Targeted Combination Therapy

Abstract

Some embodiments of the present disclosure relate to methods and compositions for treating tumors using a combination of an immunotherapeutic agent and a tumor-targeting viral capsid protein construct comprising an anti-cancer molecule conjugated to a viral capsid protein. In one aspect, a method is provided comprising: (a) administering to a subject having a tumor a composition comprising a tumor-targeting viral capsid protein construct, wherein the construct comprises a photosensitive molecule conjugated to the viral capsid protein of the construct; and (b) administering to the subject having a tumor a composition comprising an immune checkpoint inhibitor.

Description

[Background technology]

[0001] Related Applications This application claims the benefit under U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 484,693, filed April 12, 2017, which is incorporated herein by reference in its entirety.

[0002] background Although many treatments for cancer are available, many forms of cancer remain incurable, untreatable, or become resistant to standard therapies. Traditional cancer treatments are frequently accompanied by severe side effects due to the cytotoxicity caused in normal / healthy cells by anti-cancer drugs. Summary of the Invention [Means for solving the problem]

[0003] Abstract The present disclosure provides methods and compositions for selectively targeting and killing tumor cells while simultaneously presenting tumor antigens to the immune system, synergistically generating anti-tumor immunity, thereby providing a combination therapy for the treatment and prevention of cancer.Unexpectedly, experimental data have shown that tumor-targeted photosensitive molecules (which become cytotoxic upon laser activation) induce tumor antigen-specific immune responses.To boost this immune response against cancer cells, immune checkpoint inhibitors can be administered simultaneously (or sequentially) with the tumor-targeted photosensitive molecules.

[0004] Thus, in some embodiments, provided herein are methods comprising: (a) administering to a subject having a tumor a composition comprising a tumor-targeting viral capsid protein construct, the construct comprising a photosensitive molecule conjugated to a viral capsid protein of the construct; and (b) administering to the subject having the tumor a composition comprising an immune checkpoint inhibitor.

[0005] In some embodiments, an immune checkpoint inhibitor is administered to a subject with a tumor who is undergoing treatment with a tumor-targeting viral capsid protein construct, the construct comprising a photosensitive molecule conjugated to a viral capsid protein of the construct.

[0006] Also provided herein is a composition comprising an immune checkpoint inhibitor and a tumor-targeting viral capsid protein construct, wherein the construct comprises a photosensitive molecule conjugated to a viral capsid protein of the construct.

[0007] The above summary is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features and uses of the technology disclosed herein. Other embodiments, advantages, features and uses of the technology disclosed herein will be apparent from the detailed description, drawings, examples, and claims. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 is a schematic diagram illustrating the mechanism of action of the VLP conjugate of the present disclosure. The VLP conjugate comprises a tumor-targeting virus-like particle (VLP) with a photosensitive molecule (IRDye® 700DX) conjugated to a primary amine on the surface of the VLP. The VLP conjugate is inactive in the absence of irradiation. The VLP conjugate preferentially targets tumor cells. Infrared irradiation at 689 nm activates the VLP conjugate. The L1 protein of the viral capsid specifically binds to heparan sulfate proteoglycans (HSPGs) on the tumor surface in a multivalent manner. The activated drug then disrupts the tumor cell membrane, resulting in immunogenic-promoting tumor cell damage and necrosis.

[0009] [Figure 2]Figure 2 is a graph showing the survival curve of mice in the TC-1 model. After tumors of approximately 50 mm3 formed (day 0), the mice were injected with the VLP conjugate or phosphate-buffered saline (PBS) and then treated with light 12 and 24 hours later. The protocol was repeated on day 3.

[0010] [Figure 3] Figure 3 is a graph showing the survival curve of mice in the TC-1 model. After tumors of approximately 100-300 mm3 formed (day 0), the mice were injected with the VLP conjugate or PBS and then treated with light 12 hours later. The protocol was repeated on day 3. Blood was collected on days 0 and 17 to measure tumor-specific T cell responses.

[0011] [Figure 4] Figure 4 shows tumor antigen-specific CD8+ T cells detected in the blood of experimental mice on day 17. Tumor volume, HPV16 E7 tetramer+ CD8+ gating, and HPV16 E7 peptide restimulation-IFN-γ, CD8+ gating are shown.

[0012] [Figure 5] Figure 5 illustrates that light treatment alone does not generate tumor antigen-specific CD8+ T cells. Tumor volume, HPV16 E7 tetramer+ CD8+ gating, and HPV16 E7 peptide restimulation-IFN-γ, CD8+ gating are shown.

[0013] [Figure 6] FIG. 6 is a schematic diagram illustrating the combination of NIR-activated tumor-targeted drug conjugates and checkpoint inhibitors to prevent tumor growth and recurrence.

[0014] [Figure 7] FIG. 7 is a schematic diagram of the experiment performed in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description To specifically deliver cancer / tumor treatment drugs to tumors, certain viral capsid proteins can be assembled and / or chemically modified to carry therapeutic molecules without losing their tumor targeting ability or structural stability. These "viral capsid protein constructs" can selectively bind to tumor cell surfaces, for example, via binding to heparan sulfate proteoglycans (HSPGs) on the tumor cell surface, and enter the tumor cells, where the therapeutic molecules (e.g., photosensitive molecules) can produce targeted cytotoxic effects and / or anti-tumor immunity, resulting in tumor cell death and prevention of tumor recurrence. The use of such virus-like particles and constructs in cancer treatment is described, for example, in PCT Publication WO2015042325 (filed September 18, 2014, the entire contents of which are incorporated herein by reference).

[0016] Some aspects of the present disclosure are based, at least in part, on unexpected results showing that photosensitive molecules delivered to tumor cells via tumor-targeting viral capsid protein constructs generate antitumor immunity. Tumor cells undergoing immunogenic-promoting cell death stimulate antigen-presenting cells (APCs) to take up tumor antigens, process them, and release immunogenic factors that induce tumor antigen-specific T cell immune responses against the tumor. Such tumor antigen-specific T cell responses, combined with the necrotic effect of the treatment, selectively and effectively destroy tumor cells without affecting normal cells, demonstrating limitless possibilities for generating antitumor immunity and preventing tumor recurrence.

[0017] Tumor cells frequently exploit mechanisms employed by the immune system to prevent self-attack. Cancer immunotherapeutics have been developed to reduce or eliminate the ability of tumor cells to evade immune system attacks. The use of such immunotherapeutics to treat cancer is described, for example, in U.S. Patent Application Publications US20160024469, US20130202645, US20100189641, U.S. Patent Nos. 5,478,556, 5,290,551, and 5,126,129 (the entireties of which are incorporated herein by reference). In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. Thus, the present disclosure encompasses compositions and methods for combined cancer / tumor therapy that utilize the unexpected synergistic effects generated by targeting and activating cytotoxic photosensitive molecules in tumor cells and administering immune checkpoint inhibitors (or other immunotherapeutic agents) simultaneously or sequentially. Such compositions and methods are more effective in controlling tumor growth, inducing anti-tumor immunity, and preventing tumor recurrence when compared to traditional chemotherapy and tumor immunotherapy strategies.

[0018] Thus, some aspects of the present disclosure provide compositions comprising a tumor-targeting viral capsid protein construct, the construct comprising a photosensitive molecule conjugated to a viral capsid protein of the construct.

[0019] A "viral capsid protein assemblage" is any particle or assembly formed from viral capsid proteins. Examples of viral capsid protein assemblages include, but are not limited to, capsomers and viral-like nanoparticles, as well as other viral pseudoviruses and viral nanoparticles. A viral capsid protein assemblage is considered "tumor targeting" because it binds tumor (e.g., cancerous) cells without binding non-tumor (e.g., non-cancerous, otherwise normal, healthy) cells. In some embodiments, the tumor-targeting viral capsid protein assemblage is a virus-like particle (VLP) having a 72-capsomer structure (e.g., comprising L1 or L1 / L2 viral capsid proteins).

[0020] In some embodiments, the viral capsid protein construct is a human papillomavirus (HPV) viral capsid protein construct (e.g., from type 16, type 31, or a modified type comprising amino acids from type 16 and amino acids from type 31). In some embodiments, the viral capsid protein construct is a non-human papillomavirus (HPV) viral capsid protein construct. Examples of non-human papillomavirus viral capsid protein constructs include, but are not limited to, bovine papillomavirus, cotton-rabbit papillomavirus, macaque papillomavirus, and murine papillomavirus.

