Compositions, combinations and related methods for photoimmunotherapy

Abstract

The present invention provides conjugates, compositions and methods for use in photoimmunotherapy, such as photoimmunotherapy induced by activation of a phthalocyanine dye conjugated to a targeting molecule that binds to a protein on a cell, such as an IR700-antibody conjugate. In some embodiments, the phthalocyanine dye conjugate may be activated by irradiation with near-infrared light. Features of the conjugates, compositions, and methods, including doses of the conjugates, provide various benefits, such as lower toxicity and / or better efficacy. In some embodiments, a dual-labeled phthalocyanine dye conjugate is also provided in which the targeting molecule is conjugated to other fluorescent dyes that can be used in photoimmunotherapy and also exhibit better performance, for example, in imaging or detection. Also provided are therapeutic methods of using the conjugates and compositions to treat diseases and conditions, including tumors or cancers.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 206,776, filed on August 18, 2015, entitled “Combination Therapy with Photoimmunotherapy and Related Methods,” and U.S. Provisional Patent Application No. 62 / 249,085, filed on October 30, 2015, entitled “Compositions and Methods for Photoimmunotherapy,” the contents of each of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing Incorporated by Reference

[0004] This application submits a sequence listing in electronic form. The sequence listing is provided as file name 751702000440seqlist.txt, was created on August 18, 2016, and is 9,934 bytes in size. The electronic form of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to a conjugate, composition and method for photoimmunotherapy, such as photoimmunotherapy induced by activation of a phthalocyanine dye, such as an IR700-antibody conjugate, conjugated to a targeting molecule that binds to a protein on a cell. The present invention relates to a combination therapy for use in conjunction with photoimmunotherapy, such as photoimmunotherapy induced by activation of a phthalocyanine dye, such as an IR700-antibody conjugate, conjugated to a targeting molecule that targets tumor cells. In some embodiments, the phthalocyanine dye conjugate can be activated by irradiation with near-infrared light. Features of the conjugates, compositions, combinations and methods, including the dosage of the conjugate, provide various benefits, such as lower toxicity and / or better efficacy. In some embodiments, the present invention also relates to a dual-labeled phthalocyanine dye conjugate, wherein the targeting molecule is conjugated to other fluorescent dyes that can be used in photoimmunotherapy and also show better performance, such as in imaging or detection. The present invention also provides therapeutic methods using the conjugates, compositions and combinations to treat diseases and conditions including tumors or cancer. Background Art

[0006] A variety of therapies are available for treating diseases such as cancer. For example, photoimmunotherapy (PIT) is a method that uses photosensitizers coupled to antibodies or other targeting molecules to target cell surface proteins, thereby achieving targeted killing of specific cells. In some cases, PIT can selectively target diseased cells, such as tumor cells, thereby selectively killing these cells while sparing healthy cells. Strategies are needed to improve photoimmunotherapy approaches, such as enhancing the efficacy of treatment. The present invention provides compositions and methods that meet these needs. Summary of the Invention

[0007] In some embodiments, a method of using photoimmunotherapy (PIT) to treat a disease or condition in a subject is provided. In some embodiments, the method comprises administering to a subject having a disease or condition a conjugate comprising 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 in a microenvironment of damage associated with the disease or condition. In some embodiments, the conjugate is administered to achieve a systemic exposure as described below that does not exceed 75% of the total therapeutically effective systemic exposure of an antibody or antigen-binding fragment that is not so conjugated for the treatment of the same disease or condition as described on a label approved for commercialization by a regulatory agency (e.g., FDA, EMA, PDMA). In some embodiments, after administration of the conjugate, the subject is exposed to light at a wavelength of 500 to 900 nanometers (nm) at a power of at least 1 joule per square centimeter (J cm -2 ) or 1 joule per centimeter of fiber length (J / cm fiber length) to irradiate the lesion, thereby treating a tumor in the subject. In some embodiments, the wavelength used for irradiation is 600nm to 850nm, such as 660nm to 740nm.

[0008] In some embodiments, the conjugate is administered in a dosing regimen in which: the conjugate is administered only once as a single injection or infusion; or the dosing regimen does not include subsequent administration of the conjugate; or the dosing regimen does not include subsequent administration of the targeting molecule, such as a macromolecule, that is not so conjugated.

[0009] In some embodiments, the conjugate is administered systemically. In some embodiments, the conjugate is administered intravenously.

