Compositions, combinations and related methods for photoimmunotherapy

Abstract

To provide a phthalocyanine dye conjugated to a targeting molecule for use in photoimmunotherapy, and a therapy employing the dye.SOLUTION: A method of treating a disease or condition in a subject comprises: a) administering to a subject having a disease or condition a conjugate comprising a phthalocyanine dye linked to a targeting molecule that binds to a protein on a surface of a cell present in the microenvironment of a lesion associated with the disease or condition, where the conjugate is administered to effect a systemic exposure that is 75% or less of the therapeutically effective systemic exposure of an antibody or antigen-binding antibody fragment that is not so conjugated for treating the same disease or condition; and b) after administering the conjugate, irradiating the lesion at a wavelength of 500 nm to 900 nm at a dose of at least 1 J cm-2 or 1 J / cm of fiber length, thereby treating the disease in the subject.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 206,776, entitled "Combination Therapy with Photoimmunotherapy and Related Methods," filed August 18, 2015, and U.S. Provisional Patent Application No. 62 / 249,085, entitled "Compositions and Methods for Photoimmunotherapy," filed October 30, 2015, the contents of each of which are incorporated by reference in their entirety.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format, which is provided as a file entitled 751702000440seqlist.txt, created on August 18, 2016, and which is 9,934 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to conjugates, compositions and methods for use in photoimmunotherapy, e.g., photoimmunotherapy induced by activation of a phthalocyanine dye conjugated to a targeting molecule that binds to a protein on a cell, e.g., an IR700-antibody conjugate. The present disclosure also relates to combination therapy for use in combination with photoimmunotherapy, e.g., photoimmunotherapy induced by activation of a phthalocyanine dye conjugated to a targeting molecule that targets tumor cells, e.g., an IR700-antibody conjugate. In some embodiments, the phthalocyanine-dye conjugate can be activated by irradiation with near-infrared light. The features of the conjugates, compositions, combinations and methods (including the dose of the conjugate) provide various advantages, such as lower toxicity and / or improved efficacy. In some embodiments, the present disclosure also relates to dual-labeled phthalocyanine-dye conjugates in which the targeting molecule is conjugated to an additional fluorescent dye, which can be used in photoimmunotherapy while exhibiting improved performance, e.g., for imaging or detection. The present disclosure also provides therapeutic methods using the conjugates, compositions and combinations for the treatment of diseases and conditions, including tumors or cancer. [Background technology]

[0004] background A variety of therapeutic methods are available for treating diseases such as cancer. For example, photoimmunotherapy (PIT) is a method that uses photosensitizers conjugated to antibodies or other targeting molecules that target cell surface proteins to allow targeted killing of specific cells. In some cases, PIT can selectively target diseased cells, such as tumor cells, thereby selectively killing such cells without damaging healthy cells. There is a need for improved strategies to improve photoimmunotherapy methods, for example, to increase the efficacy of treatment. Compositions and methods that meet such needs are provided. Summary of the Invention

[0005] overview In some embodiments, a method of treating a disease or condition in a subject is provided, comprising using photoimmunotherapy (PIT). In some embodiments, the method comprises administering to a subject having a disease or condition a conjugate containing a phthalocyanine dye linked to a targeting molecule, such as an antibody or antigen-binding fragment thereof, that binds to a protein on the surface of a cell present in the microenvironment of a lesion associated with the disease or condition. In some embodiments, the conjugate is administered to achieve a systemic exposure that is 75% or less of the total therapeutically effective systemic exposure of the antibody or antigen-binding fragment not so conjugated to treat the same disease or condition as described on the label that has been approved for commercialization by a regulatory agency (e.g., FDA, EMA, PDMA). In some embodiments, after administration of the conjugate, the lesion receives at least 1 J cm at a wavelength of 500-900 nm. -2 or irradiated at a dose of 1 J / cm of fiber length, thereby treating a tumor in a subject. In some embodiments, the wavelength for irradiation is 600 nm to 850 nm, for example 660 nm to 740 nm.

[0006] In some embodiments, the conjugate is administered according to a dosing schedule: administration of the conjugate is performed only once as a single injection or infusion; or, the dosing schedule does not include a subsequent dose of the conjugate; or, the dosing schedule does not include a subsequent dose of a targeting molecule, e.g., a macromolecule, that is not so conjugated.

[0007] In some embodiments, the conjugate is administered systemically. In some embodiments, the conjugate is administered intravenously.

[0008] In some embodiments, the conjugate is administered to achieve a systemic exposure (AUC) that is 60% or less, 50% or less, 40% or less, or 30% or less of the therapeutically effective systemic exposure of an antibody or antigen-binding fragment that is not so conjugated to treat the same disease or condition.

[0009] In some embodiments, the disease or condition is a tumor in which the antibody or antigen-binding fragment binds to a molecule on the surface of a cell present in the tumor microenvironment, and the tumor is irradiated.

[0010] In some embodiments, a method of treating a disease or condition in a subject using photoimmunotherapy (PIT) is provided, comprising measuring systemic exposure as measured by the average area under the plasma conjugate concentration-time curve from time 0 to infinity (AUC[0-inf] or AUC 0-∞ ) is between or about 250 μg / mL*h to 100,000 μg / mL*h, between or about 500 μg / mL*h to 50,000 μg / mL*h, between or about 500 μg / mL*h to 50,000 μg / mL*h, between or about 500 μg / mL*h to 18,000 μg / mL*h, or between or about 500 μg / mL*h to 10,000 μg / mL*h. In some embodiments, systemic exposure as measured by the mean area under the plasma conjugate concentration-time curve from time 0 to infinity for a patient population, e.g., a sample patient population after administration of the conjugate (AUC[0-inf] or AUC 0-∞ ) is less than or equal to 100,000μg / mL*h, less than or equal to 75,000μg / mL*h, less than or equal to 50,000μg / mL*h, less than or equal to 40,000μg / mL*h, less than or equal to 30,000μg / mL*h, less than or equal to 20,000μg / mL*h, less than or equal to 10,000μg / mL*h, less than or equal to 5,000μg / mL*h, or less than or equal to 2,500μg / mL*h.

[0011] In some embodiments, systemic exposure as measured by the mean area under the plasma conjugate concentration-time curve from time point 0 to 24 hours for a patient population, e.g., a sample patient population after administration of the conjugate (AUC[0-24] or AUC 0-24) is between or between about 100 μg / mL*h and 25,000 μg / mL*h, between or between about 200 μg / mL*h and 10,000 μg / mL*h, between or between about 500 μg / mL*h and 5,000 μg / mL*h; or systemic exposure as measured by the mean area under the plasma conjugate concentration-time curve from time point 0 to 24 hours for a patient population, e.g., a sample patient population following administration of the conjugate (AUC[0-24] or AUC 0-24 ) is less than or equal to 25,000μg / mL*h, less than or equal to 15,000μg / mL*h, less than or equal to 10,000μg / mL*h, less than or equal to 5,000μg / mL*h, less than or equal to 2,500μg / mL*h, less than or equal to 1,000μg / mL*h, or less than or equal to 500μg / mL*h.

[0012] In some embodiments, the conjugate is at least about 10 mg / m 2 (subject's body surface area), at least about 50 mg / m 2 or at least about 75 mg / m 2 and 5000 mg / m 2 Below 2000mg / m 2 Below 1000mg / m 2 In some embodiments, the conjugate is administered in a dosage range of 500 mg / m 2 Below 250mg / m 2 or less than 200 mg / m 2 In some embodiments, the conjugate is administered in a dosage range that is less than or equal to 100 mg / m 2 ~1500mg / m 2 Between 100 mg / m 2 ~1500mg / m 2 or 150 mg / m 2 ~750mg / m 2 Between 150 mg / m 2 ~750mg / m 2In some embodiments, the conjugate is administered at a dosage between 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 , or about 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 The compound is administered at a dosage of

[0013] In some embodiments, the targeting molecule is an antibody or an antigen-binding antibody fragment. In some embodiments, the antibody is a Fab, a single V H domain, a single chain variable fragment (scFv), a multivalent scFv, a bispecific scFv or an scFv-CH3 dimer.

[0014] In some embodiments, the lesion is irradiated with a wavelength of 690±50 nm or at or about 690±20 nm. -2 ~about 400J cm -2 Or about 2J cm -2 ~about 400J cm -2 In some embodiments, the lesion is irradiated with a dose of at least 2 J / cm fiber length to about 500 J / cm fiber length or from about 2 J / cm fiber length to about 500 J / cm fiber length. -2 , 5J cm -2 , 10J cm -2 , 25J cm -2 , 50J cm -2 , 75J cm -2 , 100J cm -2 , 150J cm -2 , 200J cm -2 , 300J cm -2 , 400J cm -2 , or 500 J cm -2 , or at least about 2 J cm -2 , 5J cm -2 , 10J cm-2 , 25J cm -2 , 50J cm -2 , 75J cm -2 , 100J cm -2 , 150J cm -2 , 200J cm -2 , 300J cm -2 , 400J cm -2 , or 500 J cm -2 or the lesion is irradiated with a dose of at least 2 J / cm, 5 J / cm, 10 J / cm, 25 J / cm, 50 J / cm, 75 J / cm, 100 J / cm, 150 J / cm, 200 J / cm, 250 J / cm, 300 J / cm, 400 J / cm, or 500 J / cm; or the lesion is irradiated with a dose of at least about 2 J / cm, 5 J / cm, 10 J / cm, 25 J / cm, 50 J / cm, 75 J / cm, 100 J / cm, 150 J / cm, 200 J / cm, 250 J / cm, 300 J / cm, 400 J / cm, or 500 J / cm.

[0015] In some embodiments, the irradiation is performed or accomplished between or about 30 minutes to 96 hours after administration of the conjugate. In some embodiments, the conjugate is administered on a dosing schedule in which administration of the conjugate is performed only once as a single injection or infusion. In some embodiments, the conjugate is administered on a dosing schedule in which the dosing schedule does not include a subsequent dose of the conjugate. In some embodiments, the conjugate is administered on a dosing schedule in which the dosing schedule does not include a subsequent dose of the targeting molecule that is not so conjugated. In some embodiments, the dosing schedule is repeated.

[0016] In some embodiments, the conjugate is administered systemically. In some embodiments, the conjugate is administered intravenously.

[0017] In some embodiments, the phthalocyanine dye has a maximum absorption wavelength of from 600 nm to about 850 nm or from about 600 nm to about 850 nm.

[0018] In some embodiments, the phthalocyanine dye is linked directly or indirectly to the targeting molecule. In some embodiments, the phthalocyanine dye comprises the formula: TIFF2025015674000002.tif92128 formula, L is a linker; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; and X 2 and X 3 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 It is alkylene.

[0019] In some embodiments, the phthalocyanine dye comprises the formula: TIFF2025015674000003.tif90150 formula, X 1 and X 4 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 is alkylene; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy.

[0020] In some embodiments, the phthalocyanine dye comprises IRDye 700DX (IR700).

[0021] In some embodiments, the cell surface protein is ACTHR, endothelial cell Anxa-1, aminopetidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptor BB1, BB2, BB4, calcitonin receptor, cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, Crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endosialin, EP2, EP4 , EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, family A G protein-coupled receptor (rhodopsin-like), family B G protein-coupled receptor (secretin receptor-like), family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV 16 / 18 and E6 / E7 antigens, hTERT, IL11-R, IL-13R, ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE 1, MAGE 2, MAGE 3, MAGE4, MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease cleaved collagen IV, proteinase 3, prohibitin, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 patched, RET receptor, plexin, smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptor (VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, and VEGF-3 or FLT-4), voltage-gated ion channel, VPAC1, VPAC2, Wilms' tumor 1, Y1, Y2, Y4, and Y5.

[0022] In some embodiments, the cell surface protein is HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72, Caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VGF), IL-2 receptor, ... EGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate binding protein, gangliosides (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinase, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, and SK-1 antigen.

[0023] In some embodiments, the cell surface protein is CD25, PD-1 (CD279), PD-L1 (CD274, B7-H1), PD-L2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3 (HAVCR2), 4-1BB (CD137, TNFRSF9), CXCR2, CXCR4 (CD184), CD27, CEACAM1, Galectin 9, BTL A, CD160, VISTA (PD1 homolog), B7-H4 (VCTN1), CD80 (B7-1), CD86 (B7-2), CD28, HHLA2 (B7-H7), CD28H, CD155, CD226, TIGIT, CD96, Galectin 3, CD40, CD40L, CD70, LIGHT(TNFSF14), HVEM(TNFRSF14), B7-H3(CD276), Ox40 L (TNFSF4), CD137L (TNFSF9, GITRL), B7RP1, ICOS (CD278), ICOSL, KIR, GAL9, NKG2A (CD94), GARP, TL1A, TNFRSF25, TMIGD2, BTNL2, butyrophilin family, CD48, CD244, siglec family, CD30, CSF1R, MICA (MHC class I polypeptide-related sequence A), MICB (MHC class I polypeptide-related sequence B), NKG2D, KIR family (killer cell immunoglobulin-like receptor, LILR family (leukocyte immunoglobulin-like receptor, CD85, ILT, LIR), SIRPA (signal regulatory protein alpha), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR, and VEGF.

[0024] In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), basiliximab, nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, M K-4166, dacetuzumab, lucatumumab, SEA-CD40, CP-870, CP-893, MED16469, MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C, rHIgM12B7, urocupulumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, emactuzumab, CC-90002 and MNRP1685A, or an antigen-binding fragment thereof.

[0025] In some embodiments, the conjugate is cetuximab-IR700, panitumumab-IR700, zalutumumab-IR700, nimotuzumab-IR700, tositumomab-IR700, rituximab-IR700, ibritumomab tiuxetan-IR700, daclizumab-IR700, gemtuzumab-IR700, alemtuzumab-IR700, CEA-scan Fab fragment-IR700, OC125-IR700, ab75705-IR700, B72.3-IR700, bevacizumab-IR700, basiliximab-IR700, nivolumab-IR700, pembrolizumab-IR700, pidilizumab-IR700, MK-3475-IR700, BMS-936559-IR700, MPDL3280A-IR 700, ipilimumab-IR700, tremelimumab-IR700, IMP321-IR700, BMS-986016-IR700, LAG525-IR700, urelumab-IR700, PF-05082566-IR700, TRX518-IR700, MK-4166-IR700, dacetuzumab-IR700, lucatumumab-IR700, SEA- CD40-IR700, CP-870-IR700, CP-893-IR700, MED16469-IR700, MEDI6383-IR700, MEDI4736-IR70 0, MOXR0916-IR700, AMP-224-IR700, PDR001-IR700, MSB0010718C-IR700, rHIgM12B7-IR700, Uroku The compound is selected from among plumab-IR700, BKT140-IR700, varlilumab-IR700, ARGX-110-IR700, MGA271-IR700, lirilumab-IR700, IPH2201-IR700, AGX-115-IR700, emactuzumab-IR700, CC-90002-IR700 and MNRP1685A-IR700.

[0026] In some embodiments, the targeting molecule is an antibody or antigen-binding fragment thereof that is cetuximab, or the conjugate is cetuximab-IR700.

[0027] In some embodiments, the mean area under the plasma conjugate concentration-time curve from time 0 to infinity (AUC[0-inf] or AUC 0-∞ ) is between or between about 500 μg / mL*h and 18,000 μg / mL*h, between or between about 500 μg / mL*h and 10,000 μg / mL*h, between or between about 500 μg / mL*h and 5,000 μg / mL*h, or between or between about 500 μg / mL*h and 2,500 μg / mL*h. In some embodiments, the mean area under the plasma conjugate concentration-time curve (AUC[0-24] or AUC) from time 0 to 24 hours for a patient population, e.g., a sample patient population following administration of the conjugate. 0-24 ) is between or about 500μg / mL*h and 8,000μg / mL*h, between or about 500μg / mL*h and 5,000μg / mL*h, between or about 500μg / mL*h and 5,000μg / mL*h, between or about 500μg / mL*h and 2,000μg / mL*h, or between or about 1000μg / mL*h and 4,000μg / mL*h.

[0028] In some embodiments, the conjugate is at 75 mg / m 2 (subject's body surface area) ~1500mg / m 2 Between 75 mg / m 2 (subject's body surface area) ~1500mg / m 2 Between 75 mg / m 2 ~1000mg / m 2 Between 75 mg / m 2 ~1000mg / m 2 Between 75 mg / m 2 ~500mg / m 2 Between 75 mg / m2 ~500mg / m 2 or 75 mg / m 2 ~225mg / m 2 Between 75 mg / m 2 ~225mg / m 2 In some embodiments, the conjugate is administered in a dosage range of at least about 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 , or about 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 The drug is administered in a dosage range that is

[0029] In some embodiments, a method of treating a disease lesion in a subject comprises the steps of: a) administering intravenously to a subject having a lesion associated with a disease or condition a cetuximab-IR700 conjugate, wherein the conjugate is administered at a dose of 640 mg / m 2 or about 640 mg / m 2 and b) administering to the lesion, after administration of the conjugate, an amount of at least 50 J cm at a wavelength of 690±20 nm. -2 Or about at least 50 J cm -2 Or about 50J cm -2 or at least or about at least 100 J / cm fiber length or 100 J / cm fiber length, thereby treating the disease or condition in the subject.

[0030] In some embodiments, the conjugate is administered according to a dosing schedule in which the administration of the conjugate is performed only once as a single injection or infusion; or the dosing schedule does not include a subsequent dose of the conjugate; or the dosing schedule does not include a subsequent dose of the targeting molecule that is not so conjugated.

[0031] In some embodiments, irradiation is performed 24 hours ± 3 hours, for example 24 hours ± 2 hours, after administration of the conjugate.

[0032] In some embodiments, the lesion is a tumor and the disease or condition is a tumor or cancer.

[0033] In some embodiments, the lesion is a tumor that is a superficial tumor. In some embodiments, the tumor is less than 10 mm thick. In some embodiments, the irradiation is performed using a microlens-tipped fiber for surface illumination. In some embodiments, the light irradiation dose is 5 J / cm 2 ~About 200J / cm 2 or about 5 J / cm 2 ~About 200J / cm 2 It is.

[0034] In some embodiments, a method for treating superficial tumors with photoimmunotherapy comprises administering 5 J / cm 2 of photoimmunotherapy to a superficial tumor in a subject using a microlens-tipped fiber for surface illumination. 2 ~About 200J / cm 2 or about 5 J / cm 2 ~About 200J / cm 2 In some embodiments, the light irradiation dose is 50 J / cm, where the tumor is associated with a phototoxic agent that comprises a targeting molecule bound to a cell surface molecule of the tumor. 2 or about 50 J / cm 2 It is.

[0035] In some embodiments, the lesion is a tumor that is a stromal tumor. In some embodiments, the tumor is greater than 10 mm deep or is a subcutaneous tumor. In some embodiments, the irradiation is performed using a cylindrical diffusing fiber with a diffuser length of 0.5 cm to 10 cm and spaced 1.8±0.2 cm apart. In some embodiments, the light irradiation dose is 20 J / cm fiber length to about 500 J / cm fiber length or about 20 J / cm fiber length to about 500 J / cm fiber length.

[0036] In some embodiments, a method for treating a stromal tumor with photoimmunotherapy is provided, comprising irradiating a stromal tumor in a subject with a light dose of at or about 100 J / cm fiber length, or a fluence rate of at or about 400 mW / cm, using cylindrical diffusing fibers comprising a diffuser length of 0.5 cm to 10 cm and spaced 1.8±0.2 cm apart, wherein the tumor is associated with a phototoxic agent comprising a targeting molecule bound to a cell surface molecule of the tumor.

[0037] In some embodiments, the light exposure dose is 50 J / cm to about 300 J / cm of fiber length or about 50 J / cm to about 300 J / cm of fiber length, In some embodiments, the light exposure dose is 100 J / cm of fiber length or about 100 J / cm of fiber length.

[0038] In some embodiments, the tumor is greater than 10 mm deep or is a subcutaneous tumor. In some embodiments, the cylindrical diffusing fiber is placed in a catheter positioned 1.8±0.2 cm away in the tumor. In some embodiments, the catheter is optically transparent.

[0039] In some embodiments, the phototoxic agent comprising targeting molecule is administered to the subject more than 6 hours before irradiating the tumor, and the phototoxic agent is associated with the tumor.In some embodiments, the phototoxic agent is pre-administered to the subject more than 12 hours, 24 hours, 26 hours, 48 ​​hours, 72 hours or 96 hours before irradiating the tumor, or more than about 12 hours, 24 hours, 26 hours, 48 ​​hours, 72 hours or 96 hours before irradiating the tumor.In some embodiments, the phototoxic agent is a phthalocyanine dye-targeting molecule conjugate.In some embodiments, the phthalocyanine dye is IR700.

[0040] In some embodiments, in any of the treatment methods provided herein, the dosing schedule is repeated, and steps (a) and (b) are repeated. In some embodiments, the dosing schedule is repeated if residual lesions remain after prior treatment with the conjugate. In some embodiments, the method additionally comprises evaluating the subject for the presence of residual lesions, and repeating the dosing schedule if residual lesions remain. In some embodiments, the dosing schedule is repeated if residual lesions remain at a time point more than 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year, or about 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year, or 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year after prior administration of the conjugate. In some embodiments, the dosing schedule is repeated if residual lesions remain at or about 4 weeks after prior administration of the conjugate.

[0041] In some embodiments, the conjugate contains 1-100, 1-10, or 2-5 phthalocyanine dye molecules per targeting molecule.

[0042] In some embodiments, the method does not include administering an additional therapeutic agent or anti-cancer treatment. In some embodiments, the method includes administering an additional therapeutic agent or anti-cancer treatment. In some embodiments, the anti-cancer treatment includes radiation therapy.

[0043] In some embodiments, the additional therapeutic agent is an anti-cancer agent or an immunomodulatory agent. In some embodiments, the additional therapeutic agent is an immunomodulatory agent that is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding fragment, a small molecule or a polypeptide. In some embodiments, the immune checkpoint inhibitor is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment thereof.

[0044] In some embodiments, the immunomodulatory agent is administered before irradiating lesion or tumor.In some embodiments, the immunomodulatory agent is administered 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks or more than 1 month before irradiating tumor, or about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks or more than 1 month before.

[0045] In some embodiments, the methods provided include sequential administration of an immunomodulatory agent following irradiation three times per week, twice per week, once per week, once per two weeks, once per three weeks, or once per month.

[0046] In some embodiments, a method of treating a tumor in a subject is provided, comprising: a) administering to the subject an immunomodulator; b) administering to the subject a therapeutically effective amount of a conjugate comprising a phthalocyanine dye linked to a targeting molecule capable of binding to a molecule on the surface of cells present in the tumor microenvironment; and c) irradiating the tumor with a wavelength that makes the conjugate cytotoxic more than 12 hours after administering the immunomodulator, thereby treating the tumor. In some embodiments, the immunomodulator is administered more than 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 1 month before irradiating the tumor, or about 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 1 month before.

[0047] In some embodiments, the conjugate binds to a protein on the surface of a cell present in the tumor microenvironment. In some embodiments of the provided methods, step c) of irradiating the tumor is performed either i) after administration of the immunomodulatory agent and after administration of the conjugate, or ii) only after administration of the conjugate.

[0048] In some embodiments, the conjugate is administered prior to, simultaneously with, or subsequent to administration of the immunomodulatory agent. In some embodiments, the conjugate is administered after administration of the immunomodulatory agent but prior to irradiating the tumor. In some embodiments, the conjugate is administered 12 hours to 48 hours or about 12 hours to 48 hours prior to irradiating the tumor and the immunomodulator is administered 12 hours to about 1 month or about 12 hours to about 1 month prior to irradiating the tumor.

[0049] In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding fragment, a small molecule or a polypeptide. In some embodiments, the immune checkpoint inhibitor is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment thereof of any of the foregoing.

[0050] In some embodiments, the immunomodulatory agent is a demethylating agent that upregulates expression of a tumor-associated antigen (TAA) or is a cytokine.

[0051] In some embodiments, the methods provided comprise the step of sequentially administering an immunomodulatory agent following irradiation three times per week, twice per week, once every week, once every two weeks, once every three weeks, or once a month.

[0052] In some embodiments, methods are provided for treating a tumor in a subject, the methods comprising: a) administering to the subject an immunomodulatory agent that enhances expression of a molecule on the surface of cells present in the tumor microenvironment; b) administering to the subject a therapeutically effective amount of a conjugate that includes a phthalocyanine dye linked to a targeting molecule capable of binding to the cell surface molecule; and c) more than 5 minutes after administering the conjugate, irradiating the tumor with a wavelength that renders the conjugate cytotoxic, thereby treating the tumor.

[0053] In some embodiments, the immunomodulatory agent is a cytokine or an agent that induces increased expression of a cytokine in the tumor microenvironment. In some embodiments, the cytokine is interferon gamma.

[0054] In some embodiments, the molecule on the surface of the cell is selected from CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28, and VISTA. In some embodiments, the molecule on the surface of the cell is PD-L1.

[0055] In some embodiments, the targeting molecule is an immune checkpoint inhibitor.

[0056] In some embodiments, the targeting molecule is antibody or antibody fragment, small molecule or polypeptide.In some embodiments, the targeting molecule is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224 and MSB001078C, or any of the antigen-binding fragments thereof.

[0057] In some embodiments, a method of treating a tumor in a subject is provided, comprising: a) administering to the subject a conjugate comprising a phthalocyanine dye linked to a targeting molecule capable of binding to a cell surface molecule on cells in the tumor microenvironment; b) irradiating the tumor with a wavelength that renders the conjugate cytotoxic more than 5 minutes after administering the conjugate, wherein treatment of the tumor with the conjugate and subsequent light irradiation increases the presence of immunosuppressive cells in the tumor or increases the expression of an immunosuppressive marker in the tumor; and c) administering to the subject a therapeutically effective amount of an immunomodulatory agent capable of reducing the amount or activity of immunosuppressive cells in the tumor, or blocking the activity of an immunosuppressive marker or reducing the activity of tumor-promoting cells in the tumor, or blocking the activity of a tumor-promoting marker.

[0058] In some embodiments, the phthalocyanine dye is a first dye and the immunomodulatory agent comprises a conjugate comprising a second phthalocyanine dye conjugated to an immunomodulatory agent capable of binding to immunosuppressive or tumor-promoting cells, hi some embodiments, the first and second phthalocyanine dyes are the same or different.

[0059] In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28, and VISTA.

[0060] In some embodiments, the immunomodulatory agent is an antibody or antibody fragment, a small molecule, or a polypeptide.

[0061] In some embodiments, the immunomodulatory agent is not an anti-CTLA4 antibody.

[0062] In some embodiments, the immunomodulatory agent is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment thereof of any of the foregoing.

[0063] In some embodiments, a method of treating a tumor in a subject is provided, the method comprising: a) administering to the subject a conjugate comprising a phthalocyanine dye linked to a targeting molecule capable of binding to a molecule on the surface of a cell present in the tumor microenvironment; b) irradiating the tumor with a wavelength that renders the conjugate cytotoxic more than 5 minutes after administering the conjugate, wherein treatment of the tumor with the conjugate followed by light irradiation primes immune cell activation; and c) administering to the subject a therapeutically effective amount of an immunomodulatory agent capable of increasing immune cell activity.

[0064] In some embodiments, the immune cell is an antigen-presenting cell. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immunomodulatory agent is selected from GM-CSF, CpG-ODN (CpG oligodeoxynucleotide), lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), alum, recombinant Leishmania polyprotein, imiquimod, MF59, poly I:C, poly A:U, type 1 IFN, Pam3Cys, Pam2Cys, complete Freund's adjuvant (CFA), alpha-galactosylceramide, RC-529, MDF2β, loxoribine, anti-CD40 agonist, SIRPa antagonist, AS04, AS03, flagellin, resiquimod, DAP (diaminopimelic acid), MDP (muramyl dipeptide) and CAF01 (cationic adjuvant formulation-01). In some embodiments, the immunomodulatory agent is a Toll-like receptor (TLR) agonist, adjuvant or cytokine. In some embodiments, the immunomodulatory agent is a TLR agonist, and the TLR agonist is a TLR3 agonist, a TLR4 agonist, a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist. In some embodiments, the TLR agonist is selected from among triacylated lipoproteins, diacylated lipoproteins, lipoteichoic acid, peptidoglycan, zymosan, Pam3CSK4, dsRNA, poly I:C, poly G10, poly G3, CpG, 3M003, flagellin, lipopolysaccharide (LPS), Leishmania homolog of eukaryotic ribosome elongation and initiation factor 4a (LeIF), MEDI9197, SD-101, and imidazoquinoline TLR agonists.

[0065] In some embodiments, the immunomodulatory agent is a cytokine, and the cytokine is IL-4, TNF-α, GM-CSF or IL-2. In some embodiments, the immunomodulatory agent is administered before, simultaneously with or after irradiation. In some embodiments, the immunomodulatory agent is administered within 5 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours or 24 hours after irradiation. In some embodiments, the immunomodulatory agent is administered within 5 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours or 24 hours before irradiation.

[0066] In some embodiments, the targeting molecule binds directly or indirectly to a molecule or protein. In some embodiments, the targeting molecule is a second binding molecule that binds to a first binding molecule, and the first binding molecule is capable of binding to a molecule or protein. In some embodiments, the targeting molecule is a second antibody.

[0067] In some embodiments, the phthalocyanine dye has a maximum absorption wavelength of from 600 nm to about 850 nm or from about 600 nm to about 850 nm.

[0068] In some embodiments, the phthalocyanine dye is covalently or non-covalently linked to the targeting molecule. In some embodiments, the phthalocyanine dye comprises a linker that includes a reactive group for attachment of the dye to the targeting molecule.

[0069] In some embodiments, the phthalocyanine dye comprises the formula: TIFF2025015674000004.tif92128 formula, L is a linker; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; and X 2 and X 3 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 It is alkylene.

[0070] In some embodiments, the phthalocyanine dye comprises the formula: TIFF2025015674000005.tif90150 formula, X 1 and X 4 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 is alkylene; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy.

[0071] In some embodiments, the phthalocyanine dye comprises IRDye 700DX (IR700).

[0072] In some embodiments, the conjugate is at 50 mg / m 2 ~about 5000mg / m 2 or about 50 mg / m 2 ~about 5000mg / m 2 , about 250mg / m 2 ~about 2500mg / m 2 , about 750mg / m 2 ~about 1250mg / m 2 , or about 100 mg / m 2 ~about 1000mg / m 2 is administered at a dose of

[0073] In some embodiments, the tumor is a cancer. In some embodiments, the cancer is a cancer located in the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung. In some embodiments, the tumor is a sarcoma or carcinoma. In some embodiments, the tumor is a carcinoma that is a squamous cell carcinoma, basal cell carcinoma, or adenocarcinoma. In some embodiments, the tumor is a cancer that is a carcinoma of the bladder, pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, esophagus, or head and neck.

[0074] In some embodiments, the tumor is irradiated with at least 1 J cm at wavelengths between 600 nm and 850 nm. -2 Or at a dose of at least 1 J / cm of fiber length. In some embodiments, the tumor is irradiated at a wavelength of 690 nm ± 50 nm or at or about 690 ± 20 nm.

[0075] In some embodiments, the method reduces the size or volume of the tumor by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more within one month of irradiation compared to the size or volume of the tumor before administration and irradiation.

[0076] In some embodiments, a method of PIT treatment using a phthalocyanine dye conjugate (e.g., according to any of the methods described above or provided herein) results in an improvement in a disorder or cancer-related parameter in a population of treated subjects compared to a similar population of subjects treated with an unconjugated antibody or antigen-binding antibody fragment. In some embodiments, the parameter is selected from one or more of the following: a) objective response rate (ORR); b) progression-free survival (PFS); c) overall survival (OS); d) reduced toxicity; e) tumor response; or f) quality of life.

[0077] In some embodiments, the parameter is improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more.

[0078] In some embodiments, a method of PIT treatment using a phthalocyanine dye conjugate (e.g., according to any of the methods described above or provided herein) results in an objective response rate (ORR) in a population of treated subjects that is at least 15%, at least 25%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or greater.

[0079] In some embodiments, the conjugate comprises a phthalocyanine dye as a first dye and further comprises a second fluorescent dye linked to a targeting molecule different from the first dye. In some embodiments, the second fluorescent dye exhibits one or more spectral characteristics selected from among higher fluorescence quantum yield in water, extinction coefficient, Stokes shift, absorption and emission at long wavelengths, and photostability compared to the corresponding spectral characteristics of the first dye. In some embodiments, the lesion or tumor emits a fluorescent signal from the second fluorescent dye to achieve detection of the presence of the conjugate in the lesion or tumor in the subject. In some embodiments, the method provided further comprises imaging the lesion or tumor in the subject by irradiating or illuminating the tumor with a wavelength that can be absorbed by the second dye.

[0080] In some embodiments, the first dye is IR700. In some embodiments, the second dye is not IR700. In some embodiments, the second dye is hydroxycoumarin, Cascade Blue, Dylight 405, Pacific Orange, Alexa Fluor 430, fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2.7-Dichlorofluorescein, ATTO 488, Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo 642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700, Cy 5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705 and Qdot® 800.

[0081] In some embodiments, the first dye is IR700 and the conjugate contains 1-10 or 1-5 second dye molecules per targeting molecule.

[0082] In some embodiments, the second dye exhibits a Stokes shift that is greater than 15 nm, greater than 20 nm, greater than 30 nm, greater than 40 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, greater than 90 nm, or greater than 100 nm.

[0083] In some embodiments, the second dye has a quantum yield in water of greater than 10%, greater than 15%, greater than 20% or greater than 25%, greater than 30%, greater than 40%, greater than 50% or higher.

[0084] In some embodiments, the second dye has absorption and emission wavelengths in the spectrum between or between about 650 nm and 950 nm, between or between about 700 nm and 1000 nm, or between or between about 1000 nm and 1700 nm.

[0085] In some embodiments, the first dye and the second dye do not exhibit overlapping emission and absorption spectra. In some embodiments, the second dye is selected from among ICG, IRDye 680, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800 and IRDye 800. In some embodiments, the second dye is Alexa Fluor 488, IRDye 680, IRDye 800 or Dylight 755.

[0086] In some embodiments, the method further comprises illuminating or illuminating the tumor with a wavelength that the second dye can absorb, thereby imaging the subject. In some embodiments, illuminating or illuminating the tumor is performed using a device selected from among a handheld ultraviolet lamp, a mercury lamp, a xenon lamp, a laser, a laser diode, or an imaging device. In some embodiments, the LED imaging device comprises a near-infrared (NIR) diode.

[0087] In some embodiments, compositions are provided that contain a conjugate that contains a phthalocyanine dye linked to an antibody or antigen-binding antibody fragment that binds to a molecule on the surface of a cell present in a lesion microenvironment, e.g., a tumor microenvironment. In some embodiments, the composition is formulated for single dose administration of the conjugate in an amount that is between or about 100 mg to 1200 mg. In some embodiments, the composition is formulated for single dose administration in an amount between or about 100 mg to 500 mg, or between or about 200 mg to 400 mg. In some embodiments, the composition is formulated for single dose administration in an amount between or about 500 mg to 1500 mg, between or about 800 mg to 1200 mg, or between or about 1000 mg to 1500 mg.

[0088] In some embodiments, the volume of the composition is 10mL to 500mL or about 10mL to 500mL, or 50mL to 250mL or about 50mL to 250mL. In some embodiments, the volume of the composition is at least 10mL, 20mL, 30mL, 40mL, 50mL, 75mL, 100mL, 150mL, 200mL, 250mL, 300mL, 400mL or 500mL. In some embodiments, the volume of the composition is 10mL to 1000mL or about 10mL to 1000mL, or 50mL to 500mL or about 50mL to 500mL; or the volume of the composition is at least 10mL, 20mL, 30mL, 40mL, 50mL, 75mL, 100mL, 150mL, 200mL, 250mL, 300mL, 400mL, 500mL or 1000mL.

