Near-infrared photoimmunotherapy (NIR-PIT) combination therapy for treating cancer

Abstract

Provided herein is a method of treating a subject with cancer using a therapeutically effective amount of one or more tumor-specific antibody-IR700 molecules. The method may further include administering to the subject a therapeutically effective amount of (a) one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, or a combination thereof, (b) one or more reducing agents, (c) one or more immune activators, or a combination of a, b, and c, for example, simultaneously or substantially simultaneously with the tumor-specific antibody-IR700 molecules, or sequentially (e.g., within about 0-24 hours). The method may also include administering to the subject or cancer cells in the subject (e.g., tumor or blood cancer cells) at a wavelength of 660-740 nm with at least 1 J / cm. 2 The use of one or more reducing agents can reduce edema resulting from the treatment.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of the earlier filing date of U.S. Provisional Patent Application No. 63 / 143,068, filed January 29, 2021, the entirety of which is incorporated herein by reference.

[0002] Field The present disclosure relates to methods of using antibody-IR700 conjugates in combination with other therapeutic agents, such as one or more anti-CTLA4-IR700 conjugates, anti-PD-L1-IR700 conjugates, one or more immune activators, and / or one or more reducing agents, to kill cells, e.g., cancer cells, following irradiation with near-infrared (NIR) light.

[0003] Acknowledgements for Government Support This invention was made with Government support awarded by the National Institutes of Health, National Cancer Institute under Project No. Z01 ZIA BC 011513. The Government has certain rights in this invention. [Background technology]

[0004] background Although several treatments for cancer exist, there remains a need for therapies that effectively kill tumor cells without damaging non-cancerous cells.

[0005] Molecular targeted cancer therapy has been developed to minimize the side effects of conventional cancer therapy, including surgery, radiation, and chemotherapy. Among the existing targeted therapies, monoclonal antibody (MAb) therapy has the longest history. Many therapeutic MAbs have been approved by the Food and Drug Administration (FDA) (Waldmann, Nat Med 9:269-277, 2003; Reichert et al., Nat Biotechnol 23:1073-1078, 2005). Effective MAb therapy traditionally relies on three mechanisms: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and receptor blockade, and requires multiple high doses of MAb. MAbs have also been used at lower doses as vectors to deliver therapeutics such as radionuclides (Goldenberg et al., J Clin Oncol 24, 823-834, 2006) or chemical or biological toxins (Pastan et al., Nat Rev Cancer 6:559-565, 2006). Ultimately, however, dose-limiting toxicities are associated with the biodistribution and catabolism of the antibody conjugate.

[0006] Conventional photodynamic therapy, which combines photosensitizers with the physical energy of non-ionizing light to kill cells, is not commonly used in cancer therapy because currently available non-targeted photosensitizers are also taken up by normal tissues and therefore cause side effects, even though the excitation light itself is harmless in the near infrared (NIR) range. Cancer immunotherapy, including the use of immunomodulatory antibodies, cancer vaccines, and cell-based therapies, has also become a strategy to control cancer (Chen and Mellman, Immunity 39:1-10, 2013; Childs and Carsten, Nat. Rev. Drug Discov. 14:487-498, 2015; June et al., Sci. Transl. Med. 7:280ps7, 2015; Melero et al., Nat. Rev. Cancer 15:457-472, 2015).

[0007] Near-infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that uses targeted monoclonal antibody-photoabsorber conjugates (APCs). Following antibody localization of APCs to tumor cell surface antigens, NIR light is used to induce highly selective cell lysis. NIR-PIT induces rapid necrotic cell death, which results in innate immune ligands that activate dendritic cells (DCs) in concert with immunogenic cell death (ICD). A description of how NIR-PIT kills tumor cells is provided by Sato et al. (ACS Cent. Sci. 4:1559-69, 2018). Briefly, after the antibody-IR700 conjugate binds to its target, activation by NIR light causes a physical change in the shape of the antibody-antigen complex, which induces physical stress in the cell membrane and leads to increased water flow across the membrane, which ultimately causes cell rupture and necrotic cell death. In September 2020, the first APC for human use, cetuximab-IR700 (ASP1929), was conditionally approved and registered for clinical use by the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan.

[0008] Cytotoxic T-lymphocyte antigen 4 (CTLA4) is a major immune checkpoint ligand molecule that mediates antitumor immunosuppression. The antitumor efficacy of anti-CTLA4 immunotherapy was first reported in 1996, leading to the FDA approval of the CTLA4-targeting antibody ipilimumab in 2011. In addition to CTLA4-targeting therapy, therapeutics targeting regulatory T cells (Tregs) have been shown to induce antitumor effects. There are three important mechanisms by which Tregs normally downregulate antitumor immunity: (i) promoting the CTLA4 axis, (ii) reducing the amount of IL-2 available to effector T cells, and (iii) producing immunosuppressive cytokines such as IL-10 and TGF-β. Indeed, anti-CTLA4 immunotherapy is thought to be effective not only because it interferes with the CTLA4 pathway, but also because it results in the exhaustion of intratumoral Tregs. However, systemic cancer immunotherapies, including anti-CTLA4 therapy and Treg depletion, often induce autoimmune adverse events, and therefore more tumor-specific approaches would be desirable.

[0009] Another major immune checkpoint ligand molecule that mediates antitumor immune suppression is programmed cell death ligand-1 (PD-L1), which binds to the PD-1 receptor expressed on T lymphocytes and inhibits cell function. PD-L1 is conditionally expressed on various cells, including cancer cells. Antitumor host immunity that relies on cytotoxic T cells can be suppressed in the PD-L1-expressing microenvironment. Blockade of the PD-1 / PD-L1 axis using antibodies against these molecules inhibited immune checkpoints similar to anti-CTLA4 therapy, resulting in effective immune activation against cancer cells in some proportion of patients. However, similar to anti-CTLA4 therapy, systemic cancer immunotherapy, including anti-PD-1 / PD-L1 therapy, often induces autoimmune adverse events, and therefore a more tumor-specific approach would be desirable. [Prior art documents] [Non-patent literature]

[0010]

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Summary of the Invention

Means for Solving the Problems

[0011] Summary of the Present Disclosure Provided herein are methods of treating a subject having cancer with a combination of (1) one or more cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies-IR700 molecules, one or more programmed death-ligand 1 (PD-L1) antibodies-IR700 molecules, or a combination thereof, (2) one or more reducing agents, or a combination of both (1) and (2), a tumor-specific protein antibody-IR700 molecule (antibody-photoabsorber conjugate (APC)) and / or one or more immune activators in combination with the tumor-specific protein antibody-IR700 molecule (antibody-photoabsorber conjugate (APC)) using NIR-photoimmunotherapy (PIT).

[0012] In one example, the method includes administering to a subject having cancer a therapeutically effective amount of (a)(i) one or more antibody-IR700 molecules, where the antibody specifically binds to a cancer cell surface molecule, e.g., a tumor-specific antigen, and / or (ii) one or more immune activators (e.g., IL-15, interferon gamma, or both), and (b) one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, or both. In some cases, the tumor-specific proteins include epidermal growth factor receptor (EGFR / HER1), mesothelin, prostate-specific membrane antigen (PSMA), HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), Lewis Y, tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor (VEGF), and VEGF-associated glycoprotein (VEGF-associated glycoprotein). VEGF, VEGF receptor (VEGFR), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2 (EphA2), glypican-1, glypican-2, glypican-3, gpA33, mucins (e.g., MUC1, MUC4, MUC5AC, or cancer antigen 125 (CA125)), CAIX, folate binding protein, gangliosides (e.g., GD2, GD3, GM1, or GM2), integrin αVβ3, integrin α5 beta3,1, Erb-B2 receptor tyrosine kinase 3 (ERBB3), MET proto-oncogene, receptor tyrosine kinase (MET), insulin-like growth factor 1 receptor (IGF1R), ephrin type A receptor 3 (EPHA3), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAILR1), TRAILR2, receptor activator of nuclear factor kappa-B ligand (RANKL), fibroblast activation protein (FAP), tenascin, BCR complex, gp72, HLA-DR10beta, HLA-DR antigen, IgE, CA242, polymorphic epithelial mucin (PEM) antigen, SK-1 antigen, programmed death 1 (PD-1), or programmed death ligand 2 (PD-L2).In specific examples, the tumor-specific protein is EGFR / HER1, and in some of those examples, the antibody-IR700 molecule comprises panitumumab or cetuximab. In specific examples, EGFR antibody-IR700 and CTLA4 antibody-IR700 are administered. In specific examples, IL-15 and CTLA4 antibody-IR700 are administered. In specific examples, IFN-gamma and CTLA4 antibody-IR700 are administered. In specific examples, EGFR antibody-IR700 and PD-L1 antibody-IR700 are administered. In specific examples, IL-15 and PD-L1 antibody-IR700 are administered. In specific examples, IFN-gamma and PD-L1 antibody-IR700 are administered. The molecules (a) and (b) may be administered simultaneously, substantially simultaneously, or sequentially (e.g., within about 0-24 hours of each other). In some examples, the molecules of (a) and (b) are administered intravenously. The subject or cancer cells in the subject (e.g., a tumor or cancer cells in the blood) are then administered at least 1 J / cm at a wavelength of 660-740 nm, e.g., 660-710 nm, or 680 or 690 nm. 2 (e.g., at least 10 J / cm 2 or at least 20 J / cm 2 , e.g., 10 to 60 J / cm 2 , 10J / cm 2 ~50J / cm 2 , 20~50J / cm 2 , 20J / cm 2 ~60J / cm 2 , or 20-30J / cm 2 ) is irradiated with a dose of

[0013] Methods for treating cancer in a subject are provided that include administration of one or more reducing agents to reduce unwanted side effects of NIR-PIT therapy, such as edema. Such methods may include administering to the subject a therapeutically effective amount of one or more antibody-IR700 molecules, where the antibody specifically binds to a tumor-specific protein on the surface of a cancer cell or an immune cell-specific protein on the surface of an immune cell (e.g., a T cell). In some examples, the tumor-specific protein or immune cell-specific protein is HER1 / EGFR, CTLA4, PD-L1, mesothelin, PSMA, HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, CEA, AFP, Lewis Y, TAG72, VEGF, VEGFR, EpCAM, EphA2, glypican-1, glypican-2, glypican-3, gpA33, mucins (e.g., MUC1, MUC2, MUC3, MUC4, MUC5, MUC6, MUC7, MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, MUC19, MUC20, MUC21, MUC22, MUC23, MUC24, MUC25, MUC26, MUC27, MUC28, MUC29, MUC30, MUC31, MUC32, MUC33, MUC34, MUC35, MUC36, MUC37, MUC38, MUC39, MUC40, MUC41, MUC42, MUC43, MUC44, MUC45, MUC46, MUC47, MUC48, MUC49, MUC50, MUC51, MUC52, MUC53, MUC54, MUC55, MUC56, MUC57, MUC58, MUC59, MUC59, MUC60, MUC61, MUC62, MUC63, MUC64, MUC65, MUC66, MUC67, MUC68, MUC69, MUC70, UC4, MUC5AC, or cancer antigen 125 (CA125), CAIX, folate binding protein, ganglioside (e.g., GD2, GD3, GM1, or GM2), integrin αVβ3, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, BCR complex, gp72, HLA-DR10β, HLA-DR antigen, IgE, CA242, PEM antigen, SK-1 antigen, PD-1, or PD-L2. In one example, the tumor-specific protein is or includes HER1 / EGFR, and in some of those examples, the antibody is or includes panitumumab or cetuximab. In one example, the immune cell specific protein is or includes CTLA4, and in some of those examples, the antibody is or includes ipilimumab or tremelimumab.In one example, the tumor-specific protein is or includes PD-L1, and in some of those examples, the antibody is or includes atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (embafolimab), BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737. The method further includes administering to the subject a therapeutically effective amount of one or more reducing agents, such as sodium L-ascorbate, ascorbic acid, L-cysteine, glutathione, or a combination thereof. In one example, the one or more reducing agents is or includes sodium L-ascorbate. In some examples, the method further comprises administering to the subject a therapeutically effective amount of one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, one or more immune activators, or a combination thereof. The one or more tumor-specific antibody-IR700 molecules and one or more reducing agents (and optionally one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, one or more immune activators, or a combination thereof) may be administered sequentially or simultaneously. In some examples, the antibody-IR700 molecule is administered intravenously and the reducing agent is administered intraperitoneally. The method also comprises administering at least 1 J / cm at a wavelength of 660-740 nm, e.g., 660-710 nm, or 680 or 690 nm. 2 (e.g., at least 10 J / cm 2 or at least 20 J / cm 2 , e.g., 10 to 60 J / cm 2 , 10J / cm 2 ~50J / cm 2 , 20~50J / cm 2 , 20J / cm 2 ~60J / cm 2 , or 20-30J / cm 2The method also includes irradiating the subject with a dose of 1000 mg / kg / day and / or irradiating the cancer cells in the subject with a dose of 1000 mg / kg / day. In some examples, the irradiation is performed after administration of one or more tumor-specific antibody-IR700 molecules and one or more reducing agents. In some examples, the one or more reducing agents are administered before the irradiating step. In some examples, the one or more reducing agents are administered after the irradiating step. The use of one or more reducing agents can reduce undesirable effects of NIR-PIT, such as edema, acute inflammatory response, or both. In some examples, the use of one or more reducing agents in combination with NIR-PIT reduces edema in the treated subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or even at least 95%, compared to the amount of edema without the use of one or more reducing agents with NIR-PIT. In some examples, the use of one or more reducing agents in combination with NIR-PIT reduces the acute inflammatory response in a treated subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or even at least 95% compared to the amount of acute inflammatory response without the use of one or more reducing agents with NIR-PIT.

[0014] In some examples, the methods provided herein further include selecting a subject with cancer having a tumor or cancer that expresses a cancer cell surface protein that can specifically bind to the antibody-IR700 molecule.

[0015] In some examples, the cancer or cancer cells treated by the disclosed method are breast, liver, colon, ovarian, prostate, pancreas, brain, cervix, kidney, bone, skin, head and neck, oropharynx, or blood cancer or cancer cells. In one example, the cancer is a cancer or cancer cells in the respiratory tract or mediastinum. In some examples, the cancer treated is a highly or moderately immunogenic cancer, such as melanoma, lung cancer, or renal cell carcinoma. In some examples, the cancer treated is a low immunogenic cancer, such as breast cancer, pancreatic cancer, oropharynx cancer, or oral squamous cell carcinoma.

[0016] In some examples, the disclosed methods have an abscopal effect.

[0017] In some examples, the method is a method of killing cancer cells in the blood of a subject, and the step of irradiating the cancer cells includes irradiating the cancer cells with at least 1 J / cm at a wavelength of 660-740 nm using a NIR LED. 2 (e.g., 680 nm or 690 nm, 10 to 60 J / cm 2 Dose, for example, 20-50J / cm 2 or 20~30J / cm 2 The method includes irradiating the subject with NIR LEDs (at a dose of 100 nm), where the NIR LEDs are present in a wearable device worn by the subject.

[0018] The irradiating step of the disclosed method comprises irradiating at least 1 J / cm at a wavelength of 660-740 nm. 2 For example, the two or more doses of radiation are administered within about 12 to 36 hours, e.g., within about 24 hours, of each other.

[0019] In some examples, a subject is administered two or more doses, e.g., two, three, four, five, six, seven, eight, nine, or ten doses, of one or more antibody-IR700 molecules that specifically bind to a tumor-specific protein on the surface of a cancer cell. In some examples, the two or more doses of one or more antibody-IR700 molecules that specifically bind to a tumor-specific protein on the surface of a cancer cell are separated by at least 12 hours, at least 24 hours, at least 48 hours, or at least one week, e.g., about 24-48 hours.

[0020] In some examples, the CTLA4 antibody conjugated to IR700 is or includes ipilimumab or tremelimumab.

[0021] In some examples, the PD-L1 antibody conjugated to IR700 is or comprises atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (embafolimab), BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737. In some examples, the PD-L1 antibody conjugated to IR700 is or comprises atezolizumab or avelumab. In some examples, the PD-L1 antibody conjugated to IR700 is or comprises durvalumab, KN035, or cosibelimab. In some examples, the PD-L1 antibody conjugated to IR700 comprises or consists of BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737.

[0022] In some examples, the EGFR antibody used in the EGFR antibody-IR700 molecule is or includes panitumumab or cetuximab.

[0023] In some examples, a subject is administered two or more doses, e.g., two, three, four, five, six, seven, eight, nine, or ten doses, of a CTLA4 antibody-IR700 molecule, and / or one or more PD-L1 antibody-IR700 molecules. In some examples, the two or more doses are separated by at least 12 hours, at least 24 hours, at least 48 hours, or at least one week, e.g., about 24-48 hours.

[0024] In some examples, the tumor-specific antibody binds to one or more proteins (e.g., receptors) on the surface of cancer cells, where the proteins on the surface of cancer cells are not significantly found on non-cancer cells (e.g., normal healthy cells), and thus the antibody does not significantly bind to non-cancer cells. In one example, the tumor-specific protein is HER1 / EGFR. In a specific example, the EGFR antibody specific for EGFR is or includes panitumumab or cetuximab. In one example, the tumor-specific protein is HER2 or PSMA. In one example, the tumor-specific protein is PD-L1 or CTLA4. Tumor-specific antibodies - Additional exemplary tumor-specific proteins that may be recognized by the IR700 molecule (including, for example, antibodies specific for one or more of these antigens) include mesothelin, PSMA, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, CEA, AFP, Lewis Y, TAG72, VEGF, EpCAM, EphA2, glypican-1, glypican-2, glypican-3, gpA33, mucins (e.g., MUC1, MUC4, MUC5AC, or cancer antigen 125 (CA125)), CAIX, PSMA, folate binding protein, gangliosides (e.g., GD2, GD3, GM1, or GM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5 β3,1, integrin αVβ3, integrin α5 β3,1, Erb-B2 ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, BCR complex, gp72, HLA-DR10β, HLA-DR antigen, IgE, CA242, polymorphic epithelial mucin (PEM) antigen, SK-1 antigen, PD-L1, and programmed death-ligand 2 (PD-L2). Additional exemplary tumor-specific proteins and antibodies are provided herein (including in Table 1, below).

[0025] In some examples, a therapeutically effective amount of one or more antibody-IR700 molecules (e.g., EGFR antibody-IR700) is at least 100 mg / m 2 , e.g., at least 200 mg / m2 , at least 300 mg / m 2 , at least 400 mg / m 2 , or at least 500 mg / m 2 , for example, 100 to 800 mg / m 2 , 100-500mg / m 2 , 250-700mg / m 2 , 250-500mg / m 2 , or 300-500 mg / m 2 In some examples, the therapeutically effective amount of the one or more CTLA4 antibody-IR700 molecules is at least 100 mg / m 2 , e.g., at least 200 mg / m 2 , at least 300 mg / m 2 , at least 400 mg / m 2 , or at least 500 mg / m 2 , for example, 100 to 800 mg / m 2 , 100-500mg / m 2 , 250-700mg / m 2 , 250-500mg / m 2 , or 300-500 mg / m 2 In some examples, the therapeutically effective amount of the one or more PD-L1 antibody-IR700 molecules is at least 100 mg / m 2 , e.g., at least 200 mg / m 2 , at least 300 mg / m 2 , at least 400 mg / m 2 , or at least 500 mg / m 2 , for example, 100 to 800 mg / m 2 , 100-500mg / m 2 , 250-700mg / m 2 , 250-500mg / m 2 , or 300-500 mg / m 2In some examples, the therapeutically effective amount of one or more antibody-IR700 molecules (e.g., EGFR antibody-IR700) is at least 10 mg, at least 15 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or at least 500 mg, e.g., 10-1000 mg, 15-1000 mg, 10-500 mg, 15-300 mg, or 15-100 mg. In some examples, the therapeutically effective amount of one or more CTLA4 antibody-IR700 molecules is at least 10 mg, at least 15 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or at least 500 mg, e.g., 10-1000 mg, 15-1000 mg, 10-500 mg, 15-300 mg, or 15-100 mg. In some examples, the therapeutically effective amount of the one or more PD-L1 antibody-IR700 molecules is at least 10 mg, at least 15 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or at least 500 mg, e.g., 10-1000 mg, 15-1000 mg, 10-500 mg, 15-300 mg, or 15-100 mg. In some examples, the therapeutically effective amount of the one or more reducing agents is at least 1 g, e.g., at least 5 g, at least 10 g, at least 25 g, at least 50 g, at least 100 g, at least 200 g, or at least 300 g, e.g., 1-300 g, 10-300 g, 5-50 g, 5-25 g, 5-100 g, or 100-300 g.

[0026] In some examples, the cancer cells are in the subject's blood, and the step of irradiating the cancer cells includes irradiating the blood by using a device worn by the subject, the device including a near-infrared (NIR) light emitting diode (LED).

[0027] In some examples, the method further includes selecting a subject with a cancer that expresses a tumor-specific protein that specifically binds to the antibody-IR700 molecule.

[0028] In some examples, the disclosed methods reduce the weight, volume, or size of a cancer by at least 25% compared to the absence of treatment, reduce the weight, volume, or size of a metastasis that is not irradiated with a wavelength of 660-740 nm and is located distal to the irradiated area of ​​the tumor or lesion by at least 25% compared to the absence of treatment (e.g., an abscopal effect is present), increase the subject's survival time compared to the absence of treatment, increase the subject's progression-free survival time compared to the absence of treatment, increase the subject's disease-free survival time compared to the absence of treatment, or a combination thereof.

[0029] In some examples, the disclosed methods, e.g., those utilizing a CTLA4 antibody-IR700 molecule, include CD4 + Foxp3 + CTLA4 selectively kills Tregs from all live cells and T cell populations in cancer + Selectively depletes cells within the regional lymph nodes or spleen and does not deplete CD8 + / CD4 + Foxp3 + increasing the CD8+ / Treg ratio, increasing the CD8+ / Treg ratio, decreasing intratumoral blood perfusion, or a combination thereof.

[0030] In some examples, the disclosed methods further include detecting the cancer cells by fluorescence lifetime imaging, for example, about 0-48 hours after the irradiating step.

[0031] In some examples, the disclosed methods further include administering to the subject a therapeutically effective amount of one or more immune activators, such as IL-15, interferon gamma, or both. In certain embodiments, the immune activators include one or more immune system activators and / or inhibitors of immune suppressor cells, such as antagonist PD-1 antibodies, antagonist PD-L1 antibodies, or CD25 antibody-IR700 molecules. In some examples, the inhibitors of immune suppressor cells inhibit the activity and / or kill regulatory T (Treg) cells. In other examples, the immune system activators include one or more interleukins (e.g., IL-2 and / or IL-15). The immune modulators may, in some examples, increase the production of memory T cells specific for one or more proteins expressed by the cancer cells. In some examples, the therapeutically effective amount of the one or more immune activators is at least 0.1 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, or at least 50 mg, e.g., 0.1-100 mg, 0.5-50 mg, 0.5-25 mg, 0.5-10 mg, 1-5 mg, 2-4 mg, or 1-10 mg (e.g., for iv administration), or at least 0.01 mg, at least 0.05 mg, at least 0.1 mg, or at least 1 mg, e.g., 0.01-1 mg, 0.1-1 mg, 0.01-0.075 mg, 0.5-1 mg, 0.1-0.5 mg, or 0.05-0.5 mg (e.g., for intratumoral administration).

[0032] In some examples, the one or more immune activators are administered before administration of NIR radiation. In some examples, the one or more immune activators are administered after administration of NIR radiation. In some examples, the one or more immune activators are administered before and after administration of NIR radiation.

[0033] Provided herein are methods for treating EGFR-expressing cancer in a subject. Such methods may include administering to the subject (1) a therapeutically effective amount of one or more anti-EGFR-IR700 molecules, and (2) a therapeutically effective amount of one or more CTLA4 antibody-IR700 molecules. After administration, the method may include administering 4-100 J / cm at wavelengths of 670-700 nm. 2 (e.g., 680 nm or 690 nm, 10 to 60 J / cm 2 , e.g., 20-50 J / cm 2 , or 20-30J / cm 2 The method further comprises irradiating the subject and / or irradiating the EGFR-expressing cancer cells in the subject with a dose of 1000 nm to 1000 nm, thereby treating the EGFR-expressing cancer in the subject. In some examples, the method further comprises administering to the subject a therapeutically effective amount of one or more immune activators, e.g., IL-15, interferon gamma, or both, e.g., before or after (or both) the irradiation. In some examples, the method further comprises administering to the subject a therapeutically effective amount of one or more reducing agents, e.g., sodium L-ascorbate, e.g., before or after (or both) the irradiation. The one or more anti-EGFR-IR700 molecules and the one or more CTLA4 antibody-IR700 molecules may be administered sequentially or simultaneously. In some examples, the EGFR antibody conjugated to IR700 comprises or is panitumumab or cetuximab. In some examples, the CTLA4 antibody conjugated to IR700 comprises or is ipilimumab or tremelimumab.

[0034] Provided herein are methods for treating cancer in a subject. Such methods include administering to the subject a therapeutically effective amount of one or more anti-PD-L1-IR700 molecules, administering to the subject a therapeutically effective amount of one or more immune activators (e.g., IL-15, interferon gamma, or both), followed by administering 4-100 J / cm at wavelengths of 670-700 nm. 2and / or irradiating the subject with a dose of 1000 mg / kg / day or more of the anti-PD-L1-IR700 molecule and the one or more immune activators may be administered sequentially or simultaneously. In some examples, the PD-L1 antibody conjugated to IR700 includes or is atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (embafolimab), BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737. In some examples, the method further includes administering to the subject a therapeutically effective amount of one or more reducing agents, e.g., sodium L-ascorbate, e.g., before or after (or both) the irradiation. In some examples, the method further comprises administering to the subject a therapeutically effective amount of one or more antibody-IR700 molecules, wherein the antibody specifically binds to a tumor-specific protein on the surface of a cancer cell. In some examples, the tumor-specific protein is epidermal growth factor receptor HER1 / EGFR, CTLA4, mesothelin, PSMA, HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, CEA, AFP, Lewis Y, tumor-associated glycoprotein 72 (TAG72), VEGF, VEGFR, EpCAM, Ep hA2, glypican-3, gpA33, mucin, CAIX, folate binding protein, ganglioside, integrin αVβ3, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, BCR complex, gp72, HLA-DR10β, HLA-DR antigen, IgE, CA242, PEM antigen, SK-1 antigen, PD-1, or PD-L2.

[0035] Methods for reducing edema resulting from cancer treatment in a subject, such as NIR-PIT therapy using one or more antibody-IR700 molecules, are also provided. Such methods may include administering to the subject a therapeutically effective amount of one or more antibody-IR700 molecules, where the antibody specifically binds to a tumor-specific protein on the surface of a cancer cell (e.g., EGFR or VEGF) or an immune cell (e.g., CTLA4), and administering to the subject a therapeutically effective amount of a reducing agent, such as sodium L-ascorbate. The subject and / or cancer cells may be administered a therapeutically effective amount of at least 1 J / cm at wavelengths between 660 and 740 nm. 2 The one or more antibody-IR700 molecules and the one or more reducing agents may be administered sequentially or simultaneously.