[0010] In some embodiments, the conjugate is administered to achieve a systemic exposure (AUC) that is no more than 60%, 50%, 40%, or 30% of the therapeutically effective systemic exposure of the non-so conjugated antibody or antigen-binding fragment used to treat the same disease or condition.

[0011] In some embodiments, the disease or condition is a tumor, whereby 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.

[0012] In some embodiments, a method of treating a disease or condition in a subject using photoimmunotherapy (PIT) is provided, wherein the systemic exposure (AUC[0-inf] or AUC 0-∞ ) is about 250 μg / mL*h to 100,000 μg / mL*h, about 500 μg / mL*h to 50,000 μg / mL*h, about 500 μg / mL*h to 18,000 μg / mL*h, or about 500 μg / mL*h to 10,000 μg / mL*h. In some embodiments, the systemic exposure is measured by the mean area under the plasma conjugate concentration-time curve (AUC[0-inf] or AUC 0-∞ ) is not greater than 100,000 micrograms per milliliter*hour (μg / mL*h), not greater than 75,000 μg / mL*h, not greater than 50,000 μg / mL*h, not greater than 40,000 μg / mL*h, not greater than 30,000 μg / mL*h, not greater than 20,000 μg / mL*h, not greater than 10,000 μg / mL*h, not greater than 5,000 μg / mL*h, or not greater than 2,500 μg / mL*h.

[0013] In some embodiments, the systemic exposure is measured by the mean area under the plasma conjugate concentration-time curve (AUC[0-24] or AUC 0-24 ) is about 100 μg / mL*h to 25,000 μg / mL*h, about 200 μg / mL*h to 10,000 μg / mL*h, about 500 μg / mL*h to 5,000 μg / mL*h; or, the systemic exposure as measured by the mean area under the plasma conjugate concentration-time curve (AUC[0-24] or AUC 0-24) is no more than 25,000 μg / mL*h, no more than 15,000 μg / mL*h, no more than 10,000 μg / mL*h, no more than 5,000 μg / mL*h, no more than 2,500 μg / mL*h, no more than 1,000 μg / mL*h, or no more than 500 μg / mL*h.

[0014] In some embodiments, the conjugate is administered at a concentration of at least about 10 mg / m2 (mg / m 2 ) (body surface area of ​​the subject), at least about 50 mg / m 2 or at least about 75 mg / m 2 And not more than 5000mg / m 2 , not more than 2000mg / m 2 , not more than 1000mg / m 2 In some embodiments, the conjugate is administered at a dose of no more than 500 mg / m 2 , not more than 250mg / m 2 or no more than 200 mg / m 2 In some embodiments, the conjugate is administered at a dose of about 100 mg / m 2 ~1500 mg / m 2 or 150 mg / m 2 ~750 mg / m 2 In some embodiments, the conjugate is administered at a dose of about 160 mg / m 2 , 320mg / m 2 , 640mg / m 2 or 1280 mg / m 2 Dosage administration.

[0015] In some embodiments, the targeting molecule is an antibody or an antigen-binding antibody fragment. In some embodiments, the antibody is an antigen-binding antibody fragment, which is a Fab, a single V H domain, single-chain variable region fragment (scFv), multivalent scFv, bispecific scFv or scFv-CH3 dimer.

[0016] In some embodiments, the lesion is irradiated at a wavelength of 690 ± 50 nm or about 690 ± 20 nm. In some embodiments, the lesion is irradiated at a wavelength of about 2 J cm -2 ~about 400J cm -2 In some embodiments, the lesion is irradiated with a dose of at least about 2 J / cm fiber length to about 500 J / cm fiber length. -2 、5J cm -2 、10J cm-2 、25Jcm -2 , 50J cm -2 、75J cm -2 、100J cm -2 、150J cm -2 , 200 J cm -2 、300J cm -2 , 400J cm -2 or 500 J cm -2 or irradiating the damage with a dose of 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.

[0017] In some embodiments, the irradiation is performed or implemented about 30 minutes to 96 hours after the administration of the conjugate. In some embodiments, the conjugate is administered according to a dosing regimen as described below, in which the conjugate is administered only once as a single injection or infusion. In some embodiments, the conjugate is administered according to a dosing regimen as described below, in which the dosing regimen does not include subsequent administration of the conjugate. In some embodiments, the conjugate is administered according to a dosing regimen as described below, in which the dosing regimen does not include subsequent administration of the targeting molecule that is not so conjugated. In some embodiments, the dosing regimen is repeated.