[0089] In some embodiments, an article of manufacture is provided that includes a plurality of sealed containers, each individually containing a portion of a single dose of a composition containing a conjugate that contains a phthalocyanine dye linked to an antibody or antigen-binding antibody fragment that binds to a molecule on the surface of a cell present in a lesion microenvironment, e.g., a tumor microenvironment. In some embodiments, the combined amount of the conjugate in the plurality of sealed containers is between 100 mg and 1500 mg, or about 100 mg and 1500 mg, or between 100 mg and 1200 mg, or about 100 mg and 1200 mg. In some embodiments, the article of manufacture contains packaging material and a label or insert that includes instructions for combining the contents of the plurality of vials to prepare a single dose formulation of the composition.

[0090] In some embodiments, the combined amount of the conjugates in the plurality of sealed containers is between or about 100 mg to 1200 mg. In some embodiments, the combined amount of the conjugates in the plurality of sealed containers is between or about 100 mg to 500 mg, between or about 200 mg to 400 mg, between or about 500 mg to 1500 mg, between or about 800 mg to 1200 mg, or between or about 1000 mg to 1500 mg.

[0091] In some embodiments, the lesion is a tumor.

[0092] In some embodiments, a conjugate is provided that includes a phthalocyanine dye linked to an antibody or antigen-binding fragment that is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor.

[0093] In some embodiments, the immunomodulatory agent is an antibody or antigen-binding fragment that binds to the surface of a tumor, tumor cell, or cancer cell. In some embodiments, the immunomodulatory agent specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28, and VISTA. In some embodiments, the immunomodulatory agent is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment thereof of any of the foregoing. In some embodiments, the immunomodulatory agent is an antibody or antibody fragment that binds to PD-L1.

[0094] In some embodiments, the immunomodulatory agent is an antibody selected from BMS-935559, MEDI4736, MPDL3280A, and MSB0010718C, or an antigen-binding fragment thereof.

[0095] In some embodiments, a conjugate is provided that contains a targeting molecule linked to at least a first and a second fluorescent dye. In some embodiments, the first fluorescent dye is a phthalocyanine dye capable of exhibiting phototoxicity.

[0096] In some embodiments, the conjugate has the formula: [D 1 -(L 1 ) n ] p -A-[(L 2 ) m -D 2 ] o During the ceremony, A is a targeting molecule capable of binding to a molecule on the surface of a cell; L 1 and L 2 are each independently selected linkers, which can be the same or different; n and m are independently 1 or 2; D 1 is a first dye that is a phthalocyanine dye capable of exhibiting phototoxicity; D 2 is a second dye that is a fluorescent dye, where D 2 D 1 Unlike; p is 1 to 10; and o is a number between 1 and 10.

[0097] In some embodiments, the targeting molecule is an antibody or an antigen-binding antibody fragment.

[0098] In some embodiments, the cell surface molecule comprises an antigen, a polypeptide, a lipid, or a carbohydrate, or a combination of these molecules.

[0099] In some embodiments, the cell surface molecule is ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptor BB1, BB2, BB4, calcitonin receptor, cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, Crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endosialin, EP2, EP4 , EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, family A G protein-coupled receptor (rhodopsin-like), family B G protein-coupled receptor (secretin receptor-like), family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV 16 / 18 and E6 / E7 antigens, hTERT, IL11-R, IL-13R, ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE 1, MAGE 2, MAGE 3, MAGE4, MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease cleaved collagen IV, proteinase 3, prohibitin, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 patched, RET receptor, plexin, smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptor (VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, and VEGF-3 or FLT-4), voltage-gated ion channel, VPAC1, VPAC2, Wilms' tumor 1, Y1, Y2, Y4, and Y5.

[0100] In some embodiments, the cell surface molecule is HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72, Caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), endothelial cell proliferation and / or proliferation (IFN-γ ... GF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate binding protein, gangliosides (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinase, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, and SK-1 antigen.

[0101] In some embodiments, the cell surface molecule is CD25, PD-1 (CD279), PD-L1 (CD274, B7-H1), PD-L2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3 (HAVCR2), 4-1BB (CD137, TNFRSF9), CXCR2, CXCR4 (CD184), CD27, CEACAM1, Galectin 9, BTLA, CD160, VISTA (PD1 homologue), B7-H4 (VCTN1), CD80 (B7-1), CD86 (B7-2), CD28, HHLA2 (B7-H7), CD28H, CD155, C D226, TIGIT, CD96, Galectin 3, CD40, CD40L, CD70, LIGHT(TNFSF14), HVEM(TNFRSF14), B7-H3(CD276), Ox40L (TNFSF4), CD137L (TNFSF9, GITRL), B7RP1, ICOS (CD278), ICOSL, KIR, GAL9, NKG2A (CD94), GARP, TL1A, TNFRSF25, TMIGD2, BTNL2, butyrophilin family, CD48, CD244, siglec family, CD30, CSF1R, MICA (MHC class I polypeptide-related sequence A), MICB (MHC class I polypeptide-related sequence B), NKG2D, KIR family (killer cell immunoglobulin-like receptor, LILR family (leukocyte immunoglobulin-like receptor, CD85, ILT, LIR), SIRPA (signal regulatory protein alpha), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR, and VEGF.

[0102] In some embodiments, the targeting molecule is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), basiliximab, nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, M The antibody or antigen-binding fragment is selected from among K-4166, dacetuzumab, lucatumumab, SEA-CD40, CP-870, CP-893, MED16469, MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C, rHIgM12B7, urocupulumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, emactuzumab, CC-90002, and MNRP1685A, or an antigen-binding fragment thereof.

[0103] In some embodiments, the targeting molecule is not or does not include a nanocarrier. In some embodiments, the targeting molecule is not or does not include a virus-like particle, a nanoparticle, a liposome, a quantum dot, or a combination thereof.

[0104] In some embodiments, the first dye is a phthalocyanine dye having a maximum absorption wavelength of 600 nm to 850 nm or from about 600 nm to about 850 nm.

[0105] In some embodiments, the first dye, which is a phthalocyanine dye, comprises the formula: TIFF2025015674000006.tif92128 formula, L is a linker; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; and X 2 and X 3 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 It is alkylene.

[0106] In some embodiments, the first dye, which is a phthalocyanine dye, comprises the formula: TIFF2025015674000007.tif90150 formula, X 1 and X 4 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 is alkylene; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy.

[0107] In some embodiments, the first dye that is a phthalocyanine dye comprises IRDye 700DX (IR700).

[0108] In some embodiments, the second fluorescent dye exhibits one or more spectral properties selected from among higher fluorescence quantum yield in water, extinction coefficient, Stokes shift, absorption and emission at longer wavelengths, and photostability compared to the corresponding spectral properties of the first dye.

[0109] In some embodiments, the second dye is not IR700. In some embodiments, the second dye is hydroxycoumarin, Cascade Blue, Dylight 405, Pacific Orange, Alexa Fluor 430, fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2.7-dichlorofluorescein, ATTO 488, Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo 642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700, Cy 5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705 and Qdot® 800.

[0110] In some embodiments, the first dye is IR700 and the conjugate contains 1-10 or 1-5 second dye molecules per targeting molecule.

[0111] In some embodiments, the second dye exhibits a Stokes shift that is greater than 15 nm, greater than 20 nm, greater than 30 nm, greater than 40 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, greater than 90 nm, or greater than 100 nm.

[0112] In some embodiments, the second dye has a quantum yield in water of greater than 10%, greater than 15%, greater than 20% or greater than 25%, greater than 30%, greater than 40%, greater than 50% or higher.

[0113] In some embodiments, the second dye has absorption and emission wavelengths in the spectrum between or between about 650 nm and 950 nm, between or between about 700 nm and 1000 nm, between or between about 1000 nm and 1700 nm.

[0114] In some embodiments, the first dye and the second dye do not exhibit overlapping emission and absorption spectra.

[0115] In some embodiments, the second dye is selected from among ICG, IRDye 680, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800 and IRDye 800. In some embodiments, the second dye is Alexa Fluor 488, IRDye 680, IRDye 800 or Dylight 755.

[0116] In some embodiments, a composition is provided that contains any of the conjugates described herein. In some embodiments, the composition further contains a pharma- ceutically acceptable excipient.

[0117] In some embodiments, a method of treating a disease or condition in a subject is provided, the method comprising the steps of: a) administering to the subject a therapeutically effective amount of any of the conjugates or compositions described herein, wherein the conjugate binds to cells present in the microenvironment of a lesion associated with the disease or condition; and b) after administering the conjugate, irradiating the lesion with one or more wavelengths that induce phototoxic activity of the conjugate, thereby treating the disease or condition.

[0118] In some embodiments, a method is provided for treating a disease or condition, such as a tumor, in a subject using photoimmunotherapy (PIT), comprising administering to the subject a therapeutically effective amount of any of the conjugates or compositions described herein. In some embodiments, the method provides for the administration of at least 1 J cm at wavelengths between 660 nm and 740 nm to the tumor. -2 or irradiating with a dose of 1 J / cm of fiber length, thereby treating the disease or condition in the subject.

[0119] In some embodiments, a method of treating a disease or condition in a subject is provided, comprising: a) administering to the subject a therapeutically effective amount of any of the conjugates or compositions described herein, wherein the conjugate binds to cells present in the microenvironment of a lesion associated with the disease or condition; and b) after administering the conjugate, irradiating the lesion with one or more wavelengths that induce phototoxic activity of a first dye of the conjugate and a fluorescent signal of a second dye of the conjugate.

[0120] In some embodiments, the methods provided provide for delivering at least 1 J cm to the lesion at a wavelength that is between 400 and 900 nm or between about 400 and about 900 nm. -2or 1 J / cm fiber length. In some embodiments, the methods provided include irradiating the lesion with a single wavelength. In some embodiments, the methods provided include irradiating the lesion with two different wavelengths, either simultaneously or sequentially, where one wavelength induces phototoxic activity and another wavelength induces a fluorescent signal.

[0121] In some embodiments, the disease or condition is a tumor.

[0122] In some embodiments, the methods provided provide for delivering at least 1 J cm at wavelengths between 660 nm and 740 nm to a tumor. -2 to irradiate the tumor in the subject with a dose of 0.1 mg / kg / dose, thereby treating the tumor in the subject.

[0123] In some embodiments, the tumor is a cancer. In some embodiments, the cancer is a cancer located in the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung. In some embodiments, the tumor is a sarcoma or carcinoma. In some embodiments, the tumor is a carcinoma that is a squamous cell carcinoma, basal cell carcinoma, or adenocarcinoma. In some embodiments, the tumor is a cancer that is a carcinoma of the bladder, pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, esophagus, or head and neck.

[0124] In some embodiments, the targeting molecule is administered to the subject prior to administration of the conjugate. In some embodiments, the targeting molecule is administered up to 96 hours prior to administration of the conjugate. In some embodiments, the targeting molecule is administered at a concentration of 10 mg / m 2 ~500mg / m 2 or about 10 mg / m 2 ~about 500mg / m 2 In some embodiments, the targeting molecule is an antibody or antigen-binding fragment, such as cetuximab. In some examples, cetuximab is administered to the subject prior to administration of the conjugate. [Brief description of the drawings]

[0125] [Figure 1] 1 shows dose response curves of cetuximab and cetuximab-IRDye 700DX conjugate for inhibition of EGFR phosphorylation in A431 cells. [Diagram 2] 1 shows a dose response curve for photoimmunotherapy (PIT) with cetuximab-IRDye 700DX conjugate using 8 J / cm2 of 690 nm light on A431 cells. [Diagram 3] FIG. 1 shows a comparison of dose response curves for photoimmunotherapy (PIT) with cetuximab-IRDye 700DX conjugate using 16 J / cm2 versus 32 J / cm2 of 690 nm light on BxPC3 cells. [Figure 4] FIG. 1 shows a schematic structure for the dual-labeled panitumumab-IR700DX-Alexa-488 conjugate. [Diagram 5] Figure 5 shows the progression of cell death after photoimmunotherapy (PIT) induced by different conjugates at various light doses (0-16 J / cm2) on BxPC3 pancreatic cancer cells. Figure 5A shows cell death after PIT using a single-labeled panitumumab-IR700 conjugate. Figure 5B shows cell death after PIT using a dual-labeled panitumumab-IR700-Alexa-488 conjugate. [Figure 6A] 13 shows the duration of exposure of cetuximab-IRDye 700DX to 500 lux white fluorescent or green LED light and its effect on soluble aggregate formation. [Figure 6B] 13 shows the effect of pre-exposure of Cetuximab-IRDye 700DX to white fluorescent or green LED light on BxPC3 PIT activity. [Figure 6C] FIG. 1 shows the effect of percent of Cetuximab-IRDye 700DX soluble aggregate formation on PIT activity. [Figure 7] Shown is PIT killing activity with sequential staining using cetuximab and donkey anti-human-IRDye 700DX (DxHu IR700) secondary antibody. [Figure 8A] FIG. 1 shows light-dependent killing of BxPC3 cells using biotinylated cetuximab pre-complexed with monomeric streptavidin-IRDye 700DX (mSA IR700). [Figure 8B] 1 shows the specificity of PIT using biotinylated cetuximab pre-complexed with monomeric streptavidin-IRDye 700DX (mSA IR700). [Figure 8C] FIG. 13 shows the effect of white light pre-exposure monomeric streptavidin-IRDye 700DX on PIT killing activity with biotinylated cetuximab in BxPC3 cells. [Figure 9] (A) shows antibody dose-dependent killing of 4T1 cells with directly conjugated anti-EpCAM-IRDye 700DX. (B) shows specificity of PIT killing activity of anti-EpCAM-IRDye 700DX. [Figure 10] 1 shows Fc receptor-specific killing of THP1 cells by cetuximab-IRDye 700DX. [Figure 11A] 13 shows the specificity of light-dependent killing of EGF-IRDye 700DX in A431 cells. [Figure 11B] 1 shows the effect of EGF-IRDye 700DX pre-exposed to different types of light on light-dependent killing in A431 cells. [Figure 12A] 1 shows light-dependent killing of BxPC3 cells using cholera toxin B-IRDye 700DX. [Figure 12B] 1 shows the specificity of photoactivated killing of cholera toxin B-IRDye 700DX. [Figure 12C] FIG. 1 shows the effect of pre-exposure of cholera toxin B-IRDye 700DX to different wavelengths of light on photoactivated killing in BxPC3 cells. [Figure 13] A shows light-dependent killing of Vero cells with Influenza virus (X-31)-IRDye 700DX. B shows the effect of pre-exposure of Influenza virus (X-31)-IRDye 700DX to white light versus green light on light-activated cell killing. [Figure 14] A shows the effect of light dose on the killing activity of SNA-IRDye 700DX in BxPC3 cells.B shows the effect of sialidase treatment on the specificity of SNA-IRDye 700DX binding to cells. [Figure 15] 1 shows PIT killing of S. aureus by cetuximab-IRDye 700DX in combination with laser irradiation. [Figure 16] 1 shows PIT of influenza virus particles using mouse anti-influenza virus (H3N2) pre-complexed with GtxMs Fab-IRDye 700DX. [Figure 17] FIG. 1 shows light-dependent killing of influenza virus-infected cells using mouse anti-influenza virus (H3N2) and goat anti-mouse IRDye 700DX (GtxMs-IR700). [Figure 18] 1 shows PIT killing of rat embryonic dorsal root ganglion (DRG) neurons using cholera toxin B-IRDye 700DX. [Figure 19A] The effect of IFN-gamma treatment on the mortality of BxPC3 cells is shown. [Figure 19B] 1 shows the effect of IFN-gamma treatment on PD-L1 expression in BxPC3 cells. [Figure 19C] Shows the effect of IFN gamma treatment on PIT killing activity of anti-PD-L1 IRDye 700DX in BxPC3 cells. [Figure 20A] 1 shows the effect of PIT treatment in A431 and FaDu cells using Cetuximab-IRDye 700DX on the amount of HMGB1 detected in the extracellular solution. [Figure 20B] 1 shows upregulation of dendritic cell (DC) maturation markers on immature dendritic cells (iDCs) co-cultured with tumors subjected to cetuximab-IRDye 700DX-mediated PIT. [Figure 20C]1 shows the effect of activating antigen presenting cells by A431 cells or FaDu cells treated with PIT (treated with Cetuximab-IRDye 700DX and in the presence of light irradiation) or A431 cells or FaDu cells not treated with PIT (treated with Cetuximab-IRDye 700DX but without light irradiation) as assessed by expression of the exemplary activation marker CD86. [Figure 21] FIG. 1 shows the effect on dendritic cell activation by priming dendritic cells with PIT-treated tumor cells (treated with Cetuximab-IRDye 700DX) or non-PIT-treated tumor cells (treated with Cetuximab-IRDye 700DX but without light irradiation) and subsequently stimulating them with an immunomodulatory agent (Poly I:C), as assessed by the expression of exemplary activation markers. [Figure 22] A shows the UV-Vis spectrum of Cetuximab-IRDye 700DX-Alexa Flour 488 (CTX700-ALX488), B shows the UV-Vis spectrum of CTX-700-IRDye 800CW, and C shows the UV-Vis spectrum of CTX700-Dylight 755. [Diagram 23] A shows the PIT killing activity of cetuximab-IRDye 700DX with or without DyLight 755 in BxPC3 cells at various concentrations and light doses. B shows the PIT killing activity of cetuximab-IRDye 800CW, cetuximab-Alexafluor488, or cetuximab-IRDye 700DX conjugated or not to IRDye 800CW or Alexafluor488 in BxPC3 cells. The results are shown as the percentage of PIT activity compared to cetuximab-IRDye 700DX (control). [Figure 24]Figure 1 shows the effect of pre-exposure of cetuximab-IRDye 700DX, cetuximab-IRDye 680RD, and cetuximab-IRDye 700+IRDye 680RD dual conjugates to white or green light on soluble aggregate formation (A) and Figure 2 shows the effect of pre-exposure of cetuximab-IRDye 700DX, cetuximab-IRDye 680RD, and cetuximab-IRDye 700+IRDye 680RD dual conjugates to white or green light on fluorescence normalized to monomer content (B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0126] Detailed Description I. Photoimmunotherapy Methods Conjugates, compositions, combinations and methods related to photoimmunotherapy (PIT) are provided herein.Photoimmunotherapy is a molecular targeted therapy that utilizes a target-specific photosensitizer based on a phthalocyanine dye, such as a near-infrared (NIR) phthalocyanine dye (e.g., IR700), conjugated to a targeting molecule that targets a protein, such as a cell surface protein, on a cell in a disease or pathology, such as a cell in a tumor.For example, in some cases, the phthalocyanine dye-conjugate used in photoimmunotherapy can include conjugation to a monoclonal antibody (mAb) that targets a cell surface protein receptor or a receptor expressed on a cell in the environment of a disease lesion, such as a cell in the tumor microenvironment (which can include tumor cells and other inflammatory cells).In some embodiments, activation of the dye-conjugate by irradiating it with absorbing light, such as NIR light, excites the photosensitizer, resulting in cell killing, thereby reducing or eliminating the lesion (e.g., tumor) to treat the disease or pathology. In some cases, the use of light in the NIR range leads to deeper tissue penetration, resulting in successful eradication of tumors with only a single dose of external NIR light irradiation.

[0127] In general, targeted phototoxicity appears to be mainly dependent on the binding of dye-conjugates to cell membranes via specific targeting molecules (e.g., macromolecules such as antibodies).For example, studies using exemplary antibody-IR700 molecules have shown that the conjugates must bind to cell membranes to be active, and that cell killing does not require intracellular localization to be effective (see, for example, U.S. Patent No. 8,524,239 and U.S. Published Application No. US20140120119).Photoactivation of cells to which the conjugates are bound results in rapid cell death and necrosis.

[0128] Typically, PIT mainly results in cell death in cells to which the phthalocyanine dye conjugate, such as IR700-antibody conjugate, binds after irradiating cells with NIR, while only a small number of cells that do not express the cell surface protein recognized by the targeting molecule (e.g., antibody) are killed. Thus, the therapy is specifically targeted to diseased cells, such as cells in a tumor, so that its effect is highly selective to diseased tissues compared to healthy tissues or cells. For example, targeted photosensitizers can be distributed throughout the body, but the photosensitizer is only active when strong light is applied, reducing the possibility of off-target effects. This is in contrast to non-PIT-based methods, where the activity of similar therapeutic targeting molecules (e.g., therapeutic antibodies) that are not conjugated to photosensitizers (e.g., IR700) cannot be localized, thereby resulting in significant risk of off-target side effects. In some embodiments, the phototoxic agent is a phthalocyanine dye-targeting molecule conjugate. In some embodiments, the phthalocyanine dye is IR700.

[0129] In some embodiments, the methods provided include administering a phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate) at a dosage that achieves a much lower exposure than would otherwise be required to achieve a therapeutic effect of a targeting molecule (e.g., an antibody) that is not conjugated to a phthalocyanine dye. Because PIT requires binding of the conjugated targeting molecule (e.g., an antibody) to a cell surface protein to mediate cell killing, it was thought that receptor occupancy would be a factor that correlates with the degree of PIT activity. Thus, it was thought that a similar or even higher systemic exposure of the conjugate would be required to ensure exposure at the lesion (e.g., tumor) site that achieves sufficient cell killing as is necessary to achieve a clinically acceptable therapeutic effect of a targeting molecule (e.g., an antibody) that is not so conjugated (i.e., not conjugated to a photosensitizer, e.g., IR700). For example, the dose of Erbitux® (cetuximab) approved by the U.S. Food and Drug Administration (FDA) for treating head and neck cancer is 400 mg / m 2 followed by an initial dose of 250 mg / m 2 Erbitux 400mg / m 2 The average systemic exposure (AUC, e.g., AUC 0-inf or AUC[0-inf]) was approximately 24,620 μg*h / mL, and after 1 month of treatment (400 mg / m 2 followed by an initial dose of 250 mg / m 2 The total systemic exposure for a 4-week dose of 100 mg / kg / month (1x4,620 + 3x11,812 μg*h / mL) is estimated to be approximately 60,056 μg*h / mL (cumulative systemic exposure for 4 weekly doses in 1 month, 1x24,620 + 3x11,812 μg*h / mL) (Fracasso et al. (2007) Clin. Cancer. Res., 13:986).

[0130] However, the AUC 0-inf ) or 770+ / -47.5(AUC 0-24It is found herein that cell killing by PIT could be observed after administration of a single dose of the cetuximab-IR700 conjugate in an amount that achieved a mean systemic exposure of 400 mg / m 2 This is a maximum of 13.6 or approximately 13.6-fold less systemic exposure observed following a clinical therapeutic dose of Erbitrux® at 100 mg / kg / day. Furthermore, as shown in Example 2, the results show that the mean systemic exposure (AUC 0-24 ) but at a higher dose of 640 mg / m 2 Even at 400 mg / m 2 The results showed that the AUC for Erbitux® was approximately 15% of that for Erbitux® (3,690 μg / mL*h vs. 24,740 μg / mL*h, respectively). The results further showed that patients with head and neck cancer (including those who had failed other treatments) had a 100% overall response rate (ORR) to the single-dose treatment (all patients had a complete or partial response to the treatment), demonstrating a rapid and robust response that was surprising given the low systemic exposure. In comparison, the ORR for subjects treated with Erbitux® monotherapy is usually around 15% or less, even with the multiple doses required for continuous exposure.

[0131] Moreover, most treatments with Erbitux® include combination therapy with one or more chemotherapy treatments or other anticancer treatments. As an example, in one reported study, the increase in ORR with cetuximab in combination with chemotherapy compared to chemotherapy alone was only about 16%, which is similar to the ORR provided by cetuximab monotherapy (see, e.g., Specenier and Vermorken (2013) Biologics, 7:77-90).

[0132] Thus, the provided method is based on the observation that the efficacy of phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate) for mediating PIT and cell killing is sufficiently high even when the systemic exposure of the conjugate is low. Thus, the provided method includes administration of the conjugate at a dose that achieves a systemic exposure that is a fraction of the exposure of the clinical dose in humans of the corresponding therapeutic targeting molecule (e.g., therapeutic antibody) when not so conjugated to the phthalocyanine dye (e.g., IR700). In some embodiments, the clinical dose in humans of the therapeutic molecule is the dose as described on the label approved for commercialization by regulatory agencies (e.g., FDA, EMA, PDMA). In some embodiments, the low systemic exposure achieved by such a dose of the phthalocyanine dye-targeting molecule conjugate means that the systemic exposure of the targeting molecule is not high enough to show pharmacological activity unless PIT is induced by exposure of the conjugate to light. Because the light dose can be localized to the disease lesion (e.g., tumor), the provided methods can achieve selective cell killing of only the disease lesion (e.g., tumor), while avoiding unwanted or undesirable off-target activity.

[0133] In addition to the efficacy of the response provided by the PIT method provided herein, the provided method is also particularly advantageous for treating diseases or conditions (e.g., tumors) where current therapies, such as current antibody therapies, are prone to adverse off-target side effects. For example, in some cases, it is difficult to achieve strong anti-cancer activity in a safe range using treatments that include immunomodulators. This is because, in some cases, such immunomodulators inhibit (or enhance) the same mechanisms that the body uses to combat autoimmunity. As a result, in many cases, the administration of immunomodulators can result in significant therapeutic side effects. For the safety of many immunomodulators (and other therapeutic antibodies), acceptable doses must be administered in dosage cycles to achieve or maintain continuous systemic exposure thresholds without compromising therapeutic efficacy, and / or must be administered in combination with other chemotherapeutic or anti-cancer agents that have even higher risk of adverse side effects. The provided method solves these problems, since PIT-based conjugates containing therapeutic targeting molecules (including immunomodulators and other anti-tumor antibody molecules) can be administered at doses that avoid or minimize high systemic exposure, while allowing selective cell killing at the site of disease or lesion. Therefore, the advantages of low dosing and / or low systemic exposure are highly significant in achieving safety.

[0134] In some embodiments, the fluorescent properties of the phthalocyanine dye also allow for monitoring of PIT therapy using any of a number of imaging or other methods capable of detecting fluorescent signals. In some embodiments, fluorescence assessment, such as by imaging, can be used to monitor the upload or presence of the conjugate in the lesion (e.g., tumor) prior to PIT. In some embodiments, fluorescence assessment, such as by imaging, can be used to illuminate the lesion (e.g., tumor) to ensure that PIT is directed to the site of the lesion (e.g., tumor). In some embodiments, fluorescence assessment can be used in a surgical setting, where the edge of the lesion (e.g., tumor) can be visualized with fluorescence, and then residual cancer cells at the edge can be killed with PIT.

[0135] In some embodiments, the conjugate further contains an additional fluorescent dye in addition to the phthalocyanine dye (e.g., IR700). For example, in some cases, IR700 is not one of the most ideal dyes for imaging. This is because, for example, it exhibits one or more spectral characteristics that may not be suitable for ensuring specific and efficient fluorescent labeling. In some aspects, other fluorophores that are commonly used to label proteins can exhibit higher fluorescence quantum yield, larger Stokes shift, larger extinction coefficient at the excitation wavelength and / or longer wavelength for deeper tissue penetration. Thus, in some embodiments, a dual-labeled conjugate is provided in which a targeting molecule (e.g., an antibody or an antigen-binding antibody fragment) is conjugated to a first dye that is a phthalocyanine dye (e.g., IR700) and is also conjugated to another second fluorescent dye that exhibits one or more improved or better properties from among higher quantum yield, larger Stokes shift, larger extinction coefficient and / or longer wavelength compared to the first dye. In some embodiments, the dual-labeled conjugate can be used to monitor PIT therapy as described above, or to treat with PIT by activating the phthalocyanine dye, e.g., activating IR700. In some embodiments, the first and second dyes are selected to minimize energy transfer between them, e.g., the first and second dyes are selected to avoid or minimize overlapping emission and absorption spectra.

[0136] In some embodiments, the present specification also provides a combination therapy for use in conjunction with photoimmunotherapy to treat disease or pathology in a subject.In some embodiments, the combination therapy can be used in a method for treating tumor or cancer.In some embodiments, the combination of photoimmunotherapy and administration of additional therapeutic agent, such as immunomodulatory agent, anticancer agent or other agent, can increase the effectiveness of treating tumor, which in some cases can increase the therapeutic outcome or survival of the treated subject.

[0137] In some aspects, the combination therapy methods provided utilize the cytotoxic killing and / or lysis effect induced by PIT to enhance the therapeutic outcome associated with tumor treatment.In certain aspects, one or more additional therapeutic agents can be administered to a tumor-bearing subject prior to completing PIT (performed by irradiating light to activate phthalocyanine dye-conjugate).In some embodiments, the pre-administration of additional therapeutic agents can prime the tumor microenvironment to be more sensitive to PIT or to additional therapeutic agents after subsequent irradiation of the tumor.

[0138] For example, in one embodiment, the additional therapeutic agent can be an immunomodulatory agent, which in some aspects is administered prior to irradiation to enhance immune response to PIT-induced tumor-associated agents released from lysed cells. In another example, the additional therapeutic agent can be an anti-cancer agent, which in some aspects is administered prior to irradiation to increase the systemic availability of the anti-cancer agent and enhance delivery or uptake of the anti-cancer agent to the tumor area in response to changes in tumor permeability induced by PIT. In some embodiments, the enhanced therapeutic results from the combination therapy can result in an increased reduction in tumor size (e.g., tumor volume or weight), or increased or longer survival of the subject, compared to a method that includes treatment with either treatment alone. In some embodiments, the therapeutic effect of the combination therapy can be synergistic, compared to a treatment method that includes treatment with phthalocyanine dye-conjugate / PIT alone, or a treatment that includes an additional therapeutic agent alone, such as treatment with only an immunomodulatory agent or only an anti-cancer agent.

[0139] A. Conjugates containing phthalocyanine dyes and targeting molecules The methods, compositions and combinations provided herein include a conjugate that contains a photosensitizer, such as a phthalocyanine dye, such as IR700, and a targeting molecule (e.g., an antibody or an antigen-binding fragment of an antibody) that binds to a cell surface protein. In some embodiments, the binding of the targeting molecule conjugated to a photosensitizer, such as a phthalocyanine dye (e.g., IR700), to a cell surface protein allows the conjugate to be targeted to cells involved in a disease or condition, such as a tumor or cancer, an infectious disease, an inflammatory disease or condition, a neurological disease or condition, or other disease or condition. In some embodiments, the targeted cell (e.g., a cell that expresses a cell surface protein that the targeting molecule can bind to) is present in the microenvironment of a lesion associated with a disease or condition, for example, the cell is present in a tumor microenvironment. In some embodiments, cell targeting increases the efficacy of PIT induced by localized irradiation of a lesion (e.g., a tumor) of a subject at a wavelength absorbed by the phthalocyanine dye (e.g., near-infrared (NIR) wavelengths), since cell killing is selective to cells to which the dye-targeting molecule conjugate is bound.

[0140] In some embodiments, the phthalocyanine dye conjugates for use in combination therapy provided herein comprise a dye molecule conjugated to a targeting molecule via a linker group. In one aspect, the conjugate is a conjugate of formula I: A-[(L) n -D] p (I) During the ceremony, A is a targeting molecule capable of binding to a cell or tissue; L is a linker selected independently for each p; n is 1 or 2; D is a hydrophilic phthalocyanine dye independently selected for each p; and p is independently 1, 2, 3, 4, 5 or more than 5, such as up to 1000. For example, p can be from 1 to 1000, such as typically from 1 to 10 or from 2 to 5.

[0141] In some embodiments, the phthalocyanine dye conjugate is produced by a method or process in which a phthalocyanine dye-targeting molecule conjugate, such as an IR700-targeting molecule (e.g., IR700-antibody) conjugate, is prepared under light-protected conditions. In some embodiments, the method includes 1) preparing or providing a phthalocyanine dye and a targeting molecule; 2) contacting the targeting molecule with the phthalocyanine dye under conditions that produce a conjugate with minimal exposure of the dye; and 3) formulating, purifying and / or isolating the conjugate to produce a composition containing a drug substance, where one or more of the steps, such as all of the steps in some cases, are performed with minimal exposure of the dye or the conjugate containing the dye to ambient light. In some embodiments, the phthalocyanine dye-targeting molecule conjugate, such as an IR700-targeting molecule (e.g., an IR700-antibody) conjugate, is a conjugate or is prepared using a method for producing a conjugate as described in U.S. Application No. 62 / 206,774, which is incorporated herein by reference.

[0142] 1. Phthalocyanine dyes Phthalocyanines are a group of photosensitizer compounds that have a phthalocyanine ring system. Phthalocyanines are azaporphyrins (i.e., C 32 H 16 N 8), which form stable chelates with metal and nonmetal cations. In these compounds, the ring center is occupied by a metal ion (either a diamagnetic or paramagnetic ion) which may bear one or two ligands depending on the ion. In addition, the ring periphery may be unsubstituted or substituted. The synthesis of a wide variety of phthalocyanines and their use in photodynamic therapy are described in International Publication WO 2005 / 099689 and U.S. Patent No. 7,005,518.

[0143] In some embodiments, phthalocyanines strongly absorb red or near infrared light, with an absorption peak between about 600 nm and 810 nm, which in some cases allows deep tissue penetration by light. Phthalocyanines are generally photostable. This photostability is typically advantageous in pigments and dyes, as well as in many of the other applications of phthalocyanines.

[0144] In some embodiments, the phthalocyanine dye is water-soluble and contains a luminescent fluorophore moiety with at least one water-solubilizing moiety. In some embodiments, the water-solubilizing moiety contains silicon. In some embodiments, the phthalocyanine dye has a core atom such as Si, Ge, Sn, or Al. In some embodiments, the phthalocyanine dye exists as a single core isomer essentially free of other isomers. In some embodiments, the phthalocyanine dye contains a linker with a reactive or activatable group that can form a bond between the linker and a targeting molecule. In some embodiments, the phthalocyanine dye can be tuned to fluoresce at a specific wavelength.

[0145] In some embodiments, the phthalocyanine dye contains a linker, i.e., a linker-phthalocyanine dye moiety (LD). In some embodiments, the linker contains a reactive group. In some embodiments, the phthalocyanine dye is a phthalocyanine dye of formula Ia: TIFF2025015674000008.tif99128In formula, L is selected from a direct linkage or a covalent linkage; Q is a reactive or activatable group, which is any group that can be part of the linker L and that can react to form a bond between L and the targeting molecule A; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, or a chelating ligand, if present, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each a functional group which may be independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted alkoxy; Or, in an alternative embodiment, i) R 13 and R 14 and the carbon to which they are attached, or ii) R 17 and R 18and the carbon to which they are attached, or iii) R 21 and R 22 and at least one of the carbons to which they are attached combine to form a fused ring; and X 2 and X 3 each independently represents a C which may be interrupted by a heteroatom 1 -C 10 It is alkylene.

[0146] In some embodiments, L is a covalent linkage. In some embodiments, the covalent linkage is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-60 atoms, such as 1-45 atoms or 1-25 atoms. In some cases, such atoms can be selected from C, N, P, O, and S. In some embodiments, L can have additional hydrogen atoms (in addition to the 1-60 atoms) to satisfy the valence. In general, the linkage comprises any combination of ether, thioether, amine, ester, carbamate, urea, thiourea, oxy, or amide bonds; or single, double, triple, or aromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds; or aromatic or heteroaromatic bonds.