[0036] The above and other features of the present disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0037] [Figure 1A-B]Characterization of mEERL-hEGFR cell lines and evaluation with panitumumab or CTLA4 targeted NIR-PIT. (A) Validation of panitumumab-IR700 and CTLA4-IR700 by SDS-PAGE (left: colloidal blue staining, right: 700 nm fluorescence). Respective diluted antibodies were used as controls. (B) Membrane damage of mEERL-hEGFR cells induced by NIR-PIT was measured using PI staining. Each value represents the mean ± SEM of independent experiments. (C) Metabolic activity measured by MTT assay. Control groups had no APC administration or only NIR light irradiation. Each value represents the mean (mean of % relative to control) ± SEM of independent experiments. (D) Histological evaluation by HE staining. Panitumumab NIR-PIT induced swelling and vacuolization of tumor cells within 1 hour after light exposure, whereas CTLA4 NIR-PIT induced no obvious changes. Scale bars represent 100 μm (top) or 50 μm (bottom). (E) Representative multiplex immunohistochemistry images of LL / 2-luc, MC38-luc, and mEERL-hEGFR tumors. Top: composite image of pCK and DAPI staining, bottom: composite image of CD4, FoxP3, and CD8 staining. Intratumoral CD8+ T cells were counted in the multiplex IHC images. Data are shown as cell counts per mm2. Intratumoral CD8+ T cell density was significantly lower in mEERL-hEGFR tumors than in other tumors. Intratumoral CD8ρ / Treg ratio was also lower in mEERL-hEGFR tumors compared to other tumors (images, ×200; scale bar, 100 μm. n=4; one-way ANOVA followed by Tukey's test; *, P<0.05; **, P<0.01; NS, not significant). [Figure 1C-D] Same as above. [Figure 1E] Same as above. [Figure 2A]Depletion of CTLA4-expressing cells by dual-targeted NIR-PIT. (A) CTLA4 expression of several cells in mEERL-hEGFR tumors was analyzed by flow cytometry. Representative histograms of CTLA4 expression in tumor cells (hEGFR+CD45-), CD11b+ cells, CD8+ T cells (CD3+CD8+), CD4+FoxP3- T cells, and Tregs (CD3+CD4+FoxP3+), as well as the RFI of CTLA4, are shown (n=5, one-way ANOVA followed by Tukey's test; *, P<0.05; **, P<0.01; ****, P<0.0001). (B) Representative dot plots show CTLA4 expression in live cells in mEERL-hEGFR tumors by flow cytometry 3 hours after each NIR-PIT. (C) Percentage of CTLA4+ cells among total viable cells in mEERL-hEGFR tumors, tumor-draining lymph nodes, and spleen. CTLA4-expressing cells in tumors after CTLA4 NIR-PIT or dual NIRPIT were significantly decreased. (D) Representative dot plots show Treg populations in mEERL-hEGFR tumors 3 hours after each NIR-PIT. (E) Scatter plots show the ratios of Treg / CD3ρ, non-regulatory CD4+ (CD4+FoxP3-) / Treg, and CD8+ / Treg in tumors, tumor-draining lymph nodes, and spleen. In the CTLA4 and dual NIR-PIT groups, the Treg / CD3+ ratio was significantly decreased, while the CD4+ / Treg and CD8+ / Treg ratios were increased. (n=4; one-way ANOVA followed by Tukey's test; **, P<0.01; ***, P<0.001; ns, not significant). [Figure 2B-C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 3A]In vivo IR700 fluorescence imaging of mEERL-hEGFR tumors after injection of panitumumab-IR700 or anti-CTLA4-IR700. IR700 fluorescence and white light images were acquired using the 700 nm fluorescence channel of a Pearl Imager (LI-COR Biosciences) and analyzed using Pearl Cam Software (LI-COR Biosciences). Serial dorsal fluorescence images of IR700 signal were acquired before intravenous injection of 50 μg of pan-IR700 or CTLA4-IR700 via the tail vein, and ¼, 1, 3, 6, 9, 12, 18, 24, 48, 72, 96, and 120 hours after injection. Regions of interest (ROIs) were set on the tumor and adjacent non-tumor regions (left dorsal) were set as background. The mean fluorescence intensity was calculated for each ROI. Target-to-background ratio (TBR) was calculated from tumor and background fluorescence intensities by the following formula: (mean tumor fluorescence intensity) / (mean background fluorescence intensity). (A) A series of 700 nm fluorescence images were acquired at the indicated time points. (B) Quantitative analysis of mean fluorescence intensity in mEERL-hEGFR tumors is shown. (C) Quantitative analysis of TBR is shown (B, C; n=5, mean±SEM). [Figure 3B-C] Same as above. [Figure 4] Efficacy of in vivo CTLA4-targeted NIR-PIT on mEERL-hEGFR tumors. (A) Treatment schedule. (B) 700 nm fluorescence imaging before and after NIR-PIT in mEERL-hEGFR tumor-bearing mice. (AU; arbitrary units). (C) Tumor volume curves (n=10; mean ± SEM, repeated measures two-way ANOVA followed by Tukey's test; NS, not significant). (D) Survival curves (n=10; log-rank test with Bonferroni correction; NS, not significant). [Diagram 5]Efficacy of combined NIR-PIT targeting hEGFR and CTLA4 in vivo. (A) Treatment schedule. (B) 700 nm fluorescence imaging before and after NIR-PIT in mEERL-hEGFR tumor-bearing mice. The 700 nm fluorescence of the tumors decreased immediately after light exposure, except for the IV group. The yellow circle represents the location of the tumor. (C) Tumor volume curves (n=12-13; mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; *p<0.05; **p<0.01; ***p<0.001, versus combined PIT group). (D) Survival curves (n=12-13, log-rank test with Bonferroni correction, *p<0.05; **p<0.01; NS not significant). Panitumumab NIR-PIT, CTLA4 NIR-PIT, and dual NIR-PIT eliminated tumors in 1 / 12, 3 / 12, and 5 / 13 mice, respectively. [Figure 6A-B] Efficacy of combined NIR-PIT targeting CD44 and CTLA4 in vivo. (A) Treatment schedule. (B) 700 nm fluorescence imaging before and after NIR-PIT in LL / 2-luc tumor-bearing mice. 700 nm fluorescence decreased immediately after NIR light exposure, except for group IV. Yellow circles represent tumor locations. (C) BLI before and after NIR-PIT in LL / 2-luc tumor-bearing mice. (D) Luciferase activity calculated from BLI (n=10-12; mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; *, P<0.05; NS, not significant; vs. dual NIR-PIT group). (E) Tumor volume curves (n=10–12; mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; *, p<0.05; **, p<0.01; ***, p<0.001; NS, not significant; vs. dual NIR-PIT group). (F) Survival curves (n=10–12, log-rank test with Bonferroni correction; *, p<0.05; **, p<0.01; NS, not significant; vs. dual NIR-PIT group). [Figure 6C-D] Same as above. [Figure 6E-F] Same as above. [Figure 7A-C]Dual NIR-PIT improved the immunosuppressive environment within mEERL-hEGFR tumors. (A-C) Cell populations within regional lymph nodes were analyzed 2 days after each NIR-PIT by flow cytometry in the mEERL-hEGFR model. Expression of activation markers (CD40 (A), CD8 (B), CD96 (C)) on dendritic cells was calculated. CD86 expression in the dual NIR-PIT group was significantly higher compared to all other groups. (D) Multiplex immunohistochemical staining of tumors 7 days after NIR light exposure. Right: lymphocyte marker staining for CD8 (magenta), CD4 (green), and Foxp3 (yellow). Left: merged image. Scale bar, 100 μm. (E) Examples of CD8+ cells (magenta arrows), CD4+Foxp3- cells (green arrows), and CD4+Foxp3+ cells (yellow arrows). (F) Intratumoral CD8+ cell density was significantly higher in the double-PIT group than in the control group (n=5, one-way ANOVA followed by Tukey's test, *p<0.05). (G) Intratumoral CD8+ / Treg ratio was also increased in the double-NIR-PIT group compared to all other groups (n=5, one-way ANOVA followed by Tukey's test, *p<0.05). [Fig. 7D-G] Same as above. [Figure 7H] Dual-targeted NIR-PIT activates CD8+ T cells in draining lymph nodes. Expression of activation markers (CD25+ and CD69+) in CD8+ T cells was calculated. CD25 expression was significantly increased in all NIR-PIT groups. (n=5; one-way ANOVA followed by Tukey's test; *, p<0.05; NS, not significant; vs. group IV). [Figure 8]Dual NIR-PIT resulted in long-term immunity against mEERL-hEGFR tumors. Mice that achieved complete remission by dual NIR-PIT were re-inoculated with mEERL-hEGFR tumors on the opposite flank. (A) Treatment schedule and location of re-inoculation. (B) Tumor volume curves. Control = freshly inoculated tumors; Re-inoculation = tumors re-inoculated after complete clearance by dual NIR-PIT (n=5; mean ± SEM; repeated measures two-way repeated measures ANOVA followed by Tukey's test, ****p<0.00001). (C) Survival curves (log-rank test, ****p<0.0001). [Figure 9] Efficacy of in vivo dual NIR-PIT in a bilateral tumor model. (A) Treatment schedule. (B) Diagram of NIR light exposure. Red circle indicates where NIR light was applied. NIR light was only exposed to the right tumor. Grey mark indicates the inoculated tumor. (C) 700 nm fluorescence imaging before and after NIR-PIT in mEERL-hEGFR tumor-bearing mice. 700 nm fluorescence was only decreased on the right side after NIR light exposure, while 700 nm fluorescence persisted in the contralateral tumor. (D) Tumor volume curve. Tumor growth of both tumors was significantly suppressed in the dual NIR-PIT group compared to the IV group (n=10, mean ± SEM, vs. same side only in the IV group; two-way ANOVA repeated measures followed by Tukey's test, ****p<0.0001) (E) Survival curve (n=10, log-rank test, **p<0.01; NS not significant). Dual NIR-PIT eliminated tumors in 1 / 10 mice). [Figure 10]Dual-targeted NIR-PIT impairs intratumoral blood perfusion. Post-treatment blood perfusion analysis. Bovine albumin (Thermo Fisher) was conjugated with IRDye 800CW NHS ester (IR800, LI-COR Biosciences) using the same method as used for IR700. We abbreviate the conjugate as albumin-IR800. 24 hours after NIR light exposure, 50 μg of albumin-IR800 was injected intravenously. A series of dorsal 800 nm fluorescence images were acquired in the 800 nm channel of Pearl Imager. (A) Images of bovine albumin-IR800 fluorescence-based perfusion of the tumor were acquired before and 5, 10, and 15 minutes after injection. (B) Bar graphs of target-to-background ratios are shown (n=5; repeated measures two-way ANOVA followed by Tukey's test; *, P<0.05, **, P<0.01). [Figure 11A-B] Synthesis of anti-PD-L1-IR700 (CD274-Ab-IR700) and cell killing specificity of PD-L1-targeted NIR-PIT. (A) Evaluation of anti-PD-L1-IR700 by SDS-PAGE (left: colloidal blue staining, right: 700 nm fluorescence). Unconjugated anti-PD-L1 antibody was used as a control. (B) Evaluation of anti-CTLA4-IR700 by SEC. APC showed light absorption at 280 and 689 nm and corresponding fluorescence. Thus, the purity of the conjugate is good. (C) Flow cytometry analysis of PD-L1 expression in cancer cell lines. Representative histograms of MC38-luc (top) and LL2-luc (bottom) are shown. (D) Flow cytometry analysis of PD-L1 expression in cancer cell lines MC38-luc (left) and LL2-luc (right) obtained from cell cultures and in vivo tumors with or without interferon gamma. Interferon gamma conditionally enhances PD-L1 expression. (E) In vitro PD-L1 targeting NIR-PIT showed better cell killing against MC38-luc cells treated with interferon gamma. [Figure 11C-D] Same as above. [Figure 11E] Same as above. [Figure 12]Flow cytometry analysis showing PD-L1 expression in cancer cell lines before and after stimulation with 20 ng / ml or 50 ng / ml of IFNgamma (IFNγ). [Figure 13] Flow cytometry analysis showing PD-L1 expression on tumor-infiltrating immune cells. [Figure 14] Graphs showing the effect of treatment of MC38-luc tumors with anti-PD-L1-IR700 in vivo. The effect on tumor volume (top graph) and survival (bottom graph) is shown in the presence of vehicle alone (control), anti-PD-L1-IR700 without NIR light (IV), or anti-PD-L1-IR700 with NIR light (PIT). (A) Treatment of original tumors, (B) Treatment of re-inoculated mice. [Figure 15] Effect of LL2-luc tumor treatment in vivo with anti-PD-L1-IR700 conjugates and IL15 preconditioning. Vehicle alone (control), anti-PD-L1-IR700 without NIR light (IV only), anti-PD-L1-IR700 with NIR light (PIT), IL-15 only, anti-PD-L1-IR700+IL15 preconditioning without NIR light (IL15+APC), or anti-PD-L1-IR70+IL15 preconditioning with NIR light (IL15+PIT). [Figure 16] Photolysis analysis of SiPc. Synthesis scheme of Pc 3 with IR700 coaxial ligand. [Figure 17A-C]L-NaAA accelerates the ligand release from IR700. (A) 25 μM Pc 3 with various concentrations of L-NaAA in PBS was irradiated with NIR light and imaged using 700 nm fluorescence imaging. (B) The average 700 nm fluorescence intensity decreased in a dose-dependent manner for both NIR light and L-NaAA (n=4, mean ± SEM). (C) Aggregation after NIR light exposure of Pc 3 with L-NaAA. With the change in solubility from hydrophilic to hydrophobic, the cyan color of IR700 decreased and aggregation appeared, and at the same time, the 700 nm fluorescence disappeared in a dose-dependent manner for L-NaAA. (D) 3 μM panitumumab-IR700 with various concentrations of L-NaAA in PBS was irradiated with NIR light and imaged using 700 nm fluorescence imaging. (E) With L-NaAA above 1 mM in PBS, the mean fluorescence intensity decreased in a dose-dependent manner of NIR light (n=4, mean ± SEM). (F) With 3 μM panitumumab-IR700 in PBS with any concentration of NaN3, the mean fluorescence intensity after NIR light exposure was unchanged (n=4, mean ± SEM). (G) ESR spectra of samples containing IR700 (0.5 mM) and L-NaAA (10 mM) in PBS irradiated with NIR light under argon-saturated conditions (left) or in the presence of air (right) in the following five groups: (i) control spectrum (no drug), (ii) control spectrum of a sample containing IR700 (0.5 mM) and L-NaAA (10 mM) in PBS without NIR exposure, (iii) ESR spectrum with NIR light exposure under condition (ii), (iv) control spectrum of a sample containing IR700 (0.5 mM), L-NaAA (10 mM), and NaN3 (100 mM) in PBS without NIR exposure, (v) ESR spectrum with NIR light exposure under condition (iv). △; Unwanted but negligible signal originating from the quartz ESR tube described in Example 7. (H) Percentage of IR700 (post / pre) remaining in conditions (iii) and (v). Mean values ​​are shown (n=2). [Fig. 17D-F] Same as above. [Fig. 17G-H] Same as above. [Figure 18A-C]L-cysteine ​​accelerates ligand release after NIR light exposure, while NaN3 inhibits this reaction. (A) The mean 700 nm fluorescence intensity in 25 μM Pc 3 with various concentrations of L-cysteine ​​in PBS decreased in a dose-dependent manner of both NIR light and L-cysteine ​​(n=4, mean ± SEM). (B) Aggregation after NIR light exposure in Pc 3 with L-cysteine ​​mixtures. The cyan color of IR700 decreased and aggregation appeared, and at the same time, the 700 nm fluorescence disappeared in a dose-dependent manner of L-cysteine. (C) 3 μM panitumumab-IR700 with various concentrations of L-cysteine ​​in PBS was irradiated with NIR light and imaged using 700 nm fluorescence imaging. Under conditions of L-cysteine ​​above 1 mM in PBS, the mean 700 nm fluorescence intensity decreased in a dose-dependent manner of NIR light (n=4, mean ± SEM). (D) Under NaN3 above 1 mM with 3 μM panitumumab-IR700 in PBS, the mean 700 nm fluorescence intensity after NIR light exposure was significantly decreased (n=4, mean ± SEM, vs. 0 mM NaN3; two-way ANOVA repeated measures followed by Dunnett's test; *, p<0.05; **, p<0.01; ****, p<0.0001). (E) Under 3 μM panitumumab-IR700 in PBS with both 1 mM L-NaAA and L-cysteine, NaN3 had no effect on the mean 700 nm fluorescence intensity after NIR light exposure (n=4, mean ± SEM). (F) ESR spectrum obtained from a sample containing IR700 (0.5 mM) and L-cysteine ​​(10 mM) in PBS when irradiated with NIR light under argon-saturated conditions. (i) Control spectrum of a sample containing IR700 (0.5 mM) and L-cysteine ​​(10 mM) in PBS without NIR light exposure. (ii) ESR spectrum obtained when the sample was irradiated with NIR light. (iii) The ESR spectrum shown in red was obtained by adding 100 mM NaN3 to a sample containing IR700 (0.5 mM) and L-cysteine ​​(10 mM) in PBS and irradiating them with NIR under argon-saturated conditions. The overlaid ESR spectrum in grey is identical to the ESR spectrum obtained in the absence of NaN3 shown in (ii).△; unwanted but negligible signal originating from the quartz ESR tube. Cys; L-cysteine. (G) Residual IR700% (after / before) in conditions (ii) and (iii). Mean values ​​are shown (n=2). (H) HPLC chart of cystine and cysteine ​​in the presence of IR700 in several conditions. When a solution of IR700 containing cysteine ​​was irradiated under hypoxic conditions, cysteine ​​and cystine peaks were observed, but no cystine peak was present without irradiation. On the other hand, in the case of aerobic conditions, the cysteine ​​peak almost disappeared after irradiation. [Fig. 18D-E] Same as above. [Fig. 18F-G] Same as above. [Figure 18H] Same as above. [Figure 19A-B] Efficacy of panitumumab NIR-PIT in A431 GFP-luc cells with reducing agents supplemented with NaN3 in vitro. Membrane damage in A431 GFP-luc cells induced by panitumumab NIR-PIT with L-NaAA (A) or L-cysteine ​​(B) was measured using propidium iodide (PI) staining (n=4, mean ± SEM, versus NIR-PIT without L-NaAA or L-cysteine; one-way ANOVA followed by Dunnett's test; **, p<0.01; NS, not significant). (C) Membrane damage of A431 GFP-luc cells by NIR-PIT with both L-NaAA and L-cysteine ​​was measured using PI staining (n=4, mean ± SEM, versus NIR-PIT without reducing agent; one-way ANOVA followed by Dunnett's test; **, p<0.01; ***, p<0.001; NS, not significant). Membrane damage of A431 GFP-luc cells induced by panitumumab NIR-PIT with L-NaAA (D) or L-cysteine ​​(E) under 10 mM NaN3 was measured using PI staining (n=4, mean ± SEM; one-way ANOVA followed by Tukey's test; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; NS, not significant). [Figure 19C] Same as above. [Fig. 19D-E] Same as above. [Figure 20A-B] Membrane damage of MDAMB468 GFP-luc cells induced by panitumumab NIR-PIT with L-NaAA (A) or L-cysteine ​​(B) was measured using propidium iodide (PI) staining (n=4; **, p<0.01; NS, not significant; vs. NIR-PIT without L-NaAA or L-cysteine; one-way ANOVA followed by Dunnett's test). Membrane damage of MDAMB468 GFP-luc cells induced by panitumumab NIR-PIT with L-NaAA (C) or L-cysteine ​​(E) under 10 mM NaN3 was measured by PI staining (n=4; *, p<0.05; NS, not significant; one-way ANOVA followed by Tukey's test). Each value represents the mean ± SEM of independent experiments (n=4). [Figure 20C-D] Same as above. [Figure 21A-B] Cell viability was assessed by bioluminescence imaging (BLI). Cells were exposed to D-luciferin (150 mg mL-1, 10 μL per well; Gold Biotechnology, St. Louis, MO) 1 h after NIR-PIT treatment, and BLI was performed (Photon Imager; Biospace Lab, Paris, France) using M3 Vision Software (Biospace Lab). ROIs were set to include the entire well, and the average luciferase activity was measured. Cell viability of A431 GFP-luc cells (A, B) and MDAMB468 GFP-luc cells (C, D) induced by panitumumab NIR-PIT with L-NaAA or L-cysteine ​​under 10 mM NaN3 was measured by BLI. (n=4; ****, p<0.0001; NS, not significant; one-way ANOVA followed by Tukey's test). Each value represents the mean±SEM of independent experiments (n=4). [Fig. 21C-D] Same as above. [Figure 22A-B]Effect of reducing agents on albumin-IR700 processing. (A) Treatment schedule. (B) Representative images of fluorescence with or without L-NaAA. (C) Mean fluorescence intensity before and after NIR light exposure with or without L-NaAA in athymic mice (n=4, mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; *, p<0.05; NS, not significant). (E) Representative images of fluorescence with or without L-cysteine. (E) Mean fluorescence intensity before and after NIR light exposure with or without L-cysteine ​​in athymic mice (n=4, mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; NS, not significant). [Fig. 22C-D] Same as above. [Figure 22E] Same as above. [Diagram 23] In vivo efficacy of L-NaAA alone against MC38-luc tumors. (A) Treatment schedule. (B) BLI before (day 6) and after (days 8, 10, and 12) L-NaAA administration in MC38-luc tumor-bearing mice. (C) Luciferase activity calculated from BLI (n=10, mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; NS, not significant). [Fig. 24A-B]Efficacy of L-NaAA for CD44-targeted NIR-PIT in MC38-luc tumor model. (A) Treatment schedule. (B) Fluorescence imaging and (C) mean 700 nm fluorescence intensity before and after CD44-targeted NIR-PIT with or without L-NaAA in MC38-luc tumor-bearing mice (n=10, mean ± SEM; paired t-test; NS, not significant). (D) BLI before (day 6) and after (days 8-12) CD44-targeted NIR-PIT in MC38-luc tumor-bearing mice. (E) Luciferase activity calculated from BLI (n=10; repeated measures two-way ANOVA followed by Tukey's test; ****, P<0.0001; NS, not significant). (F) Formation of edema (red arrow) was shown 1 day after CD44-targeted NIR-PIT. (G) Body weight curve (n=10, mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; NS, not significant). (H) T2WI fat-suppressed MR imaging 1 day after NIR-PIT. Red arrows indicate MC38-luc subcutaneous tumors. (I) Mean hyperintense area calculated from coronal T2WI fat-suppressed MR images (n=3, mean ± SEM; unpaired t-test; *, p<0.05). [Fig. 24C-D] Same as above. [Fig. 24E-G] Same as above. [Fig. 24H-I] Same as above. [Figure 25A-C]In vivo efficacy of CD44-targeted NIR-PIT with L-NaAA on LL / 2-luc and MOC2-luc tumors. (A) Treatment schedule. (B) Fluorescence imaging and (C) mean fluorescence intensity before and after CD44-targeted NIR-PIT with or without L-NaAA in LL / 2-luc tumor-bearing mice (n=10, mean ± SEM; paired t-test; NS, not significant). (D) BLI before (day 6) and after (days 8-11) CD44-targeted NIR-PIT in LL / 2-luc tumor-bearing mice. Luciferase activity calculated from BLI in (E) LL / 2-luc and (F) MOC2-luc tumors (n=10, mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; ***, P<0.001; ****, P<0.0001; NS, not significant). (G) Edema formation (red arrows) 1 day after CD44-targeted NIR-PIT with or without L-NaAA in LL / 2-luc and MOC2-luc tumors. (H) Body weight curves in LL / 2-luc and (I)MOC2-luc tumor-bearing mice. (n=10, mean ± SEM; one-way ANOVA followed by Tukey's test; NS, not significant). [Fig. 25D-F] Same as above. [Fig. 25G-I] Same as above. [Figure 26A-C] In vivo efficacy of panitumumab NIR-PIT with L-NaAA on A431 GFP-luc tumors. (A) Treatment schedule. (B) Fluorescence imaging and (C) mean fluorescence intensity before and after panitumumab NIR-PIT with or without L-NaAA in A431 GFP-luc tumor-bearing mice (n=8, mean ± SEM; paired t-test; NS, not significant). (D) BLI before (day 6) and after (days 8-12) panitumumab NIR-PIT in A431 GFP-luc tumor-bearing mice. (E) Luciferase activity calculated from BLI (n=8, mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; NS, not significant). [Fig. 26D-E] Same as above. [Figure 27]Magnetic resonance imaging of edema. (A) Coronal T2WI STIR images 1 day after CD44-targeted NIR-PIT with or without L-NaAA in MC38-luc tumors. (B) Mean hyperintense area calculated from coronal T2WI STIR MR images after CD44-targeted NIR-PIT (n=3, mean ± SEM; unpaired t-test; *, p<0.05). [Figure 28A-C] Imaging and quantification of reactive oxygen species (ROS) after NIR-PIT with L-NaAA. To assess ROS after NIR-PIT with or without L-NaAA, ROS were assessed by chemiluminescence imaging (CLI), where mice were intraperitoneally injected with L-012 (0.5 mg mouse-1; FUJIFILM Wako Chemicals USA Corp, Richmond, VA). Photon counts were assessed using relative light units with a Photon Imager (Biospace Lab). ROIs were placed throughout the tumor. Relative light unit counts per minute were calculated using M3 Vision Software (Biospace Lab) and converted to percentage based on that before NIR-PIT using the following formula: [(relative light units after treatment) / (relative light units before treatment)×100(%)]. Chemiluminescence images were continuously recorded 1 and 3 hours after NIR-PIT. (A) Treatment schedule. (B) CLI before and after CD44-targeted NIR-PIT in MC38-luc tumor-bearing mice. (C) Photon counts calculated from CLI in MC38-luc after CD44-targeted NIR-PIT (n=5, mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; *, p<0.05). (D) BLI before and after CTLA4-targeted NIR-PIT in MC38-luc tumor-bearing mice. (E) Photon counts calculated from BLI in MC38-luc after CTLA4-targeted NIR-PIT (n=5, mean ± SEM; repeated measures two-way ANOVA followed by Tukey's test; *, p<0.05). [Fig. 28D-E] Same as above. [Figure 29A-C]Early efficacy of L-NaAA for CTLA4-targeted NIR-PIT in MC38-luc tumor model. (A) Treatment schedule. (B) Fluorescence imaging and (D) mean fluorescence intensity before and after CTLA4-targeted NIR-PIT with or without L-NaAA in MC38-luc tumor-bearing mice (n=10, mean ± SEM; paired t-test; NS, not significant). (D) BLI before (day 6) and after (days 8-13) CTLA4-targeted NIR-PIT in MC38-luc tumor-bearing mice. (E) Luciferase activity calculated from BLI (n=10; repeated measures two-way ANOVA followed by Tukey's test; ****, p<0.0001; NS, not significant). (G) Formation of edema (red arrow) was shown 1 day after CTLA4-targeted NIR-PIT. (G) T2WI fat-suppressed MRI imaging 1 day after CTLA4-targeted NIR-PIT. Red arrows indicate MC38-luc subcutaneous tumors. (H) Mean hyperintense area calculated from coronal T2WI fat-suppressed MRI images (n=3, mean ± SEM; unpaired t-test; **, p<0.01). (I) Weight curve (n=10, mean ± SEM; one-way ANOVA followed by Tukey's test; *, p<0.05). (J) Percentage of CTLA4hi cells among total live cells 3 hours after CTLA4-targeted NIR-PIT with L-NaAA (n=4, one-way ANOVA followed by Tukey's test; *, p<0.05; NS, not significant). [Fig. 29D-E] Same as above. [Fig. 29F-H] Same as above. [Figure 29I-J] Same as above. [Figure 30A]The scheme shows the proposed mechanism of ROS quenching and accelerating ligand release of IR700 by L-NaAA. (A) Energy diagram of the light-induced ligand release reaction of IR700. IR700 accepts electrons and becomes a radical anion. L-cysteine ​​and L-NaAA act as electron donors. The ligand release reaction from the radical anion is accelerated in acidic conditions. Thus, the ligand release reaction is accelerated by L-NaAA. (B) Mechanism of L-NaAA to suppress acute edema after NIR-PIT. During NIR-PIT, ROS are also generated, causing edema. L-NaAA quenches ROS, resulting in the prevention of acute edema, but promotes light-induced ligand release. [Figure 30B] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. The singular terms "a", "an" and "the" include plural references unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprising" means "including". Thus, "comprising A or B" means "including A", or "including B", or "including A and B".

[0039] Suitable methods and materials for the practice and / or testing of embodiments of the present disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods are described in a variety of general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

[0040] All GenBank® Accession Numbers and associated sequences referenced herein are incorporated by reference with respect to the sequences available as of January 29, 2021.

[0041] All references herein are incorporated by reference in their entirety.

[0042] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[0043] Abscopal effect: treatment of tumors, e.g., metastases, in parts of the body that are not the direct target of localized therapy (e.g., NIR-PIT). For example, irradiation of a particular tumor with NIR light in combination with an appropriate antibody-IR700 conjugate can reduce the size of a different tumor (e.g., metastasis) that is not irradiated with NIR light. The unirradiated / distant tumor is in some cases (e.g., in humans) at least 3 inches away from the tumor treated with NIR light, e.g., at least 4 inches, at least 5 inches, at least 10 inches, at least 12 inches, at least 18 inches, or at least 24 inches away from the tumor treated with NIR light.

[0044] Administration: Providing or giving to a subject an agent, such as an antibody-IR700 molecule, a reducing agent, and / or an immune modulator, by any effective route. Exemplary routes of administration include, but are not limited to, local, systemic, or regional injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, intraosseous, intraprostatic, and intravenous), oral, intraocular, sublingual, rectal, transdermal, intranasal, intravaginal, and inhalation routes. In one example, administration is intravenous. In one example, administration is intraperitoneal.

[0045] Antibody: A polypeptide ligand comprising at least a light or heavy immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, e.g., a tumor-specific protein, or a protein specifically expressed on an immune cell, e.g., a T cell. Antibodies are composed of heavy and light chains, each of which is made up of a variable heavy chain (V H ) region and the variable light chain (V L ) regions. H Area and V LThe region is responsible for binding to the antigen recognized by the antibody.

[0046] Antibodies, such as those in the antibody-IR700 molecule, include intact immunoglobulins as well as antibody variants and portions, such as Fab fragments, Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which the light chain variable region of an immunoglobulin and the heavy chain variable region of an immunoglobulin are joined by a linker, while in the case of dsFv, the chains are mutated to introduce disulfide bonds to stabilize the association of the chains. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL), Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0047] Typically, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0048] Each heavy and light chain contains a constant region and a variable region (regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind antigens. The light and heavy chain variable regions contain a "framework" region separated by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework region and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is incorporated herein by reference). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, e.g., human. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0049] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, are numbered sequentially starting from the N-terminus, and are typically identified by the chain in which the particular CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas V L CDR1 is derived from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are referred to as specificity determining residues (SDRs).

[0050] "V H References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv, or Fab. LReferences to " or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

[0051] A "monoclonal antibody" (mAb) is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies. In some examples, the antibody in the antibody-IR700 molecule is a mAb, for example a humanized mAb.

[0052] A "chimeric antibody" has framework residues derived from one species, e.g., human, and CDRs (which generally confer antigen binding) derived from another species, e.g., a murine antibody that specifically binds mesothelin.

[0053] A "humanized" immunoglobulin is an immunoglobulin that includes a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor" and the human immunoglobulin providing the framework is referred to as the "acceptor". In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions may be absent, but if present, they should be substantially identical, e.g., at least about 85-90%, e.g., about 95% or more identical, to human immunoglobulin constant regions. Thus, all parts of a humanized immunoglobulin, potentially excluding the CDRs, are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody that includes a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids taken from the donor framework. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed using genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0054] A "human" antibody (also referred to as a "fully human" antibody) is an antibody that contains a human framework region and all CDRs derived from a human immunoglobulin. In one example, the framework and CDRs are derived from the same original human heavy and / or light chain amino acid sequence. However, a framework from one human antibody can be engineered to contain CDRs from a different human antibody. All parts of a human immunoglobulin are substantially identical to the corresponding parts of a natural human immunoglobulin sequence.

[0055] "Specifically bind" refers to the ability of an individual antibody to specifically immunoreact with an antigen, e.g., a tumor-specific antigen, compared to binding to unrelated proteins, e.g., non-tumor proteins, e.g., β-actin. For example, an EGFR-specific binding agent binds substantially only to EGFR protein in vitro or in vivo. As used herein, the term "tumor-specific binding agent" includes tumor-specific antibodies (and their fragments) and other agents that bind substantially only to tumor-specific proteins in their preparations.

[0056] Binding is a non-random binding reaction between antibody molecules and antigenic determinants of T cell surface molecules.The desired binding specificity is typically determined from the reference point of the antibody's ability to differentially bind T cell surface molecules and unrelated antigens, and thus distinguish between two different antigens, especially when the two antigens have unique epitopes.An antibody that specifically binds to a specific epitope is called a "specific antibody".

[0057] In some examples, the antibody (e.g., in an antibody-IR700 molecule) binds to a target (e.g., a cell surface protein, e.g., a tumor-specific protein) with a binding constant at least 10 higher than that of other molecules in the sample or subject. 3 M -1 High, 10 4 M -1 High or 10 5 M -1 Specific binding with a high binding constant. In some examples, an antibody (e.g., mAb) or fragment thereof has an equilibrium constant (Kd) of 1 nM or less. For example, an antibody binds to a target, e.g., a tumor-specific protein, with an equilibrium constant of at least about 0.1×10 -8 M, at least about 0.3 × 10 -8 M, at least about 0.5 × 10 -8 M, at least about 0.75 × 10 -8 M, at least about 1.0 × 10 -8 M, at least about 1.3 × 10 -8 M, at least about 1.5 × 10 -8 M, or at least about 2.0 × 10-8 It binds with a binding affinity of M. Kd values ​​can be determined, for example, by competitive ELISA (enzyme-linked immunosorbent assay) or using a surface plasmon resonance device, for example, a Biacore T100 available from Biacore, Inc., Piscataway, NJ.

[0058] Antibody-IR700 molecule or antibody-IR700 conjugate: A molecule that is conjugated to IR700 and includes both an antibody, e.g., a tumor-specific antibody. In some examples, the antibody is a humanized antibody (e.g., a humanized mAb) that specifically binds to a surface protein on a cancer cell, e.g., a tumor-specific antigen.

[0059] Antigen (Ag): A compound, composition, or substance capable of stimulating the production of antibodies or a T-cell response in an animal, including compositions injected or absorbed into an animal (e.g., those containing tumor-specific proteins). Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, e.g., the disclosed antigens. "Epitope" or "antigenic determinant" refers to the region of an antigen to which B cells and / or T cells respond. In one embodiment, T cells respond to an epitope when presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3 amino acids, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and nuclear magnetic resonance.

[0060] Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids that contain antigenic determinants, such as those that are recognized by immune cells.In some examples, antigens include tumor-specific proteins or peptides (e.g., those found on the surface of cells, such as cancer cells), or immunogenic fragments thereof.In some examples, antigens include immune cell-specific proteins or peptides (e.g., those found on the surface of immune cells, such as T cells, such as regulatory T cells), or immunogenic fragments thereof.

[0061] Cancer: A malignant tumor characterized by abnormal or uncontrolled cell growth. Other features often associated with cancer include metastasis, interference with the normal functioning of neighboring cells, release of abnormal levels of cytokines or other secretory products, and suppression or exacerbation of inflammatory or immune responses, infiltration of surrounding or distant tissues or organs, e.g., lymph nodes. "Metastatic disease" refers to cancer cells that leave the original tumor site and migrate to other parts of the body, e.g., via the bloodstream or lymphatic system. In one example, the cells killed by the disclosed methods are cancer cells.

[0062] CD25 (IL-2 receptor alpha chain): (e.g., OMIM 147730) A type I transmembrane protein present on activated T cells, activated B cells, some thymocytes, myeloid precursors, and oligodendrocytes. CD25 has been used as a marker to identify CD4+FoxP3+ regulatory T cells in mice. CD25 is found on the surface of some cancer cells, including B cell neoplasms, some acute nonlymphocytic leukemias, neuroblastomas, mastocytosis, and tumor-infiltrating lymphocytes. It functions as a receptor for HTLV-1, and is consequently expressed on neoplastic cells in adult T cell lymphoma / leukemia. Exemplary CD25 sequences can be found in the GenBank® database (e.g., accession numbers CAA44297.1, NP_000408.1, and NP_001295171.1). Exemplary mAbs specific for CD25 are daclizumab and basiliximab, which can be attached to IR700 to form daclizumab-IR700 or basiliximab-IR700, which can be used in the disclosed methods to target cancer cells expressing CD25 or can be used as an immune modulatory molecule (e.g., to reduce tumor infiltrating Treg cells within a tumor).

[0063] Contacting: By bringing into direct physical association, including both solid and liquid forms. Contacting may occur in vitro, e.g., on an isolated cell, e.g., a tumor cell, or in vivo by administration to a subject (e.g., a subject having a tumor, e.g., cancer).

[0064] Cytotoxic T-lymphocyte antigen 4 (CTLA4): (e.g., OMIM 123890) is a major immune checkpoint molecule that mediates antitumor immunosuppression. CTLA4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells after activation, a phenomenon that is particularly prominent in cancer. Exemplary CTLA4 sequences can be found in the GenBank® database (e.g., accession numbers AAL07473.1, AAF01489.1, and AF414120.1). An exemplary mAb specific for CTLA4 is ipilimumab, which can be attached to IR700 to form ipilimumab-IR700, which can be used in the disclosed methods to target cells that express CTLA4, such as cells that express CTLA4 in melanoma or T cells that express CTLA4.

[0065] Reduce: To decrease the quality, quantity, or intensity of something. In one example, a therapeutic composition comprising one or more antibody-IR700 molecules may be used to reduce the viability of a cell to which the antibody-IR700 molecule specifically binds by irradiating the cell with NIR (e.g., wavelengths of about 680 nm or 690 nm) at least 1 J / cm. 2 (For example, 4 to 100 J / cm 2 ) after irradiation with a dose of 100 mg / kg / day or more, e.g., compared to the response in the absence of the antibody-IR700 molecule. In some examples, such a decrease is indicative of a decrease in the number of CD4 + Foxp3 + This is evidenced by the killing of Tregs. In some instances, the reduction in cell viability is at least 20%, at least 50%, at least 75%, or even at least 90% compared to the viability observed in a composition that does not contain the antibody-IR700 molecule. In other instances, the reduction is expressed as a fold change, e.g., a 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, or even 15- or 20-fold reduction in cell viability compared to the viability observed in a composition that does not contain the antibody-IR700 molecule. Such reductions can be measured using the methods disclosed herein.

[0066] Epidermal growth factor receptor (EGFR): (e.g., OMIM 131550) a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Epidermal growth factor receptor is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). EGFR can form heterodimers with HER2 and other HER family members. Mutations affecting EGFR expression or activity can result in cancer, including adenocarcinoma of the lung, anal carcinoma, glioblastoma, and epithelial tumors of the head and neck. Exemplary EGFR sequences can be found in the GenBank® database (e.g., accession numbers NP_001333826.1, CAA55587.1, and AAF14008.1). An exemplary mAb specific for EGFR is panitumumab, which can be attached to IR700 to form panitumumab-IR700, which can be used in the disclosed methods to target cells that express EGFR.

[0067] Immune Modulator: An immune modulator is a substance that alters (e.g., increases or decreases) one or more functions of the immune system. In some examples, an immune modulator activates the immune system. In other examples, an immune modulator inhibits (or kills) the activity of immune suppressor cells. In some examples, an immune modulator is a mAb, which can be attached to IR700 to form mAb-IR700, which can be used in the disclosed methods to target cells that express a protein recognized by the mAb. Exemplary immune modulators include the immune activators IL-15 and IFN-gamma.

[0068] IR700 (IRDye® 700DX): A dye having the following formula: [ka]

[0069] It is commercially available from LI-COR (Lincoln, NE). Amino-reactive IR700 is a relatively hydrophilic dye that can be covalently conjugated to antibodies using the NHS ester of IR700. IR700 also works well with conventional photosensitizers, such as the hematoporphyrin derivative Photofrin® (1.2×10 at 630 nm). 3 M -1 cm -1 ), meta-tetrahydroxyphenyl chlorin, Foscan® (2.2 × 10 at 652 nm 4 M -1 cm -1 ), and mono-L-aspartyl chlorin e6, NPe6 / Laserphyrin® (4.0 × 10 at 654 nm) 4 M -1 cm -1 ), which has a 5-fold higher extinction coefficient (2.1 × 10 with an absorbance maximum of 689 nm) than 5 M -1 cm -1 ).

[0070] Pharmaceutical composition: A chemical compound or composition that can induce a desired therapeutic or prophylactic effect when appropriately administered to a subject. A pharmaceutical composition may contain a therapeutic agent, such as one or more antibody-IR700 molecules, a reducing agent, and / or one or more immune activators. A therapeutic or pharmaceutical agent, alone or together with additional compounds, is one that induces a desired response (e.g., induces a therapeutic or prophylactic effect when administered to a subject). In certain examples, a pharmaceutical composition contains a therapeutically effective amount of at least one antibody-IR700 molecule.

[0071] Pharmaceutically acceptable vehicles: Pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, e.g., one or more antibody-IR700 molecules, one or more reducing agents, and / or one or more immune activators.

[0072] Generally, the nature of the carrier will depend on the particular mode of administration used.For example, parenteral formulations usually contain injectable fluids, which contain pharma- ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc., as vehicles.For solid compositions (e.g., in the form of powder, pill, tablet, or capsule), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0073] Photoimmunotherapy (PIT): A molecular targeted therapy that utilizes a target-specific photosensitizer based on the near-infrared (NIR) phthalocyanine dye IRDye700DX (IR700) conjugated to a monoclonal antibody (MAb) that targets a cellular protein (e.g., on the cell surface). In one example, the cell surface protein is one that is specifically found on cancer cells or immune cells, and thus PIT can be used to kill such cells. Cell death occurs when the antibody-IR700 conjugate binds to the cell and irradiates the cell with NIR, while cells that do not express the cell surface protein recognized by the antibody-IR700 molecule are not killed in large numbers.

[0074] Programmed Death 1 (PD-1): (e.g., OMIM 600244) A type 1 membrane protein on the surface of cells that plays a role in regulating the immune system's response to cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T-cell inflammatory activity. PD-1 binds to two ligands, PD-L1 and PD-L2. Exemplary PD-1 sequences can be found in the GenBank® database (e.g., accession numbers CAA48113.1, NP_005009.2, and NP_001076975.1). Exemplary antagonist mAbs specific for PD-1 that can be used in combination with the disclosed methods include JTX-4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001, dostallimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, and AMP-514. An exemplary mAb specific for PD-1 is nivolumab, which can be used in combination with the disclosed methods.

[0075] Programmed Death Ligand 1 (PD-L1): (e.g., OMIM 605402) A type 1 membrane protein on the surface of cells that suppresses the adaptive arm of the immune system during certain events, such as pregnancy, tissue allografts, autoimmune diseases, and hepatitis. Binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via an immunoreceptor tyrosine-based switch motif (ITSM) motif. PD-L1 binds to PD-1 found on activated T cells, B cells, and myeloid cells to modulate activation or inhibition. Exemplary PD-L1 sequences can be found in the GenBank® database (e.g., accession numbers ADK70950.1, NP_054862.1, and NP_001156884.1). Antibodies that antagonize PD-L1 activity can be conjugated to IR700 (forming anti-PD-L1-IR700) and the methods provided herein can be used in combination with, for example, tumor-specific antigen Ab-IR700 molecules. Exemplary antagonist mAbs specific for PD-L1 include atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (embafolimab), BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, and MEDI-4737, any of which can be conjugated to IR700. For example, one exemplary mAb specific for PD-L1 is durvalumab, which can be attached to IR700 to form durvalumab-IR700, which can be used in the disclosed methods to target cells expressing PD-L1.

[0076] Reducing agent: An element or compound that gives up (or "donates") electrons to an electron recipient (oxidizing agent) in a redox chemical reaction. A reducing agent is thus oxidized when it loses electrons in a redox reaction. A reducing agent "reduces" (or is "oxidized" by) an oxidizing agent. Thus, one or more reducing agents can be used in combination with NIR-PIT in vitro and in vivo to reduce unwanted non-specific tissue damage caused by reactive oxygen species resulting from IR700 after exposure to NIR light. For example, a reducing agent can inactivate reactive oxygen species that are unbound or in the area of ​​a tumor and reduce unwanted acute inflammation in other areas of the body. Exemplary reducing agents that can be used in the methods provided herein include L-cysteine, L-sodium ascorbate (L-NaAA), ascorbic acid (e.g., L- or R-ascorbic acid), and glutathione. In some examples, sodium azide is not used in vivo due to its toxicity. Thus, in some examples, the reducing agent used in the disclosed methods is not sodium azide. In one example, the reducing agent used in the disclosed methods is L-NaAA.

[0077] Subject or patient: A term that includes humans and non-human mammals. In one example, the subject is a human or veterinary subject, such as a mouse, rat, dog, cat, or non-human primate. In some examples, the subject is a mammal (e.g., a human) that has cancer or is being treated for cancer.

[0078] Therapeutically effective amount: A sufficient amount of a composition, alone or together with additional therapeutic agents (e.g., anti-CTLA4-IR700 molecule, anti-PD-L1-IR700 molecule, immunomodulator, and / or reducing agent), to achieve a desired effect in a subject or cell being treated with the agent. The effective amount of an agent (e.g., antibody-IR700 molecule, alone or in combination with other agents) may depend on several factors, including, but not limited to, the subject or cell being treated, the particular therapeutic agent, and the mode of administration of the therapeutic composition. In one example, a therapeutically effective amount or concentration of an antibody-IR700 molecule and / or immunomodulator is one that is sufficient to prevent, delay the progression of, or cause the regression of a disease (e.g., metastasis), or to reduce symptoms caused by a disease, e.g., cancer. In one example, a therapeutically effective amount or concentration of an antibody-IR700 molecule and / or immunomodulator is one that is sufficient to increase the survival time of a patient with a tumor. In one example, a therapeutically effective amount or concentration of a reducing agent is one that is sufficient to reduce edema, an acute inflammatory response, or both, in a subject treated with NIR-PIT.

[0079] In one example, the desired response is to reduce or inhibit one or more symptoms associated with cancer. One or more symptoms do not need to be completely eliminated for the composition to be effective. For example, treatment with the modalities provided herein in some examples reduces the size of a tumor (e.g., the volume or weight of a tumor or tumor metastasis) by, for example, at least 20%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% compared to the tumor size in the absence of treatment (or only with a subset of therapies provided herein). In one particular example, treatment with the modalities provided herein kills a population of cells (e.g., cancer cells) by, for example, killing at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% of the cells compared to the cell killing in the absence of treatment (or only with a subset of therapies provided herein). In one particular example, treatment with a modality provided herein increases the survival of a patient with a tumor (or one who has recently had a tumor removed), e.g., increases survival by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at least 200%, or at least 500%, compared to survival in the absence of treatment (or with only a subset of the therapies provided herein). In some examples, treatment with a modality provided herein increases the amount of memory T cells in a subject, e.g., by an increase of at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at least 200%, or at least 500%, compared to the amount of memory T cells in the absence of treatment (or with only a subset of the therapies provided herein).In some examples, treatment with the modalities provided herein increases the amount of polyclonal antigen-specific TIC response to MHC type I-restricted tumor-specific antigen in a subject, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at least 200%, or at least 500%, compared to the amount of polyclonal antigen-specific TIC response to MHC type I-restricted tumor-specific antigen in the absence of treatment (or only with a subset of the therapies provided herein). In some examples, treatment with the modalities provided herein increases the amount of Tregs (e.g., FOXP3) in the targeted tumor. + CD25 + CD4 + Treg cells), for example, by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100% compared to the amount of Tregs in the targeted tumor in the absence of treatment (or only with a subset of the therapies provided herein). In some examples, treatment with the modalities provided herein increases the CD8+ / Treg ratio in the treated subject, for example, by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at least 200%, or at least 500% compared to the CD8+ / Treg ratio in the absence of treatment (or only with a subset of the therapies provided herein). In some examples, a combination of these effects is achieved by the disclosed methods.

[0080] The effective amount of a therapeutic agent administered to a human or veterinary subject may vary depending on several factors associated with the subject, such as the subject's general health. The effective amount of an agent can be determined by varying the dosage of the composition and measuring the resulting therapeutic response, such as tumor regression. The effective amount can also be determined through various in vitro, in vivo, or in situ immunoassays. The disclosed agents can be administered in a single dose or multiple doses as necessary to obtain the desired response. However, the effective amount can depend on the treatment being applied, the subject being treated, the severity and type of the condition being treated, and the mode of administration.

[0081] In certain examples, a therapeutically effective dose of the antibody-IR700 molecule, e.g., when administered iv, is a dose of at least 0.5 milligrams per 60 kilograms (mg / kg), at least 5 mg / 60 kg, at least 10 mg / 60 kg, at least 20 mg / 60 kg, at least 30 mg / 60 kg, at least 50 mg / 60 kg, e.g., 0.5 to 50 mg / 60 kg, e.g., 1 mg / 60 kg, 2 mg / 60 kg, 5 mg / 60 kg, 20 mg / 60 kg, or 50 mg / 60 kg. In another example, a therapeutically effective dose of the antibody-IR700 molecule is, for example, when administered intratumorally or ip, at least 10 μg / kg, e.g., at least 100 μg / kg, at least 500 μg / kg, or at least 500 μg / kg, e.g., a dose between 10 μg / kg and 1000 μg / kg, e.g., 100 μg / kg, 250 μg / kg, about 500 μg / kg, 750 μg / kg, or 1000 μg / kg. In one example, a therapeutically effective dose of the antibody-IR700 molecule is at least 1 μg / ml, e.g., at least 500 μg / ml, e.g., between 20 μg / ml and 100 μg / ml, e.g., 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, or 100 μg / ml, administered in a topical solution. However, a person skilled in the art will recognize that higher or lower dosages may also be used, e.g., depending on the particular antibody-IR700 molecule. In certain examples, such daily dosages are administered in one or more divided doses (e.g., 2, 3, or 4 doses) or in a single formulation. The disclosed antibody-IR700 molecules can be administered alone, in the presence of a pharma- ceutically acceptable carrier, in the presence of other therapeutic agents (e.g., other anti-neoplastic agents).

[0082] In general, a suitable radiation dose following administration of one or more antibody-IR700 molecules (and in some cases also a reducing agent) is at least 1 J / cm at a wavelength of 660-740 nm. 2 , for example, at least 10 J / cm at wavelengths of 660 to 740 nm 2, at least 20 J / cm at wavelengths of 660 to 740 nm 2 , at least 25 J / cm at wavelengths of 660 to 740 nm 2 , at least 50 J / cm at wavelengths of 660 to 740 nm 2 , or at least 100 J / cm at wavelengths between 660 and 740 nm 2 For example, 1 to 500 J / cm at a wavelength of 660 to 740 nm 2 In some examples, the wavelength is 660-710 nm. In specific examples, a suitable radiation dose after administration of the antibody-IR700 molecule is at least 1 J / cm at a wavelength of 680 or 690 nm. 2 , for example at least 10 J / cm at a wavelength of 680 or 690 nm 2 , at least 20 J / cm at a wavelength of 680 or 690 nm 2 , at least 25 J / cm at a wavelength of 680 or 690 nm 2 , at least 50 J / cm at a wavelength of 680 or 690 nm 2 or at least 100 J / cm at a wavelength of 680 or 690 nm 2 , for example, 4 to 50 J / cm at a wavelength of 680 or 690 nm 2 or 10~25J / cm 2 In certain instances, multiple irradiations (e.g., at least two, at least three, or at least four irradiations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate administrations) are administered after administration of one or more antibody-IR700 molecules (and in some instances after administration of the immune modulator and / or reducing agent).

[0083] Treating: A term used to refer to the treatment of cells or tissues with a therapeutic agent, including contacting or incubating one or more agents (e.g., one or more antibody-IR700 molecules, and in some cases also one or more immune activators and / or one or more reducing agents) with a cell or tissue, and / or administering one or more agents to a subject, e.g., a subject with cancer. Treated cells are cells that have been contacted with a desired composition in an amount and under conditions sufficient for a desired response. In one example, treated cells are cells that have been exposed to antibody-IR700 molecules under conditions sufficient for the antibody to bind to a surface protein on the cell, optionally contacted with a reducing agent, and irradiated with NIR light until sufficient cell killing is achieved. In another example, treated subjects are subjects that have been administered one or more antibody-IR700 molecules under conditions sufficient for the antibody to bind to a surface protein on the cell, optionally administered with one or more reducing agents, and irradiated with NIR light until sufficient cell killing is achieved.

[0084] Tumor, neoplasia, malignancy, or cancer: A neoplasm is an abnormal growth of tissue or cells resulting from excessive cell division. Neoplastic growth can result in a tumor. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign." A tumor that can invade surrounding tissues and / or metastasize is referred to as "malignant." A "non-cancerous tissue" is a tissue from the same organ in which a malignant neoplasm has formed but does not have the pathology characteristic of a neoplasm. Generally, non-cancerous tissue appears histologically normal. A "normal tissue" is a tissue from an organ in which the organ is not afflicted by cancer or another disease or disorder of that organ. A "cancer-free" subject has not been diagnosed with cancer in that organ and does not have detectable cancer.

[0085] Tumors include original (primary) tumors, recurrent tumors, and metastatic (secondary) tumors. Tumor recurrence is the return of a tumor at the same site as the original (primary) tumor, for example after the tumor has been removed by surgery, drugs, or other treatment, or has otherwise disappeared. Metastasis is the spread of a tumor from one part of the body to another. Tumors formed from the spread cells are called secondary tumors and contain cells like those in the original (primary) tumor. There can be a recurrence of either the primary tumor or metastasis.

[0086] Exemplary tumors, e.g., cancers, that can be treated with the disclosed methods include solid tumors, e.g., breast cancer (e.g., lobular and ductal carcinoma), sarcoma, carcinoma of the lung (e.g., non-small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma), pulmonary mesothelioma, colorectal adenocarcinoma, head and neck cancer (e.g., adenocarcinoma, squamous cell carcinoma, metastatic squamous cell, e.g., cancers caused by HPV or Epstein-Barr virus, e.g., HPV16, including cancers of the oral cavity, tongue, cheek, nasopharynx, throat, hypopharynx, oropharynx, larynx, and trachea), Gastric cancer, prostate adenocarcinoma, ovarian cancer (e.g., serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular cancer and germ cell tumors, pancreatic adenocarcinoma, bile duct adenocarcinoma, hepatocellular carcinoma, bladder cancer (including, e.g., transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma), renal cell adenocarcinoma, endometrial cancer (including, e.g., adenocarcinoma and mixed Mullerian tumors (carcinosarcoma)), endocervical, epicervical, and vaginal cancer (including, e.g., their respective adenocarcinoma and squamous cell carcinoma), tumors of the skin (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin adnexal tumors, These include sarcoma, Kaposi's sarcoma, cutaneous lymphoma, skin adnexal tumor, and various types of sarcoma and Merkel cell carcinoma, esophageal cancer, nasopharyngeal and oropharyngeal cancer (including squamous cell carcinoma and adenocarcinoma thereof), salivary gland cancer, brain and central nervous system tumors (including tumors of glial, neuronal, and meningeal origin), peripheral nerve tumors, soft tissue sarcomas, and sarcomas of bone and cartilage, and lymphatic tumors (including B-cell and T-cell malignant lymphomas). In one example, the tumor is an adenocarcinoma. In one example, the tumor is an oropharyngeal cancer. In one example, the tumor is a squamous cell carcinoma, for example, of the head and neck or skin.

[0087] The method can also be used to treat liquid tumors (e.g., hematological malignancies), such as lymphatic, leukemia, or other types of leukemia. In a specific example, the tumor to be treated is a hematological tumor, such as leukemia (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia), lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), and myeloma.

[0088] Under conditions sufficient: A phrase used to describe any environment that allows for a desired activity. In one example, "under conditions sufficient for" includes administering to a subject an antibody-IR700 molecule sufficient to allow the antibody-IR700 molecule to bind to its targeted cell surface protein (e.g., a tumor-specific antigen or an immune cell-specific antigen). In a particular example, the desired activity is killing of cells to which the antibody-IR700 molecule is bound following therapeutic irradiation of the cells.

[0089] Untreated: Untreated cells are cells that have not been contacted with a therapeutic agent, such as an antibody-IR700 molecule and / or irradiation. In some instances, untreated cells are cells that receive a vehicle in which a therapeutic agent is delivered. Similarly, an untreated subject is a subject to which a therapeutic agent, such as an antibody-IR700 molecule and / or irradiation, has not been administered. In some instances, an untreated subject is a subject that receives a vehicle in which a therapeutic agent is delivered.

[0090] The disclosure of a particular specific example does not imply the exclusion of other embodiments. Additionally, any treatment described herein does not necessarily exclude other treatments and may be combined with other bioactive agents or treatment modalities.

[0091] Overview of the technology Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective cancer treatment that utilizes monoclonal antibodies (mAbs) conjugated to a light-absorbing silicon-phthalocyanine dye derivative called IRDye700DX (IR700) and NIR light to induce necrotic / immunogenic cell death. Approximately one day after intravenous administration, the antibody-photoabsorber conjugate (APC, also referred to herein as antibody-IR700 molecule) binds to cancer cell surface markers. Application of NIR light after APC binding causes the release of the ligand in the dye, a dramatic change in the solubility of the APC-antigen complex, and rapid irreversible cell membrane damage of cancer cells in a highly selective manner, resulting in highly immunogenic cell death. Clinically, this process results in post-treatment edema mediated by reactive oxygen species (ROS).

[0092] CTLA4 is a major immune checkpoint molecule mediating antitumor immunosuppression. CTLA4-targeted NIR-PIT (CTLA4 in tumors, many of which are regulatory T cells (Tregs)) + Provided herein is data showing synergistic effects on tumor treatment using CTLA4-targeted NIR-PIT (using tumor-specific antibodies conjugated to IR700, for example) in combination with tumor-targeted NIR-PIT (using tumor-specific antibodies conjugated to IR700, for example). Thus, CTLA4-targeted NIR-PIT can effectively treat tumors by blocking the CTLA4 axis, as well as by eliminating immune suppressor cells expressing CTLA4, resulting in the induction of potential T cell-mediated antitumor immunity. Depletion of CTLA4-expressing cells locally in the tumor bed using NIR-PIT, for example, in combination with tumor-specific antigen antibody-IR700 conjugates, can be used to treat a variety of tumor types.

[0093] Data is also provided showing that anti-PD-L1-IR700 can be positioned as a cancer + immunotherapy with NIR-PIT, as it targets cancer cells and blocks the immunosuppressive PD1-PDL1 axis, which has cured some mouse patients. In addition, further immune activation, for example in combination with IL-15, interferon gamma, or both, can improve the complete response (CR) rate and be used to treat tumors and increase survival.

[0094] To minimize transient acute inflammatory edema without compromising the therapeutic effect, reducing agents, such as sodium L-ascorbate (L-NaAA) or L-cysteine, were administered to quench harmful ROS and accelerate the ligand release reaction. L-NaAA and L-cysteine ​​suppressed acute edema by reducing ROS after NIR-PIT, but did not alter the therapeutic effect. NIR-PIT could be safely performed in the presence of L-NaAA or L-cysteine ​​without side effects caused by unnecessary ROS production. Thus, reducing agents, such as L-cysteine ​​and sodium L-ascorbate, can be used in combination with NIR-PIT to reduce unwanted nonspecific tissue damage caused by reactive oxygen species, such as edema (e.g., transient acute edema) and acute inflammatory reactions. In some cases, the reducing agent is an electron donor that quenches ROS but accelerates the ligand release reaction of IR700 induced by NIR light. In one example, the tumor to be treated is located near critical structures, such as the cervical or mediastinal airways, where edema can cause side effects such as airway obstruction and coughing, and can be treated with the disclosed methods.

[0095] Based on these observations, methods of treating cancer in a subject using NIR-PIT are provided herein, for example, administering (1) one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, or a combination thereof, (2) one or more tumor-specific antibody-IR700 conjugates, one or more immune activators, or both, and optionally (3) one or more reducing agents. In some examples, the methods include administering NIR-PIT to treat cancer in a subject, for example, administering (1) one or more tumor-specific antibody-IR700 conjugates, (2) one or more reducing agents, and optionally (3) one or more immune activators. In some examples, the methods include administering NIR-PIT to treat EGFR-expressing cancer in a subject, for example, administering (1) one or more CTLA4 antibody-IR700 molecules, (2) one or more EGFR antibody-IR700 conjugates, and optionally (3) one or more reducing agents, one or more immune activators, or both. In some examples, the methods include treating cancer in a subject using NIR-PIT, e.g., administering (1) one or more PD-L1 antibody-IR700 molecules, (2) one or more immune activators, and optionally (3) one or more reducing agents, one or more tumor-specific antibody-IR700 conjugates, or both.

[0096] Such a method kills cancer cells at the site of irradiation with NIR light as well as in non-irradiated distant metastases. The presence of a reducing agent can remove or reduce reactive oxygen species generated by irradiation with IR700 molecules, thereby reducing unwanted inflammation and edema. Such a method provides highly effective treatment of various cancers both locally and even in distant metastases far from the treated site (abscopal effect).

[0097] Methods for Treating Cancer The present disclosure provides a method for treating a subject with cancer, for example, a subject with a solid tumor or hematological malignancy. The method includes administering to the subject an antibody conjugated to the dye IR700 (referred to herein as an antibody-IR700 molecule or an antibody-photoabsorber conjugate (APC)), where the antibody specifically binds to a cancer (e.g., tumor) cell surface protein (referred to herein as a tumor-specific antigen or protein). A tumor-specific protein is a protein that is unique to cancer cells or is much more abundant in cancer cells compared to other cells, for example, normal cells. For example, HER2 is commonly found in breast cancer, while HER1 is typically found in adenocarcinomas, which can be found in multiple organs, for example, pancreas, breast, prostate, and colon. In some examples, the antibody of the APC specifically binds to a protein on an immune cell, for example, a T cell, for example, a regulatory cell. In one example, the immune cell-specific protein that specifically binds to the antibody is CTLA4. The method may further include (a) administering to the subject a therapeutically effective amount of one or more immune activators (e.g., IL-15 or IFN-gamma); (b) administering to the subject a therapeutically effective amount of one or more reducing agents (e.g., to reduce edema), or both (a) and (b).

[0098] The subject is administered a therapeutically effective amount of one or more tumor-specific antibody-IR700 molecules (e.g., in the presence of a pharma- ceutically acceptable carrier, e.g., a pharma- ceutically and physiologically acceptable fluid), under conditions that allow the antibody to specifically bind to cancer cell surface proteins. For example, the antibody-IR700 molecule can be present in a pharma- ceutically effective carrier, e.g., water, saline, balanced salt solution (e.g., PBS / EDTA), aqueous dextrose, sesame oil, glycerol, ethanol, combinations thereof, and the like, as a vehicle. The carrier and composition can be sterile, and the formulation is suitable for the mode of administration. In a specific example, the antibody-IR700 molecule is an EGFR antibody-IR700. In some instances, the tumor-specific antibody is selected from the group consisting of HER1 / EGFR, PD-L1, mesothelin, PSMA, HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, CEA, AFP, Lewis Y, TAG7, VEGF, VEGFR, EpCAM, EphA2, glypican-1, glypican-2, and glypican-1. Specific for can-3, gpA33, mucin, CAIX, folate binding protein, ganglioside, integrin αVβ3, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, BCR complex, gp72, HLA-DR10β, HLA-DR antigen, IgE, CA242, PEM antigen, SK-1 antigen, PD-1, or PD-L2.

[0099] In some examples, the subject is further administered a therapeutically effective amount of one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, or a combination thereof (e.g., in the presence of a pharma- ceutically acceptable carrier, e.g., a pharma- ceutical and physiologically acceptable fluid), under conditions that allow the antibody to specifically bind to the cancer cell surface protein. For example, the CTLA4 antibody-IR700 molecule, PD-L1 antibody-IR700 molecule, or a combination thereof, may be present in a pharma- ceutical effective carrier, e.g., water, saline, balanced salt solution (e.g., PBS / EDTA), aqueous dextrose, sesame oil, glycerol, ethanol, combinations thereof, and the like, as a vehicle.

[0100] In some examples, the tumor-specific antibody-IR700 molecule and the CTLA4 antibody-IR700 molecule, the PD-L1 antibody-IR700 molecule, or a combination thereof are present in a single composition and are administered simultaneously. In other examples, the tumor-specific antibody-IR700 molecule and the CTLA4 antibody-IR700 molecule, the PD-L1 antibody-IR700 molecule, or a combination thereof are present in separate compositions and are administered simultaneously or contemporaneously or sequentially (e.g., at least 1 minute, at least 5 minutes, at least 30 minutes, or at least 60 minutes apart).

[0101] In some examples, the subject is further administered a therapeutically effective amount of one or more immune activators, e.g., in combination with a therapeutically effective amount of one or more CTLA4 antibody-IR700 molecules and / or one or more PD-L1 antibody-IR700 molecules. The immune activators include immune system activators and / or one or more inhibitors of immune suppressor cells (e.g., in the presence of a pharma- ceutically acceptable carrier, e.g., a pharma- ceutically and physiologically acceptable fluid). In a specific example, the immune activator agent is IL-15. In another specific example, the immunomodulator is interferon gamma. In some examples, the one or more immune activators are administered to the subject simultaneously (e.g., simultaneously or substantially simultaneously) with the one or more antibody-IR700 molecules, e.g., in the same composition. In other examples, the one or more antibody-IR700 molecules and the one or more immune activators are administered to a subject sequentially (in either order), for example, within at least about 1 hour (e.g., within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 2 hours, about 12 hours, about 24 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days) of each other).

[0102] In some cases, the subject is further administered a therapeutically effective amount of one or more reducing agents, for example, to reduce edema that may result from NIR-PIT. In some cases, the antibody-IR700 molecule and the reducing agent are present in a single composition and are administered simultaneously. In other cases, the antibody-IR700 molecule and the reducing agent are present in separate compositions and are administered simultaneously or contemporaneously, or sequentially (e.g., the tumor-specific antibody-IR700 molecule followed by the reducing agent, for example, at least 1 minute, at least 5 minutes, at least 30 minutes, at least 60 minutes, at least 12 hours, or at least 24 hours apart).

[0103] In some examples, the tumor-specific antibody-IR700 molecule is administered intravenously. In some examples, the CTLA4 antibody-IR700 molecule, the PD-L1 antibody-IR700 molecule, or a combination thereof is administered intravenously. In some examples, the one or more reducing agents are administered intraperitoneally. In some examples, the tumor-specific antibody-IR700 molecule and the CTLA4 antibody-IR700 molecule, the PD-L1 antibody-IR700 molecule, or a combination thereof are administered intravenously. In some examples, the tumor-specific antibody-IR700 molecule is administered intravenously and the one or more reducing agents are administered intraperitoneally. In some examples, the one or more immune activators (e.g., IL15) are administered intravenously. In one example, the one or more immune activators is IL15, for example, administered intravenously at a dose of 1-10 mg. In some examples, the one or more immune activators (e.g., IFNγ) are administered intratumorally. In one example, the one or more immune activators is IFNγ, administered intratumorally at a dose of, for example, not more than 1 mg, for example, 0.01 to 1 mg.

[0104] After administration of one or more antibody-IR700 molecules (e.g., tumor-specific antibody-IR700 molecules), the one or more antibody-IR700 molecules can accumulate within the targeted tumor or immune cells of the tumor. Similarly, after administration of one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, or a combination thereof, such molecules can bind to their respective proteins (CTLA4 or PD-L1, respectively), e.g., to cells expressing such proteins, e.g., to cells expressing CTLA4 or PD-L1 present in the tumor bed. The tumor cells and other cells in the tumor bed (or subject with cancer) can then be subjected to at least 1 J / cm at a wavelength of, e.g., 660-740 nm (e.g., 600-710 nm) under conditions that permit killing of the cells to which the antibody-IR700 conjugate is bound. 2 (e.g., 680 nm or 690 nm, 10 to 60 J / cm 2 Dose, for example, 20-50J / cm 2or 20~30J / cm 2 The radiation is irradiated with a dose of 100 u.

[0105] In one example, there is at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, or at least about 48 hours (e.g., about 1-4 hours, 30 minutes to 1 hour, 10 minutes to 60 minutes, 30 minutes to 8 hours, 2-10 hours, 12-24 hours, 18-36 hours, or 24-48 hours) between administration of the antibody-IR700 conjugate and irradiation. In one example, one or more antibody-IR700 conjugates are administered (e.g., iv) and the tumor (or subject) is irradiated at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, or at least about 48 hours (e.g., about 1-4 hours, 30 minutes to 1 hour, 10 minutes to 60 minutes, 30 minutes to 8 hours, 2-10 hours, 12-24 hours, 18-36 hours, or 24-48 hours, e.g., about 12 hours or about 24 hours). One or more immune activators may be administered (e.g., systemically, e.g., iv or ip) before or after the one or more antibody-IR700 conjugates and before or after the irradiation. In some examples, the one or more immune activators are administered before and after irradiation, for example, at least one dose of immune activator before irradiation and at least one dose of immune activator after irradiation (e.g., 24 hours before irradiation and one or more of 24 hours, 48 ​​hours, 72 hours, 96 hours, or more after irradiation). In additional examples, the doses of immune activator may also be administered on the same day as at least one irradiation treatment. The one or more reducing agents may be administered (e.g., systemically, e.g., iv or ip) before or after the one or more antibody-IR700 conjugates, but before irradiation (e.g., at least 5 minutes, at least 10 minutes, at least 15 minutes, or at least 30 minutes, e.g., 5-60 minutes before irradiation).

[0106] In some examples, multiple doses of one or more of the antibody-IR700 molecules are administered to a subject, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In some examples, multiple doses of one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, or a combination thereof are administered to a subject, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In some examples, multiple doses of one or more immune activators are administered to a subject, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In some examples, multiple doses of one or more reducing agents are administered to the subject, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In some examples, multiple doses of radiation with NIR are administered to the subject, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In a specific example, the subject is administered at least one dose of one or more of the antibody-IR700 molecules, at least two doses of one or more immune activators, and at least two separate administrations of NIR radiation.

[0107] NIR excitation light wavelengths allow penetration into tissues of at least several centimeters. For example, by using a fiber-coupled laser diode with a diffuser tip, NIR light can be delivered to several centimeters inside tumors that are otherwise inaccessible deep to the body surface. In addition to treating solid cancers, circulating tumor cells (including but not limited to hematological malignancies) can be targeted because they can be excited as they travel through superficial blood vessels (e.g., using the NIR LED wearable device described herein).

[0108] In one example, the disclosed methods can be used to improve the survival of treated target cells (e.g., cancer cells expressing a tumor-specific antigen, or immune cells within a tumor bed, e.g., T cells expressing PD-L1 in a tumor bed or CTLA4 in a tumor bed, e.g., CD4+ T cells expressing CTLA4 in a tumor bed) compared to, for example, no treatment with the disclosed methods (e.g., no administration of one or more tumor-specific antibody-IR700 molecules in combination with (a) one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, or a combination thereof, (b) one or more reducing agents, (c) one or more immune activators, or a combination of a, b, and c, and no NIR irradiation). + Foxp3 + The method further comprises killing at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or more of the total Tregs.

[0109] In one example, the disclosed method selectively kills cells that express a protein (e.g., a tumor-specific antigen, CTLA4, or PD-L1) that specifically binds to the antibody of the antibody-IR700 conjugate used, thereby treating the tumor. For example, by selectively killing tumor cells relative to normal cells, the method can kill tumor cells more effectively than normal cells, such as, for example, cells that do not express a protein (e.g., a tumor-specific antigen) that specifically binds to the antibody administered. In some examples, the disclosed method reduces tumor weight, size, and / or volume, slows tumor growth, reduces or slows tumor recurrence, reduces or slows tumor metastasis (e.g., by reducing the number of metastases or reducing the weight / volume / size of metastases), or a combination thereof. For example, the disclosed methods, in some examples, reduce tumor size (e.g., tumor weight or volume) by, e.g., at least 10%, at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100%, e.g., compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules in combination with (a) one or more reducing agents, (b) one or more immune activators, or a combination thereof, and / or without NIR irradiation). In some examples, the disclosed methods reduce the weight, volume, or size of metastases by, for example, at least 10%, at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100%, for example, as compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules in combination with (a) one or more reducing agents, (b) one or more immune activators, or a combination thereof, and / or without NIR irradiation of the primary tumor). In some examples, the disclosed methods have an abscopal effect, which itself provides at least 1 J cm at wavelengths between 660 and 740 nm. -2 of radiation (e.g., at least 4 J cm-2 or 4~50J cm -2 (a) a dose of 1000 mg / kg / day (or more than 100 mg / kg / day) of IR700 or IR700+ (a) a dose of 1000 mg / kg / day (or more than 100 mg / kg / day) of ...

[0110] Compared to other immune cells, Treg cells (e.g., CD4 + Foxp3 + By selectively killing Tregs, the method can kill irradiated Treg cells more effectively than other immune cells, such as Tregs not within the tumor bed. In one example, the disclosed method (e.g., using CTLA4 antibody-IR700) reduces Tregs (e.g., FOXP3 in the tumor bed). + CD4 + (e.g., in instances where a CTLA4 antibody-IR700 or a PD-L1 antibody-IR700 is administered), in some instances, FOXP3 in the tumor bed. + CD4 + The number of Treg cells is reduced by at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or more, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation). In some examples, the disclosed methods reduce the number of Treg cells by at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or more, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation). + Foxp3 - Cell:CD4 +Foxp3 + In some examples, the disclosed methods increase the ratio of FOXP3 cells in the spleen or regional lymph nodes of a tumor by at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 200%, at least 300%, or more, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation). + CD4 + The number of Treg cells is not substantially reduced, for example reduced by less than 10%, less than 5%, less than 1%, or less than 0.5%, compared to a state without treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation).

[0111] In some examples, the disclosed methods reduce intratumoral vascular perfusion by at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or more, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation).

[0112] In some examples, the disclosed methods reduce one or more symptoms associated with tumors, recurrences, and / or metastatic tumors. In one example, the disclosed methods slow down tumor growth, for example, by at least 10%, for example, by at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, or more, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation). In one example, the disclosed methods reduce or eliminate tumor recurrence, for example, by at least 10%, for example, by at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100%, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation).

[0113] In some examples, the disclosed methods increase the survival time of a subject (e.g., a subject having a tumor or from whom a tumor has previously been removed), for example, as compared to a state without treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation). In some examples, the survival time of the subject is increased by at least 20%, at least 25%, at least 40%, at least 50%, at least 80%, at least 90%, or more, for example, as compared to a state without treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation). In some examples, the subject's survival time is increased by at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or more, for example, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation). For example, the disclosed methods in some examples increase the subject's progression-free or disease-free survival (e.g., lack of recurrence of a primary tumor or lack of metastasis) by at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, at least 60 months, or more, compared to the average survival time in the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 molecules and / or without NIR irradiation).

[0114] In some cases, the use of one or more reducing agents in combination with NIR-PIT reduces edema in a treated subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or even at least 95% compared to the amount of edema without the use of one or more reducing agents with NIR-PIT. In some cases, the use of one or more reducing agents in combination with NIR-PIT reduces acute inflammatory response in a treated subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or even at least 95% compared to the amount of acute inflammatory response without the use of one or more reducing agents with NIR-PIT.

[0115] In one example, a combination of the above-listed advantages is achieved in the disclosed method.

[0116] The disclosed method can be used to treat fixed tumors in the body, as well as hematological malignancies and / or circulating tumors (e.g., leukemia cells, metastases, and / or circulating tumor cells). However, circulating cells, by their nature, cannot be exposed to light for very long. Thus, if the cells to be killed are those that are circulating throughout the body, the method can be accomplished by using a device that can be worn or that covers parts of the body. For example, such devices can be worn for long periods of time. Everyday wearable articles (e.g., watches, jewelry (e.g., necklaces or bracelets), blankets, clothing (e.g., underwear, socks, and shoe insoles), and other everyday wearable articles) that incorporate NIR-emitting light-emitting diodes (LEDs) and battery packs can be used. Such devices generate light on the skin under the device for long periods of time, resulting in continuous light exposure to superficial blood vessels for long periods of time. Circulating tumor cells are exposed to light as they pass through the area under the device. As an example, a watch or bracelet version of this device can include a series of NIR LEDs with a battery power pack and is worn for most of the day. Following administration of one or more tumor-specific antibody-IR700 molecules (e.g., systemically, e.g., iv and / or ip, in combination with one or more CTLA4 antibody-IR700 molecules, one or more PD-L1 antibody-IR700 molecules, one or more reducing agents, one or more immune activators, or a combination thereof), circulating cells will bind to the antibody-IR700 conjugate and become susceptible to killing by PIT. As these cells flow within blood vessels adjacent to LEDs present in everyday wearable articles (e.g., bracelets or watches), they will be exposed to NIR light, making them susceptible to cell killing. Light doses can be adjustable depending on the diagnosis and cell type.

[0117] In some examples, the method further comprises monitoring the therapy, e.g., tumor cell killing, Treg killing, or both. In such examples, the subject is administered one or more antibody-IR700 molecules (e.g., tumor-specific antibody-IR700 molecules and / or immune cell-specific antibody-IR700 molecules), e.g., in combination with one or more reducing agents, one or more immune activators, or a combination thereof, and irradiated as described herein. However, lower doses of antibody-IR700 conjugates and NIR light may be used for monitoring (only monitoring of therapy, since cell killing may not be required). In one example, the amount of antibody-IR700 conjugate administered for monitoring is at most half (e.g., at most one-third, one-quarter, one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, or one-tenth of the therapeutic dose). In one example, the amount of antibody-IR700 conjugate administered for monitoring is at least 20% or at least 25% lower than the therapeutic dose.In one example, the amount of NIR light used for monitoring is at least 1 / 1000 or at least 1 / 10,000 of the therapeutic dose.This allows detection of cells that are being treated.For example, by using such a method, tumor size and metastasis can be monitored.

[0118] In some cases, the method is useful during surgery, such as endoscopy. For example, after antibody-IR700 molecules (e.g., tumor-specific antibody-IR700 molecules and / or immune cell-specific antibody-IR700 molecules) are administered, for example, in combination with one or more reducing agents, one or more immune activators, or a combination thereof, and the cells / subjects are irradiated as described above, this not only results in cell killing, but also allows the surgeon or other medical personnel to visualize the borders of the tumor, helping to ensure that the tumor (e.g., skin, breast, lung, colon, head and neck, or prostate tumor) is completely removed and the borders are clear. In some cases, a lower dose of antibody-IR700 conjugate, such as at most half (e.g., at most one-third, one-quarter, one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, or one-tenth of the therapeutic dose), can be used for visualization.

[0119] One or more antibody-IR700 molecules (e.g., tumor-specific antibody-IR700 molecules and / or immune cell-specific antibody-IR700 molecules), one or more reducing agents, and one or more immune activators can be administered locally or systemically, for example, to a subject having a tumor, e.g., cancer, or a subject from which a tumor has previously been removed (e.g., by surgery). Although specific examples are provided, one of ordinary skill in the art will understand that alternative methods of administration of the disclosed therapeutic agents may be used. Such methods may include, for example, the use of a catheter or an implantable pump that provides a continuous infusion over a period of several hours to several days to a subject in need of treatment.

[0120] In one example, one or more antibody-IR700 molecules (e.g., tumor-specific antibody-IR700 molecules and / or immune cell-specific antibody-IR700 molecules), one or more reducing agents, and one or more immune activators are administered by parenteral means, including direct injection or infusion into a tumor (intratumorally) or organ (e.g., prostate). In some examples, one or more antibody-IR700 molecules (e.g., tumor-specific antibody-IR700 molecules and / or immune cell-specific antibody-IR700 molecules), one or more reducing agents, and one or more immune activators are administered to a tumor by applying the agent to the tumor, for example, by local injection of the therapeutic agent, bathing the tumor in a solution containing the therapeutic agent, or pouring the therapeutic agent into the tumor.

[0121] Additionally or alternatively, the disclosed compositions can be administered systemically, for example, intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, subcutaneously, or orally, to a subject with a tumor (e.g., cancer). One or more antibody-IR700 molecules (e.g., tumor-specific antibody-IR700 molecules and / or immune cell-specific antibody-IR700 molecules), one or more reducing agents, and one or more immune activators can be administered by the same or different routes. In one example, one or more antibody-IR700 molecules are administered iv and one or more reducing agents are delivered intraperitoneally. In one example, one or more antibody-IR700 molecules and one or more immune activators are administered iv and / or intratumorally and one or more reducing agents are delivered intraperitoneally. In another example, one or more antibody-IR700 molecules (e.g., tumor-specific antibody-IR700 molecules and / or immune cell-specific antibody-IR700 molecules), one or more reducing agents, and one or more immune activators are administered systemically (e.g., intravenously or intraperitoneally). In one example, one or more antibody-IR700 molecules (e.g., tumor-specific antibody-IR700 molecules and / or immune cell-specific antibody-IR700 molecules), one or more reducing agents, and one or more immune activators are administered intraperitoneally. In one example, one or more tumor-specific antibody-IR700 molecules, one or more immune activators, and one or more reducing agents are administered intravenously.

[0122] The dosage of the therapeutic agent provided herein to be administered to a subject is not subject to absolute limits and may depend on the nature of the composition, its active ingredients and its possible unwanted side effects (e.g., immune response to antibodies), the subject being treated and the type of condition being treated, and the mode of administration. Generally, the dose will be a therapeutically effective amount, e.g., an amount sufficient to achieve a desired biological effect, e.g., an amount effective to reduce the size (e.g., volume and / or weight) of a tumor or attenuate further growth of a tumor or reduce an undesired symptom of a tumor.

[0123] For intravenous administration of antibody-IR700 molecules (including tumor-specific antibody-IR700 molecules, as well as CTLA4 and PD-L1 antibody-IR700 molecules), exemplary dosages for administration to a subject in a single treatment may range from 0.5 to 200 mg / 60 kg body weight, 1 to 100 mg / 60 kg body weight, 1 to 50 mg / 60 kg body weight, 1 to 20 mg / 60 kg body weight, for example, about 1 or 2 mg / kg body weight. In yet another example, a therapeutically effective amount of antibody-IR700 molecules (including tumor-specific antibody-IR700 molecules, as well as CTLA4 and PD-L1 antibody-IR700 molecules) administered intraperitoneally or intratumorally is 10 μg to 5000 μg of antibody-IR700 molecules per kg body weight, for example, 10 μg / kg to 1000 μg / kg, 10 μg / kg to 500 μg / kg, or 100 μg / kg to 1000 μg / kg. In one example, the dose of antibody-IR700 molecules (including tumor-specific antibody-IR700 molecules, as well as CTLA4 and PD-L1 antibody-IR700 molecules) administered to a human patient is at least 50 mg, e.g., at least 100 mg, at least 300 mg, at least 500 mg, at least 750 mg, or even 1 g.

[0124] For intravenous administration of the reducing agent, exemplary dosages for administration to a subject in a single treatment can range from 0.5 to 300 g / 60 kg body weight, 1 to 300 g / 60 kg body weight, 1 to 50 g / 60 kg body weight, 1 to 20 g / 60 kg body weight, 1 to 10 g / 60 kg body weight, 10 to 300 g / 60 kg body weight, e.g., 1, 2, 5, 10, 20, 50, 100, 200, or 300 g / 60 kg body weight. In yet another example, a therapeutically effective amount of reducing agent administered intraperitoneally or intratumorally is 10 mg to 5000 mg of reducing agent per kg body weight, e.g., 10 mg / kg to 1000 mg / kg, 10 mg / kg to 500 mg / kg, or 100 mg / kg to 1000 mg / kg. In one example, the dose of the reducing agent administered to a human patient is at least 1 g, at least 10 g, at least 20 g, at least 50 g, at least 100 g, at least 200 g, at least 300 g, e.g., 1, 2, 5, 10, 20, 50, 100, 200, or 300 g.

[0125] For intravenous administration of an immune activator, e.g., IL-15, an exemplary dosage for administration to a subject in a single treatment can range from 0.1 to 100 mg, e.g., 1 to 10 mg or 0.5 to 20 mg. For intratumoral administration of an immune activator, e.g., IFN gamma, an exemplary dosage for administration to a subject in a single treatment can range from 0.01 to 1 mg, e.g., 0.1 to 0.5 mg or 0.05 to 5 mg.

[0126] Treatment with the disclosed antibody-IR700 molecules (including tumor-specific antibody-IR700 molecules, and CTLA4 and PD-L1 antibody-IR700 molecules), one or more reducing agents, and / or one or more immune activators may be completed in one day, or may be repeated over multiple days using the same or different dosages. Repeated treatments may be performed on the same day, on consecutive days, or every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or at longer intervals. In some examples, the antibody-IR700 molecules, one or more reducing agents, and / or one or more immune activators are administered on the same day. In other examples, the antibody-IR700 molecule, the one or more reducing agents, and / or the one or more immune activators are administered on different days, e.g., the one or more immune activators are administered the day before the antibody-IR700 molecule, or the antibody-IR700 molecule is administered the day before the one or more reducing agents). In one non-limiting example, the one or more antibody-IR700 molecules and the one or more immune activators are administered to the subject on the same day, and repeat doses of the one or more immune activators (at the same or different dosage levels) are administered to the subject on consecutive days, or every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or at even longer intervals (e.g., 1, 2, 3, 4, 5, or more additional doses of the one or more immune activators). In some examples, the amount of the repeat doses of the one or more immune activators is reduced (e.g., reduced by 50%) compared to the initial dose.

[0127] Exemplary treatment combinations are provided in Table 1. [Table 1-1] [Table 1-2]

[0128] In some examples, any one of Examples 7-13 and 19-38, the immune activator is IL-15. In some examples, any one of Examples 7-13 and 19-38, the immune activator is interferon gamma. In some examples, any one of Examples 14-24, 31-36, and 38-39, the reducing agent is sodium L-ascorbate.

[0129] In additional embodiments, the method also includes administering to the subject one or more additional therapeutic agents. Irradiation (e.g., at least 10 J / cm at a wavelength of 660-710 nm) can be administered as described in International Patent Application Publication No. WO 2013 / 009475, which is incorporated herein by reference in its entirety. 2 , at least 20 J / cm 2 , at least 30 J / cm 2 , at least 40 J / cm 2 , at least 50 J / cm 2 , at least 70 J / cm 2 , at least 80 J / cm 2 , or at least 100 J / cm 2 , for example, at least 10 to 100 J / cm 2There is an approximately 8 hour window following irradiation (irradiation at a dose of 100 nm or 200 nm) during which uptake of an additional agent (e.g., a nano-sized agent, e.g., at least about 1 nm in diameter, at least 10 nm in diameter, at least 100 nm in diameter, or at least 200 nm in diameter, e.g., 1-500 nm in diameter) by PIT-treated cells is enhanced. Thus, one or more additional therapeutic agents may be further administered to the subject contemporaneously or sequentially with PIT. In one example, the additional therapeutic agent is administered after irradiation, e.g., about 0-8 hours after irradiation of the cells (e.g., at least 10 minutes, at least 30 minutes, at least 60 minutes, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, or at least 7 hours after irradiation, e.g., within 10 hours, 9 hours, or within 8 hours after irradiation, e.g., 1 hour to 10 hours, 1 hour to 9 hours, 1 hour to 8 hours, 2 hours to 8 hours, or 4 hours to 8 hours). In another example, the additional therapeutic agent is administered immediately prior to irradiation (e.g., about 10 to 120 minutes prior to irradiation, e.g., 10 to 60 minutes, or 10 to 30 minutes prior to irradiation). Additional therapeutic agents that can be used are discussed below.

[0130] In additional embodiments, methods are provided that allow for detection or monitoring of cell killing in real time. Such methods are useful, for example, to ensure that a sufficient amount of antibody-IR700 molecules (including tumor-specific antibody-IR700 molecules, and CTLA4 and PD-L1 antibody-IR700 molecules), one or more reducing agents, and / or one or more immune activators, or a sufficient amount of radiation has been delivered to a cell or tumor to promote cell killing. These methods allow for detection of cell killing before morphological changes are evident. In one example, a method includes contacting a cell having a cell surface protein with a therapeutically effective amount of one or more antibody-IR700 molecules (including tumor-specific antibody-IR700 molecules, and CTLA4 and PD-L1 antibody-IR700 molecules), one or more reducing agents, and / or one or more immune activators, and contacting the cell with at least 10 J / cm at a wavelength of 660-740 nm.2 and detecting the cells by fluorescence lifetime imaging about 0-48 hours after the cells are irradiated (e.g., at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours after the cells are irradiated, e.g., 1 minute to 30 minutes, 10 minutes to 30 minutes, 10 minutes to 1 hour, 1 hour to 8 hours, 6 hours to 24 hours, or 6 hours to 48 hours after the cells are irradiated), thereby detecting cell killing in real time. The shortening of the FLT serves as an indicator of acute membrane damage induced by PIT. Thus, the cells are irradiated under conditions sufficient to shorten the IR700 FLT by at least 25%, e.g., at least 40%, at least 50%, at least 60%, or at least 75%. In one example, the cells receive at least 10 J / cm at a wavelength between 660 nm and 740 nm (e.g., between 680 nm and 700 nm, e.g., 680 or 690 nm). 2 , at least 20 J / cm 2 , at least 30 J / cm 2 , at least 40 J / cm 2 , at least 50 J / cm 2 , or at least 60 J / cm 2 , e.g., 10-60 J / cm 2 , 20~50J / cm 2 , 20~25J / cm 2 , or 30-50J / cm 2 The radiation is irradiated at a dose of .

[0131] In some examples, the method of detecting cell killing in real time includes contacting the cells with one or more additional therapeutic agents, for example, about 0-8 hours after irradiating the cells. Real-time imaging can occur before or after contacting the cells with the one or more additional therapeutic agents. For example, if insufficient cell killing occurs after administration of one or more antibody-IR700 molecules (including tumor-specific antibody-IR700 molecules, and CTLA4 and PD-L1 antibody-IR700 molecules), one or more reducing agents, and / or one or more immune activators, as determined by real-time imaging, the cells can be contacted with one or more additional therapeutic agents. However, in some examples, the cells are contacted with the antibody-IR700 molecules (including tumor-specific antibody-IR700 molecules, and CTLA4 and PD-L1 antibody-IR700 molecules), one or more reducing agents, and / or one or more immune activators, and the additional therapeutic agents before detecting cell killing in real time.

[0132] Exemplary Cells The target cell can be an unwanted cell or a cell whose growth is undesirable, such as a cancer cell (e.g., a tumor cell) or an immune cell (e.g., a T cell, e.g., a Treg). The cell can be present in the mammal to be treated, e.g., a subject with cancer (e.g., a human or veterinary subject). Any target cell can be treated with the claimed methods. In one example, the target cell expresses a cell surface protein that is substantially not found on the surface of other normal (desired) cells, and an antibody that specifically binds to such a protein can be selected and an antibody-IR700 molecule can be generated against that protein. In one example, the cell surface protein is a tumor-specific protein (e.g., an antigen), such as EGFR. In one non-limiting example, the cell surface protein is CTLA4 (e.g., CD4 in tumor beds), and the cell surface protein is a tumor-specific protein (e.g., an antigen), such as EGFR. In one non-limiting example, ... + Foxp3 + In one non-limiting example, the cell surface protein is PD-L1.

[0133] In one example, tumor cells are cancer cells, for example, cells in patients with cancer.Exemplary cells that can be killed using the disclosed method include the cells of the following tumors: hematological malignancies, for example, leukemia, including acute leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease). In another example, the cell is a solid tumor cell, e.g., a sarcoma and carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, squamous cell carcinoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), basal cell carcinoma, adenocarcinoma (e.g., adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus). In one example, the tumor is a cell derived from a human ovarian cancer, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder cancer, and a CNS cancer (e.g., glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In one example, the tumor is an oropharyngeal cancer.

[0134] In a specific example, the cell is a lung cancer cell.

[0135] In a specific example, the cell is a breast cancer cell.

[0136] In a specific example, the cell is a colon cancer cell.

[0137] In a specific example, the cell is a head and neck cancer cell.

[0138] In a particular example, the cell is a prostate cancer cell.

[0139] In a specific example, the cell is an oropharyngeal cancer cell.

[0140] In specific examples, the cells are squamous cell carcinoma cells, such as those of a head and neck or skin cancer.

[0141] In some cases, the method may have an abscopal effect, such that the cancer or cancer cells being treated are those at a site distal to the tumor being irradiated, e.g., at least 1 J / cm at wavelengths of 660-740 nm. 2 Distant metastases that are not irradiated at a dose of 1 mm, 1 inch, 2 inches, 3 inches, 4 inches, 6 inches, 12 inches, or more.

[0142] In some examples, the cancer or cancer cells to be treated are moderately or highly immunogenic and can elicit an immune response. In some examples, the NIR-PIT method comprising administration of CTLA4 antibody-IR700 and / or PD-L antibody-IR700 is used to treat moderately or highly immunogenic tumors. Examples of moderately or highly immunogenic cancers include melanoma, lung cancer (e.g., NSCLC), colon cancer, and renal cell carcinoma. In one example, the highly immunogenic cancer is prostate cancer. In some examples, the cancer or cancer cells to be treated are not highly immunogenic and cannot elicit an immune response. In some examples, NIR-PIT with CTLA4 antibody-IR700 and / or PD-L antibody-IR700 is used in combination with anti-tumor-IL700 (e.g., anti-EGFR-IR700 or anti-PSA antigen-IR700) to treat low-immunogenic tumors. Examples of low immunogenic cancers include oral squamous cell carcinoma, breast cancer, pancreatic cancer, and multiple myeloma. In some cases, tumor immunogenicity score is used to determine whether a cancer is low immunogenic, moderately immunogenic, or highly immunogenic (see, for example, Wang et al, eLife, 8:e49020, 2019).

[0143] Example Subjects In some examples, the disclosed methods are used to treat a subject with cancer or a subject with a tumor, such as a tumor described herein. In some examples, the tumor has previously been treated, e.g., surgically or chemically removed, and the disclosed methods are subsequently used to kill any remaining unwanted tumor cells that may remain in the patient and / or to reduce tumor recurrence or metastasis.

[0144] The disclosed methods can be used to treat any mammalian subject (e.g., human or veterinary subject, e.g., dog or cat), e.g., human, having a tumor, e.g., cancer, or having previously been removed or treated. Subjects in need of the disclosed therapies can include human subjects having cancer, e.g., cancers that express tumor-specific proteins on the cell surface that can specifically bind to tumor-specific antibody-IR700 molecules, or cancers infiltrated with immune cells that can bind to immune cell-specific antibody-IR700 molecules (e.g., CTLA4 antibody-IR700). For example, the disclosed methods can be used as a first-line treatment for cancer, alone or in combination with radiation or other chemotherapy or surgery. The disclosed methods can also be used in patients who have failed previous radiation or chemotherapy. Thus, in some instances, the subject has undergone other therapies, but the other therapies have not provided the desired therapeutic response. The disclosed methods can also be used in patients with localized and / or metastatic cancer, and / or recurrence of primary tumors.

[0145] In some examples, the method includes selecting a subject who would benefit from the disclosed therapeutic methods, e.g., selecting a subject having a tumor that expresses a cell surface protein (e.g., a tumor-specific protein) that can specifically bind to an antibody-IR700 molecule. For example, if a subject is determined to have a breast cancer that expresses HER2, the subject may be selected to be treated with an anti-HER2-IR700 molecule, e.g., trastuzumab-IR700, in combination with, e.g., a CTLA4 antibody-IR700 molecule, a PD-L1 antibody-IR700 molecule, one or more reducing agents, one or more immune activators, or a combination thereof, and the subject is subsequently irradiated as described herein.

[0146] Exemplary Cell Surface Proteins In one example, the protein on the cell surface of the target cell to be killed is not present in significant amounts on other cells, for example, the cell surface protein may be a receptor that is only found on the target cell type.

[0147] In a specific example, the cell surface protein is a cancer or tumor-specific protein (also referred to as tumor-specific antigen or tumor-associated antigen), such as a member of the EGF receptor family (e.g., HER1, 2, 3, and 4), and a cytokine receptor (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.). Thus, in some examples, the cell surface protein is an antigen expressed on the cell membrane of a tumor cell. A tumor-specific protein is a protein that is unique to cancer cells or is much more abundant in cancer cells compared to other cells, e.g., normal cells. For example, HER2 is found primarily in breast cancer, while HER1 is found primarily in adenocarcinoma, which can be found in many organs, e.g., pancreas, breast, prostate, and colon.

[0148] Exemplary tumor-specific proteins that can be found in target cells (and for which antibodies specific for the proteins can be used to formulate antibody-IR700 molecules) include MAGE 1 (e.g., GenBank Accession Nos. M77481 and AAA03229), MAGE 2 (e.g., GenBank Accession Nos. L18920 and AAA17729), MAGE 3 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 4 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 5 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 6 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 7 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 8 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 9 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 10 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 11 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 12 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 13 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 14 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE 15 (e.g., GenBank Accession Nos. U0 Any of the various MAGEs (melanoma associated antigen E), including 4 (e.g., GenBank Accession Nos. D32075 and A06841.1), any of the various tyrosinases (e.g., GenBank Accession Nos. U01873 and AAB60319), mutant ras, mutant p53 (e.g., GenBank Accession Nos. X54156, CAA38095, and AA494311), p97 melanoma antigen (e.g., GenBank Accession Nos. M12154 and AAA59992), human milk fat globules (HMFG) associated with breast tumors (e.g., GenBank Accession Nos. S5615 and S5625), and the like. 1 and AAB19771), any of the various BAGEs (human B melanoma associated antigen E), including BAGE1 (e.g., GenBank accession number Q13072) and BAGE2 (e.g., GenBank accession numbers NM_182482 and NP_872288), any of the various GAGEs (G antigens), including GAGE1 (e.g., GenBank accession number Q13065) or any of GAGEs 2-6, various gangliosides, CD25 (e.g., GenBank accession numbers NP_000408.1 and NM_000417.2).

[0149] Other tumor-specific antigens include HPV 16 / 18 and E6 / E7 antigens associated with cervical cancer (e.g., GenBank accession numbers NC_001526, FJ952142.1, ADB94605, ADB94606, and U89349), mucins associated with breast cancer (MUC 1)-KLH antigen (e.g., GenBank Accession Nos. J03651 and AAA35756), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession Nos. X98311 and CAA66955), gp100 associated with melanoma (e.g., GenBank Accession Nos. S73003 and AAC60634), MART1 antigen associated with melanoma (e.g., GenBank Accession No. NP_005502), cancer antigen 125 (CA125, also known as mucin 16 or MUC16) associated with ovarian and other cancers (e.g., GenBank Accession Nos. NM_024690 and NP_005502), and other cancer antigens. 78966), alpha-fetoprotein (AFP) associated with liver cancer (e.g., GenBank Accession Nos. NM_001134 and NP_001125), Lewis Y antigen associated with colorectal cancer, cholangiocarcinoma, breast cancer, small cell lung cancer, and other cancers, tumor-associated glycoprotein 72 (TAG72) associated with adenocarcinoma, glypican 1 (GPC1 associated with pancreatic cancer, glioma, and breast cancer), glypican 2 (associated with neuroblastoma), and glypican 3 (associated with hepatocellular carcinoma), and PSA antigen associated with prostate cancer (e.g., GenBank Accession Nos. X14810 and CAA32915).

[0150] Other exemplary tumor-specific proteins include PMSA (prostate membrane specific antigen, e.g., GenBank Accession Nos. AAA60209 and AAB81971.1), which is associated with solid tumor angiogenesis and prostate cancer; HER-2 (human epidermal growth factor receptor 2, e.g., GenBank Accession Nos. M16789.1, M16790.1, M16791.1, M16792.1, and AAA58637), which is associated with breast, ovarian, gastric, and uterine cancer; HER-1 (e.g., G1 ... enBank accession numbers NM_005228 and NP_005219), NY-ESO-1 (e.g., GenBank accession numbers U87459 and AAB49693), which is associated with melanoma, sarcoma, testicular cancer, and other cancers, hTERT (also known as telomerase) (e.g., GenBank accession numbers NM_198253 and NP_937983 (variant 1), NM_198255 and NP_937986 (variant 2)), proteinase 3 (e.g., GenBank accession numbers M29142, M75154, M96839, X55668, NM 00277, M96628, X56606, CAA39943, and AAA36342), and Wilms tumor 1 (WT-1, e.g., GenBank accession numbers NM_000378 and NP_000369 (variant A), NM_024424 and NP_077742 (variant B), NM_024425 and NP_077743 (variant C), and NM_024426 and NP_077744 (variant D)).

[0151] In one example, the tumor-specific protein is EGFR, and in some examples, the EGFR antibody-IR700 is or includes panitumumab or cetuximab.

[0152] In one example, the tumor-specific protein is PD-L1, and in some examples, the PD-L1 antibody-IR700 is or includes atezolizumab, avelumab, durvalumab, or cosibelimab.

[0153] In one example, the tumor-specific protein is CD52, which is associated with chronic lymphocytic leukemia (e.g., GenBank Accession Nos. AAH27495.1 and CAI15846.1), CD33, which is associated with acute myeloid leukemia (e.g., GenBank Accession Nos. NM_023068 and CAD36509.1), and CD20, which is associated with non-Hodgkin's lymphoma (e.g., GenBank Accession Nos. NP_068769 NP_031667).

[0154] In a specific example, tumor-specific protein is CD44 (e.g., OMIM 107269, GenBank accession number ACI46596.1 and NP_000601.3).CD44 is a marker of cancer stem cell-like cells and various types of cancer, and is involved in cell-cell adhesion, cell migration, cell spatial arrangement, and promoting matrix-derived survival signals.High expression of CD44 on tumor plasma membrane can be associated with tumor aggressiveness and poor prognosis.

[0155] Thus, the disclosed methods can be used to treat any cancer that expresses a tumor-specific protein.

[0156] Exemplary Antibody-IR700 Molecules Because cell surface protein sequences are publicly available (e.g., as described above), making or purchasing antibodies (or other small molecules that can be conjugated to IR700) specific to such proteins can be accomplished. For example, if the tumor-specific protein HER2 is selected as the target, an antibody specific to HER2 (e.g., trastuzumab) can be purchased or generated and attached to the IR700 dye. Other specific examples are provided in Table 2 and elsewhere herein. In one example, the antibody conjugated to IR700 is a humanized monoclonal antibody.

[0157] In one example, the antibody-IR700 molecule is a PD-L1 antibody-IR700 molecule, such as atezolizumab-IR700, avelumab-IR700, durvalumab-IR700, cosibelimab-IR700, KN035-IR700, BMS935559-IR700, MEDI-4736-IR700, MEDfI-4737-IR700, BMS-936559-IR700, MPDL-3280A-IR700, or CK-301-IR700.

[0158] In one example, the antibody-IR700 molecule is an EGFR antibody-IR700 molecule, such as panitumumab-IR700 or cetuximab-IR700.

[0159] Antibody-IR700 molecules can be generated using methods such as those described in WO 2013 / 009475, which is incorporated herein by reference in its entirety. [Table 2-1] [Table 2-2] [Table 2-3]

[0160] Additional antibodies that can be conjugated to IR700 and used in the disclosed methods include 3F8, abagovomab, afutuzumab, alacizumab, altumomab pentetate, anatumomab mafenatox, apolizumab, bectumomab, belimumab, besilesomab, capromab pendetide, catumaxomab, sitatuzumab bogatox, detumomab, ecloneximab, eculizumab, edrecolomab, epratuzumab, eritumaxomab, galectinib ... Examples of such agents include liximab, glenbatumumab vedotin, igovomab, imcilomab, lumiliximab, mepolizumab, metelimu- mab, mitumomab, morolimumab, nacolomab butafenatox, naptumomab estafenatox, nofetumomab merpentane, pintumomab, satumomab pendetide, sonepcizumab, taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), TNX-650, or tremelimumab.

[0161] In some examples, the cell surface protein recognized by the antibody conjugated to IR700 is found on immune cells, such as Tregs. In one example, the protein is CTLA4. In one example, the antibody-IR700 molecule is a CTLA4 antibody-IR700 molecule, such as ipilimumab-IR700 or tremelimumab-IR700.

[0162] In one example, a patient is treated with at least two different antibody-IR700 molecules specific for cancer cell surface antigens. In one example, the two different antibody-IR700 molecules are specific for the same protein (e.g., HER-2), but different epitopes of the protein (e.g., epitope 1 and epitope 2 of HER-2). In another example, the two different antibody-IR700 molecules are specific for two different proteins or antigens. For example, anti-HER1-IR700 and anti-HER2-IR700 may be injected together as a cocktail to promote the killing of cells bearing either HER1 or HER2.

[0163] Immune modulator / immune activator The immune activators used in the disclosed methods include agents or compositions that activate the immune system and / or inhibit immune suppressor cells (also referred to herein as suppressor cells). Inhibition of immune suppressor cells and / or activation of immune response can increase tumor cell killing and lead to the production of memory T cells, which can provide a "vaccine" effect against recurrence and / or distant tumors or metastasis. In some examples, one or more immune activators are used in combination with one or more tumor-specific antibodies-IR700 molecules, one or more reducing agents, or both.

[0164] In some embodiments, the immune activator is an inhibitor of immune suppressor cells, e.g., an agent that inhibits or reduces the activity of immune suppressor cells. In some cases, the immune modulator kills immune suppressor cells. In some cases, the immune suppressor cells are regulatory T (Treg) cells. In some cases, not all of the suppressor cells are killed in vivo, as this may lead to the development of autoimmunity. Thus, in some cases, the method reduces the activity or number of immune suppressors in an area of ​​the subject, e.g., an area of ​​a tumor or an area that previously had a tumor, by at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or at least 95%. In some cases, the method reduces the total number of suppressor cells in the subject by at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or at least 95%.

[0165] Inhibitors of immune suppressor cells include tyrosine kinase inhibitors (e.g., sorafenib, sunitinib, and imatinib), chemotherapeutic agents (e.g., cyclophosphamide or interleukin-toxin fusions, such as denileukin diftitox (IL2-diphtheria toxin fusion), or anti-CD25 antibodies (e.g., daclizumab or basiliximab), or other antibodies that bind to suppressor cell surface proteins (e.g., those described below). In other examples, inhibitors of immune suppressor cells include immune checkpoint inhibitors, such as anti-PD-L1 antagonizing antibodies (herein referred to as PD-1 / PD-L1 antagonizing antibodies), which thereby prevent binding of PD-L1 to PD-1. These include mAb-mediated immune checkpoint blockade (ICB). Thus, in some examples, the immune modulator is a PD-L1 antagonizing antibody, such as atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (embafolimab), BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737 (or other examples provided herein). Checkpoint inhibitors also include CTLA-4 antibodies, including ipilimumab and tremelimumab. The inhibitor of immune suppressor cells can also be LAG-3 or B7-H3 antagonists, such as BMS-986016 and MGA271. In some examples, two or more of the inhibitors of immune suppressor cells can be administered to the subject. In one non-limiting example, the subject is administered anti-PD1 and anti-LAG-3 antibodies.

[0166] In some examples, agents that inhibit or reduce the activity of suppressor cells include one or more antibody-IR700 molecules, where the antibody specifically binds to a suppressor cell surface protein (e.g., CD25, CD4, CXC chemokine receptor type 4 (CXCR4), CC chemokine receptor type 4 (CCR4), cytotoxic T lymphocyte-associated protein 4 (CTLA4), glucocorticoid-inducible TNF receptor (GITR), OX40, folate receptor 4 (FR4), CD16, CD56, CD8, CD122, CD23, CD163, CD206, CD11b, Gr-1, CD14, interleukin-4 receptor alpha chain (IL-4Ra), interleukin-1 receptor alpha (IL-1Ra), interleukin-1 decoy receptor, CD103, fibroblast activation protein (FAP), CXCR2, CD33, and CD66b).

[0167] In a non-limiting example, the immune modulator is a CD25 antibody-IR700 molecule, such as daclizumab-IR700 or basiliximab-IR700.

[0168] In other embodiments, the immune modulator is an immune system activator (immunoactivator). In some examples, the immune system activator stimulates (activates) one or more T cells and / or natural killer (NK) cells. In one example, the immune system activator includes one or more interleukins (ILs), such as IL-2, IL-15, IL-7, IL-12, and / or IL-21. In a non-limiting example, the immune modulator includes or consists of IL-15. In a non-limiting example, the immune modulator includes IL-2 and IL-15. In a non-limiting example, the immune modulator includes or consists of interferon gamma. In another example, the immune system activator includes one or more agonists for a costimulatory receptor, such as 4-1BB, OX40, or GITR. In a non-limiting example, the immune modulator includes a stimulatory anti-4-1BB antibody, an anti-OX40 antibody, and / or an anti-GITR antibody.

[0169] In some examples, one or more (e.g., 1, 2, 3, 4, 5, or more) doses of the immune modulator are administered to the subject. Thus, the step of administering the immune modulator may be completed in one day or may be repeated over multiple days using the same or different dosages (e.g., at least 2 different, 3 different, 4 different, 5 different, or 10 different administrations). In some examples, the repeated administrations are the same dose. In other examples, the repeated administrations are different doses (e.g., a subsequent dose that is higher than the preceding dose, or a subsequent dose that is lower than the preceding dose). The repeated administrations of the immune modulator may be administered on the same day, on consecutive days, every other day, every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or at longer intervals. In some examples, at least one dose of the immune modulator is administered prior to irradiation and at least one dose of the immune modulator is administered following irradiation.

[0170] irradiation After one or more antibody-IR700 molecules are administered to the subject, the subject (or the tumor in the subject) is irradiated. Only cells expressing the target protein are recognized by the antibody, so only those cells will be associated with a sufficient amount of antibody-IR700 molecules to kill the cells. This reduces the possibility of undesired side effects, such as killing non-tumor cells, because irradiation only kills cells to which the antibody-IR700 molecules are bound, and not other cells.

[0171] The subject (e.g., a tumor in a subject) is irradiated with a therapeutic radiation dose at a wavelength of 660-740 nm, e.g., 660-710 nm, 660-700 nm, 680-700 nm, 670-690 nm, e.g., 680 nm or 690 nm. In certain examples, the cell, tumor, or subject is irradiated with a therapeutic radiation dose of at least 1 J / cm. 2 , e.g., at least 10 J / cm 2 , at least 20 J / cm 2 , at least 25 J / cm 2 , at least 30 J / cm 2, at least 50 J / cm 2 , at least 100 J / cm 2 , or at least 500 J / cm 2 , for example, 1 to 1000 J / cm 2 , 1~500J / cm 2 , 1~100J / cm 2 , 4~50J / cm 2 , 30~50J / cm 2 , 10~100J / cm 2 , 20~30J / cm 2 , 20~50J / cm 2 , or 10~50J / cm 2 The radiation is irradiated at a dose of .

[0172] A subject may be irradiated once or multiple times. Thus, irradiation may be completed in one day or may be repeated over multiple days using the same or different dosages (e.g., at least 2 different, 3 different, 4 different, 5 different, or 10 different irradiations). In some examples, the repeated irradiations are at the same dose. In other examples, the repeated irradiations are at different doses (e.g., a subsequent dose higher than the preceding dose, or a subsequent dose lower than the preceding dose). Repeated irradiations may be performed on the same day, on consecutive days, every other day, every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or at longer intervals. In one example, the first irradiation is at 50 J / cm 2 and the second irradiation is 100 J / cm 2 and the irradiation is performed on consecutive days (e.g., about 24 hours apart). In one example, the first irradiation is 10-50 J / cm 2 and the second irradiation is 10 to 50 J / cm 2 and the irradiation is performed on consecutive days (e.g., about 24 hours apart).

[0173] In some examples, the illumination is provided by a wearable device incorporating NIR LED. In other examples, another type of device that can be used with the disclosed method is a flashlight-like device with NIR LED. Such a device can be used for localized treatment of lesions during surgery, or can be incorporated into an endoscope that applies NIR light to the body surface after administration of one or more PIT agents. Such a device can be used by a physician or qualified medical practitioner to guide treatment to specific targets in the body.

[0174] Treatment with wearable NIR LEDs As described herein, the disclosed method is highly specific to cancer cells (or Tregs in tumor beds, for example, when CTLA4 antibody-IR700 is used). However, to kill cells circulating in the body or present on the skin, the patient can wear a device incorporating NIR LEDs. In some cases, the patient uses at least two devices, for example, an article of clothing or jewelry during the day and a bracelet at night. In some cases, the patient uses at least two devices, for example, two articles of clothing, simultaneously. These devices allow the patient to be exposed to NIR light using portable everyday clothing and jewelry, so that the treatment remains unobtrusive and does not interfere with daily activities. In some cases, the device can be worn discreetly during the day for PIT therapy. An exemplary device incorporating NIR LEDs is disclosed in International Patent Application Publication No. WO 2013 / 009475, which is incorporated herein by reference.

[0175] In one example, a patient is administered one or more antibody-IR700 molecules, e.g., in combination with one or more reducing agents and / or one or more immune activators, using the methods described herein. The patient then wears a device incorporating NIR LEDs, allowing for long-term therapy and treatment of tumor cells present in the blood or lymph nodes or skin. In some examples, the dose is at least 1 J / cm. 2 , at least 4 J / cm 2 , at least 10 J / cm 2 , at least 20 J / cm 2 , at least 30 J / cm 2 , at least 40 J / cm 2 , or at least 50 J / cm 2 , e.g., 10-100 J / cm 2 , 10~50J / cm 2 , e.g., 20 J / cm 2 or 30 J / cm 2 In some examples, administration of one or more antibody-IR700 molecules, e.g., in combination with one or more reducing agents and / or one or more immune activators, is repeated over a period of time (e.g., every two weeks or once a month) to ensure that therapeutic levels are present in the body.

[0176] In some cases, the patient wears or uses the device or combination of devices for at least one week, for example, at least two weeks, at least four weeks, at least eight weeks, at least twelve weeks, at least four months, at least six months, or even at least one year. In some cases, the patient wears or uses the device or combination of devices for at least four hours per day, for example, at least twelve hours per day, at least sixteen hours per day, at least eighteen hours per day, or even twenty-four hours per day. It is quite possible that multiple devices of similar "everyday" nature (blankets, bracelets, necklaces, underwear, socks, shoe insoles) can be worn by the same patient during the treatment period. At night, the patient can use a NIR LED blanket or other cover.

[0177] Administration of additional treatment As discussed above, the subject may receive one or more other therapies before, during, or after administration of one or more antibody-IR700 molecules, e.g., in combination with one or more reducing agents and / or one or more immune activators, and irradiation. In one example, the subject receives one or more treatments to remove or reduce tumors, e.g., prior to administration of one or more antibody-IR700 molecules, e.g., in combination with one or more reducing agents and / or one or more immune activators, and irradiation. In other examples, the additional treatment or therapeutic agent (e.g., an anti-neoplastic agent) can be administered to the subject to be treated, for example, after irradiation, for example, about 0 to 8 hours after the cells are irradiated (e.g., at least 10 minutes, at least 30 minutes, at least 60 minutes, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, or at least 7 hours after irradiation, for example, within 10 hours, 9 hours, or within 8 hours after irradiation, for example, 1 hour to 10 hours, 1 hour to 9 hours, 1 hour to 8 hours, 2 hours to 8 hours, or 4 hours to 8 hours). In another example, the additional therapeutic agent is administered immediately before irradiation (e.g., about 10 minutes to 120 minutes before irradiation, for example, 10 minutes to 60 minutes, or 10 minutes to 30 minutes before irradiation).

[0178] In some cases, the disclosed methods can be used in combination with the disclosed methods to enhance tumor accessibility to additional therapeutic agents for about 8 hours after PIT, including, but not limited to, surgical procedures for tumor removal or reduction (e.g., surgical resection, cryotherapy, or chemoembolization), and antitumor pharmaceutical treatments that can include radiotherapy agents, anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agents, and antioxidants, kinase inhibitors, and other agents. In some cases, the additional therapeutic agent is conjugated to the nanoparticle. Specific examples of additional therapeutic agents that can be used include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and / or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators. These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination.

[0179] "Microtubule-binding agent" refers to an agent that interacts with tubulin to stabilize or destabilize microtubule formation, thereby inhibiting cell division. Examples of microtubule-binding agents that can be used in conjunction with the disclosed method include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin, and rhizoxin. Analogs and derivatives of such compounds can also be used. For example, suitable epothilones and epothilone analogs are described in International Publication No. WO 2004 / 018478. Taxoids such as paclitaxel and docetaxel, as well as analogs of paclitaxel, as taught in U.S. Patent Nos. 6,610,860, 5,530,020, and 5,912,264, can be used.

[0180] The following classes of compounds can be used with the methods disclosed herein: Suitable DNA and / or RNA transcription regulators are also suitable for use in combination with the disclosed therapeutic methods, including, without limitation, actinomycin D, daunorubicin, doxorubicin, and their derivatives and analogs. DNA intercalators and crosslinkers that can be administered to a subject include, without limitation, cisplatin, carboplatin, oxaliplatin, mitomycin, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide, and their derivatives and analogs. Suitable DNA synthesis inhibitors for use as therapeutic agents include, without limitation, methotrexate, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, and their analogs. Examples of suitable enzyme inhibitors include, without limitation, camptothecin, etoposide, formestane, trichostatin, and their derivatives and analogs. Suitable compounds that affect gene regulation include agents that increase or decrease the expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone, and derivatives and analogs thereof. Kinase inhibitors include Gleevec® (imatinib), Iressa® (gefitinib), and Tarceva® (erlotinib), which prevent phosphorylation and activation of growth factors.

[0181] Non-limiting examples of antiangiogenic agents include molecules such as proteins, enzymes, polysaccharides, oligosaccharides, DNA, RNA, and recombinant vectors, as well as small molecules that function to reduce or even inhibit blood vessel growth. Examples of suitable angiogenesis inhibitors include, without limitation, angiostatin K1-3, staurosporine, genistein, fumagillin, medroxyprogesterone, suramin, interferon-alpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, thrombospondin, endostatin, thalidomide, and derivatives and analogs thereof. For example, in some embodiments, the antiangiogenic agent is an antibody that specifically binds to VEGF (e.g., Avastin, Roche) or an antibody that specifically binds to VEGF receptor (e.g., VEGFR2 antibody). In one example, the antiangiogenic agent includes VEGFR2 antibody or DMXAA (also known as vadimezan or ASA404, and available, for example, from Sigma Corp., St. Louis, MO), or both. The antiangiogenic agent may be bevacizumab, sunitinib, antiangiogenic tyrosine kinase inhibitors (TKIs), such as sunitinib, xitinib, and dasatinib, which may be used individually or in any combination.

[0182] Other therapeutic agents, such as anti-tumor agents, which may or may not fall into one or more of the above categories, are also suitable for administration in combination with the disclosed methods. By way of example, such agents include adriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.

[0183] In some cases, subjects receiving therapeutic antibody-IR700 molecules are also administered interleukin-2 (IL-2), for example, by intravenous administration. In a particular example, IL-2 (Chiron Corp., Emeryville, CA) is administered as an intravenous bolus at a dose of at least 500,000 IU / kg, over a period of 15 minutes every 8 hours, starting the day after administration of the antibody-IR700 molecule and continuing for up to 5 days. Doses may be skipped depending on the subject's tolerance.

[0184] Exemplary additional therapeutic agents include anti-neoplastic agents, such as chemotherapeutic agents and anti-angiogenic agents or treatments, such as radiation therapy. In one example, the agent is a chemotherapy immunosuppressant (e.g., rituximab, steroids) or a cytokine (e.g., GM-CSF). Exemplary chemotherapeutic agents are provided, for example, in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc; Baltzer and Berkery. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer Knobf, and Durivage (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer.

[0185] Exemplary chemotherapeutic agents that can be used with the methods provided herein include, but are not limited to, carboplatin, cisplatin, paclitaxel, docetaxel, doxorubicin, epirubicin, topotecan, irinotecan, gemcitabine, iazofurine, gemcitabine, etoposide, vinorelbine, tamoxifen, valspodar, cyclophosphamide, methotrexate, fluorouracil, mitoxantrone, Doxil (liposome-encapsulated doxorubicin), and vinorelbine. Additional examples of chemotherapeutic agents that can be used include alkylating agents, antimetabolites, natural products, or hormones and their antagonists. Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, or dacarbazine). Specific non-limiting examples of alkylating agents are temozolomide and dacarbazine. Examples of antimetabolites include folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-FU or cytarabine), and purine analogs such as mercaptopurine or thioguanine. Examples of natural products include vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (e.g., L-asparaginase). Examples of other drugs include platinum coordination complexes (e.g., cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenal cortex suppressants (e.g., mitotane and aminoglutethimide).Examples of hormones and antagonists include corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., tamoxifen), and androgens (e.g., testosterone propionate and fluoxymesterone).

[0186] Exemplary chemotherapeutic agents include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-fluorouracil (5-FU), Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitroxamate, and the like. These include gemmustard, taxol (or other taxanes, e.g., docetaxel), velban, vincristine, VP-16, gemcitabine (Gemzar), herceptin, irinotecan (Camptosar, CPT-11), leustatin, navelbine, rituxan STI-571, taxotere, topotecan (Hycamtin), xeloda (capecitabine), zevelin, and calcitriol. Non-limiting examples of immune activators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor, Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (from Imreg, New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor, Genentech).

[0187] In some examples, the additional therapeutic agent is conjugated to (or associated with) a nanoparticle, e.g., at least 1 nm in diameter (e.g., at least 10 nm in diameter, at least 30 nm in diameter, at least 100 nm in diameter, at least 200 nm in diameter, at least 300 nm in diameter, at least 500 nm in diameter, or at least 750 nm in diameter, e.g., 1 nm to 500 nm, 1 nm to 300 nm, 1 nm to 100 nm, 10 nm to 500 nm, or 10 nm to 300 nm in diameter).

[0188] In one example, at least a portion of the tumor (e.g., a metastatic tumor) is surgically removed (e.g., by surgical resection and / or cryotherapy), irradiated (e.g., administration of radioactive material or energy to the tumor site to help eradicate or shrink the tumor (e.g., external beam radiation therapy)), chemically treated (e.g., by chemoembolization), or a combination thereof, prior to administration of the disclosed treatment (e.g., administration of one or more tumor-specific antibody-IR700 molecules, e.g., in combination with one or more reducing agents and / or one or more immune activators, and irradiation). For example, a subject with a metastatic tumor may have all or a portion of the tumor surgically resected prior to administration of the disclosed treatment. In an example, one or more chemotherapeutic agents are administered following treatment with one or more antibody-IR700 molecules, e.g., in combination with one or more reducing agents and / or one or more immune activators, and irradiation, and irradiation. In another specific example, the subject has a metastatic tumor and radiation therapy, chemoembolization therapy, or both are administered simultaneously with administration of the disclosed treatment.

[0189] In some examples, the additional therapeutic agent administered is a monoclonal antibody, e.g., 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, altumomab pentetate, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, besilesomab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide, catumaxomab, CC 49, Cetuximab, Sitaxel, Cixutumumab, Clivacizumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Eclomeximab, Ecrimumab, Edrecolomab, Epratuzumab, Elitumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicin, Girentuximab, Glenbatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin ipilimumab, iratumumab, labetuzumab, raxatumumab, lintuzumab, lorvotuzumab mertansine, lucatumumab, rumiliximab, mapatumumab, matuzumab, mepolizumab, metelimumab, milatuzumab, mitumomab, morolimumab, nacolomab butafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentane, ofatumumab, olaratumab, oportuzumab monatox, oregovomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, lobatumumab, satumomab pendetide, sibrotuzumab, sonepcizumab, tacatuzumab tetraxetan, taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, zalutamumab, or a combination thereof.

[0190] Exemplary Reducing Agents Exemplary reducing agents that can be used in the methods provided herein include agents that donate (or "donate") electrons to an electron recipient (oxidizing agent) in a redox chemical reaction. Exemplary reducing agents that can be used in the methods provided herein include L-cysteine, sodium L-ascorbate (L-NaAA), ascorbic acid (e.g., L- or R-ascorbic acid), and glutathione. In some examples, the reducing agent used in the disclosed methods is not sodium azide. In some examples, the reducing agent used in the disclosed methods is not L-cysteine. In one example, the reducing agent is L-NaAA (e.g., 5-50 g, ip). EXAMPLES

[0191] Example 1 material and method This example describes the materials and methods used to obtain the results in Example 2.

[0192] cell culture Parental mEERL cells were constructed by transduction of HPV 16 E6 / E7 and hRAS into C57BL / 6-derived oropharyngeal epithelial cells (Hoover et al., Arch OtolaryngolHead Neck Surg 2007; 133: 495-502; Spanos et al., J Virol 2008; 82:2493-500; Williams et al., Head Neck 2009; 31:911-8). mEERL-hEGFR was obtained from Dr. William C. Spanos (Sanford Research, Okada et al., EBioMedicine 2021; 67:103345). In addition, MC38 cells (colon cancer, RRID: CVCL_B288, from Claudia Palena, NCI, 2015) and MOC2 cells (oral cancer, RRID: CVCL_ZD33, from Kerafast) stably expressing luciferase (MC38-luc and MOC2-luc, generated by stable transduction with RediFect Red-Fluc lentivirus from PerkinElmer according to the manufacturer's recommendations), as well as LL / 2-luc cells (Lewis lung carcinoma, RRID: CVCL_A4CM, purchased from Imanis Life Sciences) were used. High luciferase expression in MC38-luc, MOC2-luc, and LL / 2-luc cells was confirmed up to passage 10. mEERL-hEGFR cells were modified from a previous report (Mermod et al., Int J Cancer 2018; 142:2518-28) and cultured in DMEM / F-12 (Thermo Fisher Scientific) supplemented with 10% FBS, 1% penicillin-streptomycin (Thermo Fisher Scientific) and 1× human keratinocyte growth supplement (Thermo Fisher Scientific). MC38-luc and LL / 2-luc cells were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin.MOC2-luc cells were cultured in a mixture of IMDM medium and Ham's Nutrient Mixture F12 medium (2:1 ratio, GE Health Healthcare Life Sciences) supplemented with 5% fetal bovine serum, 1% penicillin-streptomycin, 5ng / mL insulin (MilliporeSigma), 40ng / mL hydrocortisone (MilliporeSigma), and 3.5ng / mL human recombinant EGF (MilliporeSigma). All cells were cultured in a humidified incubator at 37°C under an atmosphere of 95% air and 5% CO2 and cultured for no more than 30 passages. The identity of the cell line was tested by short tandem repeat (STR) profiling. For MC38-luc, MOC2-luc, and LL2-luc, the match scores were above 80%, indicating that the identity of the cell line was reliable. For mEERL-hEGFR, the STR profile did not match any known cell line. Mycoplasma testing was performed by PCR for MC38-luc, MOC2-luc, and LL2-luc, and by MycoAlert PLUS Mycoplasma Detection Kit for mEERL-hEGFR, and all cell lines tested negative.

[0193] reagent IRDye700DX NHS ester (IR700), a water-soluble silicon phthalocyanine derivative, was obtained from LI-COR Bioscience (Lincoln, NE). Panitumumab, a fully humanized IgG2 mAb against hEGFR, was obtained from Amgen (Thousand Oaks, CA). Anti-mouse / human CD44 (clone IM7, RRID:AB_110649), anti-mouse CTLA-4 (cytotoxic T-lymphocyte antigen-4) (clone 9D9, RRID:AB_10949609) were obtained from Bio X Cell (Lebanon, NH). All other chemicals were of reagent grade.

[0194] Synthesis of panitumumab, anti-CD44, and anti-CTLA-4 conjugated to IR700 Conjugation of IR700 with monoclonal antibodies (mAbs) was performed according to previous reports. Briefly, 1 mg of either mAb was incubated with a 5-fold molar excess of IR700 NIH ester (10 mmol / L in DMSO), 0.1 mol / L Na2HPO4 (pH 8.5) at room temperature for 1 h. The mixture was purified by filtration column (Sephadex G 25 column, PD-10, GE Healthcare). The quality of APC was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4%-20% gradient polyacrylamide gel (Life Technologies). Unconjugated antibodies were used as controls. After electrophoresis at 80 V for 2.5 h, the gels were viewed on a Pearl Imager (LI-COR Biosciences) using the 700 nm fluorescence channel. The gel was then stained with colloidal blue to compare the molecular weight of the conjugates to that of the unconjugated antibodies. Panitumumab, anti-CD44 antibody, and anti-CTLA4 antibody conjugated to IR700 are abbreviated herein as pan-IR700, CD44-IR700, and CTLA4 IR700, respectively.

[0195] Animal and tumor models All procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care and Use Committee. Female C57BL / 6 mice aged 6–8 weeks (IMSR_JAX:000664) were obtained from The Jackson Laboratory. During invasive procedures, mice were anesthetized by inhalation of 3% isoflurane and / or intraperitoneal injection of 750 mg pentobarbital sodium (Nembutal Sodium Solution, Ovation Pharmaceuticals Inc.).

[0196] The lower half of the mice was shaved before NIR light irradiation and image analysis. Tumors were implanted at 1 × 10 6 The cells were constructed by subcutaneous injection. 3 Mice bearing tumors that reached a volume of 100 μg / kg were used for the experiment. Mice were monitored daily and tumor volumes (tumor volume = length × width) were calculated. 2 × 0.5) for tumor volumes of 2,000 mm 3 The tumor burden was measured twice weekly until the tumor reached a mean tumor mass of 100 mg / kg / day, at which point the mice were euthanized by inhalation of carbon dioxide gas. In the bilateral model, mice were euthanized when either tumor reached its humane endpoint. Complete response was defined as tumor disappearance for 4 weeks or longer after treatment.

[0197] In vitro NIR-PIT mEERL-hEGFR, MC38-luc, and LL / 2-luc cells (2 × 10 5 IR700 cells (cells) were seeded in 12-well plates, incubated for 24 h, and then exposed to medium containing pan-IR700 or CD44-IR700 (10 μg / mL) for 1 h at 37 °C. After washing with PBS, phenol red-free RPMI 1640 medium was added. NIR light (690 nm) was applied to the cancer cells at 150 mW / cm using an ML7710 laser system (Modulight, Tampere, Finland). 2The cells were irradiated at a power density of 1000 x g / mL. After 1 hour of NIR-PIT, the cells were harvested with trypsin, stained with propidium iodide (PI, 1 μg / mL) for 5 minutes at room temperature, and then evaluated for PI positivity on a BD FACSCalibur (BD Biosciences) using FlowJo software (FlowJo LLC) and CellQuest software (BD Biosciences). To assess cell viability, cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The cells were incubated and treated as described above. After 1 hour of NIR-PIT, the medium was removed and 500 μl of MTT reagent (SIGMA Aldrich, 0.5 mg / ml) was added to each well. After 1 hour of incubation, the supernatant was removed and 500 μL of 2-propanol was added to each well to dissolve the crystalline formazan dye. After transferring 100 μL of the supernatant to a 96-well plate, the absorbance was measured at 570 nm in a microplate reader (Synergy™ H1). For relative quantification, the absorbance values ​​in each group were normalized to those in the control group.

[0198] In vivo NIR-PIT For dual targeted NIR-PIT experiments, tumor-bearing mice were randomized into four groups and received intravenous injection of the mixed antibody / APCs followed by NIR light exposure as follows: (i) mixed 25 mg panitumumab and 25 mg anti-CTLA4 antibody (IV group), (ii) mixed 25 mg pan-IR700 and 25 mg anti-CTLA4 antibody (panitumumab NIR-PIT group), (iii) mixed 25 mg panitumumab and 25 mg CTLA4-IR700 (CTLA4 NIR-PIT group), and (iv) mixed 25 mg pan-IR700 and 25 mg CTLA4-IR700 (dual NIR-PIT group). For the CTLA4-targeted NIR-PIT monotherapy experiment, tumor-bearing mice were also randomized into three groups as follows: (i) no treatment (control group), (ii) intravenous administration of CTLA4-IR700 (25 mg) without NIR light exposure (APC IV group), and (iii) intravenous administration of CTLA4-IR700 (25 mg) followed by NIR light exposure (NIR-PIT group). The mixed antibody / APC was injected 6 days after inoculation of cancer cells into C57BL / 6 mice. 24 hours after administration, the tumor was irradiated with NIR light (690 nm, 150 mW / cm2) at 50 J / cm2 in all groups. The surface of the mice other than the tumor was covered with aluminum foil. Mice that had eliminated tumors in the double NIR-PIT group were treated with hEGFRmEERL (1 × 10 6 In the bilateral model, NIR light (690 nm, 150 mW / cm2) was applied to the right tumor only at 50 J / cm2. 2 and the remainder of the mouse (including the tumor on the left side) was covered with aluminum foil during irradiation.

[0199] Histological analysis Tumors from mEERL-hEGFR, MC38-luc, and LLC tumors were harvested, fixed in formalin, embedded in paraffin, and sectioned at 4 mm. After standard hematoxylin and eosin (HE) staining, bright-field photomicrographs were obtained using a Mantra Quantitative Pathology Workstation (PerkinElmer).

[0200] Multiplex immunohistochemistry Multiplex immunohistochemistry (IHC) was performed using the Opal 7-Color Automation IHC Kit (AKOYA Bioscience) and the BOND RXm automated stainer (Leica Biosystems). The following antibodies were used: anti-CD8 (clone EPR20305, Abcam, 1:500 dilution), anti-CD4 (clone EPR19514, Abcam, 1:1,000 dilution), anti-FoxP3 (clone 1054C, Novus Biologicals, 1:1,000 dilution), and anti-pan-cytokeratin (rabbit poly, Bioss, 1:500 dilution). Staining was performed according to the Opal 7 color protocol provided by the manufacturer with the following modifications: (i) antigen retrieval was performed using BOND ER2 solution (Leica Biosystems) for 20 min, and (ii) the ImmPRESS HRP anti-rabbit IgG (peroxidase) Polymer Detection Kit (Vector Laboratories) was used instead of the anti-mouse / human secondary antibody provided in the kit. Stained slides were mounted with VECTASHIELD Hardset Antifade Mounting Medium (Vector Laboratories) and then imaged using a Mantra Quantitative Pathology Workstation (PerkinElmer). Images were analyzed with inForm software (AKOYA Biosystems). The InForm software was trained to automatically detect tissue and cell phenotypes according to the following criteria: areas with pan-cytokeratin expression = tumor, other areas = stroma, CD4+FoxP3+ cells = Treg, CD4+FoxP3- = CD4 T cells, or CD8+ = CD8+ T cells, respectively. Cell counts for each phenotype were exported and presented as counts per megapixel area. Three tumor samples were tested for each group. Five photographs were taken for each specimen, and cell counts and tissue areas were combined for all five photographs.

[0201] Flow cytometry analysis To evaluate CTLA4 expression, mEERL-hEGFR tumor-bearing mice were cultured in cultured mice with established tumors with a volume of approximately 150 mm 3 Mice were euthanized when they reached a 5% CO2 concentration. To confirm the disappearance of cells expressing CTLA4 after NIR-PIT, tumors or spleens of mEERL-hEGFR tumor-bearing mice were harvested 3 hours after NIR light exposure. Tumor-draining lymph nodes and spleens were also analyzed to assess for systemic effects. To assess immune responses in regional lymph nodes, ipsilateral inguinal lymph nodes were harvested 2 days after NIR-PIT. Single cell suspensions derived from tumor samples were prepared using the following protocol. Whole tumors were incubated in RPMI 1640 medium (Thermo Fisher Scientific) containing collagenase type IV (1 mg / mL, Thermo Fisher Scientific) and DNase I (20 mg / mL, Millipore Sigma) at 37°C for 30 min, then gently cut with scissors and crushed with the back of a 3 mL syringe plunger. Tissues were passed through a 70 μm cell strainer (Corning, Corning, NY, USA). Splenocytes were also analyzed to determine systemic effects. A total of 3.0 × 10 6 Stained cells: 5.0 x 10 5Data for cells were collected for each tumor. Cells were immunolabeled using BioLegend [anti-CD3e (145-2C11, RRID:AB_312660), anti-CD8α (53-6.7, RRID:AB_2888883), anti-CD11b (M1 / 70, RRID:AB_312791), anti-CD11c (N418, RRID:AB_314173), anti-CD25 (PC61.5, RRID:AB_312847), anti-F4 / 80 (BM8, RRID:AB_893481), anti-CD45 (30-F11, RRID:AB_2563598), and anti-IA / IE (M5 / 114.15.2, RRID:AB_313328)] or Thermo Fisher Scientific. The cells were stained with antibodies from either Thermo Fisher Scientific [anti-CD4 (RM4-5, RRID:AB_464902), anti-CD69 (H1.2F3, RRID:AB_1210795), and anti-NK1.1 (136, RRID:AB_2534431)]. To distinguish live from dead cells, cells were also stained with LIVE / DEAD Fixable Dead Cell Stain (Thermo Fisher Scientific). To assess CTLA4 expression, anti-CTLA4 (9D9) or isotype control [mouse IgG2b (MPC-11, RRID:AB_1107791), Bio X Cell] was conjugated with Alexa Flour 647 NHS ester (Thermo Fisher Scientific). Conjugation was performed in the same manner as IR700 conjugation. For staining of FoxP3, cells were fixed and permeabilized with FoxP3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific), followed by incubation with anti-FoxP3 (FJK-16s, Thermo Fisher Scientific). Cell fluorescence was then analyzed with a flow cytometer (FACSCalibur or FACSLyric, RRID:SCR_000401, BD Biosciences) and FlowJo software (FlowJo LLC, RRID:SCR_008520).Dead cells were excluded from the analysis based on staining with FSC, SSC, and LIVE / DEAD Fixable Dead Cell Stain. Treg populations were defined by gating on CD4ρFoxP3ρ T cells among CD3ρ T cells.

[0202] statistical analysis Quantitative data were expressed as mean ± SEM. For in vitro experiments, one-way analysis of variance (ANOVA) followed by Tukey's test was used. For in vivo experiments, two-way ANOVA followed by Tukey's test was used for multiple comparisons (more than three groups). Cumulative probability of survival was analyzed by Kaplan-Meier survival curve analysis and results were compared by log-rank test. Statistical analysis was performed with GraphPad Prism version 8 (GraphPad Software). P values ​​less than 0.05 were considered statistically significant.

[0203] Example 2 NIR-PIT combining simultaneous cancer cell targeting and CTLA4 targeting results in synergistic treatment effects in a syngeneic mouse model This example describes the results of using a method to reduce Treg-mediated immune suppression by Treg cell killing using CTLA4-targeted NIR-PIT in combination with tumor-targeted NIR-PIT (e.g., using a tumor-specific antibody-IR700 conjugate, e.g., an anti-EGFR-IR700 conjugate). Combination NIR-PIT targeting cells expressing cytotoxic T-lymphocyte antigen 4 (CTLA4) and cancer cells was investigated in four tumor models, including newly constructed mouse oropharyngeal cancer cells expressing hEGFR (mEERL-hEGFR). Single molecule targeted therapy (NIR-PIT targeting hEGFR or CTLA4) did not inhibit tumor progression in poorly immunogenic mEERLhEGFR tumors, whereas dual (CTLA4 / hEGFR) targeted NIR-PIT significantly suppressed tumor growth and prolonged survival, resulting in a complete response rate of 38%. After dual-targeted NIR-PIT, depletion of CTLA4-expressing cells, mainly regulatory T cells (Tregs), and an increase in the CD8ρ / Treg ratio in the tumor bed were observed, indicating enhanced host antitumor immunity. Furthermore, dual-targeted NIR-PIT demonstrated antitumor immunity in distant untreated tumors of the same type. Thus, simultaneous cancer cell-targeted NIR-PIT and CTLA4-targeted NIR-PIT are a novel cancer treatment strategy, especially in poorly immunogenic tumors where NIR-PIT monotherapy is suboptimal.

[0204] Efficacy of panitumumab and CTLA-4 NIR-PIT on mEERL-hEGFR cells in vitro SDS-page gel electrophoresis was used to confirm the conjugation of IR700 dye and panitumumab or anti-CTLA4 antibody. pan-IR-700 and the unconjugated control panitumumab showed nearly identical molecular weights (Figure 1A). The conjugates showed strong fluorescence intensity, but no discernible aggregation was detected. Nearly identical results were shown for anti-CTLA4 IR-700.

[0205] Based on propidium iodide (PI) incorporation and MTT assays, cancer cell death was induced by NIR-PIT in a NIR light dose-dependent manner in mEERL-hEGFR mice exposed to pan-IR700 (Figure 1B, 1C). Neither NIR light alone nor panitumumab IR700 alone induced significant changes in cell viability. Also, no significant cell damage was detected in hEGFRmEERL cells when CTLA4-targeted NIR-PIT was performed. These data verified that hEGFR-targeted NIR-PIT induced target cell-specific cell death in mEERL-hEGFR cells, whereas CTLA4-targeted NIR-PIT had no effect on mEERL-hEGFR cells in vitro.

[0206] Panitumumab IR700 NIR-PIT rapidly destroys tumor cells in vivo Cell damage in mEERL-hEGFR tumors after each NIR-PIT treatment was evaluated by histological analysis. Tumors were harvested 1 hour after light exposure. HE staining showed swelling and vacuolization of tumor cells in tumors treated with panitumumab NIR-PIT, but no obvious changes were found in tumors treated with CTLA4-targeted NIR-PIT (Figure 1D). Thus, the pathological data indicated that panitumumab NIR-PIT caused immediate death of tumor cells, but CTLA4 NIR-PIT did not.

[0207] Fewer tumor-infiltrating T cells in mEERL-hEGFR tumors To compare the immunogenicity of different tumor models, CD8+ / -hEGFR tumors in three mouse syngeneic tumor models, LL / 2-luc, MC38-luc, and mEERL-hEGFR tumors, were assayed. + Tumor infiltrating lymphocytes (TILs) were examined by IHC. + The number of TILs was lower than that of the other two tumor models (Figure 1E). + / CD4 + FoxP3+ The ρ(Treg) ratio was determined because it is an indicator of a strong antitumor immune response. + The IL-1 / Treg ratio was also significantly lower in mEERL-hEGFR tumors than in the other two tumors. These results indicated that mEERL-hEGFR tumors were less immunogenic than the other syngeneic tumor models.

[0208] Tregs express CTLA4 within the tumor bed To validate CTLA4 expressing cells in vivo, CTLA4 expression in various cell types derived from mEERL-hEGFR tumors was analyzed by flow cytometry. CTLA4 expression was significantly increased in mEERL-hEGFR cells (hEGFR + CD45-), myeloid cells (CD3-CD11b + ), cytotoxic T cells (CD3 + CD8 + ), and helper T cells (CD3 + CD4 + FoxP3-, Fig. 2A) were negligible or minimal. + CD4 + FoxP3 + ), CTLA4 was highly expressed and the relative fluorescence intensity (RFI) of Tregs was significantly higher than that of other cell types.

[0209] Depletion of Tregs in tumors after CTLA4-targeted NIR-PIT Next, selective depletion of cells expressing CTLA4 was evaluated in tumors, tumor-draining lymph nodes, and spleen by flow cytometry after each NIR-PIT (Figure 2B). Treatment efficacy was compared among four groups; panitumumab and anti-CTLA4 antibody injection (IV group), pan-IR700 and anti-CTLA4 antibody injection (pan NIRPIT group), panitumumab and CTLA4-IR700 injection (CTLA4 NIRPIT group), and combination injection of pan-IR700 and CTLA4-IR700 (dual NIR-PIT group). NIR light was applied to all groups one day after injection. Cells expressing CTLA4 in tumors were reduced in the CTLA4 NIR-PIT and dual NIR-PIT groups compared to the IV group, but no significant changes were detected in the regional lymph nodes or spleen (Figure 2C). We next determined the type of T cells selectively depleted by NIR-PIT. Tregs were significantly reduced in the CTLA4 NIR-PIT and dual NIR-PIT groups (Figures 2D and 2E). CD4 + FoxP3- and CD8 + The T cell population remained intact. + FoxP3- / Tregs and CD8 + The ratio of CTLA4 / Treg increased (Figure 2E). No significant changes were observed in lymph nodes and spleen among the four groups. These results showed that CTLA4-targeted NIR-PIT, both as monotherapy and in combination with panitumumab NIR-PIT, primarily depleted Tregs, and this effect was limited to the treatment site.

[0210] In vivo 700nm fluorescence imaging of mEERL-hEGFR cells To evaluate the accumulation of each APC in the tumor, sequential fluorescence images at 700 nm were obtained. The fluorescence intensity of both pan-IR700 and anti-CTLA4-IR700 in mEERL-hEGFR tumors showed peak intensity within 1 day after APC injection, which gradually decreased over the following days (Figure 3A-3B). The target-to-background ratio showed a similar trend (Figure 3C). Thus, a 1-day incubation with APC was used to obtain the maximum difference between tumor and background normal tissue.

[0211] Efficacy of CTLA4-targeted NIR-PIT monotherapy in mEERL-hEGFR tumors The antitumor efficacy of monotherapy of CTLA4-targeted NIR-PIT against mEERL-hEGFR tumors was evaluated as shown in Figure 4A. EERL tumors are not highly immunogenic. Treatment efficacy was compared among three groups; non-treated group (control group), APC injection without NIR light irradiation (APC IV group), and APC injection followed by NIR light irradiation (NIR-PIT group). One day after iv injection of CTLA4 IR700, tumors showed higher 700 nm fluorescence intensity than tumors without CTLA4-IR700 (Figure 4B). 50 J / cm 2 After exposure to NIR light, the IR700 fluorescent signal of the tumor immediately decreased, but there was no change in the APC IV or NIR-PIT group compared with the control group, and no significant difference was detected (Figure 4C). In the survival curve, the NIR-PIT group did not show improved survival compared with the other groups (Figure 4D). These results indicated that there was no significant therapeutic effect of CTLA4-targeted NIT-PIT alone in the mEERL-hEGFR allograft model.

[0212] Simultaneous dual NIR-PIT targeting hEGFR and CTLA4 inhibits tumor growth better than either single molecule targeted therapy alone To evaluate the efficacy of simultaneous dual NIR-PIT on mEERL-hEGFR tumors, in vivo experiments using pan-IR700 and anti-CTLA4 IR-700 were performed. Four tumor groups were compared; panitumumab and CTLA4 antibody (IV only), pan-IR700 and CTLA4 antibody (panitumumab NIR-PIT), panitumumab and CTLA4 IR700 (CTLA4 NIR-PIT), and pan-IR700 and CTLA4 IR700 (dual NIR-PIT) (Figure 5A). All groups were exposed to only one NIR light exposure (50 J / cm) one day after drug administration. 2 ). 700 nm fluorescence in the tumor was detected in the APC-injected groups (panitumumab NIR-PIT, CTLA4 NIR-PIT, and dual NIR-PIT groups), and this signal immediately decreased after NIR light exposure (Figure 5B). The tumor volume in the dual NIR-PIT group was significantly reduced compared to any other group (p<0.001, each group vs. dual PIT group) (Figure 5C). Tumor progression in both panitumumab and CTLA4 NIR-PIT groups was not significantly reduced compared to the IV group. Furthermore, the dual NIR-PIT group achieved a significant prolongation of survival compared to the IV only group (p<0.01) and the panitumumab NIR-PIT group (P<0.05) (Figure 5D). Although survival in the dual NIR-PIT group was not significantly prolonged compared with the CTLA4 NIR-PIT group, a greater number of tumors were eliminated in the dual NIR-PIT group than in the CTLA4-NIR-PIT monotherapy (38% vs. 25%).

[0213] To evaluate the combinatorial effects of CTLA4-targeted NIR-PIT and other cancer cell-targeted NIR-PIT, the efficacy of dual NIR-PIT was investigated in LL / 2-luc, MC38-luc, and MOC2-luc tumors utilizing CD44 and CTLA4 antibodies (Figures 6A-6F). For CTLA4 / CD44 dual targeting NIR-PIT experiments, tumor-bearing mice were randomized into four groups and intravenously injected with mixed antibodies / APCs followed by NIR light exposure as follows: i) mixed 25 μg anti-CD44 and 25 μg anti-CTLA4 antibodies (IV group), ii) mixed 25 μg CD44-IR700 and 25 μg anti-CTLA4 antibodies (CD44 NIR-PIT group), iii) mixed 25 μg anti-CD44 and 25 μg CTLA4-IR700 (CTLA4 NIR-PIT group), and iv) mixed 25 μg CD44-IR700 and 25 μg CTLA4-IR700 (dual NIR-PIT group). Mixed antibodies / APCs were injected 6 days after inoculation of cancer cells into C57BL / 6 mice. 24 hours after administration, the mice were exposed to NIR light (690 nm, 150 mW / cm 2 ) at 50 J / cm in all groups. 2The tumor was treated with 100 mL of D-luciferin at 15 mg / mL for MC38-luc and MOC2-luc. All areas outside the tumor were covered with aluminum foil. To obtain bioluminescence images in MC38-luc, MOC2-luc, and LL / 2-luc tumor-bearing mice, D-luciferin (200 mL at 15 mg / mL for MC38-luc and MOC2-luc, 200 mL at 3 mg / mL for LL / 2-luc, Gold Biotechnology, St. Louis, MO, USA) was injected intraperitoneally into the mice. Luciferase activity was analyzed with a BLI system (Photon Imager, Biospace Lab, Nesles la Vallee, France) using relative light units. ROIs were placed throughout the tumor. The counts per minute of relative light units were calculated using M3 Vision Software (Biospace Lab) and converted to percentage based on those before NIR-PIT using the following formula: [(relative light units after treatment) / (relative light units before treatment)×100(%)]. The CTLA4 / CD44 dual NIR-PIT group significantly inhibited tumor progression and extended survival in LL / 2-luc and MC38-luc tumors compared with group IV in all tumor models. CTLA4 / CD44 dual-targeted NIR-PIT eradicated 44% and 40% of established LL / 2-luc and MC38-luc tumors, respectively, but no additional effect was observed between CTLA4-targeted NIR-PIT monotherapy and CTLA4 / CD44 dual-targeted NIR-PIT in the tumor models tested.

[0214] Dual-targeted NIR-PIT activates antitumor host immunity To evaluate the activation of dendritic cells (DCs) and CD8ρ T cells after each NIR-PIT, ipsilateral inguinal lymph nodes were harvested 2 days after NIR-PIT and analyzed by flow cytometry. The mean fluorescence intensity (MFI) of CD40 and CD80 in DCs was significantly higher in the double NIR-PIT group compared to the non-treated group (control group, Figures 7A and 7B). In CD86, the MFI in the double group was higher than that of any other group (Figure 7C). Furthermore, the CD8 in lymph nodes was significantly higher than that in the control group (Figure 7D). +CD25 in T cells + The percentage of cells was significantly higher in all groups treated with NIR-PIT compared with group IV (Figure 7H). These results indicate that DC maturation and activation of cytotoxic T cell responses against cancer cells were enhanced by activated DCs after dual-targeted NIR-PIT.

[0215] Dual-targeted NIR-PIT detects CD8 in tumor tissue + Leads to accumulation of T cells To assess lymphocyte accumulation in the TME after each treatment, TILs were analyzed by multiplex IHC (Figures 7D and 7E). + , CD4 + FoxP3 - , and CD4 + FoxP3 + T cells were counted in each specimen. + T cells had a significantly higher density in the double NIR-PIT group compared to the control group (Figure 7F), while CD8 + The / Treg ratio was significantly higher than all other groups (Figure 7G). These results indicated that dual-targeted NIR-PIT reversed the immunosuppressive TME and led to T cell activation, which potently suppressed tumor progression compared with therapies targeting any single molecule.

[0216] Dual-targeted NIR-PIT results in immune memory To test immunological memory, mice whose treated mEERL-hEGFR tumors disappeared after double NIR-PIT were re-inoculated with mEERL-hEGFR cells approximately 15 weeks after the first NIR PIT on the contralateral dorsal side (Figure 8A). All mice whose previously inoculated tumors had been eliminated by double NIR-PIT completely rejected the newly implanted mEERL-hEGFR cells, whereas no rejection was observed in control mice (Figure 8B). In addition, all mice whose previously inoculated tumors had been eliminated by double NIR-PIT survived, whereas all control mice died within approximately 50 days (Figure 8C). This indicates the development of immunological memory after double targeted NIR-PIT.

[0217] Dual-targeted NIR-PIT demonstrates abscopal efficacy in a bilateral tumor model To evaluate the development of systemic antitumor immunity, a bilateral mEERL-hEGFR tumor model was constructed and treatment efficacy was compared between the IV group and the double NIR-PIT group. The treatment and imaging regimen is shown in Figure 9A. The contralateral tumor was shielded from NIR light exposure (Figure 9B). After exposure of the ipsilateral tumor to 50 J / cm2 of NIR light, the 700 nm fluorescence signal of the NIR-illuminated [NIR light (+)] tumor in the double NIR-PIT group decreased, while the 700 nm fluorescence of the contralateral tumor [NIR light (-)] did not change (Figure 9C). Tumor growth was significantly suppressed in the double NIR-PIT group compared with the IV group for not only NIR light (+) tumors but also NIR light (-) tumors [P<0.0001 (bilateral) vs. IV group, Figure 9D]. No significant difference was observed between NIR light (+) and NIR light (-) tumors in each group. Also, the survival of the dual NIR-PIT group was significantly prolonged compared to the IV group (Figure 9E). Furthermore, complete remission of bilateral tumors using unilateral dual targeted NIR-PIT was achieved in 1 out of 10 mice.

[0218] Consideration Simultaneous CTLA4 / hEGFR dual-targeted NIR-PIT showed stronger tumor responses than either treatment alone, even at the same antibody dose. The CD8+ / Treg ratios at 3 hours and 7 days after dual-targeted NIR-PIT were significantly increased compared to other treatments. Furthermore, an abscopal effect was observed in a model with bilateral tumors after dual-targeted NIR-PIT. These results indicate that dual-targeted NIR-PIT can alter the immunosuppressive TME, resulting in a strong synergistic effect on tumor growth inhibition.

[0219] Administration of unconjugated CTLA4 and hEGFR antibody monotherapy did not show any therapeutic effect (Figures 4-6). Indeed, since there was no change in Treg numbers in the spleen (Figures 2C and D), it is unlikely that anti-CTLA4 antibodies reduced Treg numbers, as less than half the therapeutic antibody dose was used (Nagaya et al., Cancer Immunol Res 2019;7:401-13, Maruoka et al., Cancer Immunol Res 2020;8:345-55, Okada et al., Bioconjug Chem 2019;30:2624-33). However, a considerable therapeutic effect was observed with dual-targeted NIR-PIT using a lower dose of anti-CTLA4 antibody. Severe autoimmune adverse effects associated with cancer immunotherapy, including ICIs, have been frequently reported in humans with the anti-CTLA4 antibody (ipilimumab) (Hodi et al., N Engl J Med 2010;363:711-23; Weber et al., Lancet Oncol 2015;16:375-84; Weber et al., N Engl J Med 2017;377:1824-35; Robert et al., N Engl J Med 2011;364:2517-26; Quirk et al., Transl Res 2015;166:412-24). Ipilimumab induces immune-related adverse effects (irAEs) more frequently than other ICIs, in particular gastrointestinal symptoms in approximately 40% of patients receiving the 3 mg / kg dose for melanoma therapy (Dougan et al., Support Care Cancer 2020;28: 6129-43, Dougan M. Front Immunol 2017;8:1547, Eggermont et al., N Engl J Med 2016;375:1845-55, Puzanov et al., J Immunother Cancer 2017;5:95.).The irAEs of ipilimumab or pembrolizumab, which block PD-L1, are reportedly dose-dependent (Dougan et al., Support Care Cancer 2020;28: 6129-43; Puzanov et al., J Immunother Cancer 2017;5:95; Maughan et al., Front Oncol 2017;7:56). Therefore, administration of low doses of ICIs may minimize irAEs. Therefore, dual-targeting NIR-PIT has high benefits not only because of its superior therapeutic effect compared with ICIs, but also because the adverse effects can be reduced by antibody dose.

[0220] CTLA4 is expressed in FoxP3ρ Tregs and various other cells. The endocytosis of CTLA4 is extremely rapid, making it difficult to capture the expression of CTLA4 on the cell surface. Regardless, CTLA4-targeted NIR-PIT could still deplete Tregs in tumors. In addition, myeloid-derived suppressor cells and several types of cancer cells also expressed CTLA4 on the cell membrane (Yu et al., Oncoimmunology 2016;5:e1151594, Pico de Coana et al., Cancer Immunol Immunother 2014;63:977-83, Chaudhary et al., Vaccines 2016;4:28). These cells could also be depleted by CTLA4-targeted NIR-PIT, which could further enhance anti-tumor host immunity. In addition, CTLA4-targeted NIR-PIT has been reported to suppress intratumoral blood perfusion (Okada et al., Adv Ther 2021;n / a:2000269, Kurebayashi et al., Cancer Res 2021;81:3092-104). Reduction in intratumoral blood flow was observed after dual-targeted NIR-PIT, as well as after monotherapy with CTLA4-targeted NIR-PIT (Figures 10A, 10B). This early vascular effect may also contribute to the overall therapeutic efficacy.

[0221] CD25-targeted NIR-PIT also induced selective Treg depletion, but because CD25 is the IL2 receptor, the presence of remaining APCs after light exposure may block IL2 / IL2R binding in activated effector cells, resulting in their inhibition. CTLA4-targeted NIR-PIT may be better than CD25-targeted NIR-PIT because CTLA4 antibodies do not block IL2 binding, and CTLA4-IR700 may block the CTLA4 immune checkpoint pathway, resulting in enhanced host anti-tumor immunity (Okada et al., Adv Ther 2021;n / a:2000269).

[0222] Dual-targeting NIR-PIT utilizing CTLA4 in combination with CD44 as a cancer targeting agent was also evaluated, but no synergistic effects were observed in the CD44-expressing LL / 2-luc, MC38-luc, and MOC2-luc tumor models (Figures 6A-6F). There are several possible reasons why the combination of CTLA4 / CD44 in these models was less effective than CTLA4 / hEGFR in the mEERL-hEGFR model. First, MC38-luc and LL / 2-luc tumors are highly immunogenic tumors. Host tumor immunity was enhanced by CTLA4-targeting NIR-PIT alone, possibly because the tumors were already highly infiltrated with lymphocytes prior to any treatment (Figure 1E). In contrast, mEERL-hEGFR tumors appeared to be poorly immunogenic tumors, as indicated by the low infiltration of T cells (Figure 1E) and the lack of therapeutic effect of CTLA4-targeting NIR-PIT by itself. CTLA4 / hEGFR dual-targeted NIR-PIT inhibited tumor progression and enhanced DC maturation and activation of T cell responses in the mEERL-hEGFR allograft model (Figures 5 and 7). Another reason may be off-target cell killing. MOC2-luc tumors were also poorly immunogenic, but the synergistic effect of CTLA4 / CD44 dual-targeted NIR-PIT was not seen in this model. CD44 is expressed not only on cancer cells but also on some immune cells, such as effector T cells and memory T cells (Govindaraju et al., Matrix Biol 2019;75-76:314-30). The CD44-positive subset of CD8+ T cells was depleted by CD44-targeted NIR-PIT (Maruoka et al. Vaccines 2020;8:528). Thus, CD44-targeted NIR-PIT may also damage effector T cells, resulting in attenuated antitumor effects, whereas hEGFR-targeted NIR-PIT kills only hEGFR-expressing cancer cells without damaging host immune cells that do not express hEGFR.Thus, CTLA4 / hEGFR dual-targeted NIR-PIT in the mEERL-hEGFR tumor model successfully induced antitumor immune activation by exclusively targeting cancer cells and amplified the effect by eliminating cells expressing CTLA4, which is generally immunosuppressive.

[0223] In conclusion, the data show that CTLA4 / hEGFR dual targeting NIR-PIT successfully depleted CTLA4-expressing cells in cancer cells and intratumoral tissues, and had a significant effect on cell growth beyond either agent alone. This combination NIR-PIT approach can enhance the therapeutic effect, especially in cases where the efficacy of NIR-PIT monotherapy is insufficient.

[0224] Example 3 PD-L1-IR700+IL-15 preconditioning provides synergistic antitumor effects in mice The materials and methods described in Example 1 were used to show the effect of PD-L1 antibody-IR700 conjugate in PD-L1 targeted NIR-PIT, except that anti-mouse CD274 antibody (10F.9G2 monoclonal antibody) obtained from Bio X Cell (West Lebanon, NH, USA) was used instead of anti-mouse CTLA4 antibody (9D9). The method further included preconditioning with IL15 prior to administration of the PD-L1 antibody-IR700 conjugate.

[0225] IR700 conjugated with anti-PD-L1 (CD274) To synthesize APC, IR700 was conjugated to anti-PD-L1 antibody (clone 10F.9G2) and the conjugates (anti-PD-L1-IR700 or CD274Ab-IR700) were analyzed by SDS-PAGE. Anti-PD-L1 and anti-PD-L1-IR700 had approximately the same molecular weight, but only anti-PD-L1-IR700 had fluorescence at 700 nm (Figure 11A). APC was also evaluated by SEC. The majority of the protein detected as the main peak with an absorbance of 280 nm (eluted at 11.5 min) had an absorbance and fluorescence of 689 nm (excitation 689 nm, emission 700 nm) (Figure 11B). These results verified the successful conjugation of APC.

[0226] PD-L1 expression in cancer and immune cells Flow cytometric analysis of PD-L1 expression in cancer cell lines was performed. Representative histograms of MC38-luc (top) and LL2-luc (bottom) are shown in Figure 11C. Flow cytometric analysis of PD-L1 expression in cancer cell lines MC38-luc and LL2-luc was obtained from cell cultures with or without interferon gamma and in vivo tumors. As shown in Figure 11A, interferon gamma conditionally enhanced the expression of PD-L1. Microscopic images before and after PD-L1-targeting NIR-PIT on in vitro cultured cancer cells showed that the cells were changed in shape and crushed. In vitro PD-L1-targeting NIR-PIT showed better cell killing against MC38-luc cells treated with interferon gamma (Figure 11E).

[0227] As shown in Figure 12, PD-L1 expression is increased in cancer cells following exposure to IFN-gamma, and as shown in Figure 13, PD-L1 is expressed in tumor-infiltrating immune cells.

[0228] Treating Cancer with Anti-PD-L1-IR700 and IL-15 Anti-PD-L1-IR700 was administered to mice bearing MC38-luc tumors. Tumors were administered at 5×10 5 Tumors were established by sc administration of 100 MC38 cells. Eight days later, anti-PD-L1-IR700 (or control IgG-IR700) was administered iv and tumors were either subjected to NIR light (50 J / cm2) (PIT) or not. As shown in Figure 14A (left panel), anti-PD-L1-IR700 with NIR light significantly reduced tumor volume and increased survival to a greater extent than anti-PD-L1-IR700 with NIR light alone. Mice were subsequently administered a booster dose of MC38 cells and the effect on tumor volume and survival was measured. As shown in Figure 14B (right panel), mice previously treated with anti-PD-L1-IR700 did not have tumor recurrence and survived, in contrast to naive mice.

[0229] The effect of IL15 preconditioning in combination with anti-PD-L1-IR700 PIT is shown in Figure 15. Tumors were inoculated into mice at 5×10 5 Tumors were constructed by sc administration of LL2 cells. Four days later, IL15 (1ug ip) was administered, followed one day later by anti-PD-L1-IR700 (or vehicle control), followed another day later by either or not NIR light (50J / cm2). As shown in Figure 15 (left panel), anti-PD-L1-IR700 in combination with NIR light significantly reduced tumor volume and increased survival to a greater extent than anti-PD-L1-IR700 without NIR light. As shown in Figure 15 (right panel), the addition of IL-15 preconditioning substantially reduced tumor volume and increased survival to a greater extent than anti-PD-L1-IR700 with NIR.

[0230] Example 4 material and method This example describes the materials and methods used to obtain the results in Examples 5-11.

[0231] reagent IRDye700DX NHS ester, a water-soluble silica-phthalocyanine derivative, was obtained from LI-COR Biosciences (Lincoln, NE). Panitumumab, a fully humanized IgG2 monoclonal antibody (mAb) against EGFR, was obtained from Amgen (Thousand Oaks, CA). Anti-mouse CTLA4 antibody (clone; 9D9) was obtained from Bio X Cell (West Lebanon, NH). Sodium L-ascorbate (L-NaAA) and L-cysteine ​​were obtained from MilliporeSigma (Burlington, MA). Sodium azide (NaN3) was obtained from MP biomedicals, LLC (Solon, OH).

[0232] synthesis The compounds were prepared by slight modifications of a previously reported method (Figure 16) (Sato et al., ACS central science 2018, 4:1559-1569). All chemicals were of the best grade available and were supplied by FUJIFILM Wako Pure Chemical Corporation, Tokyo Chemical Industries Co, Ltd., KANTO CHEMICAL Co., INC., and Sigma-Aldrich Japan KK, and were used without further purification. 1H NMR spectra were recorded at 400 MHz on a JNM-ECX400P or JMN-ECS400 (JEOL Ltd, Tokyo, Japan) instrument and are reported relative to the deuterated solvent signal.

[0233] Silicon Phthalocyanine Dihydroxide (Pc 1) Silicon tetrachloride (1.93 g, 11.4 mmol) and 1,3-diiminoisoindoline (1.10 g, 7.59 mmol) were dissolved in quinoline (13 mL) and the mixture was refluxed under argon atmosphere for 2 h. After the mixture was cooled to room temperature (RT), 5 M aqueous NaOH solution (10 mL) was added and the mixture was refluxed for 2 h. The product was collected by filtration, washed with MeOH and dried under vacuum (970 mg, 1.69 mmol). The product was used in the next reaction without further purification.

[0234] Bis(3-aminopropyldimethylsilyloxide)silicon phthalocyanine (Pc2) Pc 1 (500 mg, 0.87 mmol) and 3-aminopropyldimethylethoxysilane (1.12 g, 6.96 mmol) were dissolved in pyridine (250 mL), the mixture was refluxed under argon overnight and concentrated by rotary evaporation, the residue was diluted, filtered, washed with HO-ethanol solution (2:1) and dried under vacuum (650 mg, 0.807 mmol, 82% yield (2 steps)). 1H NMR (400 MHz, CD3OD): δ -2.86 (s, 12 H), -2.33~-2.27 (m, 4 H), -1.28~-1.19 (m, 4 H), 1.18 (t, J = 7.2 Hz, 4 H), 8.35 (dd, J = 5.7, 2.8 Hz, 8 H), 9.66 (dd, J = 5.7, 2.8 Hz, 8 H).

[0235] Bis{3-[tris(3-sulfopropyl)]ammoniopropyldimethylsilyloxide}silicon phthalocyanine (Pc3) Pc2 (300 mg, 0.373 mmol), 1,3-propane sultone (2.27 g, 18.6 mmol), and N,N-diisopropylethylamine (DIEA, 4.82 g, 37.4 mmol) were dissolved in EtOH (15 mL) and the mixture was stirred at 50 °C for 120 h under an argon atmosphere. The product was purified by high performance liquid chromatography (HPLC) system (Shimadzu Co., Kyoto, Japan) using a reversed-phase column Inertsil ODS-3 (10 mm × 250 mm) (GL Sciences Inc., Tokyo, Japan) with eluent A (HO, 0.1 M tri-ethylammonium acetate (TEAA)) and eluent B (99% MeCN, 1% HO) (A / B = 80 / 20 to 50 / 50 in 15 min, 50 / 50 to 0 / 100 in 5 min). The product was desalted with a Sep-Pak C18 cartridge (Waters Corporation, Milford, MA) and cation exchange resin to give Pc 3 (216 mg, 0.132 mmol, 36% yield, as sodium salt). 1H NMR (400 MHz, CD3OD): δ -2.79 (s, 12 H), -2.12~-2.18 (m, 4 H), -0.88~-0.98 (m, 4 H), 1.75-1.66 (m, 12H), 2.05-1.98 (m, 4 H), 2.80-2.73 (m, 24 H) 8.52 (dd, J = 5.7, 3.0 Hz, 8 H), 9.79 (dd, J = 5.7, 3.0 Hz, 8 H).

[0236] Synthesis of bovine albumin, panitumumab, anti-CD44 antibody and anti-CTLA4 antibody conjugated to IR700 Bovine albumin (1 mg, 15.0 nmol), panitumumab (1 mg, 6.8 nmol), anti-CD44 and anti-CTLA4 antibodies (1 mg, 6.7 nmol) were added in a volume of 0.1 mol L -1The cells were incubated with a 5-fold molar excess of IR700 NHS ester in 10 mmol / L Na2HPO4 (pH 8.5) at room temperature for 1 h (albumin; 146.9 μg, 75.2 nmol; panitumumab; 66.9 μg, 34.2 nmol; anti-CD44 and anti-CTLA4 antibodies; 65.1 μg, 33.3 nmol, 10 mmol / L in DMSO). -1 ). The mixture was purified on a Sephadex G25 column (PD-10, GE Healthcare, Piscataway, NJ, USA). The concentration of IR700 was determined by absorbance at 689 nm using UV-Vis (8453 Value System, Agilent Technologies, Santa Clara, CA, USA). Protein concentration was confirmed with a Coomassie Plus protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) by measuring absorbance at 595 nm. SDS-PAGE was performed as a quality control for each conjugate. Panitumumab-IR700 is abbreviated as pan-IR700, albumin-IR700 as alb-IR700, anti-CD44-IR700 as CD44-IR700, and anti-CTLA4-IR700 as CTLA4-IR700.

[0237] 700 nm fluorescence evaluation of Pc3 or mAb-IR700 after NIR light exposure in vitro 25μM Pc3 or 3μM pan-IR700 solutions were prepared with 0, 0.1, 0.5, 1.0, and 10mM L-NaAA or L-cysteine ​​in PBS buffer (pH 7.5). For analysis mixed with NaN3, 3μM mAb-IR700 solutions were prepared with 1mM L-cysteine ​​and / or 1mM L-NaAA diluted with PBS and added with different concentrations of NaN3 (0, 0.01, 0.1, 1, 10, or 100mM). The pan-IR700 was exposed to NIR light (690nm, 150mW cm-2, 50J cm-2) at each condition (0, 5, 10, 25, 50, 100, 150, and 200J) using a ML7710 laser system (Modulight, Tampere, Finland). Before and after each irradiation, the 700 nm fluorescence intensity was acquired by a fluorescence imager (Pearl Imager, LI-COR Bioscience, Lincoln, NE, USA). Pearl Cam Software (LI-COR Biosciences) was used to analyze the fluorescence. The same region of interest (ROI) was placed in the solution of each tube, and then the average 700 nm fluorescence intensity was measured. The appearance of the tubes before and after NIR light irradiation was imaged. All experiments were performed at room temperature.

[0238] Electron spin resonance (ESR) spectroscopy Thirty microliters of 0.5 mM IR700 in 100 mM phosphate buffer (pH 7.0) was filled into a gas-permeable polymethylpentene (TPX™) tube (0.76 mm ID x 1.0 mm OD x 60 mm L, Toho Kasei Sangyo Co., Ltd., Tokyo, Japan) with or without 10 mM L-cysteine ​​or 10 mM L-NaAA, which was then sealed at both ends and the TPX tube was placed into a natural quartz ESR tube (5 mm OD x 250 mm L, S-5-EPR-250S, Norell Inc., NC). The ESR sample tube was placed in the cavity of a cylindrical TE011 mode (JEOL), and argon gas was flowed into the ESR sample tube from a long capillary glass tube to remove oxygen from the sample. After purging with argon gas for 5 min, the ESR sample tube was sealed and X-band CW-ESR measurements were performed at ambient temperature using a JEOL-RE1X spectrometer (JEOL, Tokyo, Japan). At the same time that NIR light irradiation was started, ESR scanning (scan time: 8 min / 10 mT) was started from the low magnetic field side. The ESR parameters were as follows: incident microwave power 10 mW, microwave frequency 9.150 GHz, modulation frequency 100 kHz, magnetic field modulation amplitude 0.1 mT, time constant 0.3 s, scan range 325.17 ± 5 mT, and receiver gain 5.0 × 102. The linewidth, intensity, and g value of the ESR signal were estimated using the signal of the co-mounted Mn2+ marker (ES-DM1, JEOL) in the cavity and Win-Rad software (Radical Research, Tokyo, Japan).

[0239] HPLC analysis of the formation of cystine from cysteine. A solution of 0.25 mM IR700 in 10 mM sodium phosphate buffer (pH 7.0) containing 1 mM L-cysteine ​​was prepared in a sealed cuvette, and argon was bubbled through the rubber septum cap of the cuvette for 20 min. The deoxygenated solution was then irradiated with a laser (MLL-III-690, Changchun New Industries Optoelectronics Technology Co.Ltd, Changchun, China) (690 nm, 1 W cm-2) for 1 h. The solution without argon bubbling was also irradiated as described above. The irradiated solution was filtered through a Millex-LH filter (pore size: 0.45 μm, hydrophilic, PTFE, Merck Millipore, US). The solution was diluted with 1 mL of eluent A (HO, 0.1% trifluoroacetic acid) and eluent B (99% MeCN, 1% HO) (A / B=99 / 1). -1 The solutions were analyzed by an HPLC system (Shimadzu Co., Kyoto, Japan) using a reversed-phase column Inertsil ODS-3 (4.6 mm × 250 mm) (GL Sciences Inc., Tokyo, Japan) at a flow rate of 100 Hz. The detection wavelength was 202 nm. The solutions were also co-injected with standard compounds to identify the peaks of L-cysteine ​​and L-cystine.

[0240] cell culture The luciferase-expressing mouse cancer cell lines A431 GFP-luc (EGFR-expressing epidermal cancer), MDAMB468 GFP-luc (EGFR-expressing breast cancer), MC38-luc (colon cancer), LL / 2-luc (lung cancer), and MOC2-luc (oral cancer) were used. A431 GFP-luc, MDAMB468 GFP-luc, MC38-luc, and LL / 2-luc cells were cultured in 10% fetal bovine serum, 100 I.U.mL -1 MOC2-luc cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 100 I.U.mL penicillin / streptomycin (Thermo Fisher Scientific).-1 of penicillin / streptomycin, 5 ng mL -1 of insulin (MilliporeSigma), 40 ng mL -1 of hydrocortisone (MilliporeSigma), and 3.5 ng mL -1 The cells were cultured in a mixture of IMDM medium and Ham's Nutrient Mixture F12 medium (2:1 ratio, GE Health Healthcare Life Sciences) supplemented with 100 ml of human recombinant EGF (MilliporeSigma). All cells were cultured in a humidified incubator at 37°C in an atmosphere of 95% air and 5% CO2.

[0241] In vitro NIR-PIT using reducing agents A431 GFP-luc or MDAMB468 GFP-luc cells (4 × 10 5 10 μg mL -1 The cells were exposed to medium containing L-NaAA, L-cysteine, and / or NaN3 at various concentrations for 1 h at 37°C. After aspirating the medium with APCs, phenol red-free medium containing NIR light (690 nm, 150 mW cm) was added to each well. -2 , 1 or 2 J cm for A431 GFP-luc -2 , 20 J cm for MDAMB468 GFP-luc -2 ) was irradiated onto the cancer cells. For flow cytometry analysis, the cells were harvested with trypsin after 1 h of NIR light exposure. The cells were then incubated with propidium iodide (PI, 1 μg mL ) for 5 min at room temperature. -1 The cells were stained with Fluorescence Immunosorbent Assay Kit (FACSCalibur, BD Biosciences, San Jose, Calif., USA). The fluorescence of the cells was then analyzed with a flow cytometer (FACSCalibur, BD Biosciences, San Jose, Calif., USA) and FlowJo software (BD Biosciences).

[0242] Animal and tumor models All in vivo procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council. Six- to eight-week-old female C57BL / 6 mice and homozygous athymic nude mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and Charles River Laboratories (Wilmington, MA, USA), respectively. A431 GFP-luc (1 × 10 6 pcs), MC38-luc(1×10 6 pieces), LL / 2-luc(0.5×10 6 ), or MOC2-luc (1 × 10 6 1) cells were inoculated on the dorsal right side. Hair covering the tumor site was removed before NIR-PIT and imaging of C57BL / 6 mice. Body weight was measured from the day before to 4–6 days after NIR light exposure. Mice were euthanized with CO2 to terminate all experiments, including BLI.

[0243] In vivo NIR-PIT C57BL / 6 tumor-bearing mice were randomized into three groups as follows: i) no treatment (control), ii) intravenous administration of APC without L-NaAA followed by NIR light exposure (NIR-PIT), and iii) NIR-PIT with L-NaAA. APC was injected 6 days after inoculation of cancer cells into C57BL / 6 mice. Similarly, athymic tumor-bearing mice were randomized into two groups as follows: i) NIR-PIT without L-NaAA, ii) NIR-PIT with L-NaAA. The dose of APC was 100 μg for CD44-IR700 and 50 μg for CTLA4-IR700, respectively. NIR light (690 nm, 150 mW cm-2, 50 J cm-2) was exposed the next day. L-NaAA (80 mg mouse-1) or PBS was injected intraperitoneally 15 min before NIR light exposure. During NIR light exposure, normal tissue adjacent to the tumor was covered with aluminum foil to ensure that NIR light exposure was limited to the tumor site. IR700 dorsal fluorescence images were obtained in the 700 nm fluorescence channel of a Pearl Imager (LI-COR Biosciences). Images were acquired before and after NIR-PIT. ROIs were placed on the tumor. Dorsal edema formation in treated mice 1 day after NIR light irradiation was imaged by a camera. Acute treatment efficacy was evaluated with BLI analysis, where D-luciferin (3 mg mouse-1, Gold Biotechnology) was injected intraperitoneally into mice. Luciferase activity was analyzed with a BLI system (Biospace Lab) using relative light units. ROIs were placed throughout the tumor. Relative light unit counts per minute were calculated using M3 Vision Software (Biospace Lab) and converted to a percentage based on that before NIR-PIT using the following formula: [(relative light units after treatment) / (relative light units before treatment)×100(%)]. Bioluminescence images were recorded continuously until the onset of depilation-induced skin pigmentation prevented accurate measurements.

[0244] Magnetic Resonance Imaging (MRI) Under pentobarbital anesthesia, MRI was performed on a 3-T scanner using an in-house 10-inch circular mouse receiver coil array (Elition 3T, Philips Medical Systems, Best, Netherlands) 1 day after NIR light exposure. Scout images were obtained to precisely locate the tumor. All mice underwent T2-weighted fat-suppressed imaging (T2WI fat-suppressed) and short TI inversion recovery imaging (STIR). All images were acquired in the coronal plane, and T2WI fat-suppressed images were also acquired in the axial plane. All images were analyzed using Image J software to reconstruct 3D images. The high signal intensity areas derived from each image in T2WI fat-suppressed and STIR were calculated by Image J.

[0245] Flow cytometry analysis To confirm the suppression of cells expressing CTLA4, MC38-luc tumors were harvested 3 hours after NIR light exposure. Single cell suspensions derived from tumor samples were prepared using the following protocol. Whole tumors were incubated with collagenase type IV (1 mg mL -1 , Thermo Fisher Scientific) and DNase I (20 μg mL -1 The tissue was incubated at 37°C for 30 min in RPMI 1640 medium (Thermo Fisher Scientific) containing 100 mM NaCl, Millipore Sigma, Burlington, MA), then gently dissected with scissors and crushed with the back of a 3 mL syringe plunger. The tissue was passed through a 70 μm cell strainer (Corning, Corning, NY). A total of 3.0 × 10 6 Stained cells: 5.0 x 10 5Data for cells were collected for each tumor. Cells were stained with anti-CTLA4 antibody (clone, UC10-4B9) obtained from Thermo Fisher Scientific. To distinguish live from dead cells, cells were also stained with LIVE / DEAD Fixable Dead Cell Stain (Thermo Fisher Scientific). Cell fluorescence was then analyzed with a flow cytometer (FACSLyric, BD Biosciences) and FlowJo software (FlowJo LLC). Dead cells were removed from the analysis based on staining with FSC, SSC, and LIVE / DEAD Fixable Dead Cell Stain.

[0246] statistical analysis Data are expressed as mean ± SEM. Statistical analysis was performed with GraphPad Prism (GraphPad Software, LaJolla, CA, USA). Sample size (n) for each experiment is stated in each figure legend. For single measurements, two-tailed unpaired t-test (two groups) or one-way analysis of variance (ANOVA) followed by Tukey's test or Dunnett's test (three or more groups) were used. For comparison of luciferase activity, biodistribution, and body weight, repeated measures two-way ANOVA followed by Tukey's test was used. p-values ​​less than 0.05 were considered significant.

[0247] Example 5 L-NaAA accelerates the release of axial ligands upon NIR irradiation, regardless of the presence of sodium azide (NaN3). Compound Pc 3, the phthalocyanine cyanine moiety of IR700, was synthesized according to FIG. 16. The light-induced ligand release reaction of Pc 3 after NIR light exposure was accelerated in a dose-dependent manner in the presence of the electron donor L-NaAA. The 700 nm fluorescence of Pc 3 decreased in a NIR light and L-NaAA dose-dependent manner (FIG. 17A, 17B). The disappearance of fluorescence was associated with the precipitation of Pc 3 in a L-NaAA dose-dependent manner because Pc 3 dramatically changed its solubility after light exposure (FIG. 17C). The fluorescence emission of IR700 conjugated with panitumumab (panitumumab-IR700) decreased in a NIR light and L-NaAA dose-dependent manner (FIG. 17D, 17E). In the presence of NaN3, L-NaAA did not inhibit the decrease in fluorescence of panitumumab-IR700 after NIR light irradiation (Figure 17F).

[0248] Electron spin resonance (ESR) spectroscopy was performed to identify the intermediate products of IR700 and L-NaAA radical. A broad signal was observed in the central field (Figure 17G(i)), which was also observed in an empty quartz ESR tube, indicating that this signal originated from the quartz ESR tube itself. Therefore, this signal could be subtracted and did not interfere with the ESR measurement. Under argon-saturated conditions (Figure 17G, left), a small but clear doublet signal with a g value of 2.0051 and a splitting width of 0.18 mT was observed by simply mixing IR700 with L-NaAA for a few minutes (Figure 17G(ii)). The ESR parameters of this signal are almost the same as those previously reported for the L-NaAA radical produced during the oxidation of L-NaAA. This observation indicates that the L-NaAA radical is produced by redox interactions between IR700 and L-NaAA without NIR light irradiation. When a sample containing 10 mM L-NaAA and 0.5 mM IR700 was irradiated with NIR light, a small signal with a broad line width of 0.87 mT and a g value of 2.0006 appeared, as indicated by an asterisk in the left spectrum of FIG. 17G(iii). This signal was not observed in the absence of an electron donor, e.g., L-NaAA, and since the intensity of the signal in air was much smaller than that under argon-saturated conditions in the presence of oxygen, an electrophilic molecule, this ESR signal was assigned to the IR700 anion radical produced by electron transfer from L-NaAA to triplet state IR700 induced by NIR light. In this procedure, the magnetic field scan and sample irradiation were started simultaneously and continued at 8 min / 10 mT. Thus, the central region where the signal due to the IR700 anion radical was detected occurred about 4 min from the start of the scan. This signal derived from the IR700 anion radical was observed for at least 16 minutes after NIR light irradiation, indicating that NIR light irradiation produces IR700 anion radicals in the presence of L-NaAA.In the left spectra of Figure 17G(iv) and (v), the effect of NaN3 on the formation of L-NaAA radical and IR700 anion radical induced by NIR light was examined. The results showed that the addition of 100 mM NaN3 attenuated the L-NaAA radical but did not affect the formation of IR700 anion radical produced by NIR light irradiation.

[0249] Similar experiments were performed in the presence of oxygen. As shown in the right spectra of Figure 17G(ii) and (iii), the reaction of IR700 with L-NaAA in air showed a stronger radical signal derived from the L-NaAA radical than that under argon-saturated conditions, and the L-NaAA radical was further enhanced by NIR light irradiation. In addition, it was also observed that NIR light irradiation produced IR700 anion radical in air, which was significantly reduced compared to argon-saturated conditions. The effect of NaN3 on L-NaAA radical and IR700 anion radical is shown in the right spectra of Figure 17G(iv) and (v). The addition of 100 mM NaN3 to the sample reduced the ESR signal intensity derived from the L-NaAA radical, but seemed to affect the formation of IR700 anion radical produced by NIR irradiation. Axial ligand cleavage was observed under L-NaAA-added conditions, which was not enhanced by NaN3 (Figure 17H).

[0250] Example 6 When L-cysteine ​​was added as an electron donor, NaN3 inhibited the ligand release of IR700. The light-induced ligand release responses of Pc3 and panitumumab-IR700 were evaluated under the condition of addition of L-cysteine, an alternative electron donor to L-NaAA. A similar phenomenon was observed, but at lower NIR light irradiation levels than when L-NaAA was used (Figures 18A-18C). Combining 10 mM or higher concentrations of NaN3 with 1 mM L-cysteine ​​reduced the quenching of panitumumab-IR700 fluorescence seen with L-cysteine ​​alone (Figure 18D). However, the addition of 1 mM L-NaAA to 1 mM L-cysteine ​​countered the effect of NaN3 (Figure 18E).

[0251] The control ESR spectrum of 0.5 mM IR700 and 10 mM L-cysteine ​​without irradiation is shown in FIG. 18F(i). When a sample of 0.5 mM IR700 and 10 mM L-cysteine ​​was dissolved in 100 mM phosphate buffer solution (pH 7.0) and irradiated with NIR light under argon-saturated conditions, a symmetric ESR spectrum was observed with a line width of 0.87 mT and a g value of 2.0006 assigned to the IR700 anion radical, as shown in FIG. 18F(ii). Furthermore, to evaluate the effect of NaN3 on the NIR light-induced IR700 anion radical, a similar experiment was performed in the presence of 100 mM NaN3 in the sample. The ESR spectrum in the presence of NaN3 (red) was overlaid on the spectrum in the absence of NaN3 (gray, the same spectrum as FIG. 18F(ii)), as shown in FIG. 18F(iii). These results showed that NaN3 reduced the signal of IR700 anion radical. When NaN3 was added, the ligand cleavage was reduced (FIG. 18G). This result is consistent with the ESR results in which the formation of the radical anion was inhibited.

[0252] To determine whether L-cystine is produced from L-cysteine, a solution of IR700 was irradiated in the presence of L-cysteine ​​and analyzed by high performance liquid chromatography (HPLC). When IR700 was irradiated under hypoxic conditions, L-cysteine ​​and L-cystine peaks were observed, but without NIR light irradiation, the L-cystine peak was absent (Figure 18H). On the other hand, when IR700 was irradiated with NIR light in the presence of oxygen, the L-cysteine ​​peak was greatly reduced, while the L-cystine peak was larger than that under hypoxic conditions. These results indicated that L-cysteine ​​acted as a reductant for IR700 in the excited state and was converted to L-cystine. In addition, L-cysteine ​​was consumed more efficiently in the presence of oxygen than in the absence of oxygen. This may be because oxygen quenches the triplet excited state of IR700 or the anion radical in the ground state, resulting in a more efficient cycle of excitation and reduction.

[0253] Example 7 L-NaAA counteracts the inhibition of NIR-PIT cytotoxicity caused by NaN3 To investigate the effect of reducing agents on the efficacy of NIR-PIT, the cytotoxic effect of NIR-PIT targeting cancer cells was quantitatively evaluated by cell viability assay. Under low or pharmacological concentrations, L-NaAA increased the cytotoxic damage of panitumumab-IR700 NIR-PIT (Pan-PIT). Under supraphysiological concentrations of L-NaAA (e.g., above 1 mM), the cytotoxic damage by Pan-PIT was slightly inhibited. In contrast, L-cysteine ​​slightly enhanced the cytotoxic damage of Pan-PIT, but only at very low concentrations such as 0.001 mM, while it reduced the cytotoxicity at concentrations of 0.1 mM or higher (Figures 19A-19B). When both L-cysteine ​​and L-NaAA were present at comparable concentrations, L-cysteine ​​tended to counter the effect of L-NaAA due to the higher redox potential of L-cysteine ​​compared to L-NaAA (FIG. 19A).

[0254] Next, the effect of each reducing agent on the cytotoxicity of Pan-PIT in the presence of NaN3 was evaluated. L-NaAA overcame the reduction in cytotoxicity caused by NaN3, whereas L-cysteine ​​could not reverse the effect of NaN3 (Fig. 19D, 19E). In another cell line, MDAMB468, L-NaAA also restored the cytotoxicity of Pan-PIT suppressed by NaN3 (Fig. 20A-20D). A similar trend was observed by bioluminescence imaging (BLI) (Fig. 21A-21D). These results indicate that the suppression of NIR-PIT cytotoxicity by NaN3 was overcome by L-NaAA but not by L-cysteine, despite its high redox potential, possibly because the added L-cysteine ​​formed a disulfide bond with the sulfhydryl group of L-cysteine, thereby reducing its ability to accelerate the light-induced ligand release reaction.

[0255] Example 8 L-NaAA accelerates the ligand release of IR700 after NIR light exposure To evaluate whether L-NaAA or L-cysteine ​​could successfully promote ligand release from IR700 in vivo, albumin-IR700 (alb-IR700) was used and fluorescence quenching was measured (Figure 22A). Conjugated alb-IR700 (50 μg) was injected into non-tumor-bearing athymic nude mice via the lateral tail vein 2 h prior to NIR light exposure. L-NaAA (80 mg), L-cysteine ​​(10.5 mg), or PBS was injected via the tail vein 2 min prior to NIR light irradiation of the mice. The abdomen was exposed to NIR light (690 nm, 150 mW cm). -2 , 20J cm -2 ) in the rat. Ventral fluorescence was measured before and after NIR light exposure using a Pearl Imager (LI-COR Biosciences). The cycle of NIR light illumination and fluorescence imaging was repeated five times. ROIs were placed in the liver and the illumination site. The mean fluorescence intensity of each ROI was calculated.

[0256] After alb-IR700 was exposed to NIR light, the 700 nm fluorescence signal decreased at the irradiation site, whereas the signal in the liver increased in a NIR light dose-dependent manner (Figures 22B, 22D). Under L-NaAA administration, the fluorescence was significantly decreased compared to the condition without L-NaAA, whereas there was no obvious change in fluorescence with L-cysteine ​​(Figures 22C, 22E). Because ligand release results in the disappearance of fluorescence, the decrease in 700 nm fluorescence in the liver means that high ligand release occurred in alb-IR700 after NIR light exposure. Thus, L-NaAA, but not L-cysteine, accelerated the ligand release of IR700 conjugated to albumin in vivo.

[0257] Example 9 L-NaAA did not affect the efficacy of cancer cell-targeted NIR-PIT Whether L-NaAA influenced the antitumor effect of cancer cell-targeted NIR-PIT was determined utilizing a CD44-IR700 in vivo allograft tumor model. First, it was confirmed that L-NaAA did not affect tumor growth. The treatment and imaging regimen is shown (Figure 23A). The accumulation of CD44-IR700 in MC38-luc tumors was confirmed by fluorescence imaging, which decreased after NIR light exposure regardless of the presence or absence of L-NaAA (Figures 23B, 23C). The therapeutic efficacy of CD44-targeted NIR-PIT was evaluated with BLI as a readout (Figure 24D). Regardless of the presence of L-NaAA, both NIR-PIT groups showed significantly lower intensity in BLI compared to the control group, indicating cancer cell death, and there was no significant difference between NIR-PIT with L-NaAA and NIR-PIT without L-NaAA (Figure 24E). Severe edema formation was observed in the skin over the tumor on day 1 in the NIR-PIT group without L-NaAA, which likely attenuated much of the bioluminescence signal. MC38-luc, LL / 2-luc, and MOC2-luc tumor models all showed that the antitumor effect of CD44-targeted NIR-PIT was not altered by L-NaAA (Figures 25A-25I). In athymic mice, L-NaAA also did not affect the efficacy of pan-PIT (Figures 26A-26E).

[0258] Example 10 L-NaAA inhibited the formation of edema after treatment with NIR-PIT The formation of edema was evaluated after CD44-targeted NIR-PIT with and without L-NaAA. In MC38-luc, LL / 2-luc, and MOC2-luc tumor-bearing mice, edema was observed on the dorsal surface around the subcutaneous tumor in mice 1 day after NIR-PIT without L-NaAA (Figures 24F and 25G). However, no edema was detected in mice treated by NIR-PIT with L-NaAA injection. No significant difference in body weight was observed between the two groups (Figure 24G). In other allograft models, no significant difference in body weight was detected after CD44-targeted NIR-PIT with L-NaAA (Figures 25H, 25I).

[0259] To quantify the extent of edema, magnetic resonance imaging (MRI) was performed 1 day after CD44-targeted NIR-PIT. In the non-L-NaAA group, T2-weighted and fat-suppressed images (T2WI fat-suppressed) showed widespread edema extending from the peripheral tumor site to the lower end of the thorax and proximal tail, shown as areas of high signal intensity in NIR-PIT. In contrast, minimal edema was observed only around the treated tumor in the L-NaAA group (Figure 25H). The areas of high signal intensity corresponding to post-treatment edema in T2WI fat-suppressed images were quantified, showing that the high signal area was significantly larger in the group treated without L-NaAA compared to the L-NaAA group (Figure 25I). This was also confirmed by short TI inversion recovery images (STIR) (Figures 27A-27B).

[0260] To investigate the cause of edema formation, ROS was assessed by chemiluminescence imaging (CLI) using L-012 in MC38-luc tumor-bearing mice before and after NIR-PIT (Figures 28A-28C). CD44-targeted NIR-PIT with L-NaAA showed significantly lower intensity (lower ROS) in CLI compared with NIR-PIT without L-NaAA. These data indicated that L-NaAA inhibited ROS-induced edema formation after NIR-PIT.

[0261] Example 11 L-NaAA inhibited the formation of edema after NIR-PIT targeting immunosuppressive cells without interfering with the efficacy The efficacy of CTLA4-targeted NIR-PIT with and without L-NaAA was evaluated in MC38-luc tumors. In both groups, CTLA4-IR700 fluorescence was observed 1 day after injection, before NIR-PIT, but decreased after NIR light exposure (Figures 29A-29C). The NIR-PIT group without L-NaAA had lower BLI tumor signals than the NIR-PIT group with L-NaAA on days 1 to 3 after NIR light exposure (Figure 29D). However, by day 6 after NIR-PIT, no difference was observed between the two groups, and both NIR-PIT groups showed significantly lower BLI intensity compared to the control group (Figure 29E). CTLA4-targeted NIR-PIT showed stronger edema formation than CD44-targeted NIR-PIT, and thus the NIR-PIT group without L-NaAA showed lower intensity in BLI due to skin thickening. The formation of edema in mice treated by NIR-PIT with L-NaAA was also suppressed both macroscopically and by MRI (Figures 29F-29H). On the day after NIR-PIT, a significant weight loss was observed in mice in the NIR-PIT with L-NaAA group (Figure 29I), likely due to the relatively less edema.

[0262] In CTLA4-targeted NIR-PIT, higher ROS was also detected in the NIR-PIT group without L-NaAA (Figure 28D, 28E). To confirm whether the selective cytotoxicity of CTLA4-targeted NIR-PIT was preserved in the NIR-PIT group with L-NaAA, selective depletion of cells expressing CTLA4 was evaluated 3 hours after CTLA4-targeted NIR-PIT in MC38-luc tumors (Figure 29J). CTLA4hi cells were selectively depleted in both NIR-PIT groups with and without L-NaAA. Thus, L-NaAA had no effect on the efficacy of immunosuppressive cell-targeted NIR-PIT, but suppressed edema formation by ROS.

[0263] The selective cytotoxicity induced by NIR-PIT is caused by a light-induced ligand release reaction that occurs under hypoxic electron donor-rich conditions. Ligand release causes a dramatic change in the solubility of APC-antigen complexes, leading to cell membrane damage and cell death. However, under normoxic or hyperoxic conditions, NIR light induces ROS production in APCs, which likely contributes to the nonselective cytotoxicity and local edema after NIR-PIT. Another possible cause of local edema formation could be lymphatic obstruction associated with direct lymphatic system damage caused by NIR-PIT. However, no obvious lymphatic obstruction or morphological changes due to NIR-PIT were observed. Thus, ROS increase vascular permeability, leading to the formation of edema. L-NaAA, an electron donor that should promote the NIR-PIT ligand release reaction, quenches the majority of ROS, thereby reducing edema as shown in Figures 24H and 29G by MRI. Quenching of ROS may interfere with the cytotoxicity of cancer cells, thus affecting the therapeutic effect of NIR-PIT. However, NIR-PIT with L-NaAA injection still showed acceptable cytotoxicity but without edema formation. Thus, L-NaAA administration in NIR-PIT provides an effective treatment method that is safer, for example, when edema may affect the airway or mediastinum.

[0264] Under hypoxic conditions, L-NaAA radical production was minimal, whereas under normoxic conditions, the amount of L-NaAA radical production was extremely high (Figure 17G(ii) and (iii)), indicating that oxygen molecules are involved in the production of L-NaAA radicals. Either the IR700 anion radical donates one electron to oxygen in the presence of oxygen to generate the superoxide anion radical (O2-·), or the triplet state IR700 generated by photoexcitation converts to triplet oxygen ( 3 O2) to produce singlet oxygen ( 1 It is possible that L-NaAA produces O2-· and 1It has been reported that L-NaAA reacts with O2 to form L-NaAA radicals and hydrogen peroxide. Therefore, it is possible that in the presence of oxygen, L-NaAA may react with these ROS, resulting in the generation of a large number of L-NaAA radicals. In this case, a high dose of L-NaAA can consume the generated ROS and reduce the side effects of NIR-PIT in clinical practice.

[0265] Although the IR700 anion radical is clearly observed when L-cysteine ​​is used as the reducing agent (FIG. 18F(ii)), the signal is very low when L-NaAA is used as the reducing agent (FIG. 17G(iii)). This may be due to the difference in the properties of L-cysteine ​​and L-NaAA as reducing agents. L-cysteine ​​is a simple reductant with one electron reduction capability, while L-NaAA ionizes in water to L-NaAA monoanion, which acts as a reductant in the reaction process. L-NaAA monoanion not only has one electron reduction capability, but also acts as one hydrogen donor, producing ESR-detectable L-NaAA radical with a relatively long lifetime (Njus and Kelley, Biochim Biophys Acta 1993, 1144:235-48.). Using quantum chemical calculations, it has been previously shown that in the photoreduction of IR700 in the presence of a one-electron reductant, e.g., L-cysteine, axial ligand cleavage requires hydrolysis with one electron transfer from the reductant and one proton transfer from HO, as occurs with L-NaAA (Kobayashi et al., Chempluschem 2020, 85:1959-1963). When L-cysteine ​​was used as the reducing agent, one electron was donated to IR700, and the long-lived IR700 anion radical was detected by ESR because the proton transfer from HO to the IR700 anion radical was relatively slow. However, when L-NaAA was used as the reducing agent, the donation of one electron and one proton from the L-NaAA monoanion to the IR700 molecule occurred simultaneously, so that the IR700 anion radical rapidly proceeded to the axial ligand cleavage reaction as soon as it was generated and therefore could not be detected by ESR (Figures 17G and 30A).

[0266] Previous studies have shown that NaN3, a known singlet oxygen and ROS quencher, partially inhibits the cytotoxicity of NIR-PIT. However, these experiments were performed in vitro because the required doses of NaN3 are toxic to living animals in vivo. Herein, it is shown that NaN3 directly inhibited the NIR light-induced ligand release response of IR700 by quenching IR700 radical anion formation as shown by ESR (Figures 17G and 18F). The inhibition of ligand release can be measured by IR700 fluorescence quenching, which is also equivalent to the efficacy of treatment. NaN3 partially inhibits cytotoxicity by inhibiting the NIR light-induced ligand release response of IR700. Thus, the role of ROS in the mechanism of action of NIR-PIT cytotoxicity has been exaggerated in the literature (Kishimoto et al., Free radical biology & medicine 2015, 85:24-32.; Railkar, et al., Molecular cancer therapeutics 2017, 16:2201-2214).

[0267] L-cysteine ​​is an alternative to L-NaAA as a reducing agent due to its higher redox potential and clinical use than L-NaAA. In IR700 solution, L-cysteine ​​accelerated the light-induced ligand release by electron donation, especially under hypoxic conditions. However, when IR700 is conjugated to an antibody, the NIR light-induced ligand release reaction is dominated under hypoxic conditions and is not highly affected by L-cysteine. It is possible that the sulfhydryl group on the L-cysteine ​​side chain of the antibody can donate electrons to IR700 when covalently conjugated and activated by NIR light. L-cysteine ​​addition also protected cells from NIR-PIT in vitro, presumably because L-cysteine ​​forms disulfide bonds with free sulfhydryl groups on the antibody under normoxic cell culture conditions. The formation of L-cystine prevents effective electron donation to the light-activated IR700, suppressing the ligand release reaction. L-NaAA is an electron donor and may also assist in the protonation of excited IR700, which promotes the ligand-releasing reaction. Thus, although L-cysteine ​​is a good reducing agent and may accelerate the ligand-releasing reaction in vitro, it may be difficult to use for this purpose in vivo.

[0268] In summary, L-NaAA, which acts both as a direct electron donor to IR700 and as a reducing agent of ROS in NIR-PIT, suppresses local edema while promoting the selective cytotoxicity of NIR-PIT. Based on the relatively low toxicity and safety clinical profile of L-NaAA at the doses used herein, it may serve as a useful adjuvant of NIR-PIT, improving its safety profile while not hindering its efficacy (Figure 30B). Thus, one or more reducing agents, such as L-NaAA, can be used in combination with the disclosed method utilizing IR700. One or more reducing agents significantly suppress edema in some cases, such as immediately after NIR-PIT. Clinically, edema is a clinical problem in NIR-PIT. For example, when treating tumors that arise in critical parts of the body (e.g., airways or mediastinum, e.g., behind the trachea), edema can lead to undesirable events, such as suffocation.

[0269] In view of the numerous possible embodiments to which the principles of this disclosure may be applied, it is to be understood that the illustrated embodiments are merely examples of the disclosure and should not be considered as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. 1. A combination for use in a method for treating cancer in a subject, said combination comprising a therapeutically effective amount of one or more cytotoxic T-lymphocyte associated protein 4 (CTLA4) antibodies-IR700 molecules and one or more programmed death-ligand 1 (PD-L1) antibodies-IR700 molecules; The combination (i) one or more antibody-IR700 molecules, wherein the antibody specifically binds to a tumor-specific protein on the surface of a cancer cell, the tumor-specific protein being epidermal growth factor receptor (EGFR / HER1), mesothelin, prostate-specific membrane antigen (PSMA), HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, cancer cell lineage, or tumor-specific protein. Childhood antigen (CEA), alpha-fetoprotein (AFP), Lewis Y, tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2 (EphA2), glypican-1, glypican-2, glypican-3, gpA33, mucin, CAIX, folate-binding protein, ganglioside, integrin αVβ3, integrin α5 β3,1, Erb-B2 receptor tyrosine kinase 3 (ERBB3), MET proto-oncogene, receptor tyrosine kinase (MET), insulin-like growth factor 1 receptor (IGF1R), ephrin type A receptor 3 (EPHA3), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAILR1), TRAILR2, receptor activator of nuclear factor kappa-B ligand (RANKL), fibroblast activation protein (FAP), tenascin, BCR complex, gp72, HLA-DR10β, HLA-DR antigen, IgE, CA242, polymorphic epithelial mucin (PEM) antigen, SK-1 antigen, programmed death 1 (PD-1), or programmed death ligand 2 (PD-L2), and / or (ii) one or more immune activators and The subject and / or cancer cells in the subject are then treated with at least 1 J / cm at wavelengths between 660 and 740 nm. ２ is irradiated with a dose of The combination thereby treats said cancer in said subject.

2. The combination described in claim 1, wherein the combination and the one or more antibody-IR700 molecules and / or immune activators are formulated for intravenous administration.

3. 1. A composition for use in a method for treating cancer in a subject, comprising: the composition comprises a therapeutically effective amount of one or more antibody-IR700 molecules, wherein the antibody specifically binds to a tumor-specific protein on the surface of a cancer cell or an immune cell-specific protein on the surface of an immune cell; wherein the composition is administered to the subject sequentially or simultaneously with a therapeutically effective amount of one or more reducing agents; The subject and / or cancer cells in the subject have a radiation dose of at least 1 J / cm at wavelengths between 660 and 740 nm. ２ The composition is irradiated with a dose of

4. The composition of claim 3, wherein the composition is formulated for intravenous administration and the one or more reducing agents are formulated for intraperitoneal administration.

5. 5. The composition of claim 3 or 4, wherein the irradiation is performed after both administering steps and / or the one or more reducing agents are administered prior to irradiation of the subject and / or irradiation of cancer cells in the subject.

6. The tumor-specific protein is HER1 / EGFR, mesothelin, PSMA, HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, CEA, AFP, Lewis Y, TAG7), VEGF, VEGFR, EpCAM, EphA2, glypican-1, glypican-2, glypican-3, gpA33, mucin, CAIX, folate binding protein 6. The composition of claim 3, wherein the immune cell-specific protein comprises a cytoplasmic reticulum (CRM)-specific endothelial cell (ESC ...

7. 7. The combination of claim 1 or 2, or the composition of any one of claims 3 to 6, wherein the tumor specific protein comprises EGFR, and optionally the EGFR antibody comprises panitumumab or cetuximab.

8. The composition of any one of claims 3 to 6, wherein the immune cell-specific protein comprises CTLA4 or PD-L1.

9. The combination of claim 1, 2 or 7, or the composition of any one of claims 3 to 8, wherein the cancer or cancer cells are cancer or cancer cells of the breast, liver, colon, ovary, prostate, pancreas, brain, cervix, kidney, bone, skin, head and neck, oropharynx, or blood.

10. the CTLA4 antibody comprises ipilimumab or tremelimumab, and / or The anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (embaforimab), BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737; A combination according to claim 1, 2, 7 or 9, or a composition according to any one of claims 6 to 9.

11. 11. The combination of claim 1, 2, 7, 9 or 10, or the composition of any one of claims 3 to 10, wherein the therapeutically effective amount of the one or more antibody-IR700 molecules is at least 15 mg, the therapeutically effective amount of the one or more CTLA4 antibody-IR700 molecules is at least 15 mg, the therapeutically effective amount of the one or more PD-L1 antibody-IR700 molecules is at least 15 mg, or the therapeutically effective amount of the one or more antibody-IR700 molecules is at least 15 mg and the therapeutically effective amount of the one or more reducing agents is between 5 and 50 g.

12. The subject and / or the cancer cells receive between 10 and 60 J / cm at a wavelength of 680 nm or 690 nm. ２ The combination according to claim 1, 2, 7 or 9 to 11, or the composition according to any one of claims 3 to 11, which is irradiated with a dose of

13. The composition of any one of claims 3 to 12, wherein the one or more reducing agents comprise sodium L-ascorbate, ascorbic acid, L-cysteine, glutathione, or a combination thereof.

14. The subject and / or cancer cells in the subject are then irradiated with at least 1 J / cm at a wavelength of 660-740 nm. ２ and optionally said two or more radiation doses are administered within about 12 to 36 hours, such as within about 24 hours of each other.

15. The combination of claims 1, 2, 7, 9-12 or 14, or the composition of any one of claims 3-14, characterized in that the subject is administered two or more doses of the one or more antibody-IR700 molecules that specifically bind to the tumor-specific protein on the surface of the cancer cells, optionally the two or more doses of the one or more antibody-IR700 molecules that specifically bind to the tumor-specific protein on the surface of the cancer cells being separated by about 24 to 48 hours.

16. The method of claim 1, further comprising detecting said cancer cells by fluorescence lifetime imaging about 0-48 hours after said irradiation. The combination of claim 1, 2, 7, 9-12, 14 or 15, or the composition of claim 3-15, further comprising:

17. A composition described in any one of claims 3 to 16, wherein the method further comprises the step of administering to the subject one or more immune activators.

18. The combination of claim 1 or 2, or the composition of claim 17, wherein the one or more immune activators comprise IL-15, interferon gamma, or both.

19. 1. A composition for use in reducing edema resulting from cancer treatment in a subject, said composition comprising one or more antibody-IR700 molecules, said antibody specifically binds to a tumor-specific protein on the surface of a cancer cell or binds to a protein on the surface of an immune cell, optionally wherein said protein on the surface of an immune cell is CTLA4, and said tumor-specific protein is HER1 / EGFR, PD-L1, mesothelin, PSMA, HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, CE1, HER2 / ERBB2, CD1, ...2 / ERBB2, CD2, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD2, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD2, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD2, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB2, CD1, HER2 / ERBB A, AFP, Lewis Y, tumor associated glycoprotein 72 (TAG72), VEGF, VEGFR, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, folate binding protein, ganglioside, integrin αVβ3, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, BCR complex, gp72, HLA-DR10β, HLA-DR antigen, IgE, CA242, PEM antigen, SK-1 antigen, PD-1, or PD-L2, wherein the composition is administered sequentially or simultaneously with sodium L-ascorbate; The subject and / or cancer cells in the subject have a radiation intensity of at least 1 J / cm at wavelengths between 660 and 740 nm. ２ is irradiated with a dose of The composition thereby reduces edema caused by cancer treatment in a subject.

20. A composition for use in a method for treating cancer in a subject, said composition comprising one or more cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibody-IR700 molecules; The composition, (i) one or more antibody-IR700 molecules, wherein the antibody specifically binds to a tumor-specific protein on the surface of a cancer cell, the tumor-specific protein being epidermal growth factor receptor (EGFR / HER1), mesothelin, prostate-specific membrane antigen (PSMA), HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, cancer cell lineage, or tumor-specific protein. Childhood antigen (CEA), alpha-fetoprotein (AFP), Lewis Y, tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2 (EphA2), glypican-1, glypican-2, glypican-3, gpA33, mucin, CAIX, folate-binding protein, ganglioside, integrin αVβ3, integrin α5 an antibody-IR700 molecule comprising: β3,1, Erb-B2 receptor tyrosine kinase 3 (ERBB3), MET proto-oncogene, receptor tyrosine kinase (MET), insulin-like growth factor 1 receptor (IGF1R), ephrin type A receptor 3 (EPHA3), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAILR1), TRAILR2, receptor activator of nuclear factor kappa-B ligand (RANKL), fibroblast activation protein (FAP), tenascin, BCR complex, gp72, HLA-DR10β, HLA-DR antigen, IgE, CA242, polymorphic epithelial mucin (PEM) antigen, SK-1 antigen, programmed death 1 (PD-1), or programmed death ligand 2 (PD-L2); (ii) one or more immune activators, and / or (iii) one or more Programmed Death-Ligand 1 (PD-L1) antibody-IR700 molecules and the subject and / or cancer cells in the subject are then irradiated at a wavelength of 660-740 nm with a dose of at least 1 J / cm 2 ; thereby treating said cancer in said subject.

21. A composition for use in a method for treating cancer in a subject, said composition comprising one or more programmed death-ligand 1 (PD-L1) antibody-IR700 molecules; The composition, (i) one or more antibody-IR700 molecules, wherein the antibody specifically binds to a tumor-specific protein on the surface of a cancer cell, the tumor-specific protein being epidermal growth factor receptor (EGFR / HER1), mesothelin, prostate-specific membrane antigen (PSMA), HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, cancer cell lineage, or tumor-specific protein. Childhood antigen (CEA), alpha-fetoprotein (AFP), Lewis Y, tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2 (EphA2), glypican-1, glypican-2, glypican-3, gpA33, mucin, CAIX, folate-binding protein, ganglioside, integrin αVβ3, integrin α5 an antibody-IR700 molecule comprising: β3,1, Erb-B2 receptor tyrosine kinase 3 (ERBB3), MET proto-oncogene, receptor tyrosine kinase (MET), insulin-like growth factor 1 receptor (IGF1R), ephrin type A receptor 3 (EPHA3), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAILR1), TRAILR2, receptor activator of nuclear factor kappa-B ligand (RANKL), fibroblast activation protein (FAP), tenascin, BCR complex, gp72, HLA-DR10β, HLA-DR antigen, IgE, CA242, polymorphic epithelial mucin (PEM) antigen, SK-1 antigen, programmed death 1 (PD-1), or programmed death ligand 2 (PD-L2); (ii) one or more immune activators, and / or (iii) one or more cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibody-IR700 molecules and the subject and / or cancer cells in the subject are then irradiated at a wavelength of 660-740 nm with a dose of at least 1 J / cm 2 ; thereby treating said cancer in said subject.

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