[0018] In some embodiments, the conjugate is administered systemically. In some embodiments, the conjugate is administered intravenously.

[0019] In some embodiments, the phthalocyanine dye has a maximum absorption wavelength of about 600 nm to about 850 nm.

[0020] In some embodiments, the phthalocyanine dye is directly or indirectly linked to the targeting molecule. In some embodiments, the phthalocyanine dye comprises a compound of the following formula:

[0021] in:

[0022] L is the connector;

[0023] Q is a reactive group used to link the dye to the targeting molecule;

[0024] R 2 、R 3、R 7 and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl;

[0025] 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, wherein R 4 、R 5 、R 6 、R 9 、R 10 and R 11 At least one of the compounds comprises a water-soluble group;

[0026] 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

[0027] X 2 and X 3 Each independently is C1~C 10 Alkylene groups are optionally interrupted by heteroatoms.

[0028] In some embodiments, the phthalocyanine dye comprises a compound of the formula:

[0029]

[0030] in:

[0031] X 1 and X 4 Each independently is C1~C 10 alkylene, optionally interrupted by heteroatoms;

[0032] R 2 、R 3 、R 7 and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl;

[0033] 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, wherein R 4 、R 5 、R 6 、R 9 、R 10 and R 11 At least one of comprises a water-soluble group; and

[0034] R 16 、R 17 、R 18 and R 19 Each is independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino and optionally substituted alkoxy.

[0035] In some embodiments, the phthalocyanine dye comprises IRDye 700DX (IR700).

[0036] In some embodiments, the cell surface protein is selected from ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, α-4-integrin, α-5-β-3-integrin, α-5-β-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, Cripto-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 receptors), GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, group A (rhodopsin-like) G-protein-coupled receptors, group B (secretin receptor-like) G-protein-coupled receptors, group C (metabotropic glutamate receptor-like) G-protein-coupled receptors, GD2, GP100, GP120, glypican-3, hemagglutinin, heparan sulfate, HER1, HER2, HER3, HER4, HMFG, HPV 16 / 18 and E6 / E7 antigens, hTERT, IL-2R, 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, proteolytic cleavage of collagen IV, proteolytic enzyme 3, inhibin, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (such as P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 patch, RET receptor, plexin , smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEMs, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (such as 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.

[0037] In some embodiments, the cell surface protein is selected from 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), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate-binding protein, gangliosides (such as 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.

[0038] In some embodiments, the cell surface protein is selected from 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, 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, sialic acid-binding immunoglobulin-like lectin (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, ILTs, LIRs), SIRPA (signal regulatory protein alpha), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR and VEGF.

[0039] In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, tositumomab, Rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab 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, MED I6383, MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, Emactuzumab, CC-90002 and MNRP1685A, or antigen-binding fragments thereof.

[0040] In some embodiments, the conjugate is selected from the group consisting of 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, ipilimumab-IR700, tesitumumab-IR700, IMP321-IR700, BMS-986016-IR700, LAG525-IR700, urelumab-IR700, PF-0 5082566-IR700, TRX518-IR700, MK-4166-IR700, daclizumab-IR700, ruxolitinib-IR700, SEA-CD40-IR700, CP-870-IR700, CP-893-IR700, MED16469-IR700, MEDI6383-IR700, MEDI4736-IR700, MOXR0916-IR700, AMP-224-IR700, PDR001-I R700, MSB0010718C-IR700, rHIgM12B7-IR700, ululumab-IR700, BKT140-IR700, variruzumab-IR700, ARGX-110-IR700, MGA271-IR700, lirelumab-IR700, IPH2201-IR700, AGX-115-IR700, imatinib-IR700, CC-90002-IR700, and MNRP1685A-IR700.

[0041] In some embodiments, the targeting molecule is the antibody cetuximab or an antigen-binding fragment thereof, or the conjugate is cetuximab-IR700.

[0042] In some embodiments, the average area under the plasma conjugate concentration-time curve (AUC[0-inf] or AUC 0-∞) is about 500 μg / mL*h to 18,000 μg / mL*h, about 500 μg / mL*h to 10,000 μg / mL*h, about 500 μg / mL*h to 5,000 μg / mL*h, or about 500 μg / mL*h to 2,500 μg / mL*h. In some embodiments, the mean area under the plasma conjugate concentration-time curve (AUC[0-24] or AUC 0-24 ) is about 500 μg / mL*h to 8,000 μg / mL*h, about 500 μg / mL*h to 5,000 μg / mL*h, about 500 μg / mL*h to 2,000 μg / mL*h, or about 1,000 μg / mL*h to 4,000 μg / mL*h.