[0147] In some embodiments, L is of the formula -R 1 -YX 1 -Y 1 -L, where R 1 is a divalent radical or a direct link; Y and Y 1 are each independently selected from a direct bond, oxygen, optionally substituted nitrogen, or sulfur; and X 1 is a direct bond and may be an intervening C 1 -C 10 Divalent radicals include, but are not limited to, optionally substituted alkylene, optionally substituted alkyleneoxycarbonyl, optionally substituted alkylenecarbamoyl, optionally substituted alkylenesulfonyl, and optionally substituted arylene.

[0148] Example R 1 Substituents include, but are not limited to, optionally substituted alkylene, optionally substituted alkyleneoxycarbonyl, optionally substituted alkylenecarbamoyl, optionally substituted alkylenesulfonyl, optionally substituted alkylenesulfonylcarbamoyl, optionally substituted arylene, optionally substituted arylenesulfonyl, optionally substituted aryleneoxycarbonyl, optionally substituted arylenecarbamoyl, optionally substituted arylenesulfonylcarbamoyl, optionally substituted carboxyalkyl, optionally substituted carbamoyl, optionally substituted carbonyl, optionally substituted heteroarylene, optionally substituted heteroaryleneoxycarbonyl, optionally substituted heteroarylenecarbamoyl, optionally substituted heteroarylenesulfonylcarbamoyl, optionally substituted sulfonylcarbamoyl, optionally substituted thiocarbonyl, optionally substituted sulfonyl, and optionally substituted sulfinyl.

[0149] In some embodiments, Q comprises a reactive group for optional attachment to a material such as a targeting molecule. As used herein, the term "reactive group" refers to a moiety on a compound that can chemically react with a functional group on a different material (e.g., a targeting molecule) to form a linkage, such as a covalent linkage. Typically, the reactive group is an electrophile or a nucleophile that can form a covalent linkage through exposure to a corresponding functional group that is a nucleophile or an electrophile, respectively. Alternatively, the reactive group is a photoactivatable group and only becomes chemically reactive after irradiation with light of an appropriate wavelength. Typically, the conjugation reaction between the conjugated reactive dye and the targeting molecule incorporates one or more atoms of the reactive group Q into a new linkage that attaches the dye to the conjugated targeting molecule.

[0150] In some embodiments, Q comprises a reactive group that is reactive with a carboxyl group, an amine, or a thiol group on the targeting molecule. Suitable reactive groups include, but are not limited to, activated esters, acyl halides, alkyl halides, anhydrides, carboxylic acids, carbodiimides, carbonates, carbamates, haloacetamides (e.g., iodoacetamides), isocyanates, isothiocyanates, maleimides, NHS esters, phosphoramidites, platinum complexes, sulfonate esters, and thiocyanates for any attachment to the targeting molecule. In some embodiments, the reactive group is reactive with a carboxyl group, an amine, or a thiol group on the targeting molecule. In some embodiments, the reactive group is a sulfhydryl-reactive chemical group, such as maleimides, haloacetyls, and pyridyl disulfides. In some embodiments, the reactive group is amine-reactive. In some embodiments, the reactive group is an NHS ester.

[0151] In some embodiments, R 2 , R 3 , R 7 , and R 8 are each optionally substituted alkyl, such as an optionally substituted methyl, ethyl, or isopropyl group.

[0152] In some embodiments, R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The alkyl portion of R is substituted with a water-soluble substituent. As used herein, a "water-soluble group" refers to a group that contains one or more polar and / or ionic substituents that improve the overall solubility of the molecule in aqueous media. In some cases, R 4 , R 5 , R 6 , R9 , R 10 , and R 11 At least two of the groups contain water-soluble groups. In other embodiments, three or more contain water-soluble groups. Water-soluble groups include, but are not limited to, carboxylate (-CO 2 - ) group, sulfonate (-SO 3 - ) group, sulfonyl (-SO 2 - ) group, sulfate (-SO 4 -2 ) group, hydroxyl (-OH) group, phosphate (-OPO 3 -2 ) group, phosphonate (-PO 3 -2 ) group, amine (-NH 2 ) groups and optionally substituted quaternary nitrogens (each with an optional counterion).

[0153] Suitable counterions include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino salts, or magnesium salts, or similar salts. Preferably, the counterion is a biologically acceptable counterion.

[0154] In some embodiments, R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The nitrogen atom to which is attached can be trivalent or tetravalent.

[0155] In some embodiments, R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each hydrogen.

[0156] In some embodiments, X 2 and X 3 each independently represents a C which may be interrupted by an atom 1 -C 10 In some embodiments, X is selected from alkylene. 2 and / or X 3 The nitrogen attached to the may optionally be quaternized.

[0157] In some embodiments, the phthalocyanine dye is a phthalocyanine dye of formula Ib: TIFF2025015674000009.tif97150 formula, X 1 and X 4 each independently represents a C which may be interrupted by a heteroatom 1 -C 10 alkylene; and R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 16 , R 17 , R 18 , R 19 , X 2 , and X 3 is as defined herein.

[0158] In some embodiments, the reactive group is an NHS ester. In some embodiments, the reactivity of the NHS ester is determined by the X between the NHS ester and the carbamate functional group. 4 The length of the alkylene group in X between the NHS ester and the carbamate functional group can be adjusted by changing the length of the alkylene group in X. 4 The length of the alkylene group in X is inversely proportional to the reactivity of the NHS ester. 4 is C 5 In another embodiment, X is -alkylene.4 is C 3 In some embodiments, X is -alkylene. 1 is C 6 In another embodiment, X is -alkylene. 1 is C 3 -alkylene.

[0159] In some embodiments, the phthalocyanine dye has a total charge of zero. This neutral charge can be achieved with one or more optional counterions, or with a quaternized nitrogen, in certain cases.

[0160] In some embodiments, the phthalocyanine dye has sufficient solubility in aqueous solution so that when attached to a soluble targeting molecule, the targeting molecule retains its solubility. In some embodiments, the dye is also soluble in organic media (e.g., DMSO or DMF).

[0161] In some embodiments, the phthalocyanine dye has a maximum light absorption in the near infrared (NIR range). In some embodiments, the phthalocyanine dye has a maximum light absorption wavelength between 400 nm and 900 nm, such as between 600 nm and 850 nm, such as between 680 nm and 850 nm, such as approximately 690 nm ± 50 nm or 690 ± 20 nm. In some embodiments, the phthalocyanine dye can be efficiently excited by commercially available laser diodes that emit light at these wavelengths.

[0162] In some embodiments, the phthalocyanine dye containing a reactive group is IR700 NHS ester, such as IRDye 700DX NHS ester (LiCcor 929-70010, 929-70011). Thus, in some embodiments, the dye is a compound having the formula: TIFF2025015674000010.tif106160

[0163] For purposes herein, the terms "IR700", "IRDye 700DX" or variations thereof refer to the above formula when the dye is conjugated to a targeting molecule via its reactive group. In general, IR700 has several favorable chemical properties. Amino-reactive IR700 is a relatively hydrophilic dye and can be covalently conjugated to antibodies using an NHS ester of IR700. Typically, IR700 is also available as a 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 ) with an extinction coefficient that is more than five times higher than that of conventional photosensitizers such as 5 M -1 cm -1 ).

[0164] The phthalocyanine dyes described herein can be made using commercially available starting materials. The core structure is synthesized by condensation of two or more different diiminoisoindolines. Synthetic strategies using different dinitriles or diiminoisoindolines can lead to phthalocyanines with various degrees of substitution and / or positional isomers with different degrees of distribution. An exemplary synthetic scheme for producing the dyes is described in U.S. Pat. No. 7,005,518.

[0165] In some embodiments, in any of the methods provided herein, the targeting molecule (e.g., an antibody) is directly or indirectly linked to the phthalocyanine dye (e.g., IR700). In some embodiments, the targeting molecule (e.g., an antibody) is directly or indirectly linked to the phthalocyanine dye (e.g., IR700) via a covalent bond or non-covalent interaction. In some embodiments, the covalent or non-covalent interaction or linkage is direct or indirect. In some embodiments, the attachment comprises an indirect linkage, for example, through a linker (e.g., any of the exemplary linkers described above), a binding moiety or domain, or a reactive group. In some embodiments, the linkage comprises a direct interaction between the targeting molecule and the phthalocyanine dye (e.g., IR700). In other embodiments, one or both of the targeting molecule and the phthalocyanine dye are linked to one or more linkers, and the interaction is indirect, for example, between a linker attached to one of the molecules and another molecule, or between two linkers attached to the targeting molecule or the phthalocyanine dye, respectively.

[0166] In some embodiments, the phthalocyanine dye is non-covalently linked or associated with the targeting molecule. For example, the phthalocyanine dye forms a complex with the targeting molecule through non-covalent interaction. In some embodiments, the phthalocyanine dye (e.g., IR700) contains a moiety or domain that can non-covalently interact with the attachment group of the targeting molecule. In some embodiments, the method includes incubating or combining the phthalocyanine dye (e.g., IR700) with the targeting molecule (e.g., an antibody) to form a non-covalent interaction between the dye and the targeting molecule. In some examples, the non-covalent interaction between the targeting molecule and the phthalocyanine dye includes, for example, electrostatic interaction, van der Waals force, hydrophobic interaction, π effect, ionic interaction, hydrogen bond, halogen bond and / or a combination thereof, or any interaction that relies on one or more of these forces. In some embodiments, the targeting molecule and the phthalocyanine dye are linked using interactions that mimic non-covalent molecular interactions, such as, for example, ligand-receptor interactions, antibody-antigen interactions, avidin-biotin interactions, streptavidin-biotin interactions, histidine-divalent metal ion (e.g., Ni, Co, Cu, Fe) interactions, interactions between multimerization (e.g., dimerization) domains, glutathione S-transferase (GST)-glutathione interactions, and / or any combination thereof.

[0167] In some embodiments, the non-covalent interaction moiety or domain is attached to or is part of the targeting molecule and forms a non-covalent interaction, e.g., complex, with the phthalocyanine dye (e.g., IR700). In other embodiments, the non-covalent interaction molecule or domain is attached to or is part of the targeting molecule and forms a non-covalent interaction, e.g., complex, with the phthalocyanine dye molecule. In some embodiments, the method includes incubating or contacting a targeting molecule conjugated to biotin (e.g., antibody-biotin, e.g., cetuximab-biotin) with a phthalocyanine dye conjugated to avidin or an analog thereof, or streptavidin or an analog thereof, including its monomeric form (e.g., monomeric avidin-IR700 or monomeric streptavidin-IR700). Due to the non-covalent interactions between avidin, streptavidin or analogs thereof and biotin, in some embodiments, the phthalocyanine dye (e.g., IR700) forms a non-covalent complex with the targeting molecule.

[0168] 2. Targeting molecules In some embodiments, the phthalocyanine dye is conjugated to a targeting molecule via a reactive group on the dye molecule. In some embodiments, the targeting molecule is a molecule that can target the conjugate to a cell or pathogen, for example, by binding to a cell surface molecule (e.g., a cell surface receptor) on the cell or pathogen. In some embodiments, the targeting molecule, e.g., a macromolecule, can selectively bind to a desired cell type, a cell with a particular phenotype, or a cell that displays one or more cell surface markers or antigens. In some cases, the targeting molecule binds to a cell that is a cancer cell, a tumor cell, an inflammatory cell, an immune cell, a neuronal cell, a stem cell, a proliferative cell, or a cell in a hyperplastic state. In some cases, the targeting molecule binds to a pathogen or a pathogen-infected cell. In some embodiments, the cell is an inflammatory cell, such as a white blood cell, e.g., a neutrophil, an eosinophil, a basophil, a lymphocyte, or a monocyte. In some embodiments, the cell is an immune cell, such as a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a macrophage, or a neutrophil. In some embodiments, the cell is a neuron that is a peripheral nervous system neuron or a central nervous system neuron, such as a nociceptor (e.g., a thermal nociceptor, a mechanical nociceptor, a chemical nociceptor, or a polymodal nociceptor). In some cases, the targeting molecule binds to a pathogen or a pathogenic cell, such as a virus, a bacterium, a fungus, a biofilm, or other prokaryotic cell system. In some embodiments, the targeting molecule binds to a pathogen that is a gram-negative bacterium or a gram-positive bacterium.

[0169] In some embodiments, the targeting molecule (e.g., an antibody) of the phthalocyanine dye conjugate binds to a protein on the surface of one or more cells present in the microenvironment of a lesion associated with or present as a result of a disease or condition. For example, in some embodiments, the conjugate binds to a protein on the surface of one or more cells present in the tumor microenvironment associated with or present in a tumor. In some embodiments, the conjugate binds to a protein present in the extracellular matrix in the tumor microenvironment.

[0170] As used herein, a "cell present in the microenvironment of a lesion" refers to any cell present in a cellular environment associated with a lesion, disease or disorder, such as any cell present in or directly adjacent to a tumor, such as a cell present in the tumor microenvironment, or in the extracellular matrix in the tumor microenvironment.

[0171] As used herein, "cells present in the tumor microenvironment" refers to any cell present in the cellular environment in which a tumor resides, for example, any cell present in or directly adjacent to a tumor, including proliferating tumor cells (e.g., cancer cells), tumor stroma, blood vessels, infiltrating inflammatory cells (e.g., immune cells) and various associated tissue cells (e.g., fibroblasts). Thus, it is understood that reference to a tumor refers not only to tumor cells, which may include malignant or cancer cells, but also to other cells (including immune cells) present in the tumor microenvironment that regulate tumor growth. In some cases, immune cells present in the tumor microenvironment may include T lymphocytes (including regulatory T lymphocytes (Tregs)), dendritic cells, natural killer (NK) cells, B cells, macrophages and other immune cells (Whiteside (2008) Oncogene, 27:5904-5912). In some aspects, it is recognized that many non-cancerous cells present in and around a tumor can regulate tumor cell proliferation, angiogenesis, invasion and / or metastasis, thereby promoting tumor growth. Therefore, in some cases, targeting such non-cancerous cells present in tumors, such as immune cells (e.g., T cells such as regulatory T cells), may be an effective treatment for killing tumors by PIT.

[0172] Generally, cancerous cells contain tumor-associated antigens that should be recognized by the immune system. Typically, in an active immune system, immune cells such as cytotoxic T cells attack and eradicate these cancerous cells. Under normal physiological conditions, T cell-mediated immune responses are initiated by antigen recognition by T cell receptors (TCRs) and regulated by a balance of costimulatory and inhibitory signals (e.g., immune checkpoint proteins). In particular, CD4+ and CD8+ T cells expressing TCRs can become activated by recognition of antigenic peptides presented on antigen-presenting cells on major histocompatibility complex (MHC) class I or class II molecules, respectively. In some aspects, activated CD8+ cells, or cytotoxic T cells, can kill tumor cells expressing antigens, which can be assisted by the presence of CD4+ T cells.

[0173] However, in the case of tumors, the tumor microenvironment has mechanisms to suppress the immune system, thereby evading immune recognition and preventing or reducing tumor cell killing. For example, in some cases, immune checkpoint proteins may be deregulated in tumors, thereby resulting in suppression of immune responses in the tumor microenvironment as a mechanism to evade the immune system. In some cases, tumor-infiltrating lymphocytes may contain Tregs (e.g., CD4+CD25+T cells), which are cells that can suppress the proliferation of other T cells in the microenvironment (Whiteside, TL (2008) Oncogene, 27:5904-5912). In some cases, other mechanisms may act to inhibit the access of immune cells to tumor antigens, thereby also contributing to the tumor's ability to evade the immune system.

[0174] In some embodiments, the targeting molecule is a targeting molecule that binds to cell surface proteins on tumor or cancer cells.In some embodiments, the targeting molecule binds to cell surface proteins on immune cells or other non-cancerous cells present in tumor microenvironment.In some embodiments, the targeting molecule binds to cell surface proteins on the surface of T lymphocytes, such as Treg, dendritic cells, natural killer (NK) cells, B cells, macrophages or other immune cells present in tumor microenvironment.In some cases, the tumor or cancer is located in head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum or lung.

[0175] Exemplary targeting molecules, such as those that target tumors or cancers, include, but are not limited to, any of those described in published International PCT Applications WO2014120974, WO2014176284, WO2015042325, U.S. Patent No. 8,524,239, or U.S. Patent Publication No. US20140120119.

[0176] Exemplary targeting molecules include, but are not limited to, a protein, a glycoprotein, an antibody, an antibody fragment, an antigen, an antigen-binding fragment, a peptide, a polypeptide, a small molecule, a synthetic polymer, a polymeric nanoparticle, a liposome, an enzyme substrate, a hormone, a neurotransmitter, a cellular metabolite, a viral particle, a viral capsid, a viral nanoparticle, a bacterial particle, a marker, a cell, a hapten, an avidin, a streptavidin, a monomeric streptavidin, biotin, a carbohydrate, an oligosaccharide, a polysaccharide, a nucleic acid, a deoxynucleic acid, a fragment of DNA, a fragment of RNA, a nucleotide triphosphate, an acycloterminator triphosphate, or a PNA.

[0177] In some embodiments, the targeting molecule is an amino acid, a peptide, a protein, a tyramine, a polysaccharide, an ion complexing moiety, a nucleoside, a nucleotide, an oligonucleotide, a psoralen, a drug, a hormone, a lipid, a lipid aggregate, a polymer, a polymeric microparticle, a biological cell, or a virus.In some embodiments, the targeting molecule is an antigen, a steroid, a vitamin, a drug, a metabolite, a toxin, an environmental pollutant, a nucleic acid polymer, a carbohydrate, a lipid, or glass, plastic, or other non-biological polymer.In some embodiments, the targeting molecule is a cell, a cell line, a cell fragment, or a particle smaller than a cell, such as a virus particle, a bacterial particle, a virus component, a biological cell (e.g., an animal cell, a plant cell, a bacteria, a yeast, or a protist), or a cell component.In some embodiments, the reactive dye can label functional groups on the cell surface, in the cell membrane, in the organelle, or in the cytoplasm.

[0178] In some embodiments, the targeting molecule targets or binds to an antigen (e.g., any structural target that can be bound by the targeting molecule). In some embodiments, the antigen is or is included as part of a cell surface molecule, such as a protein (e.g., a receptor) expressed on a cell surface. In some embodiments, for example, the antigen is or is included as part of a molecule expressed on the surface of a cell present in a tumor (including any cell present in the tumor microenvironment). Examples of cell surface molecules include, but are not limited to, antigens, peptides, lipids, polysaccharides, carbohydrates, or nucleic acids, including antigenic determinants (such as those recognized by immune cells). In some examples, the antigen includes a tumor-specific peptide (e.g., one found on the surface of a cancer cell) or an immunogenic fragment thereof. In some embodiments, the targeting molecule is an antibody or an antigen-binding antibody fragment thereof.

[0179] In some embodiments, the cell surface molecule is ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptor BB1, BB2, BB4, calcitonin receptor, cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, Crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endosialin, EP2, EP4 , EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, Family A G protein-coupled receptor (rhodopsin-like), Family B G protein-coupled receptor (secretin receptor-like), Family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV16 / 18 and E6 / E7 antigens, hTERT, IL11-R, IL-13R, ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE1, MAGE2, MAGE3, MAGE4, ​​MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-ESO -1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease-cleaved collagen IV, proteinase 3, prohibitin, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 It can be patched, RET receptor, plexin, smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptor (VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, and VEGF-3 or FLT-4), voltage-gated ion channel, VPAC1, VPAC2, Wilms' tumor 1, Y1, Y2, Y4, or Y5.

[0180] In some embodiments, the targeting molecule is a binding partner, such as a ligand, capable of binding to a cell surface molecule, such as a cell surface protein (e.g., a cell surface receptor). In some embodiments, the targeting molecule is a targeting molecule selected from the group consisting of adrenocorticotropic hormone (ACTH), angiotensin II, atrial natriuretic factor (ANF), bombesin, bradykinin, brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), calcitonin, cardiotrophin 1 (BMP-2), CD22, CD40, cholecystokinin (CCK), ciliary neurotrophic factor (CNTF), CCL1-CCL28, CXCL1-CCL29, CXCL20, CXCL21, CXCL22, CXCL23, CXCL24, CXCL25, CXCL26, CXCL27, CXCL28, CXCL29 ... XCL17, XCL1, XCL2, CX3CL1, Crypto-1 binding peptide, Vascular endothelial growth factor (VEGF), Epidermal growth factor (EGF), Endothelin 1, Endothelin 1 / 3, FAS-ligand, Fibroblast growth factor 1 (FGF-1), Fibroblast growth factor 2 (FGF-2), Fibroblast growth factor 4 (FGF-4), Fibroblast growth factor 5 (FGF-5), Fibroblast growth factor 6 (FGF-6), Fibroblast growth factor 1 (FGF-7), Fibroblast growth factor 1 (FGF-10), Flt-3, Gust Phosphorin, gastrin releasing peptide (GRP), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM-CSF), glucagon-like peptide (GLP-1), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN-b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 19 (IL-19),Luteinizing hormone (LH), luteinizing releasing hormone (LHRH), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neuromedin B, neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), neurotensin, neuropeptide Y, oxytocin, pituitary adenylate cyclase-activating peptide (PACAP), platelet-derived growth factor AA (PDGF- AA), platelet-derived growth factor AB (PDGF-AB), platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor CC (PDGF-CC), platelet-derived growth factor DD (PDGF-DD), netrin-1 (NTN1), netrin-2 (NTN2), netrin-4 (NTN4), netrin-G1 (NTNG1) and netrin-G2 (NTNG2), ephrin A1 (EFNA1), ephrin A2 (EFNA2), ephrin A3 (EFNA3), ephrin A4 (EFNA4), ephrin A5 (EFNA5), semaphorin 3A (SEMA3A), semaphorin 4A (SEMA4A), semaphorin 5A (SEMA5A), semaphorin 6A (SEMA6A), semaphorin 7A (SEMA7A), semaphorin 8A (SEMA8A), semaphorin 9A (SEMA9A), semaphorin 10A (SEMA9A), semaphorin 11A (SEMA9A), semaphorin 12A (SEMA9A), semaphorin 13A (SEMA9A), semaphorin 14A (SEMA9A), semaphorin 15A (SEMA9A), semaphorin 16A (SEMA9A), semaphorin 17A (SEMA9A), semaphorin 18A (SEMA9A), semaphorin 19A (SEMA9A), semaphorin 20A (SEMA9A), semaphorin 21A (SEMA9A), semaphorin 22A (SEMA9A), semaphorin 23A (SEMA9A), semaphorin 24A (SEMA9A), semaphorin 25A (SEMA9A), semaphorin 26A (SEMA9A), s Semaphorin 3B (SEMA3B), Semaphorin 3C (SEMA3C), Semaphorin 3D (SEMA3D), Semaphorin 3F (SEMA3F), Semaphorin 3G (SEMA3G), Semaphorin 4A (SEMA4A), Semaphorin 4B (SEMA4B), Semaphorin 4C (SEMA4C), Semaphorin 4D (SEMA4D), Semaphorin 4F (SEMA4F), Semaphorin 4G (SEMA4G), Semaphorin 5A (SEMA5A), Semaphorin 5B (SEMA5B), Semaphorin 6A (SEMA6A), Semaphorin 6B (SEMA A6B), semaphorin 6D (SEMA6D), semaphorin 7A (SEMA7A), SLIT1, SLIT2, SLIT3, SLIT and NTRK-like family member 1 (SLITRK1), SLIT and NTRK-like family member 2 (SLITRK2), SLIT and NTRK-like family member 3 (SLITRK3), SLIT and NTRK-like family member 4 (SLITRK4), SLIT and NTRK-like family member 5 (SLITRK5), SLIT and NTRK-like family member 6 (SLITRK6),Prostaglandin E2 (PGE2), RANTES, somatostatin-14, somatostatin-28, stem cell factor (SCF), stromal cell-derived factor 1 (SDF-1), substance P, thyroid-stimulating hormone (TSH), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-α), thrombin, vasoactive intestinal peptide (VIP), Wntl, Wnt2, Wnt2b / 13, Wnt3 , Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, WntlOa, WntlOb, Wnt11, Wnt14, Wnt15 or Wnt16, Sonic hedgehog, Desert hedgehog, and Indian hedgehog, or a binding fragment thereof capable of binding to its cognate cell surface molecule, such as a cell surface protein (e.g., a cell surface receptor).

[0181] In some embodiments, the targeting molecule can be an immunomodulatory agent that can bind to cell surface molecules or proteins on immune cells to suppress or activate the body's immune response. In some embodiments, the binding of the immunomodulatory agent to cell surface molecules or proteins can stimulate the immune response against tumors and / or pathogens, for example by inhibiting immune suppression or by enhancing immune stimulation. In some embodiments, the cell surface molecule or protein is CD25, PD-1 (CD279), PD-L1 (CD274, B7-H1), PD-L2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3 (HAVCR2), 4-1BB (CD137, TNFRSF9), CXCR2, CXCR4 (CD184), CD27, CEACAM1, Galectin 9, BTLA, CD160, VISTA (PD1 homolog), B7-H4 (VCTN1), CD80 (B7-1), CD86 (B7-2), CD28, HHLA2 (B7-H7), CD28H, CD155, CD226, TIGIT, CD96, Galecti 3, CD40, CD40L, CD70, LIGHT(TNFSF14), HVEM(TNFRSF14), B7-H3(CD276), Ox40L(TNFSF4), CD137L(TNFSF9, GITRL) , B7RP1, ICOS (CD278), ICOSL, KIR, GAL9, NKG2A (CD94), GARP, TL1A, TNFRSF25, TMIGD2, BTNL2, butyrophilin family, CD48, CD244, Siglec family, CD30, CSF1R, MICA (MHC class I polypeptide-related sequence A), MICB (MHC class I polypeptide-related sequence B), NKG2D, KIR family (killer cell immunoglobulin-like receptors, LILR family (leukocyte immunoglobulin-like receptors, CD85, ILT, LIR), SIRPA (signal regulatory protein alpha), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR, or VEGF. By way of example, the targeting molecule is an antibody or antigen-binding fragment that is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor.

[0182] In some embodiments, the cell surface molecule is selected from the group consisting of HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA 125), alpha-fetoprotein (AFP), Lewis Y, TAG72, Caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate binding protein, gangliosides (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), integrin αVβ3, integrin αVβ4, integrin β1, ... The antigen may be gulin alpha5beta1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR10beta, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinase, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, or SK-1 antigen.

[0183] In some embodiments, the targeting molecule is an antibody or an antigen-binding antibody fragment that specifically binds to an antigen that is a cell surface molecule or a part of the cell surface molecule that is expressed on the surface of the cell.Such antibodies include antibodies or antigen-binding antibody fragments that can bind to cell surface molecules such as the cell surface proteins (e.g., cell surface receptors) described herein.In some cases, the antibody can bind to the antigen of the protein expressed on cells in tumors, including tumor-specific proteins.

[0184] In some embodiments, targeting molecule directly or indirectly binds to antigen or protein.For example, in some embodiments, targeting molecule is a second binding molecule that binds to the first binding molecule that can bind to antigen or protein.For example, targeting molecule is a second antibody that binds to the first binding molecule that can bind to protein or antigen, such as cell surface protein or cell surface receptor, such as a primary antibody.Thus, in some embodiments, dye is conjugated to the secondary antibody.

[0185] An "antibody" is a polypeptide ligand that contains at least one light and / or heavy immunoglobulin variable region that specifically recognizes and binds an epitope of an antigen. Generally, antibodies are characterized by a variable heavy chain (V H ) region and the variable light chain (V L The antibody is composed of heavy and light chains, each of which has a variable region called the V H Area and V L The regions together are responsible for binding the antigen recognized by the antibody. The term antibody refers to intact antibodies as well as antigen-binding antibody fragments that exhibit antigen binding, such as Fab fragments, Fab' fragments, F(ab)' fragments, and the like. 2 Fv fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region are joined by a linker, while in dsFv, the chains are mutated to introduce a disulfide bond that stabilizes the association of the chains. The term also includes genetically engineered forms, such as modified immunoglobulins, chimeric antibodies, e.g., humanized murine antibodies, and heteroconjugate antibodies, e.g., bispecific antibodies. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J. Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0186] 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 (k). There are five major heavy chain classes, or isotypes, that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0187] Each heavy and light chain contains a constant region and a variable region, also known as a "domain." In combination, the variable regions of the heavy and light chains generally bind specifically to an antigen. The variable regions of the light and heavy chains may contain a "framework" region, which is interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs." The extent of the framework region and the CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, 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, such as humans. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0188] CDRs are typically involved in binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered consecutively starting from the N-terminus, and are also generally 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 LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities, such as different binding sites for different antigens, have different CDRs. Although it is the CDR that varies from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. These positions within the CDR are called specificity determining residues (SDRs).

[0189] "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. L References to " or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

[0190] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Other antibody fragments or multispecific antibodies formed from antibody fragments include multivalent scFvs, bispecific scFvs or scFv-CH3 dimers. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolysis of intact antibodies as well as production by recombinant host cells.

[0191] A "monoclonal antibody" 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 by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells derived from the fusion of a myeloma cell and an immune spleen cell. Monoclonal antibodies include humanized monoclonal antibodies.

[0192] A "chimeric antibody" has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds mesothelin.

[0193] 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 some embodiments, the CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if they are present, they can be substantially identical, e.g., at least about 85-90%, e.g., about 95% or more identical, to human immunoglobulin constant regions. Thus, portions of a humanized immunoglobulin (except possibly for the CDRs) are substantially identical to portions of the corresponding native 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 can have a limited number of substitutions with amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0194] A "human" antibody (also called a "fully human" antibody) is an antibody that comprises a human framework region and CDRs derived from a human immunoglobulin. In some embodiments, 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 modified to contain CDRs from a different human antibody. Portions of human immunoglobulins can be substantially identical to portions of corresponding native human immunoglobulin sequences.

[0195] "Specifically binds" refers to the ability of a molecule, such as an antibody or antigen-binding fragment, to specifically bind to an antigen, such as a tumor-specific antigen, compared to binding to an unrelated protein, such as a non-tumor protein (e.g., β-actin). In some embodiments, the antibody or fragment-like molecule (including an antibody or fragment-like molecule attached to a phthalocyanine dye molecule) binds to a target, such as a cell surface protein, with a binding constant at least 10 higher than the binding constant for other molecules in the sample or subject. 3 M -1 Large, 10 4 M -1 Greater than or equal to 10 5 M -1 In some embodiments, a molecule such as an antibody or fragment thereof specifically binds with a high binding constant of about 10 6 M -1 More than 10 7 M -1 More than 10 8 M -1 More than or about 10 9 M -1 , 10 10 M -1 , 10 11 M -1 Or 10 12 M -1 The equilibrium association constant (K A The antibody also has a 10 -6 M, 10 -7 M, 10 -8 M, 10 -10 M, 10 -11 M or 10 -12 M or lower equilibrium dissociation constant (KD In some embodiments, the equilibrium dissociation constant (K D ) can be 1 nM or less. D or K A Affinity constants such as can be estimated empirically or can be determined comparatively, e.g., by comparing the affinity of one antibody to another for a particular antigen. For example, such affinities can be readily determined using techniques known in the art, e.g., by competitive ELISA (enzyme-linked immunosorbent assay) or using a surface plasmon resonance instrument such as the Biacore T100 (available from Biacore, Inc., Piscataway, NJ), by radioimmunoassay using radiolabeled target antigen, or by another method known to those of skill in the art.

[0196] In some embodiments, the phthalocyanine dye (e.g., IR700) is conjugated to an antibody or antigen-binding antibody fragment. For example, in some aspects, the phthalocyanine dye-targeting molecule conjugate is an IR700-antibody conjugate. Exemplary antibodies to which the phthalocyanine dye (e.g., IR700) can be conjugated include, but are not limited to, cetuximab, panitumumab, zalutumumab, nimotuzumab, trastuzumab, Ado-trastuzumab emtansine, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), afatinib, axitinib, bosutinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, olaparib , palbociclib, pazopanib, pertuzumab, ramucirumab, regorafenib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, vismodegib, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab (CT-011), MK-3475, BMS- 936559, MPDL3280A, tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR0916, AMP-224, MSB00107 18C, MEDI4736, PDR001, rHIgM12B7, urocupulumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, emactuzumab, CC-90002 and MNRP1685A or an antibody-binding fragment thereof.

[0197] In some embodiments, the targeting molecule is a tissue-specific homing peptide. For example, in some embodiments, the homing peri-peptide can comprise a sequence of amino acids as set forth in any of SEQ ID NOs: 1-52. In some embodiments, the targeting molecule is an RGD polypeptide, such as an iRGD polypeptide, a Lyp-1 polypeptide, a Crypto-1 binding polypeptide, a somatostatin receptor binding polypeptide, or a prohibitin binding polypeptide, an NGR polypeptide, or an iNGR polypeptide.

[0198] In some embodiments, the targeting molecule is a virus particle, such as a virus-like particle, a virus-like nanoparticle, or a virus capsid.In some embodiments, the targeting molecule is a virus-like nanoparticle.In some embodiments, the virus-like nanoparticle is assembled from L1 capsid protein.In some embodiments, the virus-like nanoparticle is assembled from a combination of L1 capsid protein and L2 capsid protein.In some embodiments, the targeting molecule binds to and infects cells.In some embodiments, the targeting molecule is the targeting molecule described in WO2015042325.

[0199] In some embodiments, virus-like particle (VLP) refers to an organized capsid-like structure, such as a roughly spherical or cylindrical shape, that comprises a self-assembled ordered array of L1 or L1 and L2 capsomers and does not contain viral genome.In some embodiments, virus-like particles are morphologically and antigenically similar to true virions, but they lack viral genetic material, such as viral nucleic acid, making the particle non-infectious.VLP can be used to deliver drugs, such as prophylactic, therapeutic or diagnostic drugs, or encapsulated circular or linear DNA or RNA molecules to recipient cells.

[0200] In some embodiments, the VLP may have modified immunogenicity and / or antigenicity relative to wild-type VLP. The VLP may be assembled from capsomers having, for example, mutant capsid proteins with modified immunogenicity and / or antigenicity. In some embodiments, the mutant capsid proteins with modified immunogenicity and / or antigenicity are those in which amino acids are modified naturally or synthetically, for example, mutated, substituted, deleted, pegylated or inserted, to reduce or prevent recognition of the capsid protein by pre-existing (e.g., endogenous) virus serotype-specific antibodies. The mutant capsid protein may be a human papillomavirus (HPV) L1 mutant, a non-human papillomavirus L1 mutant, or a papillomavirus L1 mutant based on a combination of amino acids from different HPV serotypes.

[0201] In some embodiments, the VLP is a papillomavirus VLP. The VLP can be a human papillomavirus VLP, such as one derived from a virus capable of infecting humans, while in other embodiments, the VLP can be a non-human papillomavirus VLP. Examples of non-human VLPs include, but are not limited to, those derived from bovine papillomavirus, murine papillomavirus, cotton rabbit papillomavirus, and macaque or rhesus papillomavirus particles. In some embodiments, the VLP is a bovine papillomavirus virus-like nanoparticle, such as a type 1 virus-like nanoparticle (e.g., assembled from BPV L1 capsid protein or a combination of BPV L1 capsid protein and BPV L2 capsid protein).

[0202] In some embodiments, capsid protein refers to a protein monomer, some of which form a capsomer oligomer. In some embodiments, capsomer refers to the basic oligomeric structural unit of a viral capsid, which is an outer coating of protein that protects the genetic material of the virus. In some embodiments, capsid protein can include papillomavirus L1 major capsid protein and papillomavirus L2 minor capsid protein. In some embodiments, the VLP contains only L1 capsid protein, while in other embodiments, the VLP contains a mixture of L1 capsid protein and L2 capsid protein, or a combination thereof.