[0021] The viral capsid protein construct includes a viral capsid protein. A "capsid protein" is a protein monomer. Capsid proteins can assemble together to form a capsomer (e.g., a pentamer of capsid proteins). A "capsomer" is a subunit of a viral capsid, which is the outer protein covering that protects the genetic material of a virus (e.g., human papillomavirus (HPV)). However, it should be understood that a capsomer in the context of the present disclosure can be used as a delivery vehicle (tumor-targeting delivery vehicle) independent of the capsid. Capsid proteins of the present disclosure include papillomavirus L1 capsid protein, papillomavirus L2 capsid protein, and variants thereof (e.g., variants with reduced or altered ability to induce infection-inhibiting antibodies). In some embodiments, the viral capsid protein construct comprises only the L1 capsid protein, while in other embodiments, the viral capsid protein construct comprises a combination of the L1 and L2 capsid proteins. "External capsid protein," as used herein, refers to a capsid protein that is exposed on the surface of a VLP.

[0022] Virus-like particles, or VLPs, as used herein, refer to organized capsid-like structures (e.g., roughly spherical or cylindrical in shape), capsomers, that contain self-assembling, ordered arrays of L1, or L1 and L2, and do not contain a viral genome. In some embodiments, the virus-like particles are morphologically and antigenically similar to authentic virions, but they lack viral genetic material (e.g., viral nucleic acid), rendering the particles non-infectious.

[0023] In some embodiments, tumor-targeting viral capsid protein constructs can be modified to reduce the immunogenicity of the particles themselves, i.e., to reduce the induction of neutralizing antibodies against the construct. The viral capsid protein construct can be assembled, for example, from capsomers having variant capsid proteins with altered immunogenicity. A variant capsid protein with "altered immunogenicity" is one that is naturally or synthetically modified (e.g., mutated, substituted, deleted, PEGylated, or inserted) in amino acids to reduce or prevent recognition of the capsid protein by existing (e.g., endogenous) viral serotype-specific antibodies. The variant capsid protein can be a human papillomavirus (HPV) L1 variant, a non-human papillomavirus L1 variant, or a papillomavirus L1 variant based on a combination of amino acids from different HPV serotypes. For example, the L1 variant with altered immunogenicity can be a recombinant protein, which is described in International Publication No. WO2010120266, filed July 24, 2009, the entire contents of which are incorporated herein by reference. In some embodiments, the viral capsid protein construct comprises an L1 variant with altered immunogenicity (a variant HPV16 / 31 L1 capsid protein).

[0024] The tumor-targeting viral capsid protein constructs of the present disclosure can specifically target cancer cells, and such specificity is mediated, at least in part, by the binding of the L1 protein in the viral capsid protein construct to heparan sulfate proteoglycans (HSPGs) on the surface of the tumor cells. This process is similar to the process by which viruses attach to and infect their host cells. Because the tumor-targeting viral capsid protein constructs do not bind to or pseudo-target intact epithelial cells, they are an excellent tool for specifically delivering anti-cancer drugs to tumors without affecting healthy or normal cells.

[0025] The tumor-targeting viral capsid protein constructs of the present disclosure comprise a capsid protein conjugated to a molecule with anti-cancer activity (referred to herein as an "anti-cancer molecule"). Such anti-cancer molecules may produce a targeted cytotoxic effect against tumor cells and / or, in some cases, induce an immune response against the tumor cells. Non-limiting examples of molecules of the present disclosure include: bortezomib, imatinib, seliciclib, afatinib (Gilotrif), alectinib (Alecensa), axitinib (Inlyta), belinstat (Beleodaq), bortezomib (Velcade), bosutinib (Bosulif), cabozantinib (Cometriq), carfilzomib (Kyprolis), Ceritinib (Zykadia), cobimetinib (Cotellic), crizotinib (Xalkori), dabrafenib (Tafinlar), dasatinib (Sprycel), erlotinib (Tarceva), everolimus (Afinitor), gefitinib (Iressa), ibrutinib (Imbruvica), idelalisib (Zydelig), imatinib (Gleevec), and ixazo Mibuxostat (Ninlaro), lapatinib (Tykerb), lenvatinib (Lenvima), nilotinib (Tasigna), olaparib (Lynparza), osimertinib (Tagrisso), palbociclib (Ibrance), panobinostat (Farydak), pazopanib (Votrient), ponatinib (Iclusig), regorafenib (Stivarga), ruxolitinib (Jakafi), sipuleucel-T (Provenge), sonidegib (Odomzo), sorafenib (Nexavar), temsirolimus (Torisel), tofacitinib (Xeljanz), trametinib (Mekinist), vandetanib (Caprelsa), vemurafenib (Zelboraf), vismodegib (Erivedge), and vorinostat (Zolinza). Any anti-cancer molecule known in the art may be used in accordance with the present disclosure.

[0026] In some embodiments, the anti-cancer molecule is a photosensitive molecule or photosensitizer. A "photosensitive molecule" or "photosensitizer" is a compound that can be promoted to an excited state upon absorbing light. This species rapidly attacks any organic compound it encounters and is therefore highly cytotoxic. In some embodiments, the activated photosensitive molecule re-emits light upon photoexcitation (e.g., a fluorophore). In some embodiments, the activated photosensitive molecule can become toxic or generate toxic molecules upon photoexcitation. For example, one class of photosensitive molecules can be promoted to an excited state upon absorbing light and can undergo intersystem crossing with oxygen to generate singlet oxygen.

[0027] Surprisingly, tumor-targeting viral capsid protein constructs containing photosensitive molecules conjugated to viral capsid proteins, once activated with near-infrared light, not only induced tumor necrosis but also induced pro-immunogenic tumor cell death and activated long-lasting tumor antigen-specific T cell responses against the tumor. Thus, the immune system was activated to attack the tumor, enhancing both local (regional) and distant treatment efficacy. Unexpectedly, this mechanism of action not only reduced the size of existing tumors (i.e., inhibited tumor growth) but also prevented new tumors from forming when challenged in mice (i.e., prevented tumor recurrence).

[0028] Examples of photosensitive molecules for use in accordance with the present disclosure include, but are not limited to, fluorescent dyes, infrared absorbing dyes, near-infrared absorbing dyes, porphyrin molecules, and chlorophyll molecules.

[0029] Examples of fluorescent dyes for use 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 dyes, auramine-rhodamine dyes, benzanthrone, bimane, 9,10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthacene, bisbenzimide, blacklight paint, calcein, carboxyfluorescein, carboxyfluorescein diacetate succinimidyl ester, carboxyfluorescein succinimidyl ester, 1-chloro-9,10-bis(phenylethynyl)anthracene, 2-chloro-9,10-bis(phenylethynyl)anthracene, 2-chloro-9,10-diphenylanthracene, coumarin, DAPI, dark quencher, DiOC6, DyLight Fluor, Fluo-3, Fluo-4, FluoProbes, fluorescein, fluorescein isothiocyanate, fluorescence image-guided surgery, fluoro-jade dye, fura-2, fura-2-acetoxymethyl ester, GelGreen, GelRed, green fluorescent protein, heptamethine dye, Indian yellow, Indo-1, lucifer yellow, luciferin, MCherry, merocyanine, Nile blue, Nile red, optical brightener, perylene, phloxine, phycobilin, phycoerythrin, phycoerythrobilin, propidium iodide, pyranine, rhodamine, rhodamine 123, rhodamine 6G, RiboGreen, RoGFP, rose bengal, rubrene, (E)-stilbene, (Z)-stilbene, sulforhodamine 101, sulforhodamine B, SYBR Green I, synapto-pHluorin, tetraphenylbutadiene, tris(bathophenanthrolinedisulfonate)ruthenium(II) tetrasodium, Texas red, Titan yellow, TSQ, umbelliferone, yellow fluorescent protein, and YOYO-1.

[0030] Examples of photosensitizing dyes for use in accordance with the present disclosure include, but are not limited to, HpD, porfimer sodium (Photofrin®, Photogem®, Photosan Hemporfin®), m-THPC, temoporfin (Foscan®), verteporfin (Visudyne®), HPPH (Photochlor®), Palladium-bacteria-pheophorbide (Tookad®), 5-ALA, 5 Aminolevulinic acid (Levulan®), 5-ALA methyl ester (Metvix®), 5-ALA benzyl ester (Benzvix®), 5-ALA hexyl ester (Hexvix®), lutetium(III)-texaphyrin or motexafin-lutetium (Lutex®, Lutrin®, Angrin®, Optrin®), SnET2, ethyl etiopurpurin(IV) (Purlytin®, Photrex®), NPe6, mono-L-aspartyl chlorine e6, talaporfin sodium (Talporfin®, Laserphyrin®), BOPP, boronated protoporphyrin protoporphyrin) (BOPP®), zinc phthalocyanine (CGP55847®), silicon phthalocyanine (Pc4®), a mixture of sulfonated aluminum phthalocyanine derivatives (Photosens®), ATMPn, acetoxy-tetrakis(β-methoxyethyl-)porphycene), TH9402, and dibromorhodamine methyl ester.

[0031] Examples of photosensitive molecules for use in accordance with the present disclosure include those that can be used in fluorescence imaging (e.g., near-infrared (NIR) absorbing fluorescent dyes), such as porphyrins, chlorophylls, chlorins, phthalocyanines, bacteriochlorins, texaphyrins, Photofrin® (porfimer sodium), Visudyne® (verteporfin), Laserphyrin® / NPe6 (temoporfin), Foscan (talaporfin), methylene blue (Urolene Blue®, Swiss Blue, Basic Blue 9, Chromosmon, Methylthionium Chloride), and various derivatives of aminolevulinic acid (ALA, Metvix®, Hexvix®). TM / Cysview®), or a combination thereof. In some embodiments, the photosensitive molecule conjugated to the viral capsid protein construct of the present disclosure is a near-infrared absorbing dye. In some embodiments, the near-infrared absorbing dye is IRDye® 700DX.

[0032] According to various aspects of the present disclosure, the anti-cancer molecule (e.g., IRDye® 700DX) can be conjugated to a capsid protein (e.g., papillomavirus L1 and / or L2 capsid protein) of the viral capsid protein construct. In some embodiments, the anti-cancer molecule is covalently conjugated to a capsid protein of the viral capsid protein construct. In some embodiments, the anti-cancer small molecule is covalently conjugated to a lysine residue of a capsid protein of the viral capsid protein construct. Conjugation of the anti-cancer molecule to the capsid protein does not impair binding of the viral capsid protein construct to the surface of tumor cells or to HSPGs on the surface of tumor cells. A viral capsid protein construct conjugated to the anti-cancer molecule may be referred to herein as a "conjugate."

[0033] The L1 protein of papillomaviruses contains multiple lysines accessible for chemical conjugation. Therefore, multiple photosensitive molecules (or other anti-cancer molecules) can be conjugated to one of the tumor-targeting viral capsid protein constructs of the present disclosure; i.e., the ratio of anti-cancer molecules to viral capsid protein constructs can vary. In some embodiments, the ratio of viral capsid protein construct:photosensitive molecule is about 1:10 to about 1:1000, or about 1:50 to about 1:1000. That is, in some embodiments, a viral capsid protein construct can contain about 10 to about 1,000 photosensitive molecules. In some embodiments, the ratio of viral capsid protein construct:photosensitive molecule is 1:10, 1:15, 1:20, 1:25, 1:50, 1:75, 1:100, 1:150, 1:200, 1:250, 1:500, 1:750, or 1:1000. In some embodiments, the viral capsid protein construct 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 photosensitive molecules. For example, the viral capsid protein construct may contain 10-1000, 10-500, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 100-500, 200-500, 300-500, 400-500, 100-400, 200-400, 300-400, 100-300, or 200-300 photosensitive molecules. In some embodiments, the viral capsid protein construct may contain more than 1000 photosensitive molecules or fewer than 10 anti-cancer molecules. Conjugation of photosensitive molecules to the L1 protein of the viral capsid protein construct does not affect the structural integrity or tumor-targeting ability of the construct, i.e., its ability to selectively bind HSPGs on the surface of tumor cells. Thus, another advantage of the tumor-targeting viral capsid protein constructs of the present disclosure is their ability to specifically deliver large amounts of therapeutic agents to tumors, even at very low concentrations of the molecules.

[0034] In some embodiments, the tumor-targeting viral capsid protein construct comprises a photosensitive molecule conjugated to the viral capsid protein. In some embodiments, the photosensitive molecule is IRDye® 700DX. In some embodiments, the viral particle conjugate comprises a virus-like particle, e.g., a virus-like particle assembled from a variant or modified HPV 16 / 31 L1 capsid protein and a wild-type HPV L2 capsid protein, comprising 72 capsomers conjugated to an infrared-activated molecule, e.g., IRDye® 700DX. This conjugate has been shown to selectively bind to cancer cells, and upon laser activation, the conjugate selectively disrupts the membranes of cancer cells, killing the cancer cells without damaging adjacent normal cells.

[0035] The compositions of the present disclosure further include immunotherapeutic agents for the treatment of cancer. "Immunotherapeutic agent," as used herein, refers to an agent that promotes the use of the immune system to treat cancer. Such immunotherapeutic agents typically take advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system. In some embodiments, the immunotherapeutic agent actively induces the immune system to attach to tumor cells. In some embodiments, the immunotherapeutic agent enhances existing anti-tumor responses (e.g., monoclonal antibodies, lymphocytes, and cytokines). In some embodiments, the immunotherapeutic agent molecule modulates the activity of immune checkpoints.

[0036] "Immune checkpoints" are proteins of the immune system that either enhance (costimulatory molecules) or attenuate immune response signals. Many cancers utilize inhibitory immune checkpoint proteins to protect themselves from the immune system by inhibiting T cell signals. Such inhibitory checkpoint proteins include, but are not limited to, cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death 1 receptor (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM3), lymphocyte activation gene-3 (LAG3), V-set domain-containing T cell activation inhibitor 1 (VTVN1 or B7-H4), cluster of differentiation 276 (CD276 or B7-H3), B and T lymphocyte attenuation factor (B and T lymphocyte attenuation factor (B and T lymphocyte attenuation factor (B and T lymphocyte attenuation factor (B and T lymphocyte attenuation factor (T ... Attenuator (BTLA), galectin-9 (GAL9), checkpoint kinase 1 (Chk1), adenosine A2A receptor (A2aR), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3), and V-domain Ig suppressor of T-cell activation (VISTA).

[0037] Some of these immune checkpoint proteins require their cognate binding partners, or ligands, for their immunoinhibitory activity. For example, A2AR is a receptor for adenosine A2A, and binding of A2A to A2AR activates a negative immune feedback loop. As another example, PD-1 downregulates the immune system by preventing T cell activation through association with its two ligands, PD-L1 and PD-L2. PD-1 achieves its immunoinhibitory function by promoting the programmed cell death of antigen-specific T cells in lymph nodes and simultaneously reducing the programmed cell death of suppressor T cells. As yet another example, CTLA4 is present on the surface of T cells; when bound to its binding partners, CD80 or CD86, on the surface of antigen-presenting cells (APCs), it transmits an inhibitory signal to T cells, thereby reducing their immune response.

[0038] Cancer cells are known to utilize immune checkpoint proteins to avoid being attacked by the immune system. Thus, the use of immune checkpoint inhibitors to enhance the immune response to cancer and thus treat cancer has been described. The immunotherapeutic agent in the compositions of the present disclosure can also be an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibits any one or more of the following: cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death 1 receptor (PD-1), T-cell immunoglobulin and mucin domain 3 (TIM3), lymphocyte-activation gene-3 (LAG3), V-set domain-containing T-cell activation inhibitor 1 (VTVN1 or B7-H4), cluster of differentiation 276 (CD276 or B7-H3), B- and T-lymphocyte attenuating factor (BTLA), galectin-9 (GAL9), checkpoint kinase 1 (Chk1), adenosine A2A receptor (A2aR), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte-activation gene-3 (LAG3), and V-domain Ig suppressor of T-cell activation (VISTA).

[0039] In some embodiments, "inhibit" refers to preventing or impairing the binding of an immune checkpoint protein to its cognate binding partner (e.g., PD-1, CTLA-4, or A2aR). In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the antibody comprises anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-TIM3, anti-LAG3, anti-B7-H3, anti-B7-H4, anti-BTLA, anti-GAL9, anti-Chk, anti-A2aR, anti-IDO, anti-KIR, anti-LAG3, anti-VISTA antibody, or a combination of any two or more of the foregoing antibodies. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody. In some embodiments, the immune checkpoint inhibitor comprises anti-PD1, anti-PD-L1, anti-CTLA-4, or a combination of any two or more of the foregoing antibodies. For example, the anti-PD-1 antibody is pembrolizumab (Keytruda®) or nivolumab (Opdivo®), and the anti-CTLA-4 antibody is ipilimumab (Yervoy®). Thus, in some embodiments, the immune checkpoint inhibitor comprises pembrolizumab, nivolumab, ipilimumab, or any combination of two or more of the foregoing antibodies. It should be understood that the examples described herein are not meant to be limiting, and that any immune checkpoint inhibitor known in the art, and any combination thereof, may be used in accordance with the present disclosure.