[0043] In some embodiments, the conjugate is administered at about 75 mg / m 2 (Body surface area of ​​the subject)~1500mg / m 2 , about 75mg / m 2 ~1000 mg / m 2 , about 75mg / m 2 ~500 mg / m 2 or about 75 mg / m 2 ~225 mg / m 2 In some embodiments, the conjugate is administered at a dose of at least about 160 mg / m 2 , 320mg / m 2 , 640mg / m 2 or 1280 mg / m 2 administered within the dosage range.

[0044] In some embodiments, there is provided a method of treating a disease lesion in a subject comprising: a) intravenously administering a cetuximab-IR700 conjugate to a subject having a lesion associated with a disease or condition, wherein the conjugate is administered at a dose of about 640 mg / m 2 and b) after administration of the conjugate, at a wavelength of 690 ± 20 nm, at a wavelength of at least about 50 J cm -2 The lesion is irradiated with a dose of 100 J / cm fiber length or 100 J / cm fiber length, thereby treating the disease or condition in the subject.

[0045] In some embodiments, the conjugate is administered in a dosing regimen wherein: the conjugate is administered only once as a single injection or infusion; or the dosing regimen does not include subsequent administration of the conjugate; or the dosing regimen does not include subsequent administration of the targeting molecule that is not so conjugated.

[0046] In some embodiments, the irradiation is performed 24 hours ± 3 hours, eg, 24 hours ± 2 hours, after administration of the conjugate.

[0047] In some embodiments, the lesion is a tumor and the disease or condition is a tumor or cancer.

[0048] In some embodiments, the lesion is a tumor and the tumor is superficial. In another embodiment, the thickness of the tumor is less than 10 millimeters (mm). In some embodiments, the irradiation is performed using a microlens tip fiber for surface irradiation. In some embodiments, the light irradiation dose is about 5 J / cm 2 ~about 200J / cm 2 .

[0049] In some embodiments, a method is provided for treating superficial tumors by photoimmunotherapy comprising using a microlens tip fiber for surface irradiation at about 5 J / cm 2 ~about 200J / cm 2 A light dose of about 50 J / cm2 is applied to a superficial tumor in a subject, wherein the tumor is linked to a phototoxic agent comprising a targeting molecule that binds to a cell surface molecule of the tumor. In some embodiments, the light irradiation dose is about 50 J / cm2. 2 .

[0050] In some embodiments, the lesion is a tumor, and the tumor is a mesenchymal tumor. In some embodiments, the tumor is greater than 10 mm in depth or is a subcutaneous tumor. In some embodiments, irradiation is performed using a cylindrical diffuser fiber having a diffusion length of 0.5 centimeters (cm) to 10 cm and a spacing of 1.8 ± 0.2 cm. In some embodiments, the light irradiation dose is about 20 J / cm fiber length or about 500 J / cm fiber length.

[0051] In some embodiments, a method for treating a mesenchymal tumor by photoimmunotherapy is provided, comprising irradiating a mesenchymal tumor in a subject using a cylindrical diffusing fiber comprising a diffusion length of 0.5 cm to 10 cm and a spacing of 1.8 ± 0.2 cm at a light dose of about 100 J / cm of fiber length or at a radiation energy fluence rate of about 400 mW / cm, wherein the tumor is linked to a phototoxic agent comprising a targeting molecule that binds to a cell surface molecule of the tumor.

[0052] In some embodiments, the light irradiation dose is about 50 J / cm fiber length or about 300 J / cm fiber length. In some embodiments, the light irradiation dose is about 100 J / cm fiber length.

[0053] In some embodiments, the tumor is greater than 10 mm in depth or is a subcutaneous tumor. In some embodiments, the cylindrical diffusing fiber is placed in a catheter at a distance of 1.8 ± 0.2 cm from the tumor. In some embodiments, the catheter is optically transparent.

[0054] In some embodiments, a phototoxic agent comprising a targeting molecule is administered to a subject more than 6 hours prior to tumor irradiation, wherein the phototoxic agent binds to the tumor. In some embodiments, the phototoxic agent is administered to a subject more than about 12 hours, 24 hours, 26 hours, 48 ​​hours, 72 hours, or 96 hours prior to tumor irradiation. In some embodiments, the phototoxic agent is a phthalocyanine dye-targeting molecule conjugate. In some embodiments, the phthalocyanine dye is IR700.