[0203] In some embodiments, the percentage of L1 capsid protein in the virus-like particle is greater than the percentage of L2 capsid protein in the virus-like particle.For example, in some embodiments, the percentage of L1 capsid protein in the virus-like particle is 80% to 100% of the total number of capsid proteins in the virus-like particle.In some embodiments, the percentage of L1 capsid protein in the virus-like particle is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the percentage of L2 capsid protein in the virus-like particle is 1% to 25% of the total number of capsid proteins in the virus-like particle. For example, in some embodiments, the percentage of L2 capsid protein in the virus-like particle is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0204] In some embodiments, the virus-like particle contains 12-72 L2 proteins. In some embodiments, the virus-like particle contains 360 L1 proteins and 12-72 L2 proteins. In some embodiments, the capsid protein assembles into a virus-like nanoparticle with a diameter of 20-60 nm. For example, the capsid protein can assemble into a virus-like nanoparticle with a diameter of at least or about 20, 25, 30, 35, 40, 45, 50, 55 or 60 nm.

[0205] In some embodiments, the targeting molecule is not or does not include a nanocarrier. In some embodiments, the targeting molecule is not or does not include a virus-like particle, a nanoparticle, a liposome, a quantum dot, or a combination thereof.

[0206] In some embodiments, the targeting molecule is a DARPin (designed ankyrin repeat protein). Typically, DARPins are derived from natural ankyrin repeat proteins and bind to proteins including, for example, human receptors, cytokines, kinases, human proteases, viruses and membrane proteins (Molecular Partners AG Zurich Switzerland; see Chapter 5. "Designed Ankyrin Repeat Proteins (DARPins): From Research to Therapy", Methods in Enzymology, vol 503: 101-134 (2012); and "Efficient Selection of DARPins with Sub-nanomolar Affinities using SRP Phage Display", J. Mol. Biol. (2008) 382, ​​1211-1227, the entire disclosure of which is incorporated herein by reference). In some embodiments, DARPins are antibody mimetic proteins with high specificity and high binding affinity to target proteins, which are prepared through genetic engineering. In some embodiments, DARPin has a structure that comprises at least two ankyrin repeat motifs, for example, comprises at least 3, 4 or 5 ankyrin repeat motifs.DARPin can have any suitable molecular weight depending on the number of repeat motifs.For example, DARPin that comprises 3, 4 or 5 ankyrin repeat motifs can have a molecular weight of about 10 kDa, about 14 kDa or about 18 kDa, respectively.

[0207] In some embodiments, DARPins comprise a core portion that provides structure and a target binding portion that is outside the core and binds to a target. In some embodiments, the structural core comprises a conserved amino acid sequence and the target binding portion comprises an amino acid sequence that differs depending on the target.

[0208] In some embodiments, the conjugate contains a plurality of dye moieties per targeting molecule, e.g., 1 to about 100 or about 1 to about 100, 1 to about 50 or about 1 to about 50, 1 to about 25 or about 1 to about 25, 1 to about 10 or about 1 to about 10, 1 to about 5 or about 1 to about 5. In some embodiments, the ratio of dye molecules:targeting molecules is 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, or 1000:1, or about 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, or 1000:1, :1, 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1 or 1000:1, or between or about any two of such values. In some embodiments, the targeting molecule may contain up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 dye molecules. In some embodiments, the targeting molecule may contain more than 1000 dye molecules or less than 10 dye molecules.

[0209] In some embodiments, for example, when the targeting molecule is a polypeptide such as an antibody or an antigen-binding antibody fragment, the number of dye molecules per targeting molecule can be 2 to about 5 or about 2 to about 5, for example, 2 to about 4 or about 2 to about 4, for example, about 3 or 3. In some embodiments, for example, when the targeting molecule is a nanoparticle such as a virus-like particle (VLP), the number of dye molecules and targeting molecules can be 10 to about 1000, 10 to about 500, 50 to about 500, or 50 to about 1000, or about 10 to about 1000, about 10 to about 500, about 50 to about 500, or about 50 to about 1000. Thus, in some embodiments, the targeting molecule can contain about 10 to about 1000 dye molecules.

[0210] In some embodiments, for example, when the targeting molecule is a VLP, more than one dye molecule can be conjugated to a single capsid protein. For example, a single capsid protein, such as LI or L2 capsid protein, can be conjugated to 1-5, for example, 1, 2, 3, 4 or 5 dye molecules. Thus, more than one amino acid of a capsid protein can be conjugated to a dye molecule. In some embodiments, a single capsid protein can be conjugated to 1-2, 1-3, or 2-3 dye molecules. Thus, dye molecules can be conjugated to 1, 2, 3, 4 or 5 different amino acids, for example, lysine, arginine and / or histidine, or other amino acids, of a single capsid protein.

[0211] 3. Additional dyes for imaging In some embodiments, the conjugate can optionally include an additional dye so that the targeting molecule can be conjugated to two or more different fluorescent dyes.For example, a conjugate is provided that contains a targeting molecule conjugated to a first dye, which is a phthalocyanine dye, such as any of the above (e.g., IR700), and a second fluorescent dye that is different from the first dye.In one aspect, the conjugate is a conjugate represented by formula (II): [D 1-(L 1 ) n ] p -A-[(L 2 ) m -D 2 ] o (II) During the ceremony, A is a targeting molecule capable of binding to a cell or tissue; L 1 and L 2 are each independently selected linkers for each o or p, where each L 1 and L 2 is as defined above for L; n and m are independently 1 or 2; D 1 is a hydrophilic phthalocyanine dye independently selected for each p, where D 1 is as defined above for D; D 2 is a fluorescent dye selected independently for each o; and p and o are independently 1, 2, 3, 4, 5 or greater than 5, such as up to 1000. For example, p and o can each independently be 1 to 1000, such as typically 1 to 10 or 2 to 5.

[0212] In some embodiments, the first dye D 1 is a phthalocyanine dye, e.g., a near infrared (NIR) phthalocyanine dye, e.g., any of the dyes described above. In some embodiments, the phthalocyanine dye is or includes a photosensitizer compound that is capable of exhibiting phototoxic activity upon irradiation with near infrared light. In some embodiments, D 1 is IR700.

[0213] In some embodiments, the first dye D 1 A second dye D that provides better fluorescence for visualization than IR700 (e.g., IR700) 2is selected. Thus, in some aspects, the compound represented by formula II is used for both fluorescent imaging and photoimmunotherapy. For example, irradiating the lesion or tumor will emit a fluorescent signal from the second fluorescent dye, achieving the detection of the presence of the conjugate in the lesion or tumor in the subject. In some embodiments, the conjugate is used to detect the binding of the dye to the target site (e.g., tumor) D. 2 It is also possible to monitor cells associated with a disease or pathology, such as tumor cells, by fluorescent imaging of the D 1 In some embodiments, the second dye D can be used to eradicate the tumor using photoimmunotherapy with activation of a second dye D (e.g., IR700). 2 is the corresponding D 1 In some embodiments, D exhibits one or more spectral properties selected from among a fluorescence quantum yield (e.g., in water), an extinction coefficient, a Stokes shift, absorption and emission at longer wavelengths, and photostability that are greater than the spectral properties of D. 2 is not IR700.

[0214] In some embodiments, the additional dye D 2 In some embodiments, D is a fluorescent dye. 2Examples of suitable fluorescent dyes include, but are not limited to, hydroxycoumarin, Cascade Blue, Dylight 405, Pacific Orange, Alexa Fluor 430, fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2,7-dichlorofluorescein, ATTO 488, Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 532, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo 642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700, Cy5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705, or Qdot® 800.

[0215] In some embodiments, D 2 D absorbs light and emits fluorescence at longer wavelengths where tissue autofluorescence is low, essentially nonexistent or eliminated. Thus, in some aspects, D 2 facilitates imaging of deep tissue penetration. In some embodiments, D 2is a near infrared (NIR) dye having absorption and emission wavelengths in the NIR spectrum between or about 650-1450 nm, e.g., between or about 650-900 nm, between or about 700-1000 nm, or between or about 750-950 nm. In some embodiments, D 2 is a second near infrared (NIR-II) dye having absorption and emission wavelengths in the NIR-II spectrum between or about 1000-1700 nm, e.g., between or about 1000-1400 nm. In some embodiments, D 2 is a dye having absorption and emission wavelengths in the visible spectrum between or about 300-750 nm, e.g., between or about 400-600 nm, or between or about 400-700 nm. In some embodiments, the targeting molecule conjugate contains two dyes with different emission and excitation wavelengths. In some embodiments, the additional dye has longer wavelength excitation and emission characteristics. In some embodiments, any of the methods provided can further include imaging the lesion or tumor in the subject by irradiating or illuminating the tumor with a wavelength capable of being absorbed by the second dye.

[0216] In some embodiments, the additional dye has a large extinction coefficient at the excitation wavelength. 2 is about 10,000Mol -1 cm -1 More than, for example, about 25,000 Mol -1 cm -1 Super, about 50,000Mol -1 cm -1 Super, about 75,000Mol -1 cm -1 Super, about 100,000Mol -1 cm -1 Super, about 150,000Mol -1 cm -1 Super, about 200,000Mol-1 cm -1 Super, about 250,000Mol -1 cm -1 More than or about 300,000 Mol -1 cm -1 It has an extinction coefficient in water of .

[0217] In some embodiments, the second dye D 2 When conjugated to a protein, the first dye D 1 In some embodiments, the additional dye has a high fluorescence quantum yield in water. In some embodiments, the second dye D 2 has a quantum yield in water of greater than 5%, e.g., greater than 10%, greater than 15%, greater than 20% or greater than 25%, greater than 30%, greater than 40%, or greater than 50% or higher.

[0218] In some embodiments, the second dye D 2 has a large Stokes shift (EX max ~EM max In some embodiments, the additional dye D 2 exhibits a Stokes shift that is greater than 15 nm, 20 nm, 25 nm, for example greater than 30 nm, greater than 40 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, greater than 90 nm or greater than 100 nm.

[0219] In some embodiments, the additional dye D 2 can be ICG, IRDye 680, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800, or IRDye 800. In some embodiments, the additional dye D 2 can be ICG, IRDye 680, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800, or IRDye 800. In some embodiments, the additional dye D 2can be Alexa Fluor 488, IRDye 680, IRDye 800 or Dylight 755.

[0220] In some embodiments, the conjugate comprises a plurality of second dyes D, e.g., 1 to about 100, e.g., 1 to about 100, e.g., 1 to about 50, e.g., 1 to about 25, e.g., 1 to about 10, e.g., 1 to about 5, e.g., 1 to about 5, 2 In some embodiments, the ratio of the second dye molecule:targeting molecule is 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, or 1000:1, or about 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, or 1000:1, or between or about any two of such values. In some embodiments, the ratio of second dye molecules to targeting molecules is 1-10 or 1-5 second dye molecules per targeting molecule.

[0221] II. Pharmaceutical Compositions and Articles of Manufacture Provided herein is a pharmaceutical composition containing a phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate). In some embodiments, the composition can be used in the method of PIT as described herein. The phthalocyanine dye-targeting molecule conjugate, e.g., IR700-antibody conjugate. In some embodiments, the composition can be provided in combination with another therapeutic agent (e.g., an immunomodulator or an anti-cancer agent). In some embodiments, the phthalocyanine dye-targeting molecule conjugate and another therapeutic agent, e.g., one or both of an immunomodulator or an anti-cancer agent, can be packaged as an article of manufacture as separate compositions for administration together, sequentially or intermittently. The combination can be packaged as a kit.

[0222] 1. Compositions, Formulations and Dosage Forms In some embodiments, the compound, e.g., conjugate, can be formulated in a pharma- ceutically acceptable buffer, e.g., a pharma-ceutically acceptable carrier or vehicle.Generally, pharma-ceutically acceptable carriers or vehicles, e.g., pharma-ceutically acceptable carriers or vehicles present in a pharma-ceutically acceptable buffer, are all known in the art.Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds.Pharmaceutically acceptable compositions are generally prepared in consideration of the approval of regulatory agencies or other agencies, which are made in accordance with generally accepted pharmacopoeias for use in animals and humans.

[0223] A pharmaceutical composition can include a carrier, such as a diluent, adjuvant, excipient, or vehicle, that is administered together with the compound. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions will contain a therapeutically effective amount of the compound (generally in purified form) in combination with a suitable amount of carrier to provide the form for proper administration to the patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil). Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The composition can contain, together with the active ingredient, diluents such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; lubricants such as magnesium stearate, calcium stearate, and talc; and binders such as starch, natural gums such as gum acacia, gelatin, glucose, molasses, polyvinylpyrrolidine, cellulose and its derivatives, povidone, crospovidone, and other such binders known to those skilled in the art.Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.The composition can also contain, if desired, a small amount of wetting or emulsifying agent, or pH buffering agent, such as acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.

[0224] In some embodiments, pharmaceutical preparations can be in liquid form, for example, solution, syrup or suspension.Such liquid preparations can be prepared by conventional means using pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or edible hydrogenated fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoate or sorbic acid).In some cases, pharmaceutical preparations can be presented in lyophilized form for reconstitution with water or other suitable vehicles before use.

[0225] In some embodiments, the nature of the pharma- ceutically acceptable buffer or carrier depends on the particular mode of administration employed.For example, in some embodiments, parenteral formulations may contain injection fluids that contain pharma- ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, or glycerol as vehicles.In some embodiments, for solid compositions, such as powder, pill, tablet, or capsule forms, non-toxic solid carriers may contain, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate.In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain, in some embodiments, minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0226] The compound can be formulated into suitable pharmaceutical preparations, such as oral administration solution, suspension, tablet, disintegrating tablet, pill, capsule, powder, sustained release preparation or elixir, as well as transdermal patch preparation and dry powder inhalant.Typically, compound is formulated into pharmaceutical composition using techniques and procedures well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).Generally, the mode of formulation is related to the route of administration.

[0227] The composition can be formulated for administration by any route known to those skilled in the art, including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, intravaginal, rectal, local, topical, otic, inhalation, oral (e.g., sublingual), and transdermal administration or any route. Other modes of administration are also contemplated. Administration can be local, regional, or systemic, depending on the location of treatment. Local administration to the area requiring treatment can be achieved, for example, but not limited to, by local injection during surgery, local application (e.g., with wound dressing after surgery), by injection, by catheter, by suppository, or by implant.

[0228] Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly, intratumorally, intravenously or intradermally. Injections can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid before injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical composition to be administered can also contain activating agents in the form of solvents, such as pH buffering agents, metal ion salts, or other such buffering agents. The pharmaceutical composition can also contain small amounts of other non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins. Implantation of a delayed or sustained release system, such that a constant level of dosage is maintained (see, for example, U.S. Pat. No. 3,710,795), is also contemplated herein. The percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.

[0229] Injectables are designed for local and systemic administration. Preparations for parenteral administration include sterile liquids ready for injection, sterile dry soluble products ready to be combined with a solvent immediately before use, such as lyophilized powders (including subcutaneous tablets), sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle immediately before use, and sterile emulsions. Liquids can be either aqueous or non-aqueous. When administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0230] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multi-dose containers, and include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate.

[0231] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), as well as solutions containing viscosity enhancing and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0232] The composition can be formulated for single or multiple dose administration.The drug can be formulated for direct administration.The composition can be provided as a liquid or lyophilized formulation.When the composition is provided in lyophilized form, it can be reconstituted with suitable buffer, for example, sterile saline, immediately before use.

[0233] The compositions can also be administered with other biologically active agents, either sequentially, intermittently, or in the same composition. Administration can also include controlled release systems, including controlled release formulations and controlled release devices (such as by pumps).

[0234] The most suitable route in any given case depends on various factors, such as the nature of disease, the progression of disease, the severity of disease and the specific composition used.For example, the composition is administered systemically, for example, via intravenous administration.Subcutaneous methods can also be used, but may require increased absorption time to ensure the same bioavailability compared to intravenous methods.

[0235] Pharmaceutical compositions can be formulated in dosage forms suitable for each administration route. Pharmaceutically and therapeutically active compounds and their derivatives are typically formulated and administered in unit dosage forms or multiple dosage forms. Each unit dosage contains a predetermined amount of therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the necessary pharmaceutical carrier, vehicle or diluent. Unit dosage forms include, but are not limited to, tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions, containing a suitable amount of the compound or its pharma-ceutically acceptable derivatives. Unit dosage forms can be contained ampoules and syringes, or individually packaged tablets or capsules. Unit dosage forms can be administered in divided doses or multiples thereof. Multiple dosage forms are multiple identical unit dosage forms packaged in a single container to be administered in separate unit dosage forms. Examples of multiple dose forms include vials, bottles of tablets or capsules, or bottles of pints or gallons. Thus, multiple dose forms are multiple unit doses that are not segregated in packaging. In general, dosage forms or compositions containing active ingredients in the range of 0.005% to 100% and balanced by non-toxic carriers can be prepared. Pharmaceutical compositions can be formulated in dosage forms suitable for each administration route.

[0236] The concentration of the pharmaceutically active compound is adjusted so that the injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal, as known in the art. The unit dose parenteral preparation is packaged in an ampoule, vial or syringe with a needle. The volume of the liquid solution or reconstituted powder preparation containing the pharmaceutically active compound is a function of the disease to be treated and the particular product selected for packaging. All preparations for parenteral administration must be sterile, as known and practiced in the art. In some embodiments, the composition can be provided as a lyophilized powder, which can be reconstituted for administration as a solution, emulsion and other mixture. They can also be reconstituted and formulated as a solid or gel. The lyophilized powder can be prepared from any of the above solutions.

[0237] A sterile, lyophilized powder can be prepared by dissolving the phthalocyanine dye-targeting molecule conjugate in a buffer solution that may contain excipients that improve the stability of other pharmacological components of the powder or of a reconstituted solution prepared from the powder.

[0238] In some embodiments, subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. Briefly, the lyophilized powder is prepared by dissolving an excipient, such as dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent, in a suitable buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those of skill in the art. The selected enzyme is then added to the resulting mixture and stirred until it dissolves. The resulting mixture is aseptically filtered or processed to remove particulates and ensure sterility, and dispensed into vials for lyophilization. Each vial can contain a single dose (1 mg to 1 g, typically 1 to 100 mg, e.g., 1 to 5 mg) or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature. Reconstitution of this lyophilized powder with a buffer solution provides a formulation for use in parenteral administration. The exact amount depends on the indication being treated and the compound selected. Such amounts can be determined empirically.

[0239] In some embodiments, the pH of the composition is between or about 6-10, such as between or about 6-8, between or about 6.9-7.3, such as about pH 7.1. In some embodiments, the pH of the pharma- ceutically acceptable buffer is at least or about 5, at least 6 or about 6, at least 7 or about 7, at least 8 or about 8, at least 9 or about 9, or at least 10 or about 10, or is 7.1.

[0240] The composition may be formulated for single or multiple dose administration. The agent may be formulated for direct administration.

[0241] In some embodiments, the compositions provided herein contain an amount of active compound, e.g., a phthalocyanine dye-targeting molecule conjugate, for direct administration, of 0.01 mg to about 3000 mg, or about 0.01 mg to about 3000 mg, or about 0.01 mg to about 1000 mg, about 0.01 mg to about 500 mg, about 0.01 mg to about 100 mg, about 0.01 mg to about 50 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 1 mg, about 0.01 mg to about 0.1 mg, about 0.1 mg to about 2000 mg, about 0.1 mg to about 1000 mg, about 0.1 mg to about 500 mg, about 0.1 mg to about 1 mg, It is formulated in the ranges of about 100 mg, about 0.1 mg to about 50 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 1 mg, about 1 mg to about 2000 mg, about 1 mg to about 1000 mg, about 1 mg to about 500 mg, about 1 mg to about 100 mg, about 1 mg to about 10 mg, about 10 mg to about 2000 mg, about 10 mg to about 1000 mg, about 10 mg to about 500 mg, about 10 mg to about 100 mg, about 100 mg to about 2000 mg, about 100 mg to about 1000 mg, about 100 mg to about 500 mg, about 500 mg to about 2000 mg, about 500 mg to about 1000 mg, and about 1000 mg to about 3000 mg. In some embodiments, the volume of the composition is between 0.5 mL and 1000 mL, e.g., between 0.5 mL and 100 mL, between 0.5 mL and 10 mL, between 1 mL and 500 mL, between 1 mL and 10 mL, e.g., at least 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, 50 mL or more, or about at least 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, It can be 8mL, 9mL, 10mL, 15mL, 20mL, 30mL, 40mL, 50mL or more, or about 0.5mL, 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 15mL, 20mL, 30mL, 40mL, 50mL or more, or 0.5mL, 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 15mL, 20mL, 30mL, 40mL, 50mL or more.For example, the composition is formulated for single dosage administration in an amount at or about 100 mg to 500 mg, or at or about 200 mg to 400 mg. In some embodiments, the composition is formulated for single dosage administration in an amount at or about 500 mg to 1500 mg, at or about 800 mg to 1200 mg, or at or about 1000 mg to 1500 mg. In some embodiments, the volume of the composition is between 10 mL and 1000 mL, or between about 10 mL and 1000 mL, or between 50 mL and 500 mL, or between about 50 mL and 500 mL; or the volume of the composition is at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 400 mL, 500 mL, or 1000 mL.

[0242] In some embodiments, the entire vial contents of the formulation can be removed for administration or can be divided into multiple doses for multiple administration. After removing the amount of drug for administration, the formulation can be further diluted if desired, for example, in water, saline (e.g., 0.9%) or other physiological solution.

[0243] In some embodiments, compositions containing immunomodulatory agents or anti-cancer agents are also provided, which can be prepared according to known or standard formulation guidelines as described above. In some embodiments, the immunomodulatory agent, anti-cancer agent and / or phthalocyanine dye-targeting molecule conjugate (e.g., IR700-targeting molecule, e.g., IR700-antibody conjugate) are formulated as separate compositions. In some embodiments, the immunomodulatory agent is provided as a separate composition from the phthalocyanine dye-targeting molecule conjugate, and the two compositions are administered separately. In some embodiments, the anti-cancer agent is provided as a separate composition from the phthalocyanine dye-targeting molecule conjugate, and the two compositions are administered separately. The composition can be formulated for parenteral delivery (i.e., for systemic delivery). For example, the composition or combination of compositions is formulated for subcutaneous delivery or for intravenous delivery. The agents, such as the phthalocyanine dye-targeting molecule conjugate, the immunomodulatory agent, and / or the anti-cancer agent, can be administered by different routes of administration.

[0244] 3. Packaging and Manufacture Also provided is an article of manufacture, comprising packaging material, any pharmaceutical composition or combination provided herein, and a label indicating that the composition and combination are used for the treatment of cancer.Exemplary articles of manufacture are containers, including single-chamber and dual-chamber containers.Containers include, but are not limited to, tubes, bottles, and syringes.Containers can further comprise needles for subcutaneous administration.

[0245] In some embodiments, the agent can be provided separately for packaging as an article of manufacture. In some embodiments, the article of manufacture comprises a pharmaceutical composition comprising a phthalocyanine dye-targeting molecule conjugate, such as an IR700-antibody conjugate, and an immunomodulatory agent. In some embodiments, the article of manufacture comprises a pharmaceutical composition comprising a phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-antibody conjugate) and an anti-cancer agent. In some embodiments, the article of manufacture comprises a pharmaceutical composition comprising a phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-antibody conjugate), an immunomodulatory agent, and an additional anti-cancer agent.

[0246] The products provided herein include packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those skilled in the art. For example, see U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment. The choice of packaging depends on the drug. In general, the packaging is non-reactive with the composition contained therein.

[0247] The components can be packaged in the same or different containers. For example, in some embodiments, the components are packaged separately in the same container. In general, examples of such containers include a container with a defined closed space that contains the phthalocyanine dye-targeting molecule conjugate and a separate defined closed space that contains the other component(s), with subsequent areas separated to allow the components to be administered separately. Any container or other manufactured article is contemplated, so long as the drug is separated from the other components before administration. For suitable embodiments, see, for example, the containers described in U.S. Pat. Nos. 3,539,794 and 5,171,081. In some embodiments, multiple containers are provided, each of which separately contains the phthalocyanine dye-targeting molecule conjugate, an immunomodulatory agent, or an anticancer agent. In such examples, the multiple containers can be packaged together as a kit.

[0248] In some embodiments, the container containing the phthalocyanine dye-targeting molecule conjugate is contained in a light-protecting container. In some embodiments, the container is a vial, such as a pyrogen-free glass vial. In some embodiments, the container, such as a vial, blocks certain wavelengths of light, such as the wavelengths of light absorbed by the dye or dye-targeting molecule conjugate. In some embodiments, the conjugate is protected from light using a container that protects the contents from light, or from certain wavelengths or intensities of light. For example, in some embodiments, the container has a light transmittance of 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. In some embodiments, the container is protected from the transmission of light having a wavelength between or about 500 nm and 725 nm, such as between or about 650 nm and 725 nm, or is impermeable to light with an intensity of more than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the container is green, amber, translucent, opaque, or covered with an opaque material, such as foil, for example aluminum foil. In some embodiments, the container is sterilized or depyrogenated.

[0249] In some embodiments, the conjugate is provided in a plurality of sealed containers. For example, the containers can each individually contain a portion of a single dose of a composition containing a conjugate comprising a phthalocyanine dye linked to a targeting molecule. In some embodiments, the combined amount of the conjugate in the plurality of sealed containers is 100 mg to 1500 mg or about 100 mg to 1500 mg, or 100 mg to 1200 mg or about 100 mg to 1200 mg. In some embodiments, the combined amount of conjugates in the multiple sealed containers is between or about 100 mg-500 mg, between or about 200 mg-400 mg, between or about 500 mg-1500 mg, between or about 800 mg-1200 mg, or between or about 1000 mg-1500 mg.

[0250] In some embodiments, the article of manufacture comprises packaging material and a label or insert containing instructions for combining the contents of multiple vials to prepare a single dosage formulation of the composition.

[0251] In some embodiments, the container is further packaged to protect the contents from light. In some embodiments, a packaging system is provided that includes an internal packaging material that includes a container that includes a phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-targeting molecule conjugate, e.g., an IR700-antibody conjugate) and optionally a container that includes an immunomodulatory agent or an anticancer agent. In some embodiments, the internal packaging material has a light transmittance of less than 20%, e.g., less than 15%, less than 10%, less than 5%, or less than 1%. In some embodiments, the packaging system includes an external packaging material that includes the internal packaging material. In some embodiments, the external packaging material has a light transmittance of less than 20%, e.g., less than 15%, less than 10%, less than 5%, or less than 1%. In some embodiments, the internal or external packaging material includes an opaque foil, e.g., aluminum foil. In some embodiments, the secondary packaging material is an aluminum pouch. In some embodiments, the external packaging material includes cardboard.

[0252] In some embodiments, the internal and / or external packaging material is suitable for storage of the conjugate. In some embodiments, the internal and / or external packaging material is suitable for transport of the conjugate.

[0253] The selected composition, including the article of manufacture, can also be provided as a kit. The kit can include the pharmaceutical composition described herein provided as an article of manufacture and an item for administration. The kit can optionally include instructions for application, including dosage, dosing regimen, and instructions for mode of administration. The kit can also include the pharmaceutical composition described herein and a diagnostic item.

[0254] In some embodiments, compositions used to administer agents, such as phthalocyanine dye-targeting molecule conjugates, contain an effective amount of each agent in combination with conventional pharmaceutical carriers and excipients appropriate for the type of administration intended.

[0255] In some embodiments, a single dose of a drug, such as a phthalocyanine dye-targeting molecule conjugate, is contained in a single container, such as a container in which the drug is stored.In some embodiments, a single dose of a drug is contained in multiple containers, and thus in some embodiments, multiple containers, such as vials, are combined in a container used for administering the drug, such as an intravenous (IV) bag.In some embodiments, a container used for administration, such as an IV bag, is prepared by opening one or more containers containing the drug and placing the contents into the bag, such as until the desired drug dose is achieved for administration (e.g., injection).During preparation of an administration container, such as an IV bag, photoprecautions are taken to avoid exposure of the drug to light, such as various photoprecautions described herein.

[0256] III. Method of Treatment In some embodiments, methods and uses are provided for using compositions containing phthalocyanine dye-targeting molecule conjugates (e.g., IR700-targeting molecule conjugates, e.g., IR700-antibody conjugates) for PIT. In some embodiments, the phthalocyanine dye-targeting molecule conjugates target cells or pathogens associated with a disease or condition, e.g., via binding to cell surface proteins or cell surface receptors expressed on the cells. Such methods and uses include, for example, therapeutic methods and uses that include administering molecules to a subject with a disease, condition, or disorder, and then irradiating to achieve photoimmunotherapy (PIT), thereby resulting in photolysis of such cells or pathogens to achieve treatment of the disease or disorder. In some embodiments, the methods can be used for the treatment of tumors or cancers, where the administered phthalocyanine dye-targeting molecule conjugates (IR700-targeting molecule conjugates, e.g., IR700-antibody conjugates) are targeted to cells associated with the tumor, thereby resulting in photolysis of such cells, and in some cases, resulting in treatment of the tumor. The uses include the use of the compositions in such methods and treatments, as well as the use of such compositions in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the methods and uses thereby treat a disease or condition or disorder, such as a tumor or cancer, in a subject.

[0257] In some embodiments, the method involves administering a phthalocyanine dye-targeting molecule conjugate (e.g., an IR-700 antibody conjugate) to a subject, generally under conditions in which cells targeted for killing are contacted with the conjugate. In some embodiments, the method results in binding of the targeting molecule (e.g., an antibody) portion of the conjugate to a cell surface protein associated with a tumor or cancer. After contacting with the conjugate or administering the conjugate, a localized area of ​​the subject containing the targeted cells, e.g., a cell or cells associated with a tumor, is exposed or irradiated with light absorbed by the dye, generally NIR light, thereby activating the conjugate and achieving specific cell killing. In some embodiments, the irradiation is at least 1 J cm at a wavelength of 600 nm to 850 nm. -2 Or at a dose of at least 1 J / cm of fiber length. In some embodiments, the method of administering the phthalocyanine dye-targeting molecule conjugate (e.g., IR-700 antibody conjugate) includes the methods described in U.S. Patent No. 8,524,239 or U.S. Patent Application Publication No. US2014 / 0120119.

[0258] A. Tumors and Subjects to be Treated In some embodiments, the lesion is a tumor. In some embodiments, the tumor is a cancer. In some embodiments, the cancer is a cancer of the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, lung, or blood. In some embodiments, the cancer can include a malignant tumor characterized by abnormal or uncontrolled cell growth. Other characteristics that may be associated with cancer include metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretory products, and suppression or aggravation of inflammatory or immunological responses, invasion of surrounding or distant tissues or organs such as lymph nodes, etc. Metastatic disease can refer to cancer cells that have left the original tumor site and migrated to other parts of the body, for example, via the bloodstream or lymphatic system. In some embodiments, the cells targeted by the disclosed methods are cancer cells or immune cells. In some embodiments, the cancer cells are cancer stem cells. In some embodiments, the cells targeted by the disclosed methods are cells that are cancer cells, tumor cells, inflammatory cells, immune cells, neural cells, stem cells, proliferative cells, or cells in a hyperplastic state.

[0259] The target cell can be an unwanted cell, such as a tumor or cancer cell, or a cell whose growth is undesirable. In some embodiments, the cell can be grown in culture or can be present in a mammalian subject to be treated, such as a subject with cancer. Any target cell can be treated with the claimed method. In some embodiments, the target cell expresses a cell surface protein that is not substantially found on the surface of other normal cells. In some embodiments, an antibody can be selected that specifically binds to such a protein, and a phthalocyanine dye-antibody conjugate can be generated against the protein. In some embodiments, the cell surface protein is a tumor-specific protein. In some embodiments, the cell surface protein is CD25, which can be used to target cells associated with unwanted transplant rejection.

[0260] In some embodiments, the tumor cell is a cancer cell, for example, a cell in a subject with cancer.Exemplary cells that can be targeted in the disclosed methods include cells of the following tumors: liquid tumors, for example, leukemia, including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemia (e.g., chronic myelogenous (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 some embodiments, the cells are from solid tumor cells, e.g., sarcoma or carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, 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, basal cell carcinoma, adenocarcinoma, e.g., pancreatic, colon, ovarian, lung, breast, stomach, prostate, Adenocarcinoma of the cervix or esophagus, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical carcinoma, testicular tumor, bladder carcinoma, CNS tumor, such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.In some embodiments, the cancer is squamous cell carcinoma of the head and neck.

[0261] Exemplary tumors, e.g., cancers, that can be treated with the claimed methods include solid tumors, e.g., breast carcinomas, e.g., lobular and ductal carcinomas, sarcomas, carcinomas of the lung, e.g., non-small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma, pulmonary mesothelioma, colorectal adenocarcinoma, gastric carcinoma, prostate adenocarcinoma, ovarian carcinomas, e.g., serous cystadenocarcinoma and mucinous cystadenocarcinoma, ovarian germ cell tumors, testicular carcinoma and germ cell tumors, pancreatic adenocarcinoma, bile duct adenocarcinoma, hepatocellular carcinoma, bladder carcinoma (including, e.g., transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma), renal cell adenocarcinoma, endometrial carcinoma (including, e.g., adenocarcinoma and mixed Mullerian tumor (carcinosarcoma)), endocervix, cervicovaginal, and vaginal These include carcinomas of the esophagus, such as adenocarcinomas and squamous cell carcinomas thereof, tumors of the skin, such as squamous cell carcinoma, basal cell carcinoma, malignant melanoma, tumors of the skin appendages, Kaposi's sarcoma, lymphoma of the skin, tumors of the skin adnexals, and various types of sarcomas and Merkel cell carcinomas, esophageal carcinomas, carcinomas of the nasopharynx and oropharynx (including squamous cell carcinomas and adenocarcinomas thereof), salivary gland carcinomas, tumors of the brain and central nervous system (including tumors of glial, neuronal, and meningeal origin), tumors of the peripheral nerves, soft tissue sarcomas, and sarcomas of bone and cartilage, and tumors of the lymphatic system (including B-cell and T-cell malignant lymphomas). In some embodiments, the tumor is an adenocarcinoma.

[0262] This method can also be used to treat liquid tumors, such as lymphatic leukemia, or other types of leukemia.In some embodiments, the tumor treated is blood tumors, such as leukemia, such as 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, such as Hodgkin's lymphoma and non-Hodgkin's lymphoma, and myeloma.

[0263] In some embodiments, the conjugate is targeted to a protein expressed on the surface of a cell present on the surface of a lesion or in the microenvironment of a lesion. For example, in some embodiments, the conjugate is targeted to a protein expressed on the surface of a cell in a tumor or in the microenvironment of a tumor. Exemplary such cell surface proteins are any of those described herein, including those described above.

[0264] In some embodiments, the protein on the cell surface of the targeted cell is not present in significant amounts on other cells, for example, the cell surface protein can be a receptor that is found only on the target cell type.

[0265] In some embodiments, the protein expressed in the tumor, e.g., the tumor-specific protein, is HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, Cancer Antigen 125 (CA 125), alpha-fetoprotein (AFP), Lewis Y, TAG72, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate binding protein, gangliosides (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, SK-1 antigen, or PD-L1. In some embodiments, the tumor-specific protein is PD-L1, HER1 / EGFR, HER2, CD20, CD25, CD33, CD52, prostate-specific membrane antigen (PSMA), EpCAM, EphA2, CD206, CD44, CD133, mesothelin, glypican-3, or carcinoembryonic antigen (CEA). Other cell surface proteins include any of those described above.

[0266] In some embodiments, the cell surface protein is tumor-associated, e.g., tumor-specific protein or tumor-specific antigen, such as members of the EGF receptor family (e.g., HER1, 2, 3, and 4) and members of cytokine receptors (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.). In some embodiments, tumor-specific proteins are proteins that are unique to or highly abundant on cancer cells compared to other cells, such as normal cells. For example, HER2 is commonly found in breast cancer, while HER1 is typically found in adenocarcinomas, which can be found in many organs, such as pancreas, breast, prostate, and colon.

[0267] Exemplary tumor-associated proteins that may be found on target cells and for which a phthalocyanine dye-antibody conjugate can be formulated using a targeting molecule, such as an antibody or antibody fragment specific for that protein, include, but are not limited to, any of the various MAGEs (melanoma associated antigen E), including MAGE1, MAGE2, MAGE3, and MAGE4, ​​any of the various tyrosinases, mutant Ras, mutant p53, p97 melanoma antigen, human milk fat globules (HMFG), which may be associated with breast tumors, any of the various BAGEs (human B melanoma associated antigen E), including BAGE1 and BAGE2, any of the various GAGEs (G antigens), including GAGE1, GAGE2-6, various gangliosides, and CD25.

[0268] Other tumor-associated proteins include HPV16 / 18 and E6 / E7 antigens associated with cervical cancer, mucin (MUC 1)-KLH antigen which may be associated with breast carcinoma, CEA (carcinoembryonic antigen) which may be associated with colorectal cancer, gp100 which may be associated with melanoma, MARTI antigen which may be associated with melanoma, cancer antigen 125 (CA125, also known as mucin 16 or MUC16) which may be associated with ovarian and other cancers, alpha-fetoprotein (AFP) which may be associated with liver cancer, Lewis Y antigen which may be associated with colorectal cancer, cholangiocarcinoma, breast cancer, small cell lung cancer, and other cancers, tumor-associated glycoprotein 72 (TAG72) which may be associated with adenocarcinoma, and PSA antigen which may be associated with prostate cancer.

[0269] Other exemplary tumor-associated proteins further include, but are not limited to, PMSA (prostate membrane-specific antigen), which may be associated with solid tumor angiogenesis as well as prostate cancer; HER-2 (human epidermal growth factor receptor 2), which may be associated with breast, ovarian, gastric and uterine cancer; HER-1, which may be associated with lung, anal, and glioblastoma and adenocarcinoma; NY-ESO-1, which may be associated with melanoma, sarcoma, testicular, and other cancers; hTERT (also known as telomerase), proteinase 3, and Wilms' tumor 1 (WT-1).

[0270] In some embodiments, the tumor associated protein may be CD52 and associated with chronic lymphocytic leukemia, CD33 and associated with acute myeloid leukemia, or CD20 and associated with non-Hodgkin's lymphoma.

[0271] Thus, the disclosed methods can be used to treat any cancer that expresses a tumor-specific protein. In some embodiments, the tumor therapeutic is an antibody, an antigen-binding fragment, a protein, a glycoprotein, a peptide polypeptide, a virus, a viral capsid, or a viral particle. In some embodiments, the tumor therapeutic is an antibody or an antigen-binding fragment.

[0272] In some embodiments, the subject is a human or a non-human mammal. In some embodiments, the subject is a human or an animal subject, e.g., a mouse. In some embodiments, the subject is a mammal, e.g., a human, having cancer or being treated for cancer. In some embodiments, the disclosed method is used to treat a subject having a tumor, e.g., a tumor described herein. In some embodiments, the tumor has previously been treated, e.g., surgically or chemically removed, and the disclosed method is subsequently used to kill any remaining undesirable tumor cells that may remain in the subject.

[0273] The disclosed method can be used to treat any mammalian subject, e.g., a human, having a tumor, e.g., cancer, or having a cancer that has previously been removed or treated. A subject in need of the treatment of the present disclosure can include a human subject having cancer, where the cancer cells express a tumor-specific protein on the cell surface that can specifically bind to the phthalocyanine dye-targeting molecule conjugate. For example, the disclosed method can be used as an initial treatment for cancer, either alone or in combination with radiation or other chemotherapy. The disclosed method can also be used for patients who have failed previous radiation or chemotherapy. Thus, in some embodiments, the subject is a subject who has undergone other treatments, but where the other treatments have not provided the desired therapeutic response. The disclosed method can also be used for patients with localized and / or metastatic cancer.

[0274] In some embodiments, the method includes selecting a subject who will benefit from the treatment of the present disclosure, for example, selecting a subject who has a tumor that expresses a cell surface protein, such as a tumor-specific protein, that can specifically bind to the phthalocyanine dye-targeting molecule conjugate. For example, if a subject is determined to have a breast cancer that expresses HER1, the subject may be selected to be treated with an anti-HER1-IR700 molecule, such as cetuximab-IR700.

[0275] B. Dosage and Administration The compositions provided herein containing phthalocyanine dye-targeting molecule conjugates (e.g., IR700-antibody conjugates) are administered in an amount sufficient to exert a therapeutically useful effect. Typically, the active agent is administered in an amount that does not cause undesirable side effects in the treated patient, or in an amount that minimizes or reduces observed side effects compared to the dosage and amount required for single agent treatment with one of the above agents.

[0276] Methods for determining the optimal dosage of a phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-antibody conjugate) to a patient in need thereof (either alone or in combination with one or more other agents) can be determined by standard dose-response and toxicity studies that are well known in the art.

[0277] The amount of therapeutic agent, such as a phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate), administered to a human or veterinary subject will vary depending on a number of factors associated with the subject, such as the overall health of the subject. In some embodiments, the effective amount of the agent can be determined by varying the dosage of the product and measuring the resulting therapeutic response, such as tumor regression. In some embodiments, the effective amount can be determined through various in vitro, in vivo, or in situ immunoassays. In some embodiments, the disclosed agents can be administered in a single dose or multiple doses as needed to obtain the desired response. In some embodiments, the effective amount depends on the source applied, the subject being treated, the severity and type of the condition being treated, and the mode of administration.

[0278] In some embodiments, a therapeutically effective amount is an amount of a composition that is sufficient to achieve a desired effect in a subject or cell that is being treated with the composition, alone or in combination with additional therapeutic agents such as chemotherapeutic agents.The effective amount of a therapeutic agent, such as a phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate), can depend on several factors, including but not limited to the subject or cell that is being treated, the specific therapeutic agent, and the mode of administration of the therapeutic composition.In some embodiments, a therapeutically effective amount or concentration is sufficient to prevent the progression of a disease, such as metastasis, to delay the progression of a disease, or to cause the regression of a disease, or to reduce the symptoms caused by a disease, such as cancer.In some embodiments, a therapeutically effective amount or concentration is sufficient to increase the survival time of a patient with a tumor.

[0279] In some embodiments, the therapeutically effective dose of the conjugate is 10 mg / m 2 ~5000mg / m 2 Between 10 mg / m 2 ~5000mg / m 2 between 10 mg / m 2 ~3000mg / m 2 , 10 mg / m 2 ~1500mg / m 2 , 10 mg / m 2 ~750mg / m 2 , 10 mg / m 2 ~500mg / m 2 , 10 mg / m 2 ~250mg / m 2 , 10 mg / m 2 ~200mg / m 2 , 10 mg / m 2 ~100mg / m 2 , 10 mg / m 2 ~75mg / m 2 , 10 mg / m 2 ~50mg / m 2 , 10 mg / m 2 ~25mg / m 2 , 25 mg / m 2 ~5000mg / m 2、25mg / m 2 ~3000mg / m 2 、25mg / m 2 ~1500mg / m 2 、25mg / m 2 ~750mg / m 2 、25mg / m 2 ~500mg / m 2 、25mg / m 2 ~250mg / m 2 、25mg / m 2 ~200mg / m 2 、25mg / m 2 ~100mg / m 2 、25mg / m 2 ~75mg / m 2 、25mg / m 2 ~50mg / m 2 、50mg / m 2 ~5000mg / m 2 、50mg / m 2 ~3000mg / m 2 、50mg / m 2 ~1500mg / m 2 、50mg / m 2 ~750mg / m 2 、50mg / m 2 ~500mg / m 2 、50mg / m 2 ~250mg / m 2 、50mg / m 2 ~200mg / m 2 、50mg / m 2 ~100mg / m 2 、50mg / m 2 ~75mg / m 2 、75mg / m 2 ~5000mg / m 2 、75mg / m 2 ~3000mg / m 2 、75mg / m 2 ~1500mg / m 2 、75mg / m 2 ~1000mg / m 2 、75mg / m 2 ~750mg / m 2 、75mg / m 2 ~500mg / m 2 、75mg / m2 ~250mg / m 2 、75mg / m 2 ~225mg / m 2 、75mg / m 2 ~200mg / m 2 、75mg / m 2 ~100mg / m 2 、100mg / m 2 ~5000mg / m 2 、100mg / m 2 ~3000mg / m 2 、100mg / m 2 ~1500mg / m 2 、100mg / m 2 ~750mg / m 2 、100mg / m 2 ~500mg / m 2 、100mg / m 2 ~250mg / m 2 、100mg / m 2 ~200mg / m 2 、100mg / m 2 ~150mg / m 2 、150mg / m 2 ~5000mg / m 2 、150mg / m 2 ~3000mg / m 2 、150mg / m 2 ~1500mg / m 2 、150mg / m 2 ~750mg / m 2 、150mg / m 2 ~500mg / m 2 、150mg / m 2 ~250mg / m 2 、150mg / m 2 ~200mg / m 2 、200mg / m 2 ~5000mg / m 2 、200mg / m 2 ~3000mg / m 2 、200mg / m 2 ~1500mg / m 2 、200mg / m 2 ~750mg / m 2 、200mg / m 2 ~500mg / m2 , 200 mg / m 2 ~250mg / m 2 , 250 mg / m 2 ~5000mg / m 2 , 250 mg / m 2 ~3000mg / m 2 , 250 mg / m 2 ~1500mg / m 2 , 250 mg / m 2 ~750mg / m 2 , 250 mg / m 2 ~500mg / m 2 , 500 mg / m 2 ~5000mg / m 2 , 500 mg / m 2 ~3000mg / m 2 , 500 mg / m 2 ~1500mg / m 2 , 500 mg / m 2 ~750mg / m 2 , 750 mg / m 2 ~5000mg / m 2 , 750 mg / m 2 ~3000mg / m 2 , 750 mg / m 2 ~1500mg / m 2 , 1500mg / m 2 ~5000mg / m 2 , 1500mg / m 2 ~3000mg / m 2 , and 3000 mg / m 2 ~5000mg / m 2 or about 10 mg / m 2 ~3000mg / m 2 , 10 mg / m 2 ~1500mg / m 2 , 10 mg / m 2 ~750mg / m 2 , 10 mg / m 2 ~500mg / m 2 , 10 mg / m 2 ~250mg / m 2 , 10 mg / m 2 ~200mg / m 2 , 10 mg / m 2 ~100mg / m 2、10mg / m 2 ~75mg / m 2 、10mg / m 2 ~50mg / m 2 、10mg / m 2 ~25mg / m 2 、25mg / m 2 ~5000mg / m 2 、25mg / m 2 ~3000mg / m 2 、25mg / m 2 ~1500mg / m 2 、25mg / m 2 ~750mg / m 2 、25mg / m 2 ~500mg / m 2 、25mg / m 2 ~250mg / m 2 、25mg / m 2 ~200mg / m 2 、25mg / m 2 ~100mg / m 2 、25mg / m 2 ~75mg / m 2 、25mg / m 2 ~50mg / m 2 、50mg / m 2 ~5000mg / m 2 、50mg / m 2 ~3000mg / m 2 、50mg / m 2 ~1500mg / m 2 、50mg / m 2 ~750mg / m 2 、50mg / m 2 ~500mg / m 2 、50mg / m 2 ~250mg / m 2 、50mg / m 2 ~200mg / m 2 、50mg / m 2 ~100mg / m 2 、50mg / m 2 ~75mg / m 2 、75mg / m 2 ~5000mg / m 2 、75mg / m 2 ~3000mg / m 2 、75mg / m2 ~1500mg / m 2 、75mg / m 2 ~1000mg / m 2 、75mg / m 2 ~750mg / m 2 、75mg / m 2 ~500mg / m 2 、75mg / m 2 ~250mg / m 2 、75mg / m 2 ~225mg / m 2 、75mg / m 2 ~200mg / m 2 、75mg / m 2 ~100mg / m 2 、100mg / m 2 ~5000mg / m 2 、100mg / m 2 ~3000mg / m 2 、100mg / m 2 ~1500mg / m 2 、100mg / m 2 ~750mg / m 2 、100mg / m 2 ~500mg / m 2 、100mg / m 2 ~250mg / m 2 、100mg / m 2 ~200mg / m 2 、100mg / m 2 ~150mg / m 2 、150mg / m 2 ~5000mg / m 2 、150mg / m 2 ~3000mg / m 2 、150mg / m 2 ~1500mg / m 2 、150mg / m 2 ~750mg / m 2 、150mg / m 2 ~500mg / m 2 、150mg / m 2 ~250mg / m 2 、150mg / m 2 ~200mg / m 2 、200mg / m 2 ~5000mg / m 2, 200 mg / m 2 ~3000mg / m 2 , 200 mg / m 2 ~1500mg / m 2 , 200 mg / m 2 ~750mg / m 2 , 200 mg / m 2 ~500mg / m 2 , 200 mg / m 2 ~250mg / m 2 , 250 mg / m 2 ~5000mg / m 2 , 250 mg / m 2 ~3000mg / m 2 , 250 mg / m 2 ~1500mg / m 2 , 250 mg / m 2 ~750mg / m 2 , 250 mg / m 2 ~500mg / m 2 , 500 mg / m 2 ~5000mg / m 2 , 500 mg / m 2 ~3000mg / m 2 , 500 mg / m 2 ~1500mg / m 2 , 500 mg / m 2 ~750mg / m 2 , 750 mg / m 2 ~5000mg / m 2 , 750 mg / m 2 ~3000mg / m 2 , 750 mg / m 2 ~1500mg / m 2 , 1500mg / m 2 ~5000mg / m 2 , 1500mg / m 2 ~3000mg / m 2 , and 3000 mg / m 2 ~5000mg / m 2 In some embodiments, the therapeutically effective dose of the conjugate is between 10 mg / m 2 , 50 mg / m 2 , 75 mg / m 2 , 100 mg / m 2 , 150 mg / m 2, 200 mg / m 2 , 225 mg / m 2 , 250 mg / m 2 , 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000mg / m 2 , 1250 mg / m 2 , 1500mg / m 2 , 2000mg / m 2 , 2500mg / m 2 , 3000mg / m 2 , 3500mg / m 2 , 4000mg / m 2 , 4500mg / m 2 , or 5000 mg / m 2 In some embodiments, the dose is 50 mg / m 2 ~about 5000mg / m 2 or about 50 mg / m 2 ~about 5000mg / m 2 , about 250mg / m 2 ~about 2500mg / m 2 , about 750mg / m 2 ~about 1250mg / m 2 , or about 100 mg / m 2 ~about 1000mg / m 2 In some embodiments, the dose is 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 or about 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 It is.

[0280] In some embodiments, the therapeutically effective dose of the conjugate is from 0.25 mg / kg to 150 mg / kg, from 0.25 mg / kg to 100 mg / kg, from 0.25 mg / kg to 75 mg / kg, from 0.25 mg / kg to 60 mg / kg, from 0.25 mg / kg to 50 mg / kg, from 0.25 mg / kg to 25 mg / kg, from 0.25 mg / kg to 10 mg / kg, from 0.25 mg / kg to 7.5 mg / kg, from 0.25 mg / kg to 5.0 mg / kg, from 0.25 mg / kg to 2.5 mg / kg, from 0.25 mg / kg to 1.0 mg / kg, from 0.25 mg / kg to 0.5 mg / kg g, 0.50mg / kg~150mg / kg, 0.50mg / kg~100mg / kg, 0.50mg / kg~75mg / kg, 0.50mg / kg~60mg / kg, 0.50mg / kg~50mg / kg, 0.50mg / kg~25mg / kg, 0.50mg / kg~10m g / kg, 0.50mg / kg~7.5mg / kg, 0.50mg / kg~5.0mg / kg, 0.50mg / kg~2.5mg / kg, 0.50mg / kg~1.0mg / kg, 1.0mg / kg~150mg / kg, 1.0mg / kg~100mg / kg, 1.0mg / kg ~75mg / kg, 1.0mg / kg~60mg / kg, 1.0mg / kg~50mg / kg, 1.0mg / kg~25mg / kg, 1.0mg / kg~10mg / kg, 1.0mg / kg~7.5mg / kg, 1.0mg / kg~5.0mg / kg, 1.0mg / kg~2. 5mg / kg, 2.5mg / kg~150mg / kg, 2.5mg / kg~100mg / kg, 2.5mg / kg~75mg / kg, 2.5mg / kg~60mg / kg, 2.5mg / kg~50mg / kg, 2.5mg / kg~25mg / kg, 2.5mg / kg~10mg / kg, 2.5mg / kg~7.5mg / kg, 2.5mg / kg~5.0mg / kg, 5.0mg / kg~150mg / kg, 5.0mg / kg~100mg / kg, 5.0mg / kg~75mg / kg, 5.0mg / kg~60mg / kg, 5.0mg / kg~50mg / kg , 5.0mg / kg~25mg / kg, 5.0mg / kg~10mg / kg, 5.0mg / kg~7.5mg / kg, 7.5mg / kg~150mg / kg, 7.5mg / kg~100mg / kg, 7.5mg / kg~75mg / kg, 7.5mg / kg~60mg / kg, 7.Between 5 mg / kg to 50 mg / kg, 7.5 mg / kg to 25 mg / kg, 7.5 mg / kg to 10 mg / kg, 10 mg / kg to 150 mg / kg, 10 mg / kg to 100 mg / kg, 10 mg / kg to 75 mg / kg, 10 mg / kg to 60 mg / kg, 10 mg / kg to 50 mg / kg, 10 mg / kg to 25 mg / kg, 25 mg / kg to 150 mg / kg, 25 mg / kg to 100 mg / kg, 25 mg / kg to 75 mg / kg, 25 mg / kg to 60 mg / kg, 25 mg / kg to 50 mg / kg, 50 mg / kg to 150 mg / kg, 50 mg / kg to 100 mg / kg, 50 mg / kg to 75 mg / kg, 50 mg / kg to 60 mg / kg, 60 mg / kg to 150 mg / kg, 60 mg / kg to 100 mg / kg, 60 mg / kg to 75 mg / kg, 75 mg / kg to 150 mg / kg, 75 mg / kg to 100 mg / kg, and 100 mg / kg to 150 mg / kg, or between approximately 0.25 mg / kg to 150 mg / kg, 0.25 mg / kg to 100 mg / kg, 0.25 mg / kg to 75 mg / kg, 0.25 mg / kg to 60 mg / kg, 0.25 mg / kg to 50 mg / kg, 0.25 mg / kg to 25 mg / kg, 0.25 mg / kg to 10 mg / kg, 0.25 mg / kg to 7.5 mg / kg, 0.25 mg / kg to 5.0 mg / kg, 0.25 mg / kg to 2.5 mg / kg, 0.25 mg / kg to 1.0 mg / kg, 0.25 mg / kg to 0.5 mg / kg, 0.50 mg / kg to 150 mg / kg, 0.50 mg / kg to 100 mg / kg, 0.50 mg / kg to 75 mg / kg, 0.50 mg / kg to 60 mg / kg, 0.50 mg / kg to 50 mg / kg, 0.50 mg / kg to 25 mg / kg, 0.50 mg / kg to 10 mg / kg, 0.50 mg / kg to 7.5 mg / kg, 0.50 mg / kg to 5.0 mg / kg, 0.50 mg / kg to 2.5 mg / kg, 0.50 mg / kg to 1.0 mg / kg, 1.0 mg / kg to 150 mg / kg, 1.0 mg / kg to 100 mg / kg, 1.0 mg / kg to 75 mg / kg, 1.0 mg / kg to 60 mg / kg, 1.0 mg / kg to 50 mg / kg, 1.0 mg / kg to 25 mg / kg, 1.0 mg / kg to 10 mg / kg, 1.0 mg / kg to 7.5 mg / kg, 1.0 mg / kg to 5.0 mg / kg, 1.0 mg / kg to 2.5mg / kg, 2.5mg / kg~150mg / kg, 2.5mg / kg~100mg / kg, 2.5mg / kg~75mg / kg, 2.5mg / kg~60mg / kg, 2.5mg / kg~50mg / kg, 2.5 mg / kg~25mg / kg, 2.5mg / kg~10mg / kg, 2.5mg / kg~7.5mg / kg, 2.5mg / kg~5.0mg / kg, 5.0mg / kg~150mg / kg, 5.0mg / kg~100m g / kg, 5.0mg / kg~75mg / kg, 5.0mg / kg~60mg / kg, 5.0mg / kg~50mg / kg, 5.0mg / kg~25mg / kg, 5.0mg / kg~10mg / kg, 5.0mg / k g~7.5mg / kg, 7.5mg / kg~150mg / kg, 7.5mg / kg~100mg / kg, 7.5mg / kg~75mg / kg, 7.5mg / kg~60mg / kg, 7.5mg / kg~50mg / kg, 7.5mg / kg~25mg / kg, 7.5mg / kg~10mg / kg, 10mg / kg~150mg / kg, 10mg / kg~100mg / kg, 10mg / kg~75mg / kg, 10mg / kg~60mg / kg, 10mg / kg~50mg / kg, 10mg / kg~25mg / kg, 25mg / kg~150mg / kg, 25mg / kg~100mg / kg, 25mg / kg~75mg / kg, 25mg / kg~60mg / In some embodiments, the therapeutically effective dose of the conjugate is between 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, 6.0 mg / kg, 7.0 mg / kg, 8.0 mg / kg, 9.0 mg / kg, 10.0 mg / kg, 11.0 mg / kg, 12.0 mg / kg, 13.0 mg / kg, 14.0 mg / kg, 15.0 mg / kg, 16.0 mg / kg, 17.0 mg / kg, 18.0 mg / kg, 19.0 mg / kg, 20.0 mg / kg, 21.0 mg / kg, 22.0 mg / kg, 23.0 mg / kg, 24.0 mg / kg, 25 ...The dose is less than 0mg / kg, 15mg / kg, 20mg / kg, 25mg / kg, 30mg / kg, 40mg / kg, 50mg / kg, 60mg / kg, 70mg / kg, 75mg / kg, 80mg / kg, 90mg / kg, 100mg / kg, 125mg / kg or 150mg / kg.

[0281] In some embodiments, the therapeutically effective amount is at least or at least about 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 2000 mg, 3000 mg or more.

[0282] In some embodiments, the method comprises administering to a subject having a disease or condition a therapeutically effective amount of a phthalocyanine dye-targeting molecule conjugate, such as an IR700-antibody conjugate. In some embodiments, the phthalocyanine dye-targeting molecule conjugate is targeted to cells present in the microenvironment of a tumor, lesion, or hyperplasia. In some embodiments, a therapeutically effective dose of the conjugate is administered intravenously. In some embodiments, a therapeutically effective dose of the conjugate is administered intratumorally.

[0283] In some embodiments, the dose of the conjugate is 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 of 10 μg / kg to 1000 μg / kg, e.g., about 100 μg / kg, about 250 μg / kg, about 500 μg / kg, about 750 μg / kg, or about 1000 μg / kg, e.g., when administered intratumorally or intraperitoneally (IP). In some embodiments, the dose is at least 1 μg / ml, such as at least 500 μg / ml, for example between 20 μg / ml and 100 μg / ml, such as about 10 μg / ml, about 20 μg / ml, about 30 μg / ml, about 40 μg / ml, about 50 μg / ml, about 60 μg / ml, about 70 μg / ml, about 80 μg / ml, about 90 μg / ml or about 100 μg / ml (e.g., administered in a topical solution).

[0284] In some embodiments, a therapeutically effective dose is a dose that would be administered to a human, hi some embodiments, an average human weighs between 60 and 85 kg, such as about or approximately 75 kg.

[0285] In some embodiments, a therapeutically effective dose is a therapeutically effective dose at which an administered conjugate containing a phthalocyanine dye conjugated to a targeting molecule (e.g., an antibody or antigen-binding antibody fragment) achieves a systemic exposure that does not exceed the therapeutically effective systemic exposure of the targeting molecule (e.g., an antibody or antigen-binding antibody fragment) that is not so conjugated (e.g., that occurs when a clinically acceptable dose of the drug targeting molecule is administered as the drug alone).

[0286] The term "systemic exposure" refers to the actual body exposure of a drug targeting molecule in plasma (blood or serum) after administration of the drug targeting molecule, and can be expressed as the area under the plasma drug concentration-time curve (AUC) as determined by pharmacokinetic analysis after administration of a dose of the drug targeting molecule. In some cases, the AUC is expressed in mg*h / L or its corresponding units (e.g., μg*h / L). In some embodiments, the AUC is measured as the average AUC in a patient population, such as a sample patient population, e.g., the average AUC from one or more patients. In some embodiments, the systemic exposure is expressed as the area under the curve (AUC) from 0 to infinity (inf or ∞), including all measured data and data extrapolated from the measured pharmacokinetic (PK) parameters (AUC 0-∞ or AUC[0-inf]), e.g., refers to the average AUC from a patient population, such as a sample patient population. In some embodiments, AUC 0-∞ is predicted based on one month of PK information. In some embodiments, systemic exposure is predicted based on the experimentally measured AUC from 0 to the last time point (AUC 0-last ). In some embodiments, the systemic exposure is the exposure occurring at the time of light irradiation or illumination (AUC), since the PIT depends on the dose or amount of the conjugate in the tumor at the time illumination or illumination occurs. In some embodiments, the light irradiation or illumination occurs within or about 24 hours ± 3 hours, e.g., within or about 24 hours ± 2 hours, or about 24 hours ± 2 hours, after administration of the conjugate. Thus, in some embodiments, the systemic exposure is the area under the curve (AUC) from 0 to 24 hours (AUC 0-24 or AUC[0-24]). In some embodiments, systemic exposure refers to the average area under the curve (AUC) from 0 to 24 hours (AUC 0-24 or AUC[0-24]).

[0287] In some embodiments, a therapeutically effective dose is determined by determining whether an administered conjugate containing a phthalocyanine dye conjugated to a targeting molecule (e.g., an antibody or antigen-binding antibody fragment) is greater than or equal to the AUC of a clinically tolerated dose of the targeting molecule that is not so conjugated. 0-∞ Substantially lower AUC 0-24 In some cases, because the therapeutically effective systemic exposure of the conjugate achieved at the time of PIT (time of light irradiation or illumination) is the period of PIT activity as described above. In contrast, in some cases, for a therapeutically effective systemic exposure to achieve, for example, pharmacological activity for a targeting molecule that is not so conjugated (e.g., unconjugated cetuximab), the targeting molecule (e.g., an antibody) must be present at a very high exposure. For example, under the FDA guidelines for Erbitux (cetuximab), the antibody is administered at 400 mg / m 2 at a weekly dose of 250 mg / m 2 In some cases, patients may require continuous treatment with unconjugated targeting molecules (e.g., antibodies) for more than one month.

[0288] The term "therapeutically effective systemic exposure" refers to the systemic exposure achieved by the dose of a drug targeting molecule (e.g., an antibody) with respect to pharmacological activity that achieves a therapeutic effect while being considered clinically acceptable and / or having an acceptable safety profile. It is within the skill of a person skilled in the art to determine or identify the dose of a drug targeting molecule (e.g., an antibody) that achieves a therapeutic effect while being clinically acceptable and / or having an acceptable safety profile. In some embodiments, the clinically acceptable dose of a drug targeting molecule is determined as a result of clinical trials in animals, particularly humans, such as those conducted by the U.S. Food and Drug Administration (FDA) or other regulatory agencies (e.g., EMA, PDMA). In some embodiments, therapeutically effective systemic exposure includes the systemic exposure resulting from either a single dose of a drug targeting molecule (e.g., an antibody) or repeated administration of a drug targeting molecule (e.g., a drug) in an administration cycle, such as daily, weekly, weekly, or monthly dosing.

[0289] Exemplary FDA approved clinically acceptable dosing schedules for exemplary antibody drugs are shown in Table 1. In some embodiments, the therapeutically effective systemic exposure of a drug targeting molecule, including a drug targeting molecule not conjugated to a phthalocyanine dye, can be determined or is known from pharmacokinetic studies of a subject population administered at a clinically acceptable or approved dose as a single dose of an initial dose or by repeated administration in a dosing cycle (see, e.g., Fracasso et al. (2007) Clin. Cancer. Res., 13:986 for observed systemic exposures after single dose administration of a dose of cetuximab within the FDA approved dosing range).

[0290] Table 1. FDA approved doses of exemplary drug antibody targeting molecules TIFF2025015674000011.tif157164 Available from www.accessdata.fda.gov / scripts / cder / drugsatfda / index.cfm

[0291] In some embodiments, the therapeutically effective dose of the conjugate is a therapeutically effective dose in which the administered conjugate containing a phthalocyanine dye (e.g., IR700) conjugated to a targeting molecule (e.g., an antibody or an antigen-binding antibody fragment) achieves an average systemic exposure (e.g., AUC) for a patient population such as a sample patient population that is 75% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 25% or less, 20% or less of the therapeutically effective average systemic exposure for a patient population such as a sample patient population of the corresponding targeting molecule (e.g., an antibody or an antigen-binding antibody fragment) that is not so conjugated. Typically, the systemic exposure of the conjugate is high enough to be phototoxic by PIT.

[0292] In some embodiments, the conjugate is administered in a patient population, e.g., a sample patient population after administration of the conjugate, with the area under the plasma conjugate concentration-time curve from time 0 to infinity (AUC[0-inf] or AUC 0-∞), between 250μg / mL*h (used interchangeably with μg*h / mL) and 100,000μg / mL*h or between about 250μg / mL*h and 100,000μg / mL*h, between 500μg / mL*h and 50,000μg / mL*h or between about 500μg / mL*h and 50,000μg / mL*h, between 500μg / mL*h and 25,000μg / mL*h or between about 500μg / mL*h and 25,000μg / mL*h, and between 500μg / mL*h and 18,000μg / mL In some embodiments, the conjugate is administered to achieve a mean systemic exposure that is between or between about 500 μg / mL*h and 18,000 μg / mL*h, between or between about 500 μg / mL*h and 10,000 μg / mL*h, between or between about 500 μg / mL*h and 5,000 μg / mL*h, or between or between about 500 μg / mL*h and 5,000 μg / mL*h, or between or between about 500 μg / mL*h and 2,500 μg / mL*h. In some embodiments, the conjugate is administered to achieve a mean systemic exposure that is between or between about 500 μg / mL*h and 18,000 μg / mL*h, between or between about 500 μg / mL*h and 10,000 μg / mL*h, ... 0-∞ ), to achieve a systemic exposure that is ≤100,000 μg / mL*h, ≤75,000 μg / mL*h, ≤50,000 μg / mL*h, ≤40,000 μg / mL*h, ≤30,000 μg / mL*h, ≤20,000 μg / mL*h, ≤10,000 μg / mL*h, ≤5,000 μg / mL*h, or ≤2,500 μg / mL*h.

[0293] In some embodiments, the conjugate is administered as a plasma conjugate concentration versus area under the curve (AUC[0-24] or AUC 0-24), the mean systemic exposure between or between about 100 μg / mL*h and 25,000 μg / mL*h, between or between about 200 μg / mL*h and 10,000 μg / mL*h, between or between about 500 μg / mL*h and 5,000 μg / mL*h; or the AUC 0-24 In some embodiments, the plasma conjugate AUC following administration of the conjugate is administered to achieve an average systemic exposure of 25,000 μg / mL*h or less, 15,000 μg / mL*h or less, 10,000 μg / mL*h or less, 5,000 μg / mL*h or less, 2,500 μg / mL*h or less, 1,000 μg / mL*h or less, or 500 μg / mL*h or less, as measured by 0-24 is between or about 500μg / mL*h to 8,000μg / mL*h, between 500μg / mL*h to 5,000μg / mL*h or about 500μg / mL*h to 5,000μg / mL*h, between 500μg / mL*h to 2,000μg / mL*h or about 500μg / mL*h to 2,000μg / mL*h, or between or about 1000μg / mL*h to 4,000μg / mL*h.

[0294] In some embodiments, the therapeutically effective dose of a phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-antibody conjugate) is less than the therapeutically effective dose of a single administration of the corresponding targeting molecule (e.g., an antibody or antigen-binding antibody fragment) that is not so conjugated, e.g., is 75% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 25% or less, 20% or less of the therapeutically effective dose of a single administration of the corresponding targeting molecule that is not so conjugated.

[0295] In some embodiments, the therapeutically effective dose of the phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-antibody conjugate) is less than the initial dose in a repeat dose schedule of a therapeutically effective dose of the corresponding targeting molecule (e.g., an antibody or antigen-binding antibody fragment) that is not so conjugated, e.g., 75% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 25% or less, 20% or less of the initial dose in a repeat dose schedule of a therapeutically effective dose of the corresponding targeting molecule that is not so conjugated.

[0296] In some embodiments, the therapeutically effective dose of a phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-antibody conjugate) is less than the average dose of a repeated administration schedule of a therapeutically effective dose of a corresponding targeting molecule (e.g., an antibody or antigen-binding antibody fragment) that is not so conjugated, e.g., is 75% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 25% or less, 20% or less of the average dose of a repeated administration schedule of a therapeutically effective dose of a corresponding targeting molecule that is not so conjugated.

[0297] In some embodiments, the therapeutically effective dose of the conjugate is 400 mg / m 2 Less than 300 mg / m 2 Less than 250 mg / m 2 Less than 225 mg / m 2 Less than 200 mg / m 2 Less than 180 mg / m 2 Less than 100 mg / m 2 Less than or equal to 50 mg / m 2 In some embodiments, the therapeutically effective dose of the conjugate is less than 50 mg / m 2 ~400mg / m 2 or about 50 mg / m 2 ~400mg / m 2 , 100 mg / m 2 ~300mg / m 2 or about 100 mg / m 2~300mg / m 2 , 100 mg / m 2 ~250mg / m 2 or about 100 mg / m 2 ~250mg / m 2 , or 100 mg / m 2 ~160mg / m 2 or about 100 mg / m 2 ~160mg / m 2 In some embodiments, the therapeutically effective dose of the conjugate is between 80 mg / m 2 ~240mg / m 2 , 80 mg / m 2 ~220mg / m 2 , 80 mg / m 2 ~200mg / m 2 , 80 mg / m 2 ~180mg / m 2 , 80 mg / m 2 ~160mg / m 2 , 80 mg / m 2 ~140mg / m 2 , 80 mg / m 2 ~120mg / m 2 , 80 mg / m 2 ~100mg / m 2 , 100 mg / m 2 ~240mg / m 2 , 100 mg / m 2 ~220mg / m 2 , 100 mg / m 2 ~200mg / m 2 , 100 mg / m 2 ~180mg / m 2 , 100 mg / m 2 ~160mg / m 2 , 100 mg / m 2 ~140mg / m 2 , 100 mg / m 2 ~120mg / m 2 , 120 mg / m 2 ~240mg / m 2 , 120 mg / m 2 ~220mg / m 2 , 120 mg / m 2 ~200mg / m 2 , 120 mg / m 2~180mg / m 2 , 120 mg / m 2 ~160mg / m 2 ,120mg / m 2 ~140mg / m 2 , 140 mg / m 2 ~240mg / m 2 , 140 mg / m 2 ~220mg / m 2 , 140 mg / m 2 ~200mg / m 2 , 140 mg / m 2 ~180mg / m 2 , 140 mg / m 2 ~160mg / m 2 , 160 mg / m 2 ~240mg / m 2 , 160 mg / m 2 ~220mg / m 2 , 160 mg / m 2 ~200mg / m 2 , 160 mg / m 2 ~180mg / m 2 , 180 mg / m 2 ~240mg / m 2 , 180 mg / m 2 ~220mg / m 2 , 180 mg / m 2 ~200mg / m 2 , 200 mg / m 2 ~220mg / m 2 or 200 mg / m 2 ~240mg / m 2 or about 80 mg / m 2 ~240mg / m 2 , 80 mg / m 2 ~220mg / m 2 , 80 mg / m 2 ~200mg / m 2 , 80 mg / m 2 ~180mg / m 2 , 80 mg / m 2 ~160mg / m 2 , 80 mg / m 2 ~140mg / m 2 , 80 mg / m 2 ~120mg / m 2 , 80 mg / m2 ~100mg / m 2 、100mg / m 2 ~240mg / m 2 、100mg / m 2 ~220mg / m 2 、100mg / m 2 ~200mg / m 2 、100mg / m 2 ~180mg / m 2 、100mg / m 2 ~160mg / m 2 、100mg / m 2 ~140mg / m 2 、100mg / m 2 ~120mg / m 2 、120mg / m 2 ~240mg / m 2 、120mg / m 2 ~220mg / m 2 、120mg / m 2 ~200mg / m 2 、120mg / m 2 ~180mg / m 2 、120mg / m 2 ~160mg / m 2 ,120mg / m 2 ~140mg / m 2 、140mg / m 2 ~240mg / m 2 、140mg / m 2 ~220mg / m 2 、140mg / m 2 ~200mg / m 2 、140mg / m 2 ~180mg / m 2 、140mg / m 2 ~160mg / m 2 、160mg / m 2 ~240mg / m 2 、160mg / m 2 ~220mg / m 2 、160mg / m 2 ~200mg / m 2 、160mg / m 2 ~180mg / m 2 、180mg / m 2 ~240mg / m 2, 180 mg / m 2 ~220mg / m 2 , 180 mg / m 2 ~200mg / m 2 , 200 mg / m 2 ~220mg / m 2 or 200 mg / m 2 ~240mg / m 2 It is between.

[0298] In some embodiments, the therapeutically effective dose of the conjugate is less than 12 mg / kg, less than 10 mg / kg, less than 8 mg / kg, less than 6 mg / kg, less than 4 mg / kg, less than 2 mg / kg, or less than 1 mg / kg. In some embodiments, the therapeutically effective dose of the conjugate is between or about 1 mg / kg to 12 mg / kg, 2 mg / kg to 10 mg / kg, 2 mg / kg to 6 mg / kg, or 2 mg / kg to 4 mg / kg. In some embodiments, the therapeutically effective dose of the conjugate is from 2.0 mg / kg to 6.5 mg / kg, 2.0 mg / kg to 6.0 mg / kg, 2.0 mg / kg to 5.0 mg / kg, 2.0 mg / kg to 4.0 mg / kg, 2.0 mg / kg to 3.0 mg / kg, 3.0 mg / kg to 6.5 mg / kg, 3.0 mg / kg to 6.0 mg / kg, 3.0 mg / kg to 5.0 mg.kg, 3.0 mg / kg to 4.0 mg / kg, 4.0 mg / kg to 6.5 mg / kg, 4.0 mg / kg to 6.0 mg / kg, 4.0 mg / kg to 5.0 mg / kg, 5.0 mg / kg to 6.5 mg / kg, 5.0 mg / kg to 6.0 mg / kg, and 6.0 mg / kg to 6.5mg / kg, or between about 2.0mg / kg-6.5mg / kg, 2.0mg / kg-6.0mg / kg, 2.0mg / kg-5.0mg / kg, 2.0mg / kg-4.0mg / kg, 2.0mg / kg-3.0mg / kg, 3.0mg / kg-6.5mg / kg, 3.0mg / kg-6.0mg / kg, 3.0mg / kg-5.0mg.kg, 3.0mg / kg-4.0mg / kg, 4.0mg / kg-6.5mg / kg, 4.0mg / kg-6.0mg / kg, 4.0mg / kg-5.0mg / kg, 5.0mg / kg-6.5mg / kg, 5.0mg / kg-6.0mg / kg and 6.0mg / kg-6.5mg / kg.

[0299] In some embodiments, the therapeutically effective amount is between about 75 mg to 500 mg, 75 mg to 400 mg, 75 mg to 400 mg, 75 mg to 300 mg, 75 mg to 200 mg, 75 mg to 150 mg, 150 mg to 500 mg, 150 mg to 400 mg, 150 mg to 300 mg, 150 mg to 200 mg, 200 mg to 500 mg, 200 mg to 400 mg, 200 mg to 300 mg, 300 mg to 500 mg, 300 mg to 400 mg, or 400 mg to 500 mg.

[0300] In some embodiments, the conjugate is IR700-cetuximab. In some embodiments, the therapeutically effective amount of the IR700-cetuximab conjugate is at least 160 mg / m 2 , 320 mg / m 2 or 640 mg / m 2 , or about at least 160 mg / m 2 , 320 mg / m 2 or 640 mg / m 2 , or 160 mg / m 2 , 320 mg / m 2 or 640 mg / m 2 , or about 160 mg / m 2 , 320 mg / m 2 or 640 mg / m 2 In some embodiments, the therapeutically effective amount of the IR700-cetuximab conjugate is at least, or about, 4.3 mg / kg, 8.6 mg / kg, or 17 mg / kg, or at least, or about, 4.3 mg / kg, 8.6 mg / kg, or 17 mg / kg.

[0301] In some embodiments, the therapeutically effective dose of the conjugate is for single dose administration. In some embodiments, the therapeutically effective dose is administered as a single injection or infusion in a dosing schedule or cycle, e.g., administered only once during the dosing schedule or cycle. For example, in a dosing schedule or cycle, no subsequent dose of the conjugate is administered. In some embodiments, the dosing schedule can be repeated. In some embodiments, the repeated dose, e.g., the repeated single dose, is administered at the time when the first dose is cleared from the subject (in some cases, at the time when the systemic exposure of the conjugate is undetectable). Thus, in some embodiments, the dosing of the conjugate is not administered to achieve continuous systemic exposure of the conjugate, which is different from many current therapies (including antibody therapies) that require repeated dosing in a dosing schedule or cycle to maintain continuous systemic exposure. In some embodiments, the dosing schedule or cycle is repeated once a week, every two weeks, once a month, twice a year, once a year, or less frequently as needed.

[0302] In some embodiments, in any of the treatment methods provided herein, the dosing schedule is repeated if residual lesions remain after prior treatment with conjugate.In some embodiments, the method additionally comprises the step of evaluating the subject for the presence of residual lesions, and if residual lesions remain, the dosing schedule is repeated.In some embodiments, the dosing schedule is repeated if residual lesions remain at a time point 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or more than 1 year, about 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year, or 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year after prior administration of conjugate.In some embodiments, the dosing schedule is repeated if residual lesions remain at 4 weeks or about 4 weeks after prior administration of conjugate.

[0303] In some embodiments, no subsequent dose of a targeting molecule, e.g., a therapeutic targeting molecule (e.g., a therapeutic antibody), that is not so conjugated to a photosensitizer (e.g., IR700) is administered in a dosing schedule or cycle. For example, in some embodiments, a dose of a phthalocyanine dye-targeting molecule conjugate is not followed by a dose of the targeting molecule alone.

[0304] Those skilled in the art will recognize that higher or lower dosages of the phthalocyanine dye-targeting molecule conjugate can also be used, for example, depending on the specific drug.In some embodiments, the dosage, for example, the daily dosage, is administered in one or more divided doses, for example, 2, 3, or 4 doses, or in a single formulation.The phthalocyanine dye-targeting molecule conjugate can be administered alone, in the presence of a pharma- ceutically acceptable carrier, or in the presence of other therapeutic agents, such as immunomodulators, anticancer agents, or other anti-cancer agents.

[0305] In some embodiments, the phthalocyanine dye-targeting molecule conjugate may be administered either systemically or locally to the organ or tissue to be treated. Exemplary routes of administration include, but are not limited to, topical, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), oral, sublingual, rectal, transdermal, intranasal, intravaginal, and inhalation routes. In some embodiments, the phthalocyanine dye-targeting molecule conjugate is administered intravenously. In some embodiments, the phthalocyanine dye-targeting molecule conjugate is administered parenterally. In some embodiments, the phthalocyanine dye-targeting molecule conjugate is administered enterally. In some embodiments, the conjugate is administered by local injection. In some embodiments, the conjugate is administered as a topical application.

[0306] The composition comprising the phthalocyanine dye-targeting molecule conjugate can be administered locally or systemically using any method known in the art, for example, to a subject having a tumor, e.g., cancer, or a subject from which a tumor has previously been removed, e.g., via surgery. Although specific examples are provided, one of ordinary skill in the art will understand that alternative methods of administration of the disclosed agents can be used. Such methods can include, for example, the use of a catheter or implantable pump that provides continuous infusion over a period of hours to days to a subject in need of treatment.

[0307] In some embodiments, the phthalocyanine dye-targeting molecule conjugate is administered by parenteral means, including direct injection or infusion into the tumor, e.g., intratumorally. In some embodiments, the phthalocyanine dye-targeting molecule conjugate is administered to the tumor by applying the agent to the tumor, e.g., by bathing the tumor in a solution containing the agent, e.g., the phthalocyanine dye-targeting molecule conjugate, or by pouring the agent into the tumor.

[0308] Additionally or alternatively, the disclosed compositions can be administered systemically, e.g., intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, subcutaneously, or orally, to a subject having a tumor, e.g., cancer.

[0309] The dosage of the phthalocyanine dye-targeting molecule conjugate administered to a subject is not subject to an absolute limit, but will depend on the nature of the composition and its active ingredients and its unwanted side effects, such as immune response to the agent, the subject being treated, and the type of condition being treated and the mode of administration. Generally, the dosage 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 to attenuate further growth of a tumor, or to reduce undesirable symptoms of a tumor.

[0310] In some embodiments, the composition used for administration of a drug, such as a phthalocyanine dye-targeting molecule conjugate, contains an effective amount of the drug in combination with conventional pharmaceutical carriers and excipients suitable for the intended type of administration.For example, in some embodiments, a parenteral formulation may contain a sterile aqueous solution or suspension of the conjugate.In some embodiments, a composition for enteral administration may contain an effective amount of the phthalocyanine dye-targeting molecule conjugate in an aqueous solution or suspension that may optionally contain a buffer, a surfactant, a thixotropic agent, and a flavoring agent.

[0311] C. Dosage Regimens and Photoimmunotherapy PIT involves the administration of a composition containing a phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-antibody conjugate) followed by irradiation. In some embodiments, the method includes irradiating the tumor.

[0312] In some embodiments, cells are irradiated after contacting with phthalocyanine dye-targeting molecule conjugate.The method of irradiating is known in the art.Because typically only cells that express cell surface protein are recognized by targeting molecule, generally only such cells will have sufficient amount of conjugate to bind to said protein.Irradiation can only kill the cells that conjugate is bound to, and generally cannot kill other cells, so it can reduce the possibility of undesirable side effects, such as killing normal cells.

[0313] In some embodiments, the cells are irradiated in vivo, e.g., to a subject to which the phthalocyanine dye-targeting molecule conjugate has previously been administered. In some embodiments, the subject is irradiated, e.g., to a tumor in the subject.

[0314] In some embodiments, the irradiation is accomplished after administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the irradiation or illumination is performed or accomplished between or about 30 minutes to 96 hours after administration of the phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate), such as between 30 minutes to 48 hours, 30 minutes to 24 hours, or 12 hours to 48 hours, such as generally at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or more, after administration of the conjugate. For example, the irradiation can be performed within about 24 hours after administration of the conjugate. In some embodiments, the conjugate that comprises targeting molecule and associates with tumor is administered to the subject more than 6 hours before the tumor is irradiated or illuminated.In some embodiments, the conjugate is pre-administered to the subject more than 12 hours, 24 hours, 26 hours, 48 ​​hours, 72 hours or 96 hours before the tumor is irradiated or illuminated, or about 12 hours, 24 hours, 26 hours, 48 ​​hours, 72 hours or 96 hours before the tumor is irradiated or illuminated.

[0315] In some embodiments, at the time of irradiation or after irradiation, the subject can receive one or more other treatments (e.g., immunomodulatory agents or anti-cancer agents) as described herein. In some cases, one or more other treatments are therefore also administered after administration of the phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate). In some embodiments, the additional treatment is administered within 0-24 hours or about 0-24 hours of irradiation, for example, within 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours of irradiation.

[0316] In some embodiments, prior to irradiation, the subject may receive one or more other treatments as described herein. In some cases, the one or more other treatments may be administered before, during, or after administration of the phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate), and generally prior to irradiation of the subject. In some embodiments, an additional therapeutic agent (e.g., an immunomodulatory agent or an anti-cancer agent) may be administered during or simultaneously with administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, an additional therapeutic agent (e.g., an immunomodulatory agent or an anti-cancer agent) may be administered after or subsequent to administration of the phthalocyanine dye-targeting molecule conjugate. For example, in some embodiments, the conjugate is administered prior to one or more other treatments, and the conjugate and one or more other treatments are each administered prior to irradiation of the tumor. In some embodiments, the conjugate is administered subsequent to one or more other treatments, and the conjugate and one or more other treatments are each administered prior to irradiation of the tumor. In some embodiments, irradiation occurs after administration of an additional therapeutic agent (eg, an immunomodulatory or anti-cancer agent) and a phthalocyanine dye-targeting molecule conjugate.

[0317] In some embodiments, the additional therapeutic agent is an immunomodulatory agent, and the immunomodulatory agent is administered 6 hours to 4 weeks prior to irradiation, e.g., generally, 12 hours, 24 hours, 36 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, or more than 4 weeks prior to irradiation, or about 12 hours, 24 hours, 36 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, or more than 4 weeks prior to irradiation. In some embodiments, the phthalocyanine dye-targeting molecule (e.g., IR700 antibody conjugate) is administered 6 hours to 96 hours prior to irradiation, e.g., generally, within 6 hours, 12 hours, 24 hours, 36 hours, 72 hours, or 96 hours prior to irradiation, or about 6 hours, 12 hours, 24 hours, 36 hours, 72 hours, or 96 hours prior to irradiation.

[0318] In some embodiments, prior to irradiation, the subject may receive an immunomodulatory agent, such as an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is generally administered prior to irradiation of the subject. In some embodiments, the immunomodulatory agent is administered between 12 hours and 2 months, or between about 12 hours and 2 months, such as between 12 hours and 1 month, 12 hours and 3 weeks, 12 hours and 2 weeks, 12 hours and 1 week, and 1 week and 1 month, for example, generally at least 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 1 month prior to irradiating the tumor. In some embodiments, the immunomodulatory agent may be administered prior to, subsequent to, or simultaneously with the conjugate, so long as both the immunomodulatory agent and the conjugate are administered prior to irradiation. In some embodiments, the immunomodulatory agent may be administered prior to administration of the phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate). In some embodiments, the immunomodulatory agent can be administered during or simultaneously with the administration of the phthalocyanine dye-targeting molecule conjugate.In some embodiments, the immunomodulatory agent can be administered following or after the administration of the phthalocyanine dye-targeting molecule conjugate.For example, the immunomodulatory agent following irradiation is administered three times a week, twice a week, once every week, once every two weeks, once every three weeks, or once a month.

[0319] For example, in some cases, the conjugate is administered at least 12 hours, e.g., 12 hours to 48 hours or about 12 hours to 48 hours, prior to irradiation, and the immunomodulatory agent is administered at least 12 hours, e.g., 12 hours to 1 month or about 12 hours to about 1 month, prior to irradiation. In some embodiments, the conjugate is administered within 36 hours, 24 hours, 18 hours, or 12 hours, or within about 36 hours, 24 hours, 18 hours, or 12 hours, prior to irradiation, and the immunomodulatory agent is administered more than 12 hours, e.g., generally more than 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 1 month prior to irradiation.

[0320] In some embodiments, the immunomodulatory agent is a conjugate itself, containing a phthalocyanine dye, such as a phthalocyanine dye linked to an antibody or antigen-binding fragment that is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an IR700-antibody conjugate that includes an immunomodulatory antibody (e.g., a checkpoint inhibitor) that binds to a checkpoint protein (e.g., PD-L1) on tumor cells. In some embodiments, the immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulatory agent) is administered, for example, between 12 hours and 2 months prior to administration of the phthalocyanine dye-targeting molecule conjugate, for example, generally at least 12 hours, at least 24 hours, at least 48 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month prior to administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulator) is administered during or simultaneously with administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulator) is administered between, for example, 12 hours and 2 months after administration of the phthalocyanine dye-targeting molecule conjugate, for example, typically at least 12 hours, at least 24 hours, at least 48 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month after administration of the phthalocyanine dye-targeting molecule conjugate.

[0321] In some embodiments, irradiation is performed or accomplished after administration of an immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulator) and a phthalocyanine dye-targeting molecule conjugate. In some embodiments, irradiation is accomplished after administration of a phthalocyanine dye-targeting molecule conjugate.

[0322] In some embodiments, the combination therapy method includes two irradiations or illuminations. In some embodiments, the combination therapy method includes a first irradiation of the tumor after administration of an immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulator) and a second irradiation of the tumor after administration of a phthalocyanine dye-targeting molecule conjugate. In some embodiments, each irradiation is performed within 6 to 48 hours after administration of the respective conjugate, for example, generally at least about 6 hours, 12 hours, 24 hours, or 36 hours after administration of each conjugate.

[0323] In some embodiments, the method of combination therapy including an immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulator) and a phthalocyanine dye-targeting molecule conjugate only includes a single dose of irradiation. In some embodiments, the phthalocyanine dye-targeting molecule conjugate is administered at least 12 hours after administration of the immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulator) and within 6-48 hours prior to irradiating the tumor.

[0324] In some embodiments, the immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulator) and the phthalocyanine dye-targeting molecule conjugate are administered by the same route of administration. In some embodiments, both the immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulator) and the phthalocyanine dye-targeting molecule conjugate are administered systemically. In other embodiments, both conjugates are administered intravenously.

[0325] In some embodiments, prior to irradiation, the subject may receive an anti-cancer agent. In some embodiments, the anti-cancer agent is generally administered prior to irradiation of the subject. In some embodiments, irradiation is performed or accomplished between 5 minutes and 2 weeks, or between about 5 minutes and 2 weeks, such as between 5 minutes and 1 week, 5 minutes and 3 days, 5 minutes and 48 hours, 5 minutes and 24 hours, 5 minutes and 12 hours, 5 minutes and 6 hours, and 5 minutes and 1 hour after administration of the anti-cancer agent, such as generally at least about 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours prior to irradiating the tumor. In some embodiments, the anti-cancer agent may be administered prior to, subsequent to, or simultaneously with the conjugate, so long as both the anti-cancer agent and the conjugate are administered prior to irradiation. In some embodiments, the anti-cancer agent may be administered prior to administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, an anti-cancer agent can be administered during or simultaneously with administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, an anti-cancer agent can follow administration of the phthalocyanine dye-targeting molecule conjugate.

[0326] For example, in some cases, the conjugate is administered at least 12 hours, e.g., 12 to 48 hours or about 12 to 48 hours, prior to irradiation, and the anti-cancer agent is administered at least 5 minutes, e.g., 5 to 24 hours or about 5 to 24 hours, prior to irradiation. In some embodiments, the conjugate is administered within 36, 24, 18, or 12 hours, or within about 36, 24, 18, or 12 hours, prior to irradiation, and the anti-cancer agent is administered more than 5 minutes prior to irradiation, generally within 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours prior to irradiation.

[0327] In some embodiments, cells, e.g., tumors, are irradiated with a therapeutic dose of radiation having a wavelength in the range of 400 nm to about 900 nm, e.g., 500 nm to about 900 nm, e.g., 600 nm to about 850 nm, e.g., 600 nm to about 740 nm, e.g., about 660 nm to about 740 nm, about 660 nm to about 710 nm, about 660 nm to about 700 nm, about 670 nm to about 690 nm, about 680 nm to about 740 nm, or about 690 nm to about 710 nm. In some embodiments, cells, e.g., tumors, are irradiated with a therapeutic dose of radiation having a wavelength in the range of 600 nm to 850 nm, e.g., 660 nm to 740 nm. In some embodiments, cells, e.g., tumors, are irradiated with a wavelength of at least, or about, 600 nm, 620 nm, 640 nm, 660 nm, 680 nm, 700 nm, 720 nm or 740 nm, e.g., 690±50 nm, e.g., about 680 nm.

[0328] In some embodiments, the cells, e.g., tumors, have a density of at least 1 J cm -2 , e.g. at least 10 J cm -2 , at least 30J cm -2 , at least 50 J cm -2 , at least 100 J cm -2 , or at least 500 J cm -2 In some embodiments, the dose of irradiation is from 1 to about 1000 or from about 1 to about 1000 J cm. -2 , about 1~500J cm -2 , about 5~200J cm -2 , about 10~100J cm -2 , or about 10 to about 50 J cm -2 In some embodiments, the cells, e.g., tumors, have a cytotoxicity of at least 2 J cm -2 , 5J cm -2 , 10J cm -2 , 25J cm -2 , 50J cm-2 , 75J cm -2 , 100J cm -2 , 150J cm -2 , 200J cm -2 , 300J cm -2 , 400J cm -2 , or 500 J cm -2 , or at least about 2 J cm -2 , 5J cm -2 , 10J cm -2 , 25J cm -2 , 50J cm -2 , 75J cm -2 , 100J cm -2 , 150J cm -2 , 200J cm -2 , 300J cm -2 , 400J cm -2 , or 500 J cm -2 The radiation is irradiated at a dose of .

[0329] In some embodiments, the cells, e.g., tumors, are irradiated or illuminated with a dose of at least 1 J / cm fiber length, e.g., at least 10 J / cm fiber length, at least 50 J / cm fiber length, at least 100 J / cm fiber length, at least 250 J / cm fiber length, or at least 500 J / cm fiber length. In some embodiments, the dose of irradiation is 1 to about 1000 or about 1 to about 1000 J / cm fiber length, about 1 to about 500 J / cm fiber length, about 2 to about 500 J / cm fiber length, about 50 to about 300 J / cm fiber length, about 10 to about 100 J / cm fiber length, or about 10 to about 50 J / cm fiber length. In some embodiments, cells, e.g., tumors, are irradiated with a dose of at least, or at least about, 2 J / cm, 5 J / cm, 10 J / cm, 25 J / cm, 50 J / cm, 75 J / cm, 100 J / cm, 150 J / cm, 200 J / cm, 250 J / cm, 300 J / cm, 400 J / cm, or 500 J / cm.

[0330] In some embodiments, the dose of irradiation or illumination in a human subject is from 1 to about 400 J cm -2 Or about 1 to about 400 J cm -2 , about 2~400J cm -2 , about 1~300J cm -2 , about 10~100J cm -2 , or about 10 to about 50 J cm -2 and for example at least 10 J cm -2 Or at least about 10 J cm -2 or 10J cm -2 or 10J cm -2Within or about 10J cm -2 or less than 10J cm -2 or about 10 J cm -2 , at least 30J cm -2 , at least 50 J cm -2 , at least 100 J cm -2 In some embodiments, the dose of irradiation in a human subject is between 1 and 300 J / cm fiber length or about 1 and 300 J / cm fiber length, between 10 and 100 J / cm fiber length or about 10 and 100 J / cm fiber length, or between 10 and 50 J / cm fiber length or about 10 and 50 J / cm fiber length, e.g., at least 10 J / cm fiber length or at least about 10 J / cm fiber length, or within 10 J / cm fiber length or within about 10 J / cm fiber length, or about 10 J / cm fiber length, at least 30 J / cm fiber length, at least 50 J / cm fiber length, at least 100 J / cm fiber length. In some cases, it is found that the dose of irradiation in a human subject that achieves PIT can be less than the dose required for PIT in mice. For example, in some cases, light dosimetry in an in vivo tumor mouse model is less than 50 J / cm fiber length. 2 (50J cm -2 ) is not effective against PIT, which is in contrast to what the inventors have been able to observe in clinical human patients.

[0331] In some embodiments, the dose of radiation following administration of a composition comprising a phthalocyanine dye-targeting molecule conjugate is at least 1 J cm at a wavelength of 660-740 nm. -2 or 1 J / cm of fiber length, e.g., at least 10 J cm at wavelengths of 660-740 nm -2 or 10 J / cm of fiber length, at least 50 J cm at wavelengths from 660 to 740 nm -2 or 50 J / cm of fiber length, or at least 100 J cm at wavelengths between 660 and 740 nm -2or 100 J / cm of fiber length, e.g., 1.0-500 J cm at wavelengths of 660-740 nm -2 or 1.0-500 J / cm of fiber length. In some embodiments, the wavelength is 660-710 nm. In some embodiments, the dose of radiation following administration of a composition comprising a phthalocyanine dye-targeting molecule conjugate is at least 1.0 J cm at a wavelength of 680 nm. -2 or 1 J / cm of fiber length, e.g., at least 10 J cm at a wavelength of 680 nm -2 or 10 J / cm of fiber length, at least 50 J cm at a wavelength of 680 nm -2 or 50 J / cm of fiber length, or at least 100 J cm at a wavelength of 680 nm -2 or 100 J / cm of fiber length, e.g., 1.0 to 500 J cm at a wavelength of 680 nm -2 or 1.0-500 J / cm of fiber length. In some embodiments, multiple irradiations are performed, e.g., at least 2, at least 3, or at least 4 irradiations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate administrations. Exemplary irradiation following administration of a conjugate or composition provided herein is administered to a tumor at a wavelength of 660 nm to 740 nm with at least 1 J cm -2 or irradiating with a dose of 1 J / cm of fiber length.

[0332] In some embodiments, light or laser can be applied to cells containing dye molecules, such as conjugates, for about 5 seconds to about 5 minutes. For example, in some embodiments, light or laser is applied for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 seconds, or for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 seconds, or within a range between two such values, to activate dye molecules. In some embodiments, light or laser is applied for 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 minutes, or for about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 minutes, or longer, or within a range between any two of such values. In some embodiments, the length of time that light or laser is applied can vary, for example, depending on the energy, e.g., wattage, of the light or laser. For example, a lower wattage light or laser can be applied for a longer period of time to activate the dye molecules.

[0333] In some embodiments, the light or laser can be applied about 30 minutes to about 48 hours after administration of the conjugate. For example, in some embodiments, the light or laser is applied at or about 30, 35, 40, 45, 50, or 55 minutes after administration of the conjugate, or within a range between any two of such values. In some embodiments, the light or laser is applied at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after administration of the conjugate, or is administered within a range between or about any two of such values. In some embodiments, the light or laser is applied for at or about 1-24 hours, e.g., at or about 1-12 hours, at or about 12-24 hours, at or about 6-12 hours, or may be administered more than 24 hours after administration of the conjugate, hi some embodiments, the light or laser is administered 36 or 48 hours after administration of the conjugate.

[0334] In some embodiments, cells or subjects can be irradiated once or multiple times. Thus, irradiation can be completed in one day or repeated over several days with the same or different doses, for example, at least 2 separate, 3 separate, 4 separate, 5 separate or 10 separate irradiations. In some embodiments, repeated irradiation can 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 even longer intervals.

[0335] In some embodiments, the dose or method of radiation varies depending on the type or morphology of the tumor.

[0336] In some embodiments, the lesion is a tumor that is a superficial tumor. In some embodiments, the tumor is less than 10 mm thick. In some embodiments, the irradiation is performed using a microlens-tipped fiber for surface illumination. In some embodiments, the light irradiation dose is 5 J / cm 2 ~About 200J / cm 2 or about 5 J / cm 2 ~About 200J / cm 2 It is.

[0337] In some embodiments, the methods provided involve administering 5 J / cm to a superficial tumor in a subject using a microlens-tipped fiber for surface illumination. 2 ~About 200J / cm 2 or about 5 J / cm 2 ~About 200J / cm 2 In some embodiments, the light irradiation dose is 50 J / cm, where the tumor is associated with a phototoxic agent that comprises a targeting molecule bound to a cell surface molecule of the tumor. 2 or about 50 J / cm 2 It is.

[0338] In some embodiments, the lesion is a tumor that is a stromal tumor. In some embodiments, the tumor is greater than 10 mm deep or is a subcutaneous tumor. In some embodiments, the irradiation is performed using a cylindrical diffusing fiber with a diffuser length of 0.5 cm to 10 cm and spaced 1.8±0.2 cm apart. In some embodiments, the light irradiation dose is 20 J / cm fiber length to about 500 J / cm fiber length or about 20 J / cm fiber length to about 500 J / cm fiber length.

[0339] In some embodiments, the method provided includes irradiating a stromal tumor in a subject with a light dose of at or about 100 J / cm2 of fiber length or a fluence rate of at or about 400 mW / cm2 using a cylindrical diffusing fiber with a diffuser length of 0.5 cm to 10 cm and placed 1.8±0.2 cm apart, where the tumor is associated with a phototoxic agent that includes a targeting molecule bound to a cell surface molecule of the tumor. In some embodiments, the tumor is greater than 10 mm deep or is a subcutaneous tumor. In some embodiments, the cylindrical diffusing fiber is placed in a catheter positioned 1.8±0.2 cm apart in the tumor. In some embodiments, the catheter is optically transparent.

[0340] In some embodiments, the methods provided include irradiation at one or more wavelengths. In some examples, after administration of the conjugate, the lesion or tumor is irradiated at one or more wavelengths to induce phototoxic activity of a first dye of the conjugate and a fluorescent signal of a second dye of the conjugate. For example, in methods employing a first and second dye conjugated to a targeting molecule with different excitation wavelengths, two different wavelengths can be used for irradiation. In some embodiments, the methods provided include irradiating the lesion with a single wavelength. In some embodiments, the methods provided include irradiating the lesion with two different wavelengths, either simultaneously or sequentially, where one wavelength induces phototoxic activity and the other wavelength induces a fluorescent signal. For example, in some embodiments, the methods provided include irradiating the lesion with at least 1 J cm at one or more wavelengths that are between 400 and about 900 nm or about 400 and about 900 nm. -2 or irradiating the lesion with a dose of 1 J / cm of fiber length.

[0341] D. Additional Therapeutic Agents In some embodiments, another therapeutic agent, such as an immunomodulatory agent or an anti-cancer agent, is administered together with a photoimmunotherapy agent, such as a phthalocyanine dye conjugate, such as an IR700-antibody conjugate. In some embodiments, the combination therapy can include administration of a phthalocyanine dye conjugate, such as an IR700-antibody conjugate, in combination with an anti-cancer agent or an immunomodulatory agent, as described in U.S. Provisional Patent Application No. 62 / 206,776 (herein incorporated by reference).

[0342] In some embodiments, other or additional drug(s) can be administered at a sufficient time before irradiation is performed to increase the therapeutic effect on tumor treatment. In some embodiments, before irradiation in photoimmunotherapy method, one or more other therapeutic agents, such as immunomodulators (e.g., immune checkpoint inhibitors) or anticancer drugs (e.g., antimetabolites), are administered to the subject. In one embodiment, the immunomodulator can be administered at a sufficient time before irradiation, such as generally at least 12 hours before irradiation, to make the immune system responsive to tumor-associated agents released by tumor cell lysis after photoimmunotherapy. In another embodiment, the anticancer drug can be administered at a sufficient time before irradiation, such as generally at least 5 minutes before irradiation, to achieve systemic availability of the anticancer drug so that it can be delivered immediately to tumors by the change in vascular permeability after photoimmunotherapy.

[0343] One or more other agents, such as immunomodulatory agents or anti-cancer agents, can be administered prior to the phthalocyanine dye-targeting molecule conjugate, simultaneously with the phthalocyanine dye-targeting molecule conjugate, subsequent to the phthalocyanine dye-targeting molecule conjugate, or intermittently with the phthalocyanine dye-targeting molecule conjugate. In some embodiments, activation of the phthalocyanine dye photosensitizer of the conjugate by irradiation with light is not achieved until a time after administration of the other therapeutic agent, such as a therapeutic agent as described herein. In some embodiments, activation of the phthalocyanine dye photosensitizer of the conjugate by irradiation with light is performed prior to administration of the other therapeutic agent, such as a therapeutic agent as described herein.

[0344] In some embodiments, the combined effect of photoimmunotherapy in combination with one or more other agents can be synergistic compared to treatments involving only photoimmunotherapy with a phthalocyanine dye-targeting molecule conjugate or treatments involving monotherapy with other therapeutic agents. In some embodiments, the methods provided herein provide an increase or improvement in the desired anti-tumor therapeutic effect, such as an increase or improvement in the reduction or inhibition of one or more symptoms associated with cancer, over photoimmunotherapy or monotherapy alone.

[0345] Treatment with the phthalocyanine dye-targeting molecule conjugate, and optionally an additional therapeutic immunomodulatory or anticancer agent, may each independently be completed in one day or may be repeated over several days at the same or different dosages. Repeated treatments may occur 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.

[0346] In some embodiments, the combination therapy comprises administering to the subject a therapeutically effective amount of an immunomodulatory agent, such as an immune checkpoint inhibitor. The immunomodulatory agent is administered in an amount between 0.01 mg and 1000 mg or about 0.01 mg and 1000 mg, for example at a dose of at least 0.01 mg, 0.1 mg, 1 mg, 10 mg, 1000 mg, 2000 mg, 3000 mg or more. In exemplary embodiments, the immunomodulatory agent, such as an immune checkpoint inhibitor, may be administered at about 0.3 mg / kg to 10 mg / kg, or at a maximum tolerated dose, for example at least 0.5 mg / kg, or at least 1 mg / kg, or at least 2 mg / kg, or at least 3 mg / kg, or at least 5 mg / kg, or at least 8 mg / kg. In some cases, the dose can be administered as a single dose or in multiple doses. Alternatively, the immunomodulator, such as an immune checkpoint inhibitor, may be administered by an ascending dosage regimen, comprising administering a first dose at about 3 mg / kg, a second dose at about 5 mg / kg, and a third dose at about 9 mg / kg. Alternatively, the ascending dosage regimen comprises administering a first dose of the immunomodulator at about 5 mg / kg and a second dose at about 9 mg / kg. Another stepwise ascending dosage regimen escalation may comprise administering a first dose of the immunomodulator at about 3 mg / kg, a second dose of 3 mg / kg, a third dose of about 5 mg / kg, a fourth dose of about 5 mg / kg, and a fifth dose of about 9 mg / kg. In another aspect, the stepwise ascending dosage regimen may comprise administering a first dose of 5 mg / kg, a second dose of 5 mg / kg, and a third dose of 9 mg / kg. In some embodiments, a particular dosage can be administered twice a week, once a week, once every two weeks, once every three weeks, or once or more per month. In some cases, the dosage can be administered in a series of cycles that can be repeated, for example, for one month, two months, three months, six months, a year, or more.

[0347] In some embodiments, the combination therapy includes administering to the subject a therapeutically effective amount of an anti-cancer agent, such as any of those described herein. In some embodiments, the therapeutically effective dose can be 0.01 mg to 1000 mg or about 0.01 mg to 1000 mg, such as at least 0.01 mg, 0.1 mg, 1 mg, 10 mg, 1000 mg, 2000 mg, 3000 mg or more. In some embodiments, the therapeutically effective dose of the anti-cancer agent is 0.01 mg / kg to about 50 mg / kg or about 0.01 mg / kg to about 50 mg / kg, such as about 0.1 mg / kg to about 20 mg / kg, about 0.1 to about 10 mg / kg, about 0.3 to about 10 mg / kg, about 0.5 mg / kg to about 5 mg / kg, or about 0.5 mg / kg to about 1 mg / kg.

[0348] In some embodiments, the dose of the immunomodulator (e.g., immune checkpoint inhibitor) or anticancer drug is continued or repeated according to its clinical dosing schedule after PIT treatment.Thus, in some embodiments, in the dosing schedule or cycle of administration according to the provided method, the phthalocyanine dye conjugate (e.g., IR700 antibody conjugate) can be administered only once for PIT, for example, in a single dose or injection, but the administration of the immunomodulator is continued or repeated more than once during the dosing schedule or cycle of administration, for example, 3 times a week, 2 times a week, 1 time a week, 1 time every 2 weeks, 1 time every 3 weeks, or 1 time a month.In some embodiments, the dosing schedule or cycle of administration is 28 days or 4 weeks or about 28 days or about 4 weeks.

[0349] In some embodiments, the other or additional agent(s) administered, or the additional agent in the combination therapy, is an unconjugated targeting molecule. In some embodiments, the unconjugated targeting molecule is the same or substantially the same targeting molecule as the targeting molecule of the conjugate. For example, in some embodiments, a targeting molecule that targets a protein or antigen, such as an unconjugated antibody, is administered to the subject prior to administration of the conjugate. In some embodiments, the targeting molecule is administered up to 96 hours prior to administration of the conjugate. In some embodiments, the targeting molecule is administered at a dose of 10 mg / m 2 ~about 500mg / m 2 or about 10 mg / m 2 ~about 500mg / m 2 For example, the targeting molecule is cetuximab, and the cetuximab is administered to the subject up to 96 hours prior to administration of the conjugate.

[0350] 1. Immunomodulators The present disclosure provides an immunomodulator that can be administered in combination with the PIT method that employs phthalocyanine dye conjugates.Thus, the combination therapy provided herein (including the combination and method of use) includes an immunomodulator.An immunomodulator, or immunomodulator, is a substance that suppresses or activates the body's immune response.For example, an immunomodulator that stimulates the immune response to tumors and / or pathogens may be used in combination with photoimmunotherapy.

[0351] Generally, cancerous cells contain tumor-specific antigens that should be recognized by the immune system. Typically, in an active immune system, immune cells such as cytotoxic T cells attack and eradicate these cancerous cells. Under normal physiological conditions, T cell-mediated immune responses are initiated by antigen recognition by T cell receptors (TCRs) and regulated by a balance of costimulatory and inhibitory signals (e.g., immune checkpoint proteins). In particular, CD4+ and CD8+ T cells expressing TCRs can become activated by recognition of antigenic peptides presented on antigen-presenting cells on major histocompatibility complex (MHC) class I or class II molecules, respectively. In some aspects, activated CD8+ cells, or cytotoxic T cells, can kill tumor cells expressing antigens, which can be assisted by the presence of CD4+ T cells. In some embodiments, the immune cells are antigen-presenting cells. In some embodiments, the immune cells are dendritic cells.

[0352] However, in the case of tumors, the tumor microenvironment has mechanisms to suppress the immune system, thereby evading immune recognition and preventing or reducing tumor cell killing.For example, in some cases, immune checkpoint proteins can be deregulated in tumors, thereby resulting in the suppression of immune responses in the tumor microenvironment as a mechanism to evade the immune system.In some cases, other mechanisms can act to inhibit the access of immune cells to tumor antigens, thereby also contributing to the tumor's ability to evade the immune system.The combination therapy provided herein addresses both of these evasion mechanisms to provide a more robust immune response to tumors while killing tumor cells by photolysis mechanisms.

[0353] In some embodiments of the combination therapy methods provided herein, an immunomodulatory agent is administered to a subject to inhibit immunosuppressive signaling or enhance immunostimulatory signaling. For example, inhibitory checkpoint protein antagonists and / or agonists of costimulatory receptors can stimulate the host's intrinsic antitumor immune response by amplifying antigen-specific T cell responses. In aspects of the provided methods, photoimmunotherapy can also be performed, which can result in tumor cell killing and thereby release tumor antigens. By performing photoimmunotherapy in combination with administration of an immunomodulatory agent, the subsequent PIT-induced release of antigens can provide a source of antigen stimulation for T cells whose response is amplified or stimulated by the immunomodulatory agent. Thus, in some aspects, the enhanced immune response generated by treatment with an immunomodulatory agent is primed and ready to respond to tumor antigens exposed by the lysis of cells after PIT. Thus, in some aspects, the combination therapy provided herein accommodates natural evasion mechanisms that can exist in the tumor microenvironment to provide a more robust immune response to tumors while still killing tumor cells by photolytic mechanisms.

[0354] Generally, in the provided methods, PIT-mediated cell killing is performed by irradiation and activation of the administered phthalocyanine dye-conjugate at a time after the immune response is stimulated or enhanced after administration of the immunomodulator.In some embodiments, the immunomodulator is administered at a sufficient time before irradiation of the administered phthalocyanine dye-conjugate, so that the amplification of T cell response occurs before PIT-induced cell lysis.Thus, generally, the immunomodulator is administered at a sufficient time before irradiation to be therapeutically effective in amplifying T cell response, for example, at least 12 hours before performing irradiation, for example, generally at least 24 hours, at least 48 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month, to induce PIT-mediated cell killing through activation of the administered phthalocyanine dye-conjugate (e.g., IR700-targeting molecule conjugate, e.g., IR700-antibody dye conjugate).

[0355] In some embodiments, the conjugate is administered prior to, simultaneously with, or subsequent to administration of the immunomodulatory agent. In some embodiments, the conjugate is administered after administration of the immunomodulatory agent but before irradiating the tumor. In some embodiments, the conjugate is administered 12 hours to 48 hours or about 12 hours to 48 hours before irradiating the tumor and the immunomodulatory agent is administered 12 hours to 1 month or about 12 hours to about 1 month before irradiating the tumor. In some embodiments, the immunomodulatory agent is administered 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or more than 1 month before irradiating the tumor.

[0356] In some embodiments, irradiation of the tumor occurs either i) after administration of the immunomodulatory agent and after administration of the conjugate, or ii) only after administration of the conjugate.

[0357] Exemplary dosage regimens and schedules for administering the immunomodulatory agent, the phthalocyanine dye-conjugate (e.g., an IR700-targeting molecule conjugate, such as an IR700-antibody dye conjugate), and for performing the irradiation are described elsewhere herein.

[0358] In some embodiments, the method of combination therapy can be carried out with any immunomodulatory agent that can stimulate, amplify and / or otherwise enhance anti-tumor immune response, for example by inhibiting immunosuppressive signaling or enhancing immunostimulatory signaling.In some embodiments, the immunomodulatory agent is a peptide, a protein or a small molecule.In some embodiments, the protein can be a fusion protein or a recombinant protein.In some embodiments, the immunomodulatory agent binds to an immunological target, for example, a cell surface receptor, expressed on immune cells, for example, T cells, B cells or antigen-presenting cells.For example, in some embodiments, the immunomodulatory agent is an antibody or antigen-binding antibody fragment, a fusion protein, a small molecule or a polypeptide.

[0359] In some embodiments, the immunomodulator inhibits immune checkpoint pathway.The immune system has multiple inhibitory pathways that are involved in maintaining self-tolerance and regulate immune response.It is known that tumors can use certain immune checkpoint pathways as a major mechanism of immune tolerance, especially against T cells that are specific for tumor antigens (Pardoll, 2012, Nature Reviews Cancer 12:252-264).Many such immune checkpoints are initiated by ligand-receptor interactions, so they can be easily blocked by antibodies against ligands and / or their receptors.

[0360] Therefore, treatment with antagonist molecules that block immune checkpoint pathways, such as small molecules, nucleic acid inhibitors (e.g., RNAi) or antibody molecules, is becoming a promising avenue for immunotherapy of cancer and other diseases. In contrast to most anticancer drugs, checkpoint inhibitors do not need to directly target tumor cells, but rather target lymphocyte receptors or their ligands, to enhance the intrinsic antitumor activity of the immune system (Pardoll, 2012, Nature Reviews Cancer 12:252-264).

[0361] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, prevents or regulates one or more checkpoint proteins.Checkpoint proteins control the activation or function of T cells.These proteins are responsible for the co-stimulatory or inhibitory interactions of T cell responses.Immune checkpoint proteins control and maintain self-tolerance and the duration and magnitude of physiological immune responses.

[0362] Immune checkpoint inhibitors include any agent that statistically significantly blocks or inhibits an inhibitory pathway of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies, or antigen-binding fragments thereof, that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary immune checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, PD1 (CD279), PDL1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, Ox40 (CD134, TNFRSF4), CXCR2, tumor-associated antigen (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules and expressed on all NK, gamma delta, and memory CD8+ (alpha beta) T cells), CD160 (also referred to as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, or antigen-binding fragments thereof, or other binding proteins that bind to and block or inhibit the activity of one or more of PD1, PDL1, PDL2, CTLA-4, LAG3, TIM3, 4-1BB, 4-1BBL, GITR, CD40, Ox40, CXCR2, TAA, B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor).

[0363] Programmed cell death 1 (PD1) is an immune checkpoint protein expressed in B cells, NK cells, and T cells (Shinohara et al., 1995, Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45; Finger et al., 1997, Gene 197:177-87; Pardoll, 2012, Nature Reviews Cancer 12:252-264). The main role of PD1 is to limit the activity of T cells in peripheral tissues during inflammation in response to infection, as well as to limit autoimmunity (Pardoll, 2012, Nature Reviews Cancer 12:252-264). PD1 expression is induced in activated T cells, and binding of PD1 to one of its endogenous ligands acts to inhibit T cell activation by inhibiting stimulatory kinases (Pardoll, 2012, Nature Reviews Cancer 12:252-264). PD1 also acts to inhibit TCR "stop signals" (Pardoll, 2012, Nature Reviews Cancer 12:252-264). PD1 is strongly expressed on Treg cells and can increase their proliferation in the presence of ligand (Pardoll, 2012, Nature Reviews Cancer 12:252-264). Anti-PD1 antibodies have been used to treat melanoma, non-small cell lung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck cancer, triple-negative breast cancer, leukemia, lymphoma, and renal cell carcinoma (Topalian et al., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85).Exemplary anti-PD1 antibodies include nivolumab (Opdivo by BMS), pembrolizumab (Keytruda by Merck), pidilizumab (CT-011 by Cure Tech), lambrolizumab (MK-3475 by Merck), and AMP-224 (Merck).

[0364] PD-L1 (also known as CD274 and B7-H1) and PD-L2 (also known as CD273 and B7-DC) are ligands of PD1 found on activated T cells, B cells, myeloid cells, macrophages, and some types of tumor cells. Antitumor therapy focuses on anti-PD-L1 antibodies. The complex of PD1 and PD-L1 inhibits the proliferation of CD8+ T cells and reduces the immune response (Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65). Anti-PD-L1 antibodies have been used to treat non-small cell lung cancer, melanoma, colorectal cancer, renal cell carcinoma, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, and hematological malignancies (Brahmer et al., N Eng J Med 366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res 19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer Res 14:13044-51). Exemplary anti-PD-L1 antibodies include MDX-1105 (Medarex), MEDI4736 (Medimmune), MPDL3280A (Genentech), BMS-935559 (Bristol-Myers Squibb), and MSB0010718C.

[0365] Cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152, is a co-inhibitory molecule that functions to regulate T-cell activation. CTLA-4 is a member of the immunoglobulin superfamily that is expressed exclusively on T cells. CTLA-4 has been reported to act to inhibit T-cell activation and to inhibit the activity of helper T cells and enhance the immunosuppressive activity of regulatory T cells (Pardoll, 2012, Nature Reviews Cancer 12:252-264). Although the exact mechanism of action of CTLA-4 is under investigation, it has been suggested that CTLA-4 inhibits T-cell activation by outcompeting CD28 for binding to CD80 and CD86 and by actively delivering an inhibitor signal to T cells (Pardoll, 2012, Nature Reviews Cancer 12:252-264). Anti-CTLA-4 antibodies are being used in clinical trials for the treatment of melanoma, prostate cancer, small cell lung cancer, and non-small cell lung cancer (Robert & Ghiringhelli, 2009, Oncologist 14:848-61; Ott et al., 2013, Clin Cancer Res 19:5300; Weber, 2007, Oncologist 12:864-72; Wada et al., 2013, J Transl Med 11:89). A striking feature of anti-CTLA-4 is the kinetics of the antitumor effect, which requires a period of up to 6 months for a physiological response after initial treatment (Pardoll, 2012, Nature Reviews Cancer 12:252-264). In some cases, tumors may actually increase in size after treatment begins, but then a reduction is seen (Pardoll, 2012, Nature Reviews Cancer 12:252-264). Exemplary anti-CTLA-4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer). Ipilimumab was recently approved by the FDA for the treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11:89). In some embodiments, the immunomodulatory agent is not an anti-CTLA-4 antibody.

[0366] Lymphocyte activation gene-3 (LAG-3), also known as CD223, is another immune checkpoint protein. LAG-3 is associated with the inhibition of lymphocyte activity and in some cases with the induction of lymphocyte anergy. LAG-3 is expressed on various cells in the immune system, including B cells, NK cells, and dendritic cells. LAG-3 is a natural ligand for the MHC class II receptor, which is substantially expressed on melanoma inflammatory T cells, including those with potent immunosuppressive activity. An exemplary anti-LAG-3 antibody is BMS-986016. IMP321 is a soluble version of the immune checkpoint molecule LAG-3, which activates dendritic cells to increase antigen presentation.

[0367] First identified on activated Th1 cells, T cell immunoglobulin domain and mucin domain-3 (TIM-3) has been shown to be a negative regulator of immune responses. Blockade of TIM-3 promotes T cell-mediated antitumor immunity and has antitumor activity in a wide range of mouse tumor models. Combination of TIM-3 blockade with other immunotherapeutics, such as TSR-042, an anti-CD137 antibody, can be additive or synergistic in increasing antitumor efficacy. TIM-3 expression has been associated with a number of different tumor types, including melanoma, NSCLC, and renal cancer, and additionally, intratumoral TIM-3 expression has been shown to correlate with poor prognosis across a wide range of tumor types, including NSCLC, cervical cancer, and gastric cancer. Blockade of TIM-3 is also of interest in promoting increased immunity to a number of chronic viral diseases. TIM-3 has also been shown to interact with a number of ligands, including galectin-9, phosphatidylserine, and HMGB1, although it is currently unclear which, if any, of these are involved in controlling antitumor responses.

[0368] 4-1BB, also known as CD137, is a transmembrane glycoprotein that belongs to the TNFR superfamily. 4-1BB receptors are present on activated T cells and B cells as well as monocytes. An exemplary anti-4-1BB antibody is urelumab (BMS-663513), which has potential immunostimulatory and antineoplastic activity.

[0369] Glucocorticoid-induced TNFR family related gene (GITR) is also a member of the TNFR superfamily. GITR is upregulated on activated T cells, which enhances the immune system. An exemplary anti-GITR antibody is TRX518.

[0370] Cluster of differentiation 40 (CD40) is also a member of the TNFR superfamily. CD40 is a costimulatory protein found on antigen-presenting cells and mediates a wide range of immune and inflammatory responses. CD40 is also expressed on some malignant tumors, where it promotes their proliferation. Exemplary anti-CD40 antibodies are dacetuzumab (SGN-40), lucatumumab (Novartis, antagonist), SEA-CD40 (Seattle Genetics), and CP-870,893.

[0371] Tumor necrosis factor receptor superfamily member 4 (TNFRSF4), also known as OX40 and CD134, is another member of the TNFR superfamily. OX40 is not constitutively expressed on resting naive T cells and acts as a secondary costimulatory immune checkpoint molecule. Exemplary anti-OX40 antibodies are MEDI6469 and MOXR0916 (RG7888, Genentech).

[0372] In some embodiments, the immunomodulatory agent is an antibody or an antigen-binding antibody fragment thereof. Exemplary such antibodies include, but are not limited to, daclizumab (Zenapax), bevacizumab (Avastin®), basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (atezolizumab), tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX51 8, MK-4166, dacetuzumab (SGN-40), lucatumumab (HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR0916, AMP-224, MSB0010718C (Avelumab), MEDI4736, PDR001, rHIgM12B7, urocupulumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, ARGX-115, emactuzumab, CC-90002 and MNRP1685A or an antibody-binding fragment thereof.

[0373] CXCR2 is a chemokine receptor expressed on myeloid-derived suppressor cells (MDSCs). CXCR2 contributes to tumor immune evasion. It has been shown that anti-CXCR2 monoclonal antibody therapy enhanced anti-PD1 antibody-induced anti-tumor immune responses and anti-tumor efficacy.

[0374] In some embodiments, the immunomodulator is a cytokine. In some embodiments, the immunomodulator is a cytokine or an agent that induces increased expression of cytokine in tumor microenvironment. "Cytokine" refers to a collective term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines are growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors, such as NGF-beta; platelet growth factor; transforming growth factors (TGFs), such as TGF- Cytokines include alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); bone morphogenetic factor; interferons, such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors, such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.For example, the immunomodulatory agent is a cytokine, and the cytokine is IL-4, TNF-α, GM-CSF, or IL-2.

[0375] In some embodiments, the immunomodulatory agent is selected from among GM-CSF, CpG-ODN (CpG oligodeoxynucleotide), lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), alum, recombinant Leishmania polyprotein, imiquimod, MF59, poly I:C, poly A:U, type 1 IFN, Pam3Cys, Pam2Cys, complete Freund's adjuvant (CFA), alpha-galactosylceramide, RC-529, MDF2β, loxoribine, anti-CD40 agonist, SIRPa antagonist, AS04, AS03, flagellin, resiquimod, DAP (diaminopimelic acid), MDP (muramyl dipeptide), and CAF01 (cationic adjuvant formulation-01). In some embodiments, the immunomodulatory agent is a Toll-like receptor (TLR) agonist, adjuvant, or cytokine. In some embodiments, the immunomodulatory agent is a TLR agonist, and the TLR agonist is a TLR4 agonist, a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist. In some embodiments, the TLR agonist is selected from among triacylated lipoproteins, diacylated lipoproteins, lipoteichoic acid, peptidoglycan, zymosan, Pam3CSK4, dsRNA, poly I:C, poly G10, poly G3, CpG, 3M003, flagellin, lipopolysaccharide (LPS), Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), MEDI9197, SD-101, and imidazoquinoline TLR agonists.

[0376] In some embodiments, the immunomodulatory agent can include one or more interleukins or other cytokines. For example, the interleukins can include Leukocyte Interleukin Injection (Multikine), which is a combination of natural cytokines.

[0377] In some embodiments, the immunomodulatory agent is a Toll-like receptor (TLR) agonist. In some embodiments, such agonists can include TLR4 agonists, TLR8 agonists, or TLR9 agonists. Such agonists can be selected from peptidoglycan, poly I:C, CpG, 3M003, flagellin, and Leishmania homolog of eukaryotic ribosome elongation and initiation factor 4a (LeIF).

[0378] In some embodiments, the immunomodulatory agent can be one that enhances the immunogenicity of tumor cells, such as patupilone (epothilone B), epidermal growth factor receptor (EGFR)-targeted monoclonal antibody 7A7.27, histone deacetylase inhibitors (e.g., vorinostat, romidepsin, panobinostat, belinostat, and entinostat), n3-polyunsaturated fatty acid docosahexaenoic acid, proteasome inhibitors (e.g., bortezomib), shikonin (the main component of the root of Lithospermum erythrorhizon) and oncolytic viruses, such as TVec (talimogene laherparepvec). In some embodiments, immunomodulators such as anthracyclines (doxorubicin, mitoxantrone), BK channel agonists, bortezomib, bortezomib+mitomycin C+hTert-Ad, cardiac glycosides+non-ICD inducers, cyclophosphamide, GADD34 / PP1 inhibitors+mitomycin, LV-tSMAC, and oxaliplatin activate the immunogenic cell death of cancer or tumor. In some embodiments, the immunomodulator can be epigenetic therapy, such as DNA methyltransferase inhibitors (e.g., decitabine, 5-aza-2'-deoxycytidine).

[0379] For example, in some embodiments, the immunomodulatory agent can be a DNA methyltransferase inhibitor, which can control the expression of tumor-associated antigens (TAA). TAA is an antigenic substance produced in tumor cells that triggers immune responses. TAA is often downregulated in tumors by DNA methylation to evade the immune system. Reversal of DNA methylation restores TAA expression and increases the immunogenicity of tumor cells. For example, demethylating agents such as decitabine (5-aza-2'-deoxycytidine) can upregulate the expression of TAA in tumor cells and increase immune recognition of cancerous cells. Photoimmunotherapy will further expose TAA to the immune system by destroying cells.

[0380] In some embodiments, the immunomodulatory agent itself can be an antibody conjugate containing a phthalocyanine dye linked to an antibody or antigen-binding antibody fragment that is an immunomodulatory agent, such as an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent targets or binds to an immunosuppressive molecule, such as an immune checkpoint molecule, on the surface of tumor cells. For example, PD-L1 is an immunosuppressive molecule that is constitutively expressed or induced on many tumor cells and can prevent T cell activation through interaction with its receptor PD-1 expressed on immune cells. In some aspects, a phthalocyanine-dye conjugate containing an immunomodulatory agent that binds to an immunosuppressive molecule (e.g., PD-L1) on tumor cells can be administered to both enhance the immune response and act to specifically kill cancer cells that express the immunosuppressive molecule, thereby reversing immunosuppression in the tumor microenvironment. In particular, irradiation of tumor cells to which the conjugate is bound can result in its activation and mediate PIT-induced cell killing of PD-L1 cancer cells, which would also act to specifically eliminate cancer cells in tumors that control T cell suppression in the tumor microenvironment.

[0381] Thus, provided herein is a conjugate containing a phthalocyanine dye (e.g., IR700) linked to an immunomodulatory agent that binds to an immunosuppressive molecule expressed on a tumor cell. For example, in some embodiments, the immunosuppressive molecule expressed on a tumor cell can be an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule expressed on a tumor cell is PD-L1. In some embodiments, the immunomodulatory agent that is part of the conjugate is an immune checkpoint inhibitor, such as an antibody or antigen-binding antibody fragment that binds to PD-L1. For example, provided herein is a conjugate containing a phthalocyanine dye (e.g., IR700) linked to an antibody or antigen-binding antibody fragment that binds to PD-L1. Exemplary immune checkpoint inhibitors (including antibodies or antigen-binding antibody fragments) against PD-L1 are described above, and any can be included in the provided conjugate. Exemplary anti-PD-L1 antibodies include, but are not limited to, BMS-935559, MEDI4736, MPDL3280A, and MSB0010718C, or antigen-binding antibody fragments thereof. Exemplary conjugate molecules provided herein can include, for example, IR700-BMS-935559, IR700-MEDI4736, IR700-MPDL3280A and IR700-MSB0010718C. In some embodiments, such conjugates can be used in photoimmunotherapy methods, for example by irradiation with light at a wavelength sufficient to activate the dye. Such conjugates can be used in monotherapy-based photoimmunotherapy or in combination therapy methods with other phthalocyanine dye conjugates.

[0382] For example, in some embodiments, a method of combination therapy is provided in which a first conjugate (e.g., an anti-PD-L1 antibody, e.g., an IR700-anti-PD-L1 conjugate) containing a phthalocyanine dye (e.g., IR700) linked to an immunomodulatory agent that binds to an immunosuppressive molecule expressed on cells of a tumor is administered to a subject, and then a second conjugate containing a phthalocyanine dye linked to a targeting molecule is administered to the subject. In general, the second conjugate can comprise any targeting molecule that can bind to a cell surface protein on cells in a tumor, such as cells present in the tumor microenvironment, e.g., any of the above. In some embodiments, the first conjugate and the second conjugate bind to different proteins expressed on cells in a tumor. In some embodiments, the second conjugate can comprise a phthalocyanine dye (e.g., IR700) linked to an antibody or antigen-binding antibody fragment that binds to a cell surface protein expressed on cells in a tumor.Exemplary antibodies or antigen-binding antibody fragments of the second conjugate include, but are not limited to, bevacizumab, cetuximab, panitumumab, zalutumumab, nimotuzumab, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragments, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), as well as basiliximab, nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518 , MK-4166, dacetuzumab, lucatumumab, SEA-CD40, CP-870, CP-893, MED16469, MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C, rHIgM12B7, urocupulumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, emactuzumab, CC-90002 and MNRP1685A, or an antibody binding fragment thereof.

[0383] In some embodiments, for example, when treatment of tumor with the conjugate and subsequent light irradiation increases the presence of immunosuppressive cells in the tumor or increases the expression of immunosuppressive markers in the tumor, a therapeutically effective amount of an immunomodulatory agent can be administered that can reduce the amount or activity of immunosuppressive cells in the tumor, or block the activity of an immunosuppressive marker or reduce the activity of tumor-promoting cells in the tumor, or block the activity of a tumor-promoting marker.For example, in some embodiments, a conjugate having a first dye that is a phthalocyanine dye administered in combination with an immunomodulatory agent comprises a conjugate that includes a second phthalocyanine dye conjugated to an immunomodulatory agent, and the immunomodulatory agent is capable of binding to and modulating the activity of immunosuppressive cells or tumor-promoting cells.In some embodiments, the first and second phthalocyanine dyes are the same or different.

[0384] Thus, in some embodiments, the immunomodulatory agent is itself a conjugate that contains a phthalocyanine dye, e.g., a phthalocyanine dye linked to an antibody or antigen-binding antibody fragment that is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an IR700-antibody conjugate that includes an immunomodulatory antibody (e.g., a checkpoint inhibitor) that binds to a checkpoint protein (e.g., PD-L1) on tumor cells. In some embodiments, the immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulatory agent) is administered between 12 hours and 2 months prior to administration of the phthalocyanine dye-targeting molecule conjugate, e.g., generally at least 12 hours, at least 24 hours, at least 48 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month prior to administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the immunomodulatory conjugate (e.g., an IR700-antibody conjugate that is an immunomodulatory agent) is administered during or simultaneously with administration of the ph...

Claims

1. (a) administering to a subject having a disease or condition a conjugate comprising a phthalocyanine dye linked to a targeting molecule that binds to a protein on the surface of a cell present in the microenvironment of a lesion associated with the disease or condition, wherein the conjugate is administered to achieve a systemic exposure that is 75% or less of the therapeutically effective systemic exposure of an antibody or antigen-binding antibody fragment that is not so conjugated to treat the disease or condition; and (b) After administration of the conjugate, the lesion is exposed to at least 1 J cm at wavelengths between 500 nm and 900 nm. -2 or irradiating with a dose of 1 J / cm of fiber length, thereby treating the disease in the subject. A method of treating a disease or condition in a subject, comprising:

2. 2. The method of claim 1, wherein the wavelength is between 600 nm and 850 nm.

3. 3. The method of claim 1 or claim 2, wherein the wavelength is between 660 nm and 740 nm.

4. The conjugate is administered according to the following dosing schedule: The administration of the conjugate is performed only once as a single injection or infusion; or the dosing schedule does not include a subsequent dose of the conjugate; or The dosing schedule does not include a subsequent dose of the macromolecule that is not so conjugated. The method according to any one of claims 1 to 3.

5. The method of any one of claims 1 to 4, wherein the conjugate is administered systemically.

6. The method of any one of claims 1 to 5, wherein the conjugate is administered intravenously.

7. The method of any one of claims 1 to 6, wherein the conjugate is administered to achieve a systemic exposure (AUC) that is 60% or less, 50% or less, 40% or less, or 30% or less of the therapeutically effective systemic exposure of the antibody or antigen-binding antibody fragment that is not so conjugated for treating said disease or condition.

8. The method of any one of claims 1 to 7, wherein the disease or condition is a tumor, the antibody or antigen-binding antibody fragment binds to a molecule on the surface of a cell present in the tumor microenvironment, and the tumor is irradiated.

9. systemic exposure as measured by the mean area under the plasma conjugate concentration-time curve from time 0 to infinity for the patient population following administration of the conjugate (AUC[0-inf]) of between or between about 250 μg / mL*h and 100,000 μg / mL*h, between or between about 500 μg / mL*h and 50,000 μg / mL*h, between or between about 500 μg / mL*h and 50,000 μg / mL*h, between or between about 500 μg / mL*h and 18,000 μg / mL*h; between or between about 500 μg / mL*h and 10,000 μg / mL*h; or systemic exposure as measured by the mean area under the plasma conjugate concentration-time curve from time 0 to infinity for the patient population following administration of the conjugate (AUC[0-inf]) is ≤100,000 μg / mL*h, ≤75,000 μg / mL*h, ≤50,000 μg / mL*h, ≤40,000 μg / mL*h, ≤30,000 μg / mL*h, ≤20,000 μg / mL*h, ≤10,000 μg / mL*h, ≤5,000 μg / mL*h, ≤2,500 μg / mL*h; The method according to any one of claims 1 to 8.

10. systemic exposure as measured by the mean area under the plasma conjugate concentration-time curve from time point 0 to 24 hours for the patient population following administration of the conjugate (AUC[0-24]) of between or between about 100 μg / mL*h and 25,000 μg / mL*h, between or between about 200 μg / mL*h and 10,000 μg / mL*h, between or between about 500 μg / mL*h and 5,000 μg / mL*h; or systemic exposure as measured by the mean area under the plasma conjugate concentration-time curve from time point 0 to 24 hours (AUC[0-24]) for the patient population following administration of the conjugate is ≤25,000 μg / mL*h, ≤15,000 μg / mL*h, ≤10,000 μg / mL*h, ≤5,000 μg / mL*h, ≤2,500 μg / mL*h, ≤1,000 μg / mL*h, or ≤500 μg / mL*h; The method of any one of claims 1 to 9.

11. The conjugate is at least about 10 mg / m 2 (subject's body surface area), at least about 50 mg / m 2 or at least about 75 mg / m 2 and 5000 mg / m 2 Below 2000mg / m 2 Below, 1000mg / m 2 Below, 500mg / m 2 Below 250mg / m 2 or less than 200 mg / m 2 The method of any one of claims 1 to 10, wherein the dosage range is administered as follows:

12. The conjugate is 100 mg / m 2 ~1500mg / m 2 Between 100 mg / m 2 ~1500mg / m 2 or 150 mg / m 2 ~750mg / m 2 Between 150 mg / m 2 ~750mg / m 2 The method of any one of claims 1 to 11, wherein the dosage is between

13. The conjugate is 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 , or about 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 The method of any one of claims 1 to 12, wherein the dosage is

14. The method of any one of claims 1 to 13, wherein the targeting molecule is an antibody or an antigen-binding antibody fragment.

15. The antibody is a Fab, a single V H The method of claim 14, wherein the antigen-binding antibody fragment is a domain, a single chain variable fragment (scFv), a multivalent scFv, a bispecific scFv or an scFv-CH3 dimer.

16. 16. The method of any one of claims 1 to 15, wherein the irradiation is performed between or about 30 minutes to 96 hours after administering the conjugate.

17. 17. The method of any one of claims 1 to 16, wherein the lesion is irradiated with a wavelength of 690±50 nm or at or about 690±20 nm.

18. The lesion is 2J cm -2 ~Approx. 400J cm -2 Or about 2J cm -2 ~Approx. 400J cm -2 or from about 2 J / cm to about 500 J / cm of fiber length or from about 2 J / cm to about 500 J / cm of fiber length.

19. The lesion has a mass of at least or at least about 2 J cm -2 , 5J cm -2 , 10J cm -2 , 25J cm -2 , 50J cm -2 , 75J cm -2 , 100J cm -2 , 150J cm -2 , 200J cm -2 , 300J cm -2 , 400J cm -2 , or 500 J cm -2 or the lesion is irradiated with a dose of at least or at least about 2 J / cm fiber length, 5 J / cm fiber length, 10 J / cm fiber length, 25 J / cm fiber length, 50 J / cm fiber length, 75 J / cm fiber length, 100 J / cm fiber length, 150 J / cm fiber length, 200 J / cm fiber length, 250 J / cm fiber length, 300 J / cm fiber length, 400 J / cm fiber length, or 500 J / cm fiber length; 19. The method of any one of claims 1 to 18.

20. 20. The method of any one of claims 1 to 19, wherein the phthalocyanine dye has a maximum absorption wavelength of from 600 nm to about 850 nm or from about 600 nm to about 850 nm.

21. 21. The method of any one of claims 1 to 20, wherein the phthalocyanine dye is directly or indirectly linked to a targeting molecule.

22. The phthalocyanine dye has the following formula: wherein L is a linker; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; and X 2 and X 3 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 is alkylene, 22. The method of any one of claims 1 to 21.

23. The phthalocyanine dye has the following formula: wherein X 1 and X 4 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 is alkylene; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; 23. The method of any one of claims 1 to 22.

24. 24. The method of any one of claims 1 to 23, wherein the phthalocyanine dye comprises IRDye 700DX (IR700).

25. Cell surface proteins include ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptors BB1, BB2, BB4, calcitonin receptor, and cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, Crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endosialin, EP2, EP4 , EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, family A G protein-coupled receptor (rhodopsin-like), family B G protein-coupled receptor (secretin receptor-like), family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV 16 / 18 and E6 / E7 antigens, hTERT, IL11-R, IL-13R, ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE 1, MAGE 2, MAGE 3, MAGE4, MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease-cleaved collagen IV, proteinase 3, prohibitin, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 25. The method of any one of claims 1-24, wherein the receptor is selected from among patched, RET receptor, plexin, smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptor (VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, and VEGF-3 or FLT-4), voltage-gated ion channel, VPAC1, VPAC2, Wilms' tumor 1, Y1, Y2, Y4, and Y5.

26. Cell surface proteins include HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72, caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), 26. The method of any one of claims 1 to 25, wherein the antigen is selected from among CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate binding protein, gangliosides (such as GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), integrin alphaVbeta3, integrin alpha5beta1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10beta, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinases, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, and SK-1 antigen.

27. Cell surface proteins include CD25, PD-1 (CD279), PD-L1 (CD274, B7-H1), PD-L2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3 (HAVCR2), 4-1BB (CD137, TNFRSF9), CXCR2, CXCR4 (CD184), CD27, CEACAM1, Galectin 9, BTLA, CD160, VISTA ( PD1 homolog), B7-H4 (VCTN1), CD80 (B7-1), CD86 (B7-2), CD28, HHLA2 (B7-H7), CD28H, CD155, CD226, TIGIT, CD9 6, Galectin 3, CD40, CD40L, CD70, LIGHT (TNFSF14), HVEM (TNFRSF14), B7-H3 (CD276), Ox40L (TNFSF4), CD137L (TNFSF9, GITRL), B7RP1, ICOS (CD278), ICOSL, KIR, GAL9, NKG2A (CD94), GARP, TL1A, TNFRSF25, TMIGD2, BTNL2, butyrophilin family, CD48, CD244, Siglec family, CD30, CSF1R, MICA (MHC class I polypeptide-related sequence A), MICB (MHC class I polypeptide-related sequence B), NKG2D, KIR family (killer cell immunoglobulin-like receptor), LILR family (leukocyte immunoglobulin-like receptor, CD85, ILT, LIR), SIRPA (signal regulatory protein alpha), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR, and VEGF.

28. The antibody or antigen-binding antibody fragment may be selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), basiliximab, nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, M 28. The method of any one of claims 1 to 27, wherein the antibody is selected from among K-4166, dacetuzumab, lucatumumab, SEA-CD40, CP-870, CP-893, MED16469, MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C, rHIgM12B7, urocupulumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, emactuzumab, CC-90002 and MNRP1685A, or an antigen-binding antibody fragment thereof.

29. The conjugates are cetuximab-IR700, panitumumab-IR700, zalutumumab-IR700, nimotuzumab-IR700, tositumomab-IR700, rituximab-IR700, ibritumomab tiuxetan-IR700, daclizumab-IR700, gemtuzumab-IR700, alemtuzumab-IR700, CEA-scan Fab fragment-IR700, OC125-IR700, ab75705-IR700, B72.3-IR700, bevacizumab-IR700, basiliximab-IR700, nivolumab-IR700, pembrolizumab-IR700, pidilizumab-IR700, MK-3475-IR700, BMS-936559-IR700, MPDL3280A-IR700, I Pilimumab-IR700, Tremelimumab-IR700, IMP321-IR700, BMS-986016-IR700, LAG525-IR700, Urelumab-IR700, PF-05082566-IR700, TRX518-IR700, MK-4166-IR700, Dacetuzumab-IR700, Lucatumumab-IR700, SEA-CD40-IR700 , CP-870-IR700, CP-893-IR700, MED16469-IR700, MEDI6383-IR700, MEDI4736-IR700, MOXR0916-IR 700, AMP-224-IR700, PDR001-IR700, MSB0010718C-IR700, rHIgM12B7-IR700, urokupulumab-IR700, BKT1 40-IR700, varlilumab-IR700, ARGX-110-IR700, MGA271-IR700, lirilumab-IR700, IPH2201-IR700, AGX-115-IR700, emactuzumab-IR700, CC-90002-IR700 and MNRP1685A-IR700.

30. 30. The method of claim 29, wherein the targeting molecule is an antibody or antigen-binding antibody fragment thereof that is cetuximab, or the conjugate is cetuximab-IR700.

31. 31. The method of claim 30, wherein the mean area under the plasma conjugate concentration-time curve from time 0 to infinity (AUC[0-inf]) for the patient population following administration of the conjugate is between or between about 500 μg / mL*h and 18,000 μg / mL*h, between or between about 500 μg / mL*h and 10,000 μg / mL*h, between or between about 500 μg / mL*h and 10,000 μg / mL*h, between or between about 500 μg / mL*h and 5,000 μg / mL*h, or between or between about 500 μg / mL*h and 2,500 μg / mL*h.

32. 31. The method of claim 30, wherein the mean area under the plasma conjugate concentration-time curve from time point 0 to 24 hours (AUC[0-24]) for the patient population following administration of the conjugate is between or between about 500 μg / mL*h and 8,000 μg / mL*h, between or between about 500 μg / mL*h and 5,000 μg / mL*h, between or between about 500 μg / mL*h and 5,000 μg / mL*h, between or between about 500 μg / mL*h and 2,000 μg / mL*h, or between or between about 1000 μg / mL*h and 4,000 μg / mL*h.

33. The conjugate is 75 mg / m 2 (subject's body surface area) ~1500mg / m 2 Between 75 mg / m 2 (subject's body surface area) ~1500mg / m 2 Between 75 mg / m 2 ~1000mg / m 2 Between 75 mg / m 2 ~1000mg / m 2 Between 75 mg / m 2 ~500mg / m 2 Between 75 mg / m 2 ~500mg / m 2 or 75 mg / m 2 ~225mg / m 2 Between 75 mg / m 2 ~225mg / m 2 between; or At least about 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 , or about 160 mg / m 2 , 320 mg / m 2 , 640 mg / m 2 or 1280 mg / m 2 is administered in a dosage range 33. The method of any one of claims 30 to 32.

34. (a) administering intravenously to a subject having a lesion associated with a disease or condition a cetuximab-IR700 conjugate, wherein the conjugate is administered at a concentration of 640 mg / m 2 or about 640 mg / m 2 and (b) After administration of the conjugate, the lesion is irradiated with at least or about at least or about 50 J cm at a wavelength of 690±20 nm. -2 or irradiating with a dose of 100 J / cm of fiber length, thereby treating the disease or condition in the subject.

23. A method of treating a disease pathology in a subject, comprising:

35. The conjugate is administered according to the following dosing schedule: The administration of the conjugate is performed only once as a single injection or infusion; or the dosing schedule does not include a subsequent dose of the conjugate; or The dosing schedule does not include a subsequent dose of the macromolecule that is not so conjugated.

35. The method of claim 34.

36. 36. The method of any one of claims 1 to 35, wherein the irradiation is performed 24 hours ± 3 hours after administration of the conjugate.

37. 37. The method of any one of claims 34 to 36, wherein the lesion is a tumor and the disease or condition is a tumor or a cancer.

38. 38. The method of any one of claims 1 to 37, wherein the lesion is a tumor that is a superficial tumor.

39. 40. The method of claim 38, wherein the tumor is less than 10 mm thick.

40. 40. The method of claim 38 or claim 39, wherein the illumination is performed using a microlens-tipped fiber for surface illumination.

41. The light exposure dose is 5 J / cm 2 ~About 200J / cm 2 or about 5 J / cm 2 ~About 200J / cm 2 The method of any one of claims 1 to 40, wherein

42. A microlens-tipped fiber was used to illuminate the surface of the subject's superficial tumor at 5 J / cm 2 ~About 200J / cm 2 or about 5 J / cm 2 ~About 200J / cm 2 4. A method for treating a superficial tumor with photoimmunotherapy comprising the step of irradiating the tumor with a light dose of 0.1 mg / kg / day, wherein the tumor is associated with a phototoxic agent comprising a targeting molecule bound to a cell surface molecule of the tumor.

43. The light exposure dose is 50 J / cm 2 or about 50 J / cm 2 43. The method of claim 41 or claim 42, wherein

44. 41. The method of any one of claims 1 to 40, wherein the lesion is a tumor that is a stromal tumor.

45. 45. The method of claim 44, wherein the tumor is greater than 10 mm deep or is a subcutaneous tumor.

46. 46. ​​The method of claim 44 or claim 45, wherein the irradiation is performed using cylindrical diffusing fibers comprising a diffuser length of 0.5 cm to 10 cm and spaced 1.8±0.2 cm apart.

47. 47. The method of any one of claims 1-37 and 44-46, wherein the light exposure dose is from 20 J / cm fiber length to about 500 J / cm fiber length or from about 20 J / cm fiber length to about 500 J / cm fiber length.

48. 1. A method for treating stromal tumors with photoimmunotherapy comprising irradiating a stromal tumor in a subject with a cylindrical diffusing fiber comprising a diffuser length of 0.5 cm to 10 cm and spaced 1.8±0.2 cm apart with a light dose of at or about 100 J / cm / cm of fiber length or a fluence rate of at or about 400 mW / cm, wherein the tumor is associated with a phototoxic agent comprising a targeting molecule bound to a cell surface molecule of the tumor.

49. 49. The method of claim 47 or claim 48, wherein the light exposure dose is between 50 J / cm fiber length and about 300 J / cm fiber length or between about 50 J / cm fiber length and about 300 J / cm fiber length.

50. 50. The method of any one of claims 47 to 49, wherein the light exposure dose is at or about 100 J / cm of fiber length.

51. The method of any one of claims 48-50, wherein the tumor is greater than 10 mm deep or is a subcutaneous tumor.

52. The method of any one of claims 47 to 51, wherein the cylindrical diffusing fiber is placed in a catheter positioned 1.8±0.2 cm away in the tumor.

53. 53. The method of claim 52, wherein the catheter is optically transparent.

54. The method of any one of claims 42, 43 and 48-53, wherein a phototoxic agent comprising a targeting molecule is administered to the subject more than 6 hours prior to irradiating the tumor, wherein the phototoxic agent associates with the tumor.

55. 55. The method of claim 54, wherein the phototoxic agent is pre-administered to the subject more than or about 12, 24, 26, 48, 72 or 96 hours prior to irradiating the tumor.

56. The method of any one of claims 42, 43 and 48-55, wherein the phototoxic agent is a phthalocyanine dye-targeting molecule conjugate.

57. 57. The method of claim 56, wherein the phthalocyanine dye is IR700.

58. The method of any one of claims 1-41, 44-47, 49, 50 and 52-54, wherein said dosing schedule is repeated to repeat steps (a) and (b).

59. 59. The method of claim 58, wherein the dosing schedule is repeated if residual disease remains after prior treatment with the conjugate.

60. 60. The method of claim 58 or claim 59, comprising evaluating the subject for the presence of residual disease and repeating the dosing schedule if residual disease remains.

61. 61. The method of any one of claims 58-60, wherein the dosing schedule is repeated if residual disease remains at more than, or about, or 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year after initiation of the prior administration of the conjugate.

62. 62. The method of any one of claims 58-61, wherein the dosing schedule is repeated if residual lesions remain at or about 4 weeks after initiation of the prior administration of the conjugate.

63. 63. The method of any one of claims 1-62, wherein the conjugate comprises 1-100, 1-10, or 2-5 phthalocyanine dye molecules per macromolecule.

64. 64. The method of any one of claims 1 to 63, wherein the method does not include administration of an additional therapeutic agent or anti-cancer treatment.

65. 64. The method of any one of claims 1 to 63, comprising administering an additional therapeutic agent or anti-cancer treatment.

66. 66. The method of claim 65, wherein the anti-cancer treatment comprises radiation therapy.

67. 67. The method of claim 66, wherein the additional therapeutic agent is an anti-cancer agent or an immunomodulatory agent.

68. 68. The method of claim 67, wherein the additional therapeutic agent is an immunomodulatory agent that is an immune checkpoint inhibitor.

69. The method of claim 68, wherein the immune checkpoint inhibitor specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

70. The method of claim 68 or claim 69, wherein the immune checkpoint inhibitor is an antibody or antigen-binding fragment, a small molecule, or a polypeptide.

71. 71. The method of any one of claims 68-70, wherein the immune checkpoint inhibitor is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment thereof.

72. The method of any one of claims 68-71, wherein an immunomodulatory agent is administered prior to irradiating the lesion or tumor.

73. The method of claim 72, wherein the immunomodulatory agent is administered at or about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks or more than 1 month prior to irradiating the tumor.

74. 74. The method of any one of claims 67-73, comprising continuous administration of the immunomodulatory agent following irradiation three times per week, twice per week, once every week, once every two weeks, once every three weeks, or once a month.

75. (a) administering an immunomodulatory agent to a subject; (b) administering to a subject a therapeutically effective amount of a conjugate comprising a phthalocyanine dye linked to a targeting molecule capable of binding to a molecule on the surface of a cell present in the tumor microenvironment; and (c) more than 12 hours after administration of the immunomodulatory agent, irradiating the tumor with a wavelength that renders the conjugate cytotoxic, thereby treating the tumor.

23. A method for treating a tumor in a subject, comprising:

76. 76. The method of claim 75, wherein the immunomodulatory agent is administered more than 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks or 1 month prior to irradiating the tumor, or about 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks or 1 month prior to irradiating the tumor.

77. The method of claim 75 or claim 76, wherein the conjugate binds to a protein on the surface of a cell present in the tumor microenvironment.

78. 78. The method of any one of claims 75-77, wherein step (c) of irradiating the tumor is performed either (i) after administration of the immunomodulatory agent and after administration of the conjugate, or (ii) only after administration of the conjugate.

79. The method of any one of claims 68-78, wherein the conjugate is administered prior to, simultaneously with, or following administration of the immunomodulatory agent.

80. The method of any one of claims 68-79, wherein the conjugate is administered after administration of the immunomodulatory agent but before irradiating the tumor.

81. The method of any one of claims 68-80, wherein the conjugate is administered 12 hours to about 48 hours, or about 12 hours to about 48 hours, prior to irradiating the tumor, and the immunomodulatory agent is administered 12 hours to about one month, or about 12 hours to about one month, prior to irradiating the tumor.

82. The method of any one of claims 75-81, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

83. The method of claim 82, wherein the immune checkpoint inhibitor specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

84. The method of claim 82 or claim 83, wherein the immune checkpoint inhibitor is an antibody or antigen-binding fragment, a small molecule, or a polypeptide.

85. The method of any one of claims 82-84, wherein the immune checkpoint inhibitor is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment of any of them.

86. The method of any one of claims 75-81, wherein the immunomodulatory agent is a demethylating agent that upregulates expression of a tumor-associated antigen (TAA) or is a cytokine.

87. 87. The method of any one of claims 75-86, comprising continuous administration of the immunomodulatory agent following irradiation three times per week, twice per week, once every week, once every two weeks, once every three weeks, or once a month.

88. (a) administering to a subject an immunomodulatory agent that enhances expression of a molecule on the surface of cells present in the tumor microenvironment; (b) administering to the subject a therapeutically effective amount of a conjugate comprising a phthalocyanine dye linked to a targeting molecule capable of binding to the cell surface molecule; and (c) more than 5 minutes after administering the conjugate, irradiating the tumor with a wavelength that renders the conjugate cytotoxic, thereby treating the tumor.

23. A method for treating a tumor in a subject, comprising:

89. The method of claim 88, wherein the immunomodulatory agent is a cytokine or an agent that induces increased expression of a cytokine in the tumor microenvironment.

90. 90. The method of claim 88 or claim 89, wherein the cytokine is interferon gamma.

91. The method of any one of claims 88-90, wherein the molecule on the surface of the cell is selected from CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

92. The method of any one of claims 88 to 91, wherein the cell surface molecule is PD-L1.

93. The method of any one of claims 88-92, wherein the targeting molecule is an immune checkpoint inhibitor.

94. The method of any one of claims 88 to 93, wherein the targeting molecule is an antibody or antibody fragment, a small molecule, or a polypeptide.

95. 95. The method of any one of claims 88-94, wherein the targeting molecule is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment of any of them.

96. (a) administering to a subject a conjugate comprising a phthalocyanine dye linked to a targeting molecule capable of binding to a cell surface molecule on cells in the tumor microenvironment; (b) irradiating the tumor with a wavelength that renders the conjugate cytotoxic more than 5 minutes after administration of the conjugate, wherein treatment of the tumor with the conjugate and subsequent light irradiation increases the presence of immunosuppressive cells in the tumor or increases the expression of immunosuppressive markers in the tumor; and (c) administering to the subject a therapeutically effective amount of an immunomodulatory agent capable of reducing the amount or activity of immunosuppressive cells in the tumor or blocking the activity of an immunosuppressive marker.

23. A method for treating a tumor in a subject, comprising:

97. 97. The method of claim 96, wherein the phthalocyanine dye is a first dye and the immunomodulatory agent comprises a conjugate comprising a second phthalocyanine dye conjugated to an immunomodulatory agent capable of binding to an immunosuppressive cell.

98. 98. The method of claim 97, wherein the first and second phthalocyanine dyes are the same or different.

99. The method of any one of claims 96-98, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

100. The method of any one of claims 96-99, wherein the immunomodulatory agent specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

101. The method of any one of claims 96 to 100, wherein the immunomodulatory agent is an antibody or antibody fragment, a small molecule, or a polypeptide.

102. The method of any one of claims 96-101, wherein the immunomodulatory agent is not an anti-CTLA4 antibody.

103. 103. The method of any one of claims 96-102, wherein the immunomodulatory agent is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment of any of them.

104. (a) administering to a subject a conjugate comprising a phthalocyanine dye linked to a targeting macromolecule capable of binding to a molecule on the surface of a cell present in the tumor microenvironment; (b) irradiating the tumor with a wavelength that renders the conjugate cytotoxic more than 5 minutes after administration of the conjugate, wherein treatment of the tumor with the conjugate and subsequent light irradiation primes immune cell activation; and (c) administering to the subject a therapeutically effective amount of an immunomodulatory agent capable of increasing the activity of immune cells.

23. A method for treating a tumor in a subject, comprising:

105. The method of claim 104, wherein the immune cell is an antigen-presenting cell.

106. The method of claim 105, wherein the immune cell is a dendritic cell.

107. Immunomodulatory agents include GM-CSF, CpG-ODN (CpG oligodeoxynucleotides), lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), alum, recombinant Leishmania polyprotein, imiquimod, MF59, poly I:C, poly A:U, type 1 IFN, Pam3Cys, Pam2Cys, complete Freund's adjuvant (CFA), alpha-galactosylceramide, RC-529, MDF2β, loxoribine, anti-CD40 agonist, SIRPa antagonist, AS04, AS03, flagyl. The method of any one of claims 104 to 106, wherein the therapeutic agent is selected from among rituximab, resiquimod, DAP (diaminopimelic acid), MDP (muramyl dipeptide), CAF01 (cationic adjuvant formulation-01), anthracyclines (doxorubicin, mitoxantrone), BK channel agonists, bortezomib, bortezomib + mitomycin C + hTert-Ad, cardiac glycosides + non-immunogenic cell death inducers, cyclophosphamide, GADD34 / PP1 inhibitors + mitomycin, LV-tSMAC, and oxaliplatin.

108. The method of any one of claims 104-107, wherein the immunomodulatory agent is a Toll-like receptor (TLR) agonist, an adjuvant, or a cytokine.

109. 109. The method of claim 108, wherein the immunomodulatory agent is a TLR agonist, wherein the TLR agonist is a TLR4 agonist, a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist.

110. The method of claim 108 or claim 109, wherein the TLR agonist is selected from among triacylated lipoproteins, diacylated lipopeptides, lipoteichoic acid, peptidoglycan, zymosan, Pam3CSK4, dsRNA, poly I:C, poly G10, poly G3, CpG, 3M003, flagellin, lipopolysaccharide (LPS), Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), MEDI9197, SD-101, and imidazoquinoline TLR agonists.

111. The method of any one of claims 104-107, wherein the immunomodulatory agent is a cytokine, said cytokine being IL-4, TNF-α, GM-CSF or IL-2.

112. The method of any one of claims 96-111, wherein the immunomodulatory agent is administered prior to, simultaneously with, or after irradiation.

113. 113. The method of claim 112, wherein the immunomodulatory agent is administered within 5 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours or 24 hours after irradiation.

114. The method of any one of claims 75-113, wherein the targeting molecule directly or indirectly binds to a molecule or protein.

115. 115. The method of claim 114, wherein the targeting molecule is a second binding molecule that binds to a first binding molecule, the first binding molecule being capable of binding to the molecule or protein.

116. The method of claim 114 or claim 115, wherein the targeting molecule is a secondary antibody.

117. 117. The method of any one of claims 75 to 116, wherein the phthalocyanine dye has a maximum absorption wavelength of from 600 nm to about 850 nm or from about 600 nm to about 850 nm.

118. The method of any one of claims 75-117, wherein the phthalocyanine dye is covalently or non-covalently linked to the targeting molecule.

119. The method of any one of claims 75-118, wherein the phthalocyanine dye comprises a linker comprising a reactive group for attachment of the dye to a targeting molecule.

120. The phthalocyanine dye has the following formula: wherein L is a linker; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; and X 2 and X 3 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 is alkylene, 120. The method of claim 119.

121. The phthalocyanine dye has the following formula: wherein X 1 and X 4 each independently represents a heteroatom which may be present; C 1 -C 10 is alkylene; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; 121. The method of claim 119 or claim 120.

122. 122. The method of any one of claims 75 to 121, wherein the phthalocyanine dye comprises IRDye 700DX (IR700).

123. The conjugate is 50 mg / m 2 ~Approx. 5000mg / m 2 or about 50 mg / m 2 ~Approx. 5000mg / m 2 , about 250mg / m 2 ~About 2500mg / m 2 , about 750mg / m 2 ~Approx. 1250mg / m 2 , or about 100 mg / m 2 ~Approx. 1000mg / m 2 The method of any one of claims 75 to 122, wherein the

124. The method of any one of claims 8-33 and 37-123, wherein the tumor is cancer.

125. 125. The method of claim 124, wherein the cancer is located in the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung.

126. The method of any one of claims 8-33 and 37-125, wherein the tumor is a sarcoma or a carcinoma.

127. 127. The method of claim 126, wherein the tumor is a carcinoma that is a squamous cell carcinoma, a basal cell carcinoma, or an adenocarcinoma.

128. 128. The method of claim 127, wherein the tumor is a cancer that is a bladder, pancreas, colon, ovary, lung, breast, stomach, prostate, cervical, esophageal or head and neck carcinoma.

129. Tumors receive at least 1 J cm at wavelengths between 600 nm and 850 nm. -2 or at least 1 J / cm of fiber length.

130. The method of any one of claims 75-129, wherein the tumor is irradiated at a wavelength of 690 nm ± 50 nm or at or about 690 ± 20 nm.

131. The method of any one of claims 1-130, wherein the method reduces the size or volume of the tumor by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more within one month of irradiation compared to the size or volume of the tumor prior to the administration and irradiation.

132. The method of any one of claims 1-131, wherein said method achieves an improvement in a disorder- or cancer-related parameter in a population of treated subjects compared to a similarly situated population of subjects treated with an unconjugated antibody or antigen-binding antibody fragment, said parameter being selected from one or more of: (a) objective response rate (ORR); (b) progression-free survival (PFS); (c) overall survival (OS); (d) reduced toxicity; (e) tumor response; or (f) quality of life.

133. 133. The method of claim 132, wherein the parameter is improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more.

134. The method of any one of claims 1-133, achieving an overall response rate (ORR) of at least 15%, at least 25%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more in a population of treated subjects.

135. 134. The method of any one of claims 1-133, wherein the phthalocyanine dye is a first dye and the conjugate further comprises a second fluorescent dye linked to a macromolecule different from the first dye.

136. The method of claim 135, wherein irradiating the lesion or tumor causes a fluorescent signal to be emitted from the second fluorescent dye to effect detection of the presence of the conjugate in the lesion or tumor in the subject.

137. The method of claim 135 or claim 136, further comprising imaging the lesion or tumor in the subject by irradiating or illuminating the tumor with a wavelength capable of being absorbed by the second dye.

138. The method of any one of claims 135-137, wherein the second fluorescent dye exhibits one or more spectral properties selected from among higher fluorescence quantum yield in water, extinction coefficient, Stokes shift, absorption and emission at longer wavelengths, and photostability compared to the corresponding spectral properties of the first dye.

139. The method of any one of claims 135-138, wherein the first dye is IR700.

140. 140. The method of any one of claims 135-139, wherein the second dye is not IR700.

141. The second dye may be hydroxycoumarin, Cascade Blue, Dylight 405, Pacific Orange, Alexa Fluor 430, fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2.7-dichlorofluorescein, ATTO 488, Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo 642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700, Cy 5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705 and Qdot® 800.

142. 142. The method of any one of claims 135-141, wherein the first dye is IR700 and the conjugate comprises 1 to 10 or 1 to 5 second dye molecules per macromolecule.

143. 104. The method of any one of claims 96-103, wherein the second dye exhibits a Stokes shift that is greater than 15 nm, greater than 20 nm, greater than 30 nm, greater than 40 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, greater than 90 nm or greater than 100 nm.

144. 144. The method of any one of claims 135-143, wherein the second dye has a quantum yield in water of greater than 10%, greater than 15%, greater than 20%, or greater than 25%, greater than 30%, greater than 40%, greater than 50% or higher.

145. 145. The method of any one of claims 135-144, wherein the second dye has absorption and emission wavelengths in the spectrum of between or about 650 nm to 950 nm, between or about 700 nm to 1000 nm, or between or about 1000 nm to 1700 nm.

146. 146. The method of any one of claims 135-145, wherein the first dye and the second dye do not exhibit overlapping emission and absorption spectra.

147. 147. The method of any one of claims 135-146, wherein the second dye is selected from among ICG, IRDye 680, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800 and IRDye 800.

148. The method of any one of claims 135-147, wherein the second dye is Alexa Fluor 488, IRDye 680, IRDye 800, or Dylight 755.

149. The method of any one of claims 1-148, wherein the irradiation or illumination is performed with a device selected from among a handheld ultraviolet lamp, a mercury lamp, a xenon lamp, a laser, a laser diode, or an LED imaging device.

150. 150. The method of claim 149, wherein the imaging device comprises a near-infrared (NIR) diode.

151. A composition comprising a conjugate comprising a phthalocyanine dye linked to an antibody or antigen-binding antibody fragment that binds to a molecule on the surface of a cell present in the microenvironment of a lesion, the composition being formulated for administration of a single dose of the conjugate in an amount that is between or between about 100 mg and 2000 mg.

152. The composition of claim 151, formulated for single dosage administration in an amount of between or about 100 mg and 500 mg, between or about 200 mg and 400 mg.

153. The composition of claim 151 or claim 152, formulated for single dose administration in an amount between, at or about, 500 mg to 1500 mg, 800 mg to 1200 mg, or 1000 mg to 1500 mg.

154. the volume of the composition is between or about 10 mL and 1000 mL, or between or about 50 mL and 500 mL; or the volume of the composition is at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 400 mL, 500 mL, or 1000 mL; The composition of any one of claims 151-153.

155. a plurality of sealed containers each individually containing a portion of a single dose of a composition comprising a conjugate comprising a phthalocyanine dye linked to an antibody or antigen-binding antibody fragment that binds to a molecule on the surface of a cell present in the microenvironment of a lesion, wherein the combined amount of the conjugate in the plurality of sealed containers is between or about 100 mg-1500 mg; packaging material; a label or package insert containing instructions for combining the contents of the multiple vials to prepare a single dose formulation of the composition; Manufactured goods, including.

156. 156. The article of manufacture of claim 155, wherein the combined amount of the conjugates in the plurality of sealed containers is between or about 100 mg and 1200 mg.

157. 157. The article of manufacture of claim 155 or claim 156, wherein the combined amount of the conjugates in the plurality of sealed containers is between or about 100 mg and 500 mg, between or about 200 mg and 400 mg, between or about 500 mg and 1500 mg, between or about 800 mg and 1200 mg, or between or about 1000 mg and 1500 mg.

158. The composition of any one of claims 151-154, or the article of manufacture of any one of claims 155-157, wherein the lesion is a tumor.

159. A conjugate comprising a phthalocyanine dye linked to an antibody or antigen-binding fragment that is an immunomodulator.

160. The conjugate of claim 159, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

161. The conjugate of claim 159 or claim 160, wherein the immunomodulatory agent is an antibody or antigen-binding fragment that binds to the surface of a tumor, tumor cell or cancer cell.

162. The conjugate of any one of claims 159-161, wherein the immunomodulatory agent specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

163. 163. The conjugate of any one of claims 159-162, wherein the immunomodulatory agent is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment of any of them.

164. The conjugate of any of claims 159-163, wherein the immunomodulatory agent is an antibody or antibody fragment that binds to PD-L1.

165. The conjugate of claim 164, wherein the immunomodulatory agent is an antibody selected from BMS-935559, MEDI4736, MPDL3280A and MSB0010718C, or an antigen-binding fragment thereof.

166. A conjugate comprising a targeting molecule linked to at least a first and a second fluorescent dye, the first fluorescent dye being a phthalocyanine dye capable of exhibiting phototoxicity.

167. The following formula: [D 1 -(L 1 ) n ] p -A-[(L 2 ) n -D 2 ] o wherein A is a targeting molecule capable of binding to a molecule on the surface of a cell; L 1 and L 2 are each independently selected linkers, which can be the same or different; n and m are independently 1 or 2; D 1 is a first dye that is a phthalocyanine dye capable of exhibiting phototoxicity; D 2 is a second dye that is a fluorescent dye, where D 2 D 1 Unlike; p is 1 to 10; and o is 1 to 10; The conjugate of claim 166.

168. The conjugate of claim 166 or claim 167, wherein the targeting molecule is an antibody or an antigen-binding antibody fragment.

169. The conjugate of any one of claims 166-168, wherein the cell surface molecule comprises an antigen, a polypeptide, a lipid, or a carbohydrate, or a combination of these molecules.

170. Cell surface molecules include ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptors BB1, BB2, BB4, calcitonin receptor, and cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, Crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endosialin, EP2, EP4 , EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, family A G protein-coupled receptor (rhodopsin-like), family B G protein-coupled receptor (secretin receptor-like), family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV 16 / 18 and E6 / E7 antigens, hTERT, IL11-R, IL-13R, ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE 1, MAGE 2, MAGE 3, MAGE4, MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease-cleaved collagen IV, proteinase 3, prohibitin, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 170. The conjugate of any one of claims 166-169, wherein the conjugate is selected from among patched, RET receptor, plexin, smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptor (VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, and VEGF-3 or FLT-4), voltage-gated ion channels, VPAC1, VPAC2, Wilms' tumor 1, Y1, Y2, Y4, and Y5.

171. Cell surface molecules include HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72, caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), CD 30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate binding protein, gangliosides (such as GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), integrin alphaVbeta3, integrin alpha5beta1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10beta, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinases, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, and SK-1 antigen.

172. Cell surface molecules include CD25, PD-1 (CD279), PD-L1 (CD274, B7-H1), PD-L2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3 (H AVCR2), 4-1BB (CD137, TNFRSF9), CXCR2, CXCR4 (CD184), CD27, CEACAM1, galectin 9, BTLA, CD160, VISTA (PD1 homolog body), B7-H4 (VCTN1), CD80 (B7-1), CD86 (B7-2), CD28, HHLA2 (B7-H7), CD28H, CD155, CD226, TIGIT, CD96, Galek Chin3, CD40, CD40L, CD70, LIGHT (TNFSF14), HVEM (TNFRSF14), B7-H3 (CD276), Ox40L (TNFSF4), CD137L (TNFSF 9, GITRL), B7RP1, ICOS (CD278), ICOSL, KIR, GAL9, NKG2A (CD94), GARP, TL1A, TNFRSF25, TMIGD2, BTNL2, butyrophilin family, CD48, CD244, Siglec family, CD30, CSF1R, MICA (MHC class I polypeptide-related sequence A), MICB (MHC class I polypeptide-related sequence B), NKG2D, KIR family (killer cell immunoglobulin-like receptors), LILR family (leukocyte immunoglobulin-like receptors, CD85, ILT, LIR), SIRPA (signal regulatory protein alpha), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR, and VEGF.

173. Macromolecules include cetuximab, panitumumab, zalutumumab, nimotuzumab, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), basiliximab, nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, M 173. The conjugate of any one of claims 166-172, which is an antibody or antigen-binding antibody fragment selected from among K-4166, dacetuzumab, lucatumumab, SEA-CD40, CP-870, CP-893, MED16469, MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C, rHIgM12B7, urocupulumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, emactuzumab, CC-90002 and MNRP1685A, or an antigen-binding antibody fragment thereof.

174. The conjugate of any one of claims 166-173, wherein the targeting molecule is not or does not include a nanocarrier.

175. The conjugate of any one of claims 166-174, wherein the targeting molecule is not or does not comprise a virus-like particle, a nanoparticle, a liposome, a quantum dot, or a combination thereof.

176. 176. The conjugate of any one of claims 166-175, wherein the first dye is a phthalocyanine dye having a maximum absorption wavelength of from 600 nm to about 850 nm, or from about 600 nm to about 850 nm.

177. The first dye, which is a phthalocyanine dye, has the following formula: wherein L is a linker; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; and X 2 and X 3 each independently represents a heteroatom which may be present; C 1 -C 10 is alkylene, The conjugate of any one of claims 166 to 176.

178. The first dye, which is a phthalocyanine dye, has the following formula: wherein X 1 and X 4 each independently represents an integer of 1 to 5, optionally interrupted by a heteroatom; 1 -C 10 is alkylene; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; The conjugate of any one of claims 166-177.

179. 179. The conjugate of any one of claims 166-178, wherein the first dye which is a phthalocyanine dye comprises IRDye 700DX (IR700).

180. The conjugate of any one of claims 166-179, wherein the second fluorescent dye exhibits one or more spectral properties selected from among higher fluorescence quantum yield in water, extinction coefficient, Stokes shift, absorption and emission at longer wavelengths, and photostability compared to the corresponding spectral properties of the first dye.

181. The conjugate of any one of claims 166-180, wherein the second dye is not IR700.

182. The second dye is hydroxycoumarin, Cascade Blue, Dylight 405, Pacific Orange, Alexa Fluor 430, fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2.7-dichlorofluorescein, ATTO 488, Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo 642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700, Cy 5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705 and Qdot® 800.

183. The conjugate of any one of claims 166-182, wherein the first dye is IR700 and the conjugate comprises 1 to 10 or 1 to 5 second dye molecules per macromolecule.

184. 184. The conjugate of any one of claims 166-183, wherein the second dye exhibits a Stokes shift that is greater than 15 nm, greater than 20 nm, greater than 30 nm, greater than 40 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, greater than 90 nm, or greater than 100 nm.

185. The conjugate of any one of claims 166-184, wherein the second dye has a quantum yield in water of greater than 10%, greater than 15%, greater than 20%, or greater than 25%, greater than 30%, greater than 40%, greater than 50% or higher.

186. 186. The conjugate of any one of claims 166-185, wherein the second dye has absorption and emission wavelengths in the spectrum between or between about 650 nm and 950 nm, between or between about 700 nm and 1000 nm, between or between about 1000 nm and 1700 nm.

187. The conjugate of any one of claims 166-186, wherein the first dye and the second dye do not exhibit overlapping emission and absorption spectra.

188. The conjugate of any one of claims 166-187, wherein the second dye is selected from among ICG, IRDye 680, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800 and IRDye 800.

189. The conjugate of any one of claims 166-188, wherein the second dye is Alexa Fluor 488, IRDye 680, IRDye 800, or Dylight 755.

190. 190. A composition comprising the conjugate of any one of claims 159-189.

191. 191. The composition of claim 190, further comprising a pharma- ceutically acceptable excipient.

192. (a) administering to a subject a therapeutically effective amount of a conjugate of any one of claims 159-165 or a composition of claim 190 or claim 191, wherein the conjugate binds to cells present in the microenvironment of a lesion associated with a disease or pathology; and (b) administering the conjugate and then irradiating the lesion with one or more wavelengths to induce phototoxic activity of the conjugate, thereby treating the disease or condition. A method of treating a disease or condition in a subject, comprising:

193. (a) administering to a subject a therapeutically effective amount of a conjugate of any one of claims 166-189 or a composition of claim 190 or claim 191, wherein the conjugate binds to cells present in the microenvironment of a lesion associated with a disease or pathology; and (b) administering the conjugate and then irradiating the lesion with one or more wavelengths to induce phototoxic activity of a first dye of the conjugate and a fluorescent signal of a second dye of the conjugate. A method of treating a disease or condition in a subject, comprising:

194. at least 1 J cm at a wavelength that is between 400 and about 900 nm or between about 400 and about 900 nm -2 or irradiating with a dosage of 1 J / cm of fiber length.

195. 195. The method of claim 193 or claim 194, comprising irradiating the lesion with a single wavelength.

196. The method of claim 193 or claim 195, comprising irradiating the lesion with two different wavelengths simultaneously or sequentially, one wavelength inducing phototoxic activity and another wavelength inducing a fluorescent signal.

197. The method of any one of claims 192-196, wherein the disease or condition is a tumor.

198. At wavelengths between 660 nm and 740 nm and at least 1 J cm -2 200. The method of claim 197, comprising irradiating the tumor with a dose of

199. The method of claim 197 or claim 198, wherein the tumor is cancer.

200. 200. The method of claim 199, wherein the cancer is located in the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung.

201. The method of any one of claims 197-200, wherein the tumor is a sarcoma or a carcinoma.

202. 202. The method of claim 201, wherein the tumor is a carcinoma that is a squamous cell carcinoma, a basal cell carcinoma, or an adenocarcinoma.

203. 203. The method of claim 202, wherein the tumor is a cancer that is a bladder, pancreas, colon, ovary, lung, breast, stomach, prostate, cervical, esophageal or head and neck carcinoma.

204. The method of any one of claims 1-150 and 192-203, wherein a targeting molecule is administered to the subject prior to administration of the conjugate.

205. The method of any one of claims 1-150 and 192-204, wherein the targeting molecule is administered up to 96 hours prior to administration of the conjugate.

206. The targeting molecule is 10 mg / m 2 ~Approx. 500mg / m 2 or about 10 mg / m 2 ~Approx. 500mg / m 2 The method of claim 204 or claim 205, wherein the dose is within the range of

207. The method of any one of claims 1-150 and 192-206, wherein the targeting molecule is an antibody or an antigen-binding fragment.

208. The method of claim 207, wherein the antibody is cetuximab.

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