[0040] The compositions disclosed herein, comprising tumor-targeting viral capsid protein constructs and immunotherapeutic agents, are suitable for the treatment of cancer. Thus, the present disclosure also encompasses methods for treating tumors by administering such compositions to a subject with a tumor. The compositions are administered to the subject in an amount effective to reduce tumor growth and / or prevent tumor recurrence.

[0041] "Administering" or "administration" or "administering" means providing a substance to a subject in a pharmacologically useful manner. Conventional methods known to those skilled in the medical field can be used to administer the therapeutic agent or composition to the subject, depending on the type of cancer or site of cancer to be treated. The composition can also be administered via other conventional routes (e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir). The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intravesical, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Additionally, it can be administered to a subject via an injectable depot administration route (e.g., using 1-month, 3-month, or 6-month depot injectable or biodegradable materials and methods). In some embodiments, the composition can be administered systemically, e.g., intravenously. In some embodiments, the composition is administered topically, hi some embodiments, the composition is administered by implantation.

[0042] In some embodiments, the composition of the present disclosure can be injected into the target site where tumor growth exists.The injection method that can be used according to the present disclosure includes but is not limited to intravenous injection, intralesional injection, subcutaneous injection, intravitreal injection, suprachoroidal injection, intraperitoneal injection, intraarterial injection, intrahepatic injection and intravesical injection.When injection is used, hollow needle, coated needle, mini-needle or micro-needle is used depending on the area of ​​injection.

[0043] In some embodiments, the disclosed cancer treatment method comprises administering to a subject having a tumor a composition comprising a tumor-targeting viral capsid protein construct, the composition comprising a photosensitive molecule conjugated to the viral capsid protein, and a composition comprising an immunotherapeutic agent.

[0044] In some embodiments, the tumor-targeting viral capsid protein construct and the immunotherapeutic agent are administered simultaneously. "Concurrently" means that two or more substances / agents (e.g., the tumor-targeting viral capsid protein construct and the immunotherapeutic agent) are administered to a subject at the same time. For example, compositions containing the tumor-targeting viral capsid protein construct or the immunotherapeutic agent can be combined / mixed prior to administration.

[0045] In some embodiments, the tumor-targeting viral capsid protein construct and the immunotherapeutic agent are administered sequentially. "Sequentially" means that the administration of one agent and the administration of another agent are separated in time (as two separate steps). In some embodiments, the tumor-targeting viral capsid protein construct is administered first, and the immunotherapeutic agent (e.g., a checkpoint inhibitor) is administered second. In some embodiments, the immunotherapeutic agent is administered first, and the tumor-targeting viral capsid protein construct is administered second. The time between the two administering steps can be at least 1 minute, at least 5 minutes, at least 30 minutes, at least 1 hour, at least 5 hours, at least 10 hours, at least 1 day, at least 1 week, or even at least 1 month. When the tumor-targeting viral capsid protein construct and the immunotherapeutic agent are administered sequentially, they can also be administered via different routes or to different locations. For example, the tumor-targeting viral capsid protein construct may be administered intralesionally to the exposed tumor lesion or subcutaneously to the subject's skin, while the immunotherapeutic agent may be administered systemically, e.g., intravenously.

[0046] In some embodiments, the cancer treatment methods of the present disclosure involve administering an immunotherapeutic agent to a subject having a tumor who is undergoing treatment with a tumor-targeting viral capsid protein construct comprising a molecule (e.g., a photosensitive molecule) conjugated to a viral capsid protein.

[0047] The photosensitive molecules of the present disclosure can be activated by infrared, near-infrared, or ultraviolet light, depending on the type of molecule. For example, an infrared laser, a near-infrared laser, or an ultraviolet laser can be used to activate the photosensitive molecules of the tumor-targeting viral capsid protein construct in some embodiments. The energy delivered by the laser can range from 5 joules (J) to about 150 J, or from 8 J to 36 J. In some embodiments, the energy delivered by the laser is 5 J, 6 J, 7 J, 8 J, 9 J, 10 J, 12 J, 14 J, 16 J, 18 J, 20 J, 22 J, 24 J, 26 J, 28 J, 30 J, 32 J, 34 J, 36 J, 38 J, 40 J, 50 J, 60 J, 70 J, 80 J, 90 J, 100 J, 110 J, 120 J, 130 J, 140 J, or 150 J.

[0048] Light or laser can be applied to the photosensitive molecule (or tumor-targeting viral capsid protein construct) for about 5 seconds to about 5 minutes. For example, in some embodiments, the light or laser is applied to the photosensitive molecule for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 seconds to activate the molecule. In some embodiments, the laser is applied to the photosensitive molecule for 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 minutes.

[0049] Light or a laser can be applied to the photosensitive molecule (or the tumor-targeting viral capsid protein construct) about 30 minutes to about 48 hours after administration of the tumor-targeting viral capsid protein construct. For example, in some embodiments, the light or laser is applied to the photosensitive molecule 30, 35, 40, 45, 50, or 55 minutes after administration of the tumor-targeting viral capsid protein construct. In some embodiments, the 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 administration of the tumor-targeting viral capsid protein construct. In some embodiments, the light or laser is applied to the photosensitive molecule 36 or 48 hours after administration of the tumor-targeting viral capsid protein construct.

[0050] The light or laser can, for example, be applied directly to the site of the tumor.

[0051] According to the cancer treatment method of the present disclosure, a subject with a tumor undergoing treatment with a viral capsid protein construct conjugated to a photosensitive molecule may further be administered an immunotherapeutic agent (e.g., an immune checkpoint inhibitor). Any of the immunotherapeutic agents described herein are suitable for administration to the subject. It should be understood that various immunotherapeutic agents may be administered depending on the type of tumor to be treated. Those skilled in the art will be able to identify appropriate immunotherapeutic agents. In some embodiments, an immune checkpoint inhibitor (e.g., an anti-PD-1, anti-PD-L1, and / or anti-CTLA-4 antibody) is administered.

[0052] Tumor types that can be treated using the compositions and methods of the present disclosure include, but are not limited to, premalignant neoplasms, malignant tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth such that it is considered cancerous or precancerous. The cancer can be primary or metastatic. Cancers include, but are not limited to, eye cancer, bile duct cancer, bladder cancer, pleural cancer, stomach cancer, ovarian cancer, meningeal cancer, kidney cancer, brain cancer (including glioblastoma and medulloblastoma), breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, stomach cancer, hematological neoplasms (including acute lymphocytic leukemia and myeloid leukemia), multiple myeloma, AIDS-related leukemia and adult T-cell leukemia-lymphoma, intraepithelial neoplasia (including Bowen's disease and Paget's disease), liver cancer, lung cancer, lymphoma, and the like. Cancers include Hodgkin's disease and lymphocytic lymphoma, neuroblastoma, oral cancer (including squamous cell carcinoma), ovarian cancer (including those arising from epithelial, stromal, germ, and mesenchymal cells), pancreatic cancer, prostate cancer, rectal cancer, sarcomas (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma), skin cancer (including melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma), testicular cancer (including seminoma, non-seminoma, teratoma, choriocarcinoma, stromal tumor, and germinal tumors such as germ cell tumors), thyroid cancer (including thyroid adenocarcinoma and medullary carcinoma), and kidney cancer (including adenocarcinoma and Wilms' tumor). Commonly encountered cancers include breast cancer, prostate cancer, lung cancer, ovarian cancer, colorectal cancer, and brain cancer. In some embodiments, the tumor is a melanoma, carcinoma, sarcoma, or lymphoma.

[0053] In some embodiments, the tumor has metastasized and has cutaneous or superficial tumor lesions (e.g., Merkel cell carcinoma, head and neck squamous cell carcinoma, basal cell carcinoma, breast cancer and metastatic breast cancer, cutaneous T-cell lymphoma (Sézary syndrome), and sarcoma). In some embodiments, the tumor is accessible without surgical intervention (e.g., tumors located in the head, neck, cervix, larynx, or skin). In some embodiments, the tumor is cancerous or malignant. In some embodiments, the tumor is made accessible (made accessible to light / laser) by surgical or endoscopic intervention. In some embodiments, the tumor is metastatic. In some embodiments, the tumor is a primary tumor and has metastasized. In some embodiments, the metastatic cancer has cutaneous or superficial tumor lesions. In some embodiments, the tumor has lesions accessible to treatment with an infrared laser. In some embodiments, the metastatic cancer has tumor lesions or metastases in the eye. In some embodiments, the tumor is an eye tumor and has metastasized to the liver. If the tumor is an ocular tumor or has metastasized to the eye, the ocular tumor is located in the vitreous, choroidal space, iris, ciliary body, sclera, fovea, retina, optic nerve head, or optic nerve.

[0054] The tumor-bearing subject is, in some embodiments, a mammal (eg, a human).

[0055] The compositions and / or therapeutic agents of the present disclosure are administered to the subject in an amount effective to reduce or prevent cancer growth. As used herein, "effective amount" refers to the amount of each agent, either alone or in combination with one or more other agents, required to confer a therapeutic effect on the subject. As will be recognized by those skilled in the art, effective amounts will vary depending on the particular condition being treated, the severity of the condition, individual subject parameters (including age, health, size, sex, and weight), the duration of treatment, the nature of concurrent treatment (if any), the particular route of administration, and similar factors within the knowledge and belief of a health practitioner. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred that maximum doses of individual components or combinations thereof, i.e., their highest safe doses according to sound medical judgment, be used. However, it will be understood by those skilled in the art that subjects may persist at lower or tolerated doses for medical, psychological, or virtually any other reason.

[0056] Empirical considerations (e.g., half-life) generally contribute to the determination of the dosage. For example, an agent compatible with the human immune system (e.g., an agent containing a region derived from a humanized antibody or a fully human antibody) can be used to prolong the half-life of the compound and prevent the compound from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment, and is generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of the disease. Alternatively, a sustained continuous release formulation of the compound may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0057] In some embodiments, dosage is every day, every 2 days, every 3 days, every 4 days, every 5 days, or every 6 days.In some embodiments, administration frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, every 3 months, or longer.The progress of this treatment can be easily monitored by conventional techniques and assays.The administration regimen (including the compound used) can be varied over time.

[0058] In some embodiments, a normal weight adult subject may be administered a dose ranging from about 0.01 to 1000 mg / kg. In some embodiments, the dose is between 1 and 200 mg. The specific dosing regimen, i.e., dose, timing, and repetition, will depend on the particular subject and their medical history, as well as the characteristics of the agent (e.g., the half-life of the agent and other considerations well known in the art).

[0059] For purposes of this disclosure, the appropriate dosage of a therapeutic agent as described herein will depend on the particular agent (or composition thereof) used, the formulation and route of administration, the type and severity of the disease, whether the compound is administered for prophylactic or therapeutic purposes, previous treatments, the subject's clinical history and response to antagonists, and the judgment of the attending physician. Typically, a clinician will administer the agent until a dosage is reached that achieves the desired result. Administration of one or more agents can be continuous or intermittent, depending, for example, on the recipient's health status, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the agent can be essentially continuous over a preselected period of time, or can be in a series of spaced doses, for example, either before, during, or after the onset of the disease.

[0060] As used herein, the term "treating" refers to the application or administration of an agent or a composition containing the agent to a subject having a disease, a symptom of the disease, or a predisposition to the disease, with the intent to cure, ameliorate, alleviate, relieve, alter, cure, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition to the disease.

[0061] Alleviating a disease includes delaying the onset or progression of the disease or reducing the severity of the disease. Alleviating a disease does not necessarily require a curative result. As used herein, "delaying" the onset of a disease means putting off, preventing, slowing down, delaying, stabilizing, and / or postponing the progression of the disease. This delay can be of various lengths of time, depending on the medical history and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease or delaying the onset of the disease is a method that reduces the probability of developing one or more symptoms of the disease within a specified time frame and / or reduces the severity of the symptoms within a specified time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

[0062] "Development" or "progression" of a disease refers to the initial manifestation of the disease and / or the progression that occurs after the disease. The development of the disease can be detected and assessed using standard clinical techniques as are well known in the art. However, development also refers to progression, which may be undetectable. For purposes of this disclosure, development or progression refers to the biological course of the condition. "Development" encompasses appearance, recurrence, and onset. As used herein, "onset" or "occurrence" of a disease encompasses initial onset and / or recurrence.

[0063] The present disclosure also includes the following paragraphs: 1. The following: (a) administering to a subject having a tumor a composition comprising a tumor-targeting viral capsid protein construct, the construct comprising a photosensitive molecule conjugated to a viral capsid protein of the construct; and (b) administering to the subject having a tumor a composition comprising an immune checkpoint inhibitor; The method includes:

[0064] 2. The method of paragraph 1, wherein the viral capsid protein is a human papillomavirus capsid protein.

[0065] 3. The method of paragraph 1, wherein the viral capsid protein is a non-human papillomavirus capsid protein.

[0066] 4. The method of paragraph 3, wherein the non-human papillomavirus capsid protein is a bovine papillomavirus capsid protein or a cottontail papillomavirus protein.

[0067] 5. The method of any one of paragraphs 1 to 4, wherein the viral capsid protein comprises a papillomavirus L1 capsid protein, or a combination of a papillomavirus L1 capsid protein and a papillomavirus L2 capsid protein.

[0068] 6. The method of any one of paragraphs 1 to 5, wherein the tumor-targeting viral capsid protein construct is a capsomer or virus-like particle.

[0069] 7. The method of any one of paragraphs 1 to 6, wherein the tumor-targeting viral capsid protein construct comprises 10 to 1000 photosensitive molecules, and optionally, the tumor-targeting viral capsid protein construct comprises 200 to 500 photosensitive molecules.

[0070] 8. The method of any one of paragraphs 1 to 7, wherein the photosensitive molecule comprises a fluorescent dye, an infrared absorbing (IR) dye, a near-infrared absorbing (NIR) dye, a porphyrin, a chlorophyll, a chlorin, a phthalocyanine, a bacteriochlorin, a texaphyrin, porfimer sodium, verteporfin, temoporfin, talaporfin, methylene blue, aminolevulinic acid, or a combination thereof.

[0071] 9. The method of any one of paragraphs 1 to 8, wherein the tumor-targeting viral capsid protein construct is a virus-like particle comprising 72 capsomers assembled from a variant or modified HPV 16 / 31 L1 capsid protein and a wild-type HPV L2 capsid protein, and the photosensitive molecule comprises IRdye 700DX.

[0072] 10. The method of any one of paragraphs 1 to 9, wherein the immune checkpoint inhibitor inhibits binding of CTLA-4, PD-1, PD-L1, TIM3, LAG3, B7-H3, B7-H4, BTLA, GAL9, Chk1, or A2aR to its cognate binding partner.

[0073] 11. The method of paragraph 10, wherein the immune checkpoint inhibitor is an antibody.

[0074] 12. The method of paragraph 11, wherein the antibody is a monoclonal antibody.

[0075] 13. The method of any one of paragraphs 1 to 12, wherein the antibody is selected from an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-Chk1 antibody, an anti-A2aR antibody, and a combination of any two or more of the foregoing antibodies.

[0076] 14. The method of paragraph 13, wherein the antibody is selected from pembrolizumab, nivolumab, ipilimumab, and any combination of two or more of the foregoing antibodies.

[0077] 15. The method of any one of paragraphs 1 to 14, wherein the tumor-targeting viral capsid protein construct and the immune checkpoint inhibitor are administered simultaneously.

[0078] 16. The method of any one of paragraphs 1 to 14, wherein the tumor-targeting viral capsid protein construct and the immune checkpoint inhibitor are administered sequentially.

[0079] 17. The method of paragraph 16, wherein the tumor-targeting viral capsid protein construct is administered to the subject before the immune checkpoint inhibitor is administered to the subject.

[0080] 18. The method of paragraphs 16 and 17, wherein the tumor-targeting viral capsid protein construct is administered locally to a tumor or tumor lesion and the immune checkpoint inhibitor is administered systemically.

[0081] 19. The method of any one of paragraphs 1 to 18, wherein the tumor-targeting viral capsid protein construct and / or the immune checkpoint inhibitor is administered by injection, topically, or by implantation.

[0082] 20. The method of paragraph 19, wherein the injection is an intralesional injection, a subcutaneous injection, an intravitreal injection, a suprachoroidal injection, an intraperitoneal injection, an intraarterial injection, an intrahepatic injection, an intravesical injection, or any combination thereof.

[0083] 21. The method of any one of paragraphs 1 to 20, further comprising activating the photosensitive molecule using an infrared laser, a near-infrared laser, or an ultraviolet laser.

[0084] 22. The method of any one of paragraphs 1 to 21, wherein the tumor is cancerous.

[0085] 23. The method of paragraph 23, wherein the tumor is metastatic.

[0086] 24. The method of any one of paragraphs 1 to 23, wherein the tumor is a melanoma, carcinoma, sarcoma, or lymphoma.

[0087] 25. The method of any one of paragraphs 1 to 24, wherein the tumor has a lesion accessible to treatment with an infrared laser.

[0088] 26. The method of paragraph 25, wherein said tumor is Merkel cell carcinoma, squamous cell carcinoma, basal cell carcinoma, metastatic breast cancer, cutaneous T-cell lymphoma or sarcoma.

[0089] 27. The method of any one of paragraphs 1 to 25, wherein the tumor is a primary tumor and has metastasized.

[0090] 28. The method of any one of paragraphs 1 to 27, wherein the tumor is located in and / or has metastasized to the liver, bladder, eye, head, neck, cervix, larynx, skin, lung, pleura, pancreas, stomach, esophagus, colon, breast, ovary, prostate, testicle, brain, meninges, or kidney.

[0091] 29. The method of any one of paragraphs 1 to 27, wherein the subject is a human.

[0092] 30. A method comprising administering an immune checkpoint inhibitor to a subject having a tumor who is undergoing treatment with a tumor-targeting viral capsid protein construct, wherein the construct comprises a photosensitive molecule conjugated to a viral capsid protein of the construct.

[0093] 31. A composition comprising an immune checkpoint inhibitor and a tumor-targeting viral capsid protein construct, said construct comprising a photosensitive molecule conjugated to a viral capsid protein of said construct.

[0094] 32. The composition of paragraph 31, wherein the viral capsid protein is a human papillomavirus capsid protein.

[0095] 33. The composition of paragraph 31, wherein the viral capsid protein is a non-human papillomavirus capsid protein.

[0096] 34. The composition of paragraph 33, wherein the non-human papillomavirus capsid protein is a bovine papillomavirus capsid protein or a cottontail rabbit papillomavirus protein.

[0097] 35. The composition of any one of paragraphs 31 to 34, wherein the viral capsid protein comprises a papillomavirus L1 capsid protein, or a combination of a papillomavirus L1 capsid protein and a papillomavirus L2 capsid protein.

[0098] 36. The composition of any one of paragraphs 31 to 35, wherein the tumor-targeting viral capsid protein construct is a capsomer or virus-like particle.

[0099] 37. The composition of any one of paragraphs 31 to 36, wherein the tumor-targeting viral capsid protein construct comprises 10 to 1000 photosensitive molecules.

[0100] 38. The composition of any one of paragraphs 31 to 37, wherein the photosensitive molecule comprises a fluorescent dye, an infrared absorbing (IR) dye, a near-infrared absorbing (NIR) dye, a porphyrin, a chlorophyll, a chlorin, a phthalocyanine, a bacteriochlorin, a texaphyrin, porfimer sodium, verteporfin, temoporfin, talaporfin, methylene blue, aminolevulinic acid, or a combination thereof.

[0101] 39. The composition of any one of paragraphs 31 to 38, wherein the tumor-targeting viral capsid protein construct is a virus-like particle comprising 72 capsomers assembled from a variant or modified HPV 16 / 31 L1 capsid protein and a wild-type HPV L2 capsid protein, and the photosensitive molecule comprises IRdye 700DX.

[0102] 40. The composition of any one of paragraphs 31 to 39, wherein said immune checkpoint inhibitor inhibits binding of CTLA-4, PD-1, PD-L1, TIM3, LAG3, B7-H3, B7-H4, BTLA, GAL9, Chk1 or A2aR to its cognate binding partner.

[0103] 41. The composition of paragraph 40, wherein the immune checkpoint inhibitor is an antibody.

[0104] 42. The composition of paragraph 41, wherein the antibody is a monoclonal antibody.

[0105] 43. The composition of any one of paragraphs 31 to 42, wherein the antibody is selected from an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-Chk1 antibody, an anti-A2aR antibody, and a combination of any two or more of the foregoing antibodies.

[0106] 44. The composition of paragraph 43, wherein the antibody is selected from pembrolizumab, nivolumab, ipilimumab, and any combination of two or more of the foregoing antibodies.

[0107] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting in any way. [Example]

[0108] Example 1: Efficacy and induction of anti-tumor immune responses using intravenously administered VLP conjugates Methods. Albino C57Bl / 6 mice were treated with 5 × 10 5 TC-1 cells were injected subcutaneously. 3 Upon reaching 100 μg / kg (designated as day 0), the animals were administered 25 μg VLP conjugate (tumor-targeted VLP conjugated to IRDye® 700DX) or PBS via intravenous injection, followed 12 hours later by tumor-targeted near-IR light treatment (50 J / cm 2 ;690 nm). This protocol was repeated on day 3. Blood was collected on days 0 and 17 to measure tumor-specific T cell responses. 3 When the stool was reached, the animals were euthanized.

[0109] Results. Compared with PBS-treated animals, survival was significantly increased overall in VLP conjugate / light-treated animals. All control animals were euthanized on day 21, with a median survival of 11 days. VLP conjugate / light-treated animals survived for a median of 24 days, with two animals exhibiting complete tumor regression (Figure 3 and Table 2). Tumor-specific CD8+ T cells (E7 tetramer-positive and IFNγ-secreting after E7 peptide stimulation) were detected in these same two animals, indicating that VLP conjugates could induce antitumor immunity against neo-tumor antigens (Figure 4). Animals with smaller tumors controlled tumor growth, and E7-specific immune responses were detected on day 17. At the time of treatment, the tumor burden was higher than in the representative experiment, and the two responding animals also had minimal tumors at the start of treatment, indicating that a treatment threshold may exist. Light treatment alone was shown to be ineffective (Figure 5). Thus, in an aggressive animal model of lung cancer (immune competent), the combined treatment showed good efficacy (two complete responses). Tetramer staining and cytokine secretion in response to stimulation with E7 peptide indicated the initiation of antitumor immunity. [Table 1]

[0110] Example 2: Efficacy and induction of anti-tumor immune responses using VLP conjugates administered IV in a new formulation buffer Methods. Albino C57Bl / 6 mice were treated with 5 × 10 5 TC-1 cells were injected subcutaneously. The tumors were approximately 50 mm 3 When the tumor reached 100 μg / cm (designated as day 0), the animals were administered 100 μg VLP conjugate or PBS via intravenous injection, followed by tumor-directed near-IR light treatment (50 J / cm ) 12 and 24 hours later. 2 ; 690 nm) was performed. Two additional arms were included (VLP conjugates without treatment and without light). This protocol was repeated on day 3. Blood was taken on days 0 and 17 to measure tumor-specific T cell responses. Tumors >1500 mm in size were included. 3When the stool was reached, the animals were euthanized.

[0111] Naive mice and mice showing tumor regression were treated with 5 x 10 5 Animals were challenged subcutaneously with TC-1 cells and tumor burden was measured for one month. Animals rechallenged with TC-1 cells showed no tumor growth after rechallenge, while all rechallenged control animals had tumor growth.

[0112] Results. In an aggressive lung cancer animal model (immune competent), survival was significantly increased overall in the VLP conjugate / light group compared with all other arms (Figure 2 and Table 2). One animal in the untreated group showed spontaneous regression. Two VLP conjugate / light mice showed complete regression, and one mouse showed controlled growth in that its tumor reduced in size and did not grow or disappear. Thus, the combined treatment demonstrated good efficacy. The generated antitumor response was able to prevent new tumor outgrowth upon challenge.

[0113] E7 tetramer staining and IFNγ secretion were detectable in two animals. However, the high background staining may have masked the low positive values. The four remaining mice were then challenged with TC-1 tumors in parallel with the three naive mice. By the time the naive mice had to be euthanized due to tumor burden, three-quarters of the challenged mice remained tumor-free. The only animal that did not survive tumor challenge was the mouse that showed tumor control (no growth). The challenge tumor did not grow; however, the primary tumor was no longer controlled and began to grow again. These data indicate that an immunological response was mounted that not only eliminated the primary tumor but also prevented the development of subsequent tumors. Changes in the VLP conjugate buffer may also result in reduced stability after IV injection, resulting in fewer responders. [Table 2]

[0114] Example 3: Combining VLP conjugates with existing cancer treatments to enhance the generation of tumor-specific T cells Background: Several classes of cancer treatments drive tumor cells toward apoptotic death, a process often ignored by the host immune response. Furthermore, many tumors have evolved to evade immune recognition by the host. In light of this, a new generation of immune modulators / checkpoint inhibitors has been introduced. Specifically, antibodies against T cells and tumor cell surface antigens block inhibitory signals passing between cells (e.g., PD-1, PD-L1, CTLA-4) (Melero, 2015). Data indicate that VLP conjugates can induce antitumor immunity, likely due to acute necrosis. Combining these conjugates with treatment modalities that enhance T cell / tumor interactions significantly increases their efficacy against primary tumors and provides systemic antitumor immunity, potentially targeting unidentified distant metastases and preventing disease recurrence.

[0115] Methods: Using the TC-1 tumor model, VLP conjugate / light treatment is combined with anti-PD-1, anti-PD-L1, and / or anti-CTLA-4 (or other molecules, if appropriate). Control arms include animals receiving these antibodies or VLP conjugates alone. VLP conjugate / light treatment is highly disruptive to the tumor microenvironment immediately after treatment. This, combined with the knowledge that it takes an estimated two weeks to generate a tumor-specific T cell response, means that immune checkpoint inhibitors are applied before and / or after VLP conjugate / light treatment to assess synergistic effects on tumor responses. Measurements of viable and circulating tumor-specific T cells are read at two weekly intervals after treatment. VLP conjugate + checkpoint inhibitor treatment is more effective in controlling tumor growth and inducing antitumor immunity when compared to the same checkpoint inhibitor or its VLP conjugate as a single treatment (Figure 6).

[0116] Example 4: Subcutaneous model Mice were inoculated with TC-1 (lung) or MB49 (bladder) cancer cells (1–5 × 10 5 The tumor is transplanted into the mouse. 3 When the time reached 10 days (approximately 10 days), they were treated with 100 μg of antibody (αCTLA-4, αPD-L1, or αPD-1 and its corresponding isotype control) i.p., 100-200 μg of tumor-targeting virus-like particles (VLPs) (drug) i.v. with a photosensitive molecule (IRDye® 700DX) conjugated to a primary amine on the surface of the VLPs, and 12 hours later, irradiated with 690 nm light (50 J / cm). 2 ) is given (Figure 7).

[0117] The antibody is administered two more times, two days apart.

[0118] Drug and 690 nm light exposure is repeated 24 hours later.

[0119] A second round of drug / light and antibody treatment may be administered one week later.

[0120] Antibody - 100 μg delivered ip; obtained from BioXCell αCTLA-4 - clone 9D9 αPD-L1 - clone 10F.9G2 αPD-1 - clone RMP1-14 Isotype controls

[0121] References Feltkamp MC, Smits HL, Vierboom MP, Minnaar RP, de Jongh BM, Drijfhout JW, ter Schegget J, Melief CJ, Kast WM. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol. 1993 Sep;23(9):2242-9. Lin KY, Guarnieri FG, Staveley-O'Carroll KF, Levitsky HI, August JT, Pardoll DM, Wu TC. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 1996 Jan 1;56(1):21-6. Melero I, Berman DM, Aznar MA, Korman AJ, Perez Gracia JL, Haanen J. Evolving synergistic combinations of targeted immunotherapies to combat cancer. Nat Rev Cancer. 2015 Aug;15(8):457-72.

[0122] All references, patents, and patent applications disclosed herein are each cited and, in some cases, incorporated by reference with respect to the subject matter thereof, which may include the entire document.

[0123] The indefinite articles "a" and "an," as used in this specification and claims, should be understood to mean "at least one," unless clearly indicated otherwise. The term "or" is generally used in its sense to include "and / or," unless the context clearly dictates otherwise.

[0124] Unless expressly indicated otherwise, it should be understood that in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0125] In the claims and the above specification, all transitional phrases (e.g., "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," etc.) are to be understood to be open (including, but not limited to). Only the transitional phrases "consisting of" and "consisting essentially of," respectively, shall be closed or semi-closed transitional phrases as set forth in the U.S. Patent and Trademark Office Guidelines for Patent Examination, Section 2111.03. It is also to be understood that all open transitional phrases may be replaced with closed or semi-closed transitional phrases. Thus, the term "comprising" may be replaced with "consisting of" or "consisting essentially of." In certain embodiments, for example, the following items are provided: (Item 1) below: (a) administering to a subject having a tumor a composition comprising a tumor-targeting viral capsid protein construct, the construct comprising a photosensitive molecule conjugated to a viral capsid protein of the construct; and (b) administering to said subject having a tumor a composition comprising an immune checkpoint inhibitor; The method includes: (Item 2) 2. The method of claim 1, wherein the viral capsid protein is a human papillomavirus capsid protein. (Item 3) 2. The method of claim 1, wherein the viral capsid protein is a non-human papillomavirus capsid protein. (Item 4) 4. The method of claim 3, wherein the non-human papillomavirus capsid protein is a bovine papillomavirus capsid protein or a cottontail rabbit papillomavirus protein. (Item 5) 2. The method of claim 1, wherein the viral capsid protein comprises a papillomavirus L1 capsid protein or a combination of a papillomavirus L1 capsid protein and a papillomavirus L2 capsid protein. (Item 6) Item 10. The method of item 1, wherein the tumor-targeting viral capsid protein construct is a capsomer or virus-like particle. (Item 7) Item 1. The method according to item 1, wherein the tumor-targeting viral capsid protein construct comprises 10 to 1000 photosensitive molecules, and optionally, the tumor-targeting viral capsid protein construct comprises 200 to 500 photosensitive molecules. (Item 8) Item 10. The method of item 1, wherein the photosensitive molecule comprises a fluorescent dye, an infrared absorbing (IR) dye, a near-infrared absorbing (NIR) dye, a porphyrin, a chlorophyll, a chlorin, a phthalocyanine, a bacteriochlorin, a texaphyrin, porfimer sodium, verteporfin, temoporfin, talaporfin, methylene blue, aminolevulinic acid, or a combination thereof. (Item 9) 2. The method of claim 1, wherein the tumor-targeting viral capsid protein construct is a virus-like particle comprising 72 capsomers assembled from a variant or modified HPV 16 / 31 L1 capsid protein and a wild-type HPV L2 capsid protein, and the photosensitive molecule comprises IRdye 700DX. (Item 10) 2. The method of claim 1, wherein the immune checkpoint inhibitor inhibits binding of CTLA-4, PD-1, PD-L1, TIM3, LAG3, B7-H3, B7-H4, BTLA, GAL9, Chk1, or A2aR to its cognate binding partner. (Item 11) 11. The method of claim 10, wherein the immune checkpoint inhibitor is an antibody. (Item 12) Item 12. The method of item 11, wherein the antibody is a monoclonal antibody. (Item 13) 2. The method of claim 1, wherein the antibody is selected from an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-Chk1 antibody, an anti-A2aR antibody, and a combination of any two or more of the foregoing antibodies. (Item 14) 14. The method of item 13, wherein the antibody is selected from pembrolizumab, nivolumab, ipilimumab, and any combination of two or more of the foregoing antibodies. (Item 15) 2. The method of claim 1, wherein the tumor-targeting viral capsid protein construct and the immune checkpoint inhibitor are administered simultaneously. (Item 16) 2. The method of claim 1, wherein the tumor-targeting viral capsid protein construct and the immune checkpoint inhibitor are administered sequentially. (Item 17) 17. The method of claim 16, wherein the tumor-targeting viral capsid protein construct is administered to the subject before the immune checkpoint inhibitor is administered to the subject. (Item 18) 17. The method of claim 16, wherein the tumor-targeting viral capsid protein construct is administered locally to a tumor or tumor lesion and the immune checkpoint inhibitor is administered systemically. (Item 19) 2. The method of claim 1, wherein the tumor-targeting viral capsid protein construct and / or the immune checkpoint inhibitor are administered by injection, topically, or by implantation. (Item 20) 20. The method of item 19, wherein the injection is an intralesional injection, a subcutaneous injection, an intravitreal injection, a suprachoroidal injection, an intraperitoneal injection, an intraarterial injection, an intrahepatic injection, an intravesical injection, or any combination thereof. (Item 21) Item 10. The method of claim 1, further comprising activating the photosensitive molecule using an infrared laser, a near-infrared laser, or an ultraviolet laser. (Item 22) 2. The method of claim 1, wherein the tumor is cancerous. (Item 23) 24. The method of claim 23, wherein the tumor is metastatic. (Item 24) 2. The method of claim 1, wherein the tumor is a melanoma, carcinoma, sarcoma, or lymphoma. (Item 25) Item 10. The method of item 1, wherein the tumor has a lesion accessible to treatment with an infrared laser. (Item 26) 26. The method of claim 25, wherein the tumor is Merkel cell carcinoma, squamous cell carcinoma, basal cell carcinoma, metastatic breast cancer, cutaneous T-cell lymphoma, or sarcoma. (Item 27) 2. The method of claim 1, wherein the tumor is a primary tumor and has metastasized. (Item 28) 2. The method of item 1, wherein the tumor is located in and / or has metastasized to the liver, bladder, eye, head, neck, cervix, larynx, skin, lung, pleura, pancreas, stomach, esophagus, colon, breast, ovary, prostate, testicle, brain, meninges, or kidney. (Item 29) Item 10. The method of item 1, wherein the subject is a human. (Item 30) 1. A method comprising administering an immune checkpoint inhibitor to a subject having a tumor who is undergoing treatment with a tumor-targeting viral capsid protein construct, the construct comprising a photosensitive molecule conjugated to a viral capsid protein of the construct. (Item 31) 1. A composition comprising an immune checkpoint inhibitor and a tumor-targeting viral capsid protein construct, said construct comprising a photosensitive molecule conjugated to a viral capsid protein of said construct. (Item 32) 32. The composition of claim 31, wherein the viral capsid protein is a human papillomavirus capsid protein. (Item 33) 32. The composition of claim 31, wherein the viral capsid protein is a non-human papillomavirus capsid protein. (Item 34) 34. The composition of claim 33, wherein the non-human papillomavirus capsid protein is a bovine papillomavirus capsid protein or a cottontail rabbit papillomavirus protein. (Item 35) 32. The composition of claim 31, wherein the viral capsid protein comprises a papillomavirus L1 capsid protein or a combination of a papillomavirus L1 capsid protein and a papillomavirus L2 capsid protein. (Item 36) 32. The composition of claim 31, wherein the tumor-targeting viral capsid protein construct is a capsomer or virus-like particle. (Item 37) 32. The composition of claim 31, wherein the tumor-targeting viral capsid protein construct comprises 10 to 1000 photosensitive molecules. (Item 38) 32. The composition of claim 31, wherein the photosensitive molecule comprises a fluorescent dye, an infrared absorbing (IR) dye, a near-infrared absorbing (NIR) dye, a porphyrin, a chlorophyll, a chlorin, a phthalocyanine, a bacteriochlorin, a texaphyrin, porfimer sodium, verteporfin, temoporfin, talaporfin, methylene blue, aminolevulinic acid, or a combination thereof. (Item 39) 32. The composition of claim 31, wherein the tumor-targeting viral capsid protein construct is a virus-like particle comprising 72 capsomers assembled from a variant or modified HPV 16 / 31 L1 capsid protein and a wild-type HPV L2 capsid protein, and the photosensitive molecule comprises IRdye 700DX. (Item 40) 32. The composition of claim 31, wherein the immune checkpoint inhibitor inhibits binding of CTLA-4, PD-1, PD-L1, TIM3, LAG3, B7-H3, B7-H4, BTLA, GAL9, Chk1, or A2aR to its cognate binding partner. (Item 41) 41. The composition of claim 40, wherein the immune checkpoint inhibitor is an antibody. (Item 42) 42. The composition of claim 41, wherein the antibody is a monoclonal antibody. (Item 43) 32. The composition of claim 31, wherein the antibody is selected from an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-Chk1 antibody, an anti-A2aR antibody, and a combination of any two or more of the foregoing antibodies. (Item 44) 44. The composition of item 43, wherein the antibody is selected from pembrolizumab, nivolumab, ipilimumab, and any combination of two or more of the foregoing antibodies.

Claims

1. A combination for simultaneous, separate or sequential use in therapy, a tumor-targeting viral capsid protein construct, said construct comprising a photosensitive molecule conjugated to a human papillomavirus capsid protein of said construct; and A composition comprising an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an antibody that binds to and inhibits an immune checkpoint protein selected from CTLA-4, PD-1, and PD-L1. A combination comprising:

2. 10. The combination of claim 1 for use in tumor therapy.

3. The tumor-targeting viral capsid protein construct and the immune checkpoint inhibitor are i) simultaneously, or ii) sequentially When administered sequentially, the tumor-targeting viral capsid protein construct is optionally administered in a sequential manner. a) is administered to a subject before the immune checkpoint inhibitor is administered to the subject; or b) administered locally to the tumor or tumor lesion, and the immune checkpoint inhibitor is administered systemically.

2. The combination according to claim 1, characterized in that

4. the composition comprising the tumor-targeting viral capsid protein construct and / or the immune checkpoint inhibitor is administered by injection, topically, or by implantation; 10. The combination of claim 1, wherein the injection is optionally intralesional, subcutaneous, intravitreal, suprachoroidal, intraperitoneal, intraarterial, intrahepatic, intravesical, or any combination thereof.

5. 10. The combination of claim 1, wherein the treatment further comprises activating the photosensitive molecule using an infrared laser, a near-infrared laser, or an ultraviolet laser.

6. The tumor is a) cancerous, and optionally the tumor is metastatic; b) is a melanoma, carcinoma, sarcoma, or lymphoma; c) having a lesion accessible to treatment with an infrared laser, optionally wherein the tumor is Merkel cell carcinoma, squamous cell carcinoma, basal cell carcinoma, metastatic breast cancer, cutaneous T-cell lymphoma or sarcoma; d) It is a primary tumor that has metastasized, or e) located in and / or metastasized to the liver, bladder, eye, head, neck, cervix, larynx, skin, lung, pleura, pancreas, stomach, esophagus, colon, breast, ovary, prostate, testicle, brain, meninges, kidney; The combination according to claim 2.

7. The combination of claim 1, wherein the subject is a human.

8. 1. A composition for use in a method of treating a tumor, the composition comprising an immune checkpoint inhibitor, the method comprising administering the composition to a subject having a tumor who is undergoing treatment with a tumor-targeting viral capsid protein construct, the construct comprising a photosensitive molecule conjugated to a human papillomavirus capsid protein of the construct, and the immune checkpoint inhibitor is an antibody that binds to and inhibits an immune checkpoint protein selected from CTLA-4, PD-1, and PD-L1.

9. 2. The combination of claim 1, wherein the human papillomavirus capsid protein comprises human papillomavirus L1 capsid protein or a combination of human papillomavirus L1 capsid protein and human papillomavirus L2 capsid protein.

10. The tumor-targeting viral capsid protein construct comprises: a) is a capsomere or virus-like particle; b) comprising 10 to 1000 photosensitive molecules, optionally wherein said tumor-targeting viral capsid protein construct comprises 200 to 500 photosensitive molecules; The combination of claim 1.

11. 2. The combination of claim 1, wherein the photosensitive molecule comprises a fluorescent dye, an infrared absorbing (IR) dye, a near infrared absorbing (NIR) dye, a porphyrin, a chlorophyll, a chlorin, a phthalocyanine, a bacteriochlorin, a texaphyrin, porfimer sodium, verteporfin, temoporfin, talaporfin, methylene blue, aminolevulinic acid, or a combination thereof.

12. 2. The combination of claim 1, wherein the tumor-targeting viral capsid protein construct is a virus-like particle comprising 72 capsomers assembled from a variant or modified HPV 16 / 31 L1 capsid protein and a wild-type HPV L2 capsid protein, and the photosensitive molecule comprises IRdye 700DX.

13. The combination described in claim 1, wherein the antibody is a monoclonal antibody.

14. A combination described in claim 1 or claim 2, wherein the antibody is selected from pembrolizumab, nivolumab, ipilimumab and any combination of two or more of the aforementioned antibodies.

15. A composition for simultaneous, separate, or sequential use in therapy, comprising a tumor-targeting viral capsid protein construct comprising a photosensitive molecule conjugated to a human papillomavirus capsid protein of the construct, wherein the composition is administered in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an antibody that binds to and inhibits an immune checkpoint protein selected from CTLA-4, PD-1, and PD-L1.

16. 1. A composition for simultaneous, separate, or sequential use in therapy comprising an immune checkpoint inhibitor, wherein the composition is administered in combination with a tumor-targeting viral capsid protein construct comprising a photosensitive molecule conjugated to a human papillomavirus capsid protein of the construct, wherein the immune checkpoint inhibitor is an antibody that binds to and inhibits an immune checkpoint protein selected from CTLA-4, PD-1, and PD-L1.

Images