[0055] In some embodiments, in any of the methods of treatment provided by the present invention, the dosing regimen is repeated, thereby repeating steps (a) and (b). In some embodiments, if residual damage is still left after the previous treatment with the conjugate, the dosing regimen is repeated. In some embodiments, the method further comprises evaluating the presence of residual damage in the subject, and if residual damage is still left, the dosing regimen is repeated. In some embodiments, if residual damage is still left for more than or about 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year after the previous conjugate administration begins, the dosing regimen is repeated. In some embodiments, if residual damage is still left for about 4 weeks after the previous conjugate administration begins, the dosing regimen is repeated.

[0056] In some embodiments, the conjugate contains 1 to 100, 1 to 10, or 2 to 5 phthalocyanine dye molecules per targeting molecule.

[0057] 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.

[0058] In some embodiments, the other therapeutic agent is an anticancer agent or an immunomodulator. In some embodiments, the other therapeutic agent is an immunomodulator, which is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically binds to a molecule 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 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 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.

[0059] In some embodiments, the immunomodulator is administered prior to irradiating the lesion or tumor. In some embodiments, the immunomodulator is administered more than about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 1 month prior to irradiating the tumor.

[0060] In some embodiments, provided methods comprise continuous administration of the immunomodulator three times per week, twice per week, once per week, once every two weeks, once every three weeks, or once a month following irradiation.

[0061] In some embodiments, a method of treating a tumor in a subject is provided, comprising: a) administering an immunomodulatory agent to the subject; 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 at a wavelength that renders the conjugate cytotoxic more than 12 hours after administration of the immunomodulatory agent, thereby treating the tumor. In some embodiments, the immunomodulatory agent is administered more than about 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 1 month prior to irradiating the tumor.

[0062] In some embodiments, the conjugate binds to a protein on the surface of cells present in the tumor microenvironment.In some embodiments of the provided methods, irradiating the tumor in step c) is performed i) after administration of the immunomodulator and after administration of the conjugate, or ii) only after administration of the conjugate.

[0063] In some embodiments, the conjugate is administered before, simultaneously with, or after administration of the immunomodulator. In some embodiments, the conjugate is administered after administration of the immunomodulator and before irradiation of the tumor. In some embodiments, the conjugate is administered about 12 hours to 48 hours before irradiation of the tumor, and the immunomodulator is administered about 12 hours to about 1 month before irradiation of the tumor.

[0064] In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically binds to a molecule 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 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 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 one of the foregoing.

[0065] In some embodiments, the immunomodulator is a demethylating agent that upregulates the expression of tumor-associated antigens (TAAs), or is a cytokine.

[0066] In some embodiments, provided methods comprise continuous administration of the immunomodulator three times per week, twice per week, once per week, once every two weeks, once every three weeks, or once a month following irradiation.

[0067] In some embodiments, a method of treating a tumor in a subject is provided, 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 comprising a phthalocyanine dye linked to a targeting molecule capable of binding to the cell surface molecule; and c) more than 5 minutes after administration of the conjugate, irradiating the tumor at a wavelength that renders the conjugate cytotoxic, thereby treating the tumor.

[0068] In some embodiments, the immunomodulator is a cytokine, or an agent that induces enhanced expression of a cytokine in the tumor microenvironment. In some embodiments, the cytokine is interferon-gamma.

[0069] In some embodiments, the molecule on the cell surface 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 cell surface is PD-L1.

[0070] In some embodiments, the targeting molecule is an immune checkpoint inhibitor.

[0071] In some embodiments, the targeting molecule is an antibody or antibody fragment, a small molecule or a polypeptide. In some embodiments, the targeting molecule is selected from 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 one of the foregoing.

[0072] 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 more than 5 minutes after administration of the conjugate at a wavelength that renders the conjugate cytotoxic, wherein treatment of the tumor by 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 immunomodulator that reduces the amount or activity of immunosuppressive cells in the tumor, blocks the activity of an immunosuppressive marker, reduces the activity of tumor-promoting cells in the tumor, or blocks the activity of a tumor-promoting marker.

[0073] In some embodiments, the phthalocyanine dye is a first dye and the immunomodulator comprises a conjugate comprