) and buprenorphine (0.2 mg / kg). result

[0162] Overall, 11 animals developed histopathologically evident tumors. As previously described, two animals died unexpectedly and served as untreated controls. Nine animals with varying tumor sizes were treated with virus-like nanoparticles. One animal was excluded from evaluation due to tumor extent in the preequatorial segment, which was not amenable to laser treatment.

[0163] In animals with tumors in the back of the eye that received the full treatment (photosensitive virus-like nanoparticles + laser), a significant tumor response was observed, characterized by the following three elements: 1) induction of extensive tumor necrosis; 2) change in growth pattern from diffuse to a "sleeve-like pattern"; and 3) sparing of the adjacent retina. Table 8 Untreated control

[0164] Rabbit #14 was euthanized at week 4 due to unacceptable weight loss (>20% of initial body weight). Fundus examination was unreliable for the presence of tumor due to massive hemorrhage and retinal detachment. This animal received no treatment.

[0165] Ultrasound: This rabbit did not undergo ultrasound examination due to the timing of death.

[0166] Gross Pathology / Histopathology: Based on gross pathology, an intraocular tumor measuring 3 mm height (H) x 8 mm largest tumor dimension (LTD) was observed, and based on histopathology, an intraocular tumor measuring 2.2 mm H x 9.5 mm LTD was observed. Based on gross pathology, an extraocular tumor measuring 1.4 mm height x 9.4 mm LTD was observed. Approximately 10% of both the intraocular and extraocular tumors were necrotic. No cuffing pattern was detected. Full processing Rabbit 9

[0167] Rabbit #9 had a clinically detectable tumor of approximately 1 DD in size on the fundus at week 4 and was immediately treated. In the following week, the tumor was estimated to be 0.5 DD. In week 6, the tumor was estimated to be <0.5 DD, and in the final week, no tumor was detected.

[0168] Ultrasound: No tumor was discernible on ultrasound at week 3, but a mass measuring 1.04 mm in height was identified at week 4. Ultrasound measurements decreased over subsequent weeks until the tumor was no longer visible by week 6 and thereafter.

[0169] Gross Pathology / Histopathology: Histopathology revealed no tumor or cells. However, serial sections of the whole eye and immunohistochemistry are needed to further confirm this finding. Rabbit 6

[0170] At week four, there was clinical suspicion of a tumor (elevated subretinal mass), but subretinal hemorrhage, fluid, and retinal detachment hampered clinical size estimation for the duration of the experiment.

[0171] Ultrasound: By ultrasound, a large mass of 4.88 mm was detected at week 3, which grew to 5.29 mm at week 4, at which point we started treatment. At week 5, the tumor measured 4.88 mm, while at weeks 6 and 7, the tumors measured 4.02 mm and 4.98 mm, respectively.

[0172] Gross Pathology / Histopathology: Based on gross pathology, two unique tumors were identified: an intraocular tumor and a conjunctival tumor, the latter suspected to be caused by reflux during cell implantation. Due to its location, the conjunctival tumor could not be processed and we therefore considered it as an internal control. The intraocular tumor was disaggregated and measured 7 mm H x 11 mm LTD. An extraocular extension measuring 6 mm H x 9 LTD mm was also identified, which had a characteristic texture. Based on histopathology, the intraocular tumor measured 4.9 mm H x 8.3 LTD mm and was >70% necrotic, with the remaining majority of viable cells forming the aforementioned cuff-like pattern. Untreated conjunctival tumors exhibited much less necrosis (approximately 15%) and the cuff pattern was not obvious.

[0173] The purpose of this study was to explore the activity of photosensitive virus-like nanoparticles + NIR light in an orthotopic xenograft model of uveal melanoma in rabbit eyes. All animals that received photosensitive virus-like nanoparticles + laser treatment responded favorably to the treatment. This was particularly evident for small to medium tumors, which had significant tumor shrinkage as a response to treatment and a complete histopathological response. For example, in rabbit 9, which presented with a small tumor at week 4, the tumor was completely eradicated by the first two doses of treatment and was no longer detectable clinically or histopathologically two weeks after the second treatment. In larger tumors (e.g., rabbit 4), the tumor was largely necrotic, which was in stark contrast to the untreated control (rabbit 14), which showed necrosis in only about 10% of the tumor volume, clearly indicating the efficacy of the treatment. In addition, several animals with intraocular tumors that received the full treatment had extraocular extensions that were not laser-treated; these sections showed significantly less necrosis compared to the treated tumor sections, which is another evidence supporting the efficacy of laser-activated photosensitive virus-like nanoparticles for the treatment of uveal melanoma. The retinal area adjacent to the tumor was not affected by the treatment.

[0174] Based on the intraocular tumor response after treatment, particularly compared to controls (untreated extraocular sites), the data presented herein support the selective and potent anticancer activity of photosensitive virus-like nanoparticles for the treatment of ocular melanoma in the presence of tumors. Example 18 - In vitro potency assay comparing HPV L1 and BPV L1

[0175] The efficacy of photosensitive virus-like nanoparticles (e.g., virus-like nanoparticles comprising a combination of variant HPV16 / 31L1 protein and HPVL2 protein conjugated to IR700 dye) was determined by in vitro cell killing measurements. Uveal melanoma cells (e.g., cell lines OCM-1 or 92.1) were harvested using a solution of EDTA and trypsin by conventional methods. Once removed from tissue culture plastic, the cells were suspended in complete growth medium and allowed to recover at 37°C for approximately 30 minutes. During this recovery period, serial dilutions of photosensitive virus-like nanoparticles were prepared in PBS+2% fetal bovine serum at 1 / 2 log increments (2000pM, 600pM, 200pM, 60pM, 20pM, 6pM, 2pM, and 0.6pM). After the recovery period, the cells were counted, centrifuged, and suspended in PBS+2% FBS until the cell density was 3×10 6 An equal volume of cell suspension was added to the virus-like nanoparticle dilutions to produce 1.5×10 cells at appropriate concentrations of virus-like nanoparticles (1000 pM, 300 pM, 100 pM, 30 pM, 10 pM, 3 pM, 1 pM, and 0.3 pM). 6 These conditions (e.g., 360 μl) were incubated on ice for approximately 1.5 to 2 hours.

[0176] After this incubation, the tubes were centrifuged to collect the cells and then washed twice with PBS + 2% FBS in the absence of photosensitive virus-like nanoparticles. After the final centrifugation, the cells were suspended in 200 μl of PBS + 2% FBS. 100 μl of each sample was removed and transferred to a well of a 96-well 1 / 2 zone plate. The cells were then irradiated with 25 J / cm using a Coherent Opal Photoactivator ophthalmic laser. 2 Each sample was irradiated with near-infrared light (689 nm) (600 mW, 43 seconds). After irradiation, the cell samples were then transferred to new tubes. Both the irradiated and non-irradiated samples were placed at 37° C. for another 1 to 2 hours.

[0177] After this incubation, a final 20 μl cell sample was mixed 1:1 with AOPI stain (acridine orange and propidium iodide) and the viability of the cells was assessed using a Nexcelom Cellometer Auto 2000. Figure 20 It was shown that BPVL1 and HPVL1 had comparable effects on cell viability at half maximal effective concentration (EC50) (BPVL1 = 88 pm; HPVL1 = 60.5 pm), indicating that the potency of the photosensitizing molecules is comparable to each other.

[0178] Figure 21 Shown is the analysis of photosensitive virus-like nanoparticle binding from Figure 20 Cell samples from the killing assay described in . Cells from the killing assay were scanned on an Odyssey Clx gel / plate scanner. The Odyssey Clx is specifically designed to detect and quantify a range of infrared dyes, including IR700 dye (e.g., 700DX). Thus, in this assay, cells treated with different concentrations of photosensitive virus-like nanoparticles showed concentration-dependent amounts of cell-associated fluorescence, indicating that the cells bound both BPV-L1-IR700 and HPV-L1-IR700. Example 19 - Activity of photosensitive virus-like nanoparticles in a xenograft model of head and neck cancer.

[0179] Head and neck cancer cells were implanted into the dorsal flanks of nu / nu mice. Tumors were allowed to grow for two weeks. Once tumors reached 150 mm 3 The animals were randomly divided into 6 study groups (7 animals per group) as follows: saline; photosensitive virus-like nanoparticles (HPV16 / 31L1 / L2; 200 μg dose); saline + NIR light (50 J / cm 2 ); photosensitive virus-like nanoparticles (200 μg dose) + NIR light (50 J / cm 2 ); Photosensitive virus-like nanoparticles (100 μg dose) + NIR light (50 J / cm 2 ); and photosensitive virus-like nanoparticles (50 μg dose) + NIR light (50 J / cm 2 Drug administration and NIR light treatment were performed every 3 days. Tumor measurements were recorded every 3 to 5 days.

[0180] Although all controls showed no substantial effect on their corresponding treatments, significant tumor growth inhibition was observed in all treatment groups ( Figure 22 The observed tumor growth inhibition was dose-dependent. Tumor responses were present in the high-dose group. Two animals died in the 200 μg treatment group, which was associated with extensive cell death and potentially related to toxicity associated with tumor lysis syndrome. Example 20 - Production of Photosensitive Virus-Like Nanoparticles

[0181] In order to produce the photosensitive virus-like nanoparticles of the present disclosure, HEK293F is cultivated in suspension culture and transiently transfected with a bicistronic plasmid DNA encoding L1 (or L1 and L2) capsid protein. This induces the formation of the protocapsid (as described in Current Protocols in Cell Biology 26.1.1-26.1.19, December 2007, such as Buck). After the cell mass is recovered and fragmented, the protocapsid is subjected to benzonase treatment to remove host DNA contaminants and to subsequent in vitro maturation process to form stable virus-like nanoparticles for conjugation. After purification, the virus-like nanoparticles are chemically conjugated to produce photosensitive virus-like nanoparticles with IR700NHS ester. Figure 23 shows a schematic diagram of the production process.

[0182] The photosensitive virus-like nanoparticles produced by the process described in this example have been characterized using SDS-PAGE, SE-HPLC, and DLS and shown to be 90% to 95% pure. Histones from HEK293 cells are present as part of the virus-like nanoparticle composition and account for 10% to 15% of the total protein in the virus-like nanoparticles. sequence Variant HPV16 / 31 L1 protein nucleotide sequence (SEQ ID NO: 1) BPV1L1 nucleotide sequence (SEQ ID NO: 2)

Claims

1. Tumor-targeting virus-like particles containing a photosensitive molecule conjugated to the capsid protein. The virus-like particle of claim 1 , wherein the capsid protein is a papillomavirus capsid protein. The virus-like particle of claim 2 , wherein the papillomavirus capsid protein is a non-human papillomavirus capsid protein. The virus-like particle of claim 3 , wherein the non-human papillomavirus capsid protein is a bovine papillomavirus capsid protein.

5. The virus-like particle of claim 2, wherein the virus-like particle comprises human papillomavirus capsid protein and does not cross-react with human papillomavirus (HPV) 16, HPV 18, or existing antibodies specific for HPV. The virus-like particle of any one of claims 1 to 5 , wherein the capsid protein comprises an L1 capsid protein. The virus-like particle of claim 1 , wherein the capsid protein comprises a combination of L1 and L2 capsid proteins. The virus-like particle of claim 7 , wherein the capsid protein consists of L1 capsid protein.

9. The virus-like particle of any one of claims 1 to 8, wherein the virus-like particle has modified immunogenicity and / or antigenicity.

10. The virus-like particle of any one of claims 1 to 9, wherein the photosensitive molecule is covalently conjugated to the capsid protein. The virus-like particle of any one of claims 1 to 10 , wherein the photosensitive molecule is conjugated to a lysine residue of the capsid protein.

12. The virus-like particle of claim 10 or 11, wherein the photosensitive molecule is conjugated to the capsid protein via a covalent amide bond. 13 . The virus-like particle of claim 1 , wherein the photosensitive molecule does not disrupt the binding of the virus-like particle to the surface of tumor cells. The virus-like particle of claim 13 , wherein the photosensitive molecule does not disrupt the binding of the virus-like particle to heparan sulfate proteoglycan on the surface of tumor cells.

15. The virus-like particle of any one of claims 1 to 14, wherein the virus-like particle comprises about 10 to about 1000 photosensitive molecules.

16. The virus-like particle of claim 15, wherein the virus-like particle comprises about 50 to about 1000 photosensitive molecules.

17. The virus-like particle of claim 16, wherein the virus-like particle comprises about 100 to about 1000 photosensitive molecules.

18. The virus-like particle of any one of claims 1 to 17, wherein the photosensitive molecule comprises a fluorescent dye, an infrared dye, a near-infrared dye, a porphyrin molecule, a chlorophyll molecule, or a combination of any two or more thereof.

19. The virus-like particle of any one of claims 1 to 18, wherein the photosensitive molecule is activated by infrared light, near-infrared light, or ultraviolet light.

20. The virus-like particle of any one of claims 1 to 19, wherein the photosensitive molecule is selected from the group consisting of a phthalocyanine dye, a verteporfin molecule, and a combination of a phthalocyanine dye and a verteporfin molecule.

21. A tumor-targeting virus-like particle comprising about 50 to about 1000 photosensitive molecules. The virus-like particle of claim 21 , wherein the photosensitive molecule is conjugated to a capsid protein, and the capsid protein is a papillomavirus capsid protein. The virus-like particle of claim 22 , wherein the papillomavirus capsid protein is a non-human papillomavirus capsid protein. The virus-like particle of claim 23 , wherein the non-human papillomavirus capsid protein is a bovine papillomavirus capsid protein.

25. The virus-like particle of claim 22, wherein the virus-like particle comprises a human papillomavirus capsid protein and does not cross-react with human papillomavirus (HPV) 16, HPV 18, or existing antibodies specific for HPV. 26 . The virus-like particle of claim 21 , wherein the photosensitive molecule is conjugated to a capsid protein of the virus-like particle. The virus-like particle of claim 26 , wherein the capsid protein comprises an L1 capsid protein.

28. The virus-like particle of claim 26 or 27, wherein the capsid protein comprises a combination of L1 and L2 capsid proteins. The virus-like particle of claim 28 , wherein the capsid protein consists of L1 capsid protein.

30. The virus-like particle of any one of claims 21 to 29, wherein the virus-like particle has modified immunogenicity and / or antigenicity.

31. The virus-like particle of any one of claims 22 to 30, wherein the photosensitive molecule is covalently conjugated to the capsid protein.

32. The virus-like particle of any one of claims 22 to 31, wherein the photosensitive molecule is conjugated to a lysine residue of the capsid protein.

33. The virus-like particle of claim 31 or 32, wherein the photosensitive molecule is conjugated to the capsid protein via a covalent amide bond.

34. The virus-like particle of any one of claims 21 to 33, wherein the photosensitive molecule does not disrupt the binding of the virus-like particle to the surface of tumor cells. The virus-like particle of claim 34 , wherein the photosensitive molecule does not disrupt the binding of the virus-like particle to heparan sulfate proteoglycans on the surface of tumor cells.

36. The virus-like particle of any one of claims 21 to 35, wherein the virus-like particle comprises about 100 to about 1000 photosensitive molecules.

37. The virus-like particle of any one of claims 21 to 36, wherein the photosensitive molecule comprises a fluorescent dye, an infrared dye, a near-infrared dye, a porphyrin molecule, a chlorophyll molecule, or a combination of any two or more of the foregoing.

38. The virus-like particle of any one of claims 21 to 37, wherein the photosensitive molecule is activated by infrared light, near-infrared light, or ultraviolet light.

39. The virus-like particle of any one of claims 21 to 38, wherein the photosensitive molecule is selected from the group consisting of a phthalocyanine dye, a verteporfin molecule, and a combination of a phthalocyanine dye and a verteporfin molecule.

40. Methods including the following: Administering the virus-like particle of any one of claims 1 to 39 to a subject having a tumor.

41. The method of claim 40, further comprising activating a photosensitive molecule.

42. The method of claim 40 or 41, further comprising activating the photosensitive molecule at a wavelength of light that allows intersystem crossing of oxygen to produce cytotoxic molecules or direct energy transfer to damage cell membranes.

43. Methods including the following: A tumor-targeting virus-like particle comprising a photosensitive molecule conjugated to a capsid protein is administered to a subject having a tumor.

44. Methods including the following: A tumor-targeting virus-like particle comprising about 50 to about 1000 photosensitive molecules is administered to a subject having a tumor.

45. The method of claim 43 or 44, further comprising activating the photosensitive molecule at a wavelength that renders the molecule visible.

46. ​​The method of any one of claims 43 to 45, further comprising activating the photosensitive molecule at a wavelength that renders the molecule cytotoxic, thereby killing tumor cells.

47. The method of claim 46, wherein the photosensitive molecule is laser activated.

48. The method of claim 47, wherein the laser is an infrared laser, a near-infrared laser, or an ultraviolet laser.

49. The method of claim 48, wherein the energy delivered by the infrared laser is 5 J to 100 J.

50. The method of claim 49, wherein the energy delivered by the infrared laser is 50 J.

51. The method of any one of claims 47 to 49, wherein the laser is applied for about 5 seconds to about 5 minutes.

52. The method of any one of claims 43 to 51, wherein the photosensitive molecule is activated about 30 minutes to about 48 hours after administration of the virus-like particle.

53. The method of any one of claims 43 to 52, wherein the tumor is an ocular tumor.

54. The method of claim 53, wherein the ocular tumor is located in the vitreous, choroidal cavity, iris, ciliary body, sclera, fovea, retina, optic disc, or optic nerve.

55. The method of any one of claims 43 to 52, wherein the tumor is located in the lung, pleura, liver, pancreas, stomach, esophagus, colon, breast, ovary, prostate, brain, meninges, testicle, kidney, or bladder.

56. The method of any one of claims 43 to 53, wherein the tumor is accessible without surgical intervention.

57. The method of claim 52, wherein the tumor is located in the head, neck, cervix, larynx, or skin.

58. The method of any one of claims 43 to 57, wherein the tumor is an orphan or rare disease.

59. The method of any one of claims 43 to 58, wherein the tumor is cancerous or malignant.

60. The method of claim 59, wherein the tumor is metastatic, precancerous, dysplastic, or has cells suspicious for growth indicative of malignant transformation.

61. The method of any one of claims 43 to 60, wherein the virus-like particle is administered by injection.

62. The method of claim 61, wherein the virus-like particle is administered using a hollow needle or coated needle, a microneedle, or a micromanipulation needle.

63. The method of any one of claims 43 to 60, wherein the virus-like particles are administered topically, intraocularly, intravitreally, or suprachoroidally.

64. The method of any one of claims 43 to 60, wherein the virus-like particle is administered by implantation.

65. The method of any one of claims 43 to 64, wherein the capsid protein is a papillomavirus capsid protein.

66. The method of claim 65, wherein the papillomavirus capsid protein is a non-human papillomavirus capsid protein.

67. The method of claim 66, wherein the non-human papillomavirus capsid protein is a bovine papillomavirus capsid protein.

68. The method of any one of claims 43 to 64, wherein the virus-like particle comprises human papillomavirus capsid protein and does not cross-react with antibodies induced by human papillomavirus (HPV) 16, HPV 18 VLPs or existing antibodies induced by HPV infection.

69. The method of any one of claims 43 to 68, wherein the photosensitive molecule is conjugated to a capsid protein of the virus-like particle.

70. The method of claim 69, wherein the capsid protein comprises an L1 capsid protein.

71. The method of claim 69 or 70, wherein the capsid protein comprises a combination of L1 and L2 capsid proteins.

72. The method of claim 71, wherein the capsid protein consists of L1 capsid protein.

73. The method of any one of claims 43 to 72, wherein the virus-like particle has modified immunogenicity and / or antigenicity.

74. The method of any one of claims 43 to 73, wherein the photosensitive molecule is covalently conjugated to the capsid protein.

75. The virus-like particle of any one of claims 43 to 74, wherein the photosensitive molecule is conjugated to a lysine residue of the capsid protein.

76. The virus-like particle of claim 74 or 75, wherein the photosensitive molecule is conjugated to the capsid protein via a covalent amide bond.

77. The virus-like particle of any one of claims 43 to 76, wherein the photosensitive molecule does not disrupt the binding of the virus-like particle to the surface of tumor cells.

78. The virus-like particle of claim 77, wherein the photosensitive molecule does not disrupt the binding of the virus-like particle to heparan sulfate proteoglycans on the surface of tumor cells.

79. The method of any one of claims 43, 46 to 78, wherein the virus-like particle comprises about 10 to about 1000 photosensitive molecules.

80. The method of any one of claims 43, 46 to 79, wherein the virus-like particle comprises about 50 to about 1000 photosensitive molecules.

81. The method of any one of claims 43 to 80, wherein the virus-like particle comprises about 100 to about 1000 photosensitive molecules.

82. The method of any one of claims 43 to 81, wherein the photosensitive molecule comprises a fluorescent dye, an infrared dye, a near-infrared dye, a porphyrin molecule, a chlorophyll molecule, or a combination of any two or more of the foregoing.

83. The method of any one of claims 43 to 82, wherein the photosensitive molecule is selected from the group consisting of a phthalocyanine dye, a verteporfin molecule, and a combination of a phthalocyanine dye and a verteporfin molecule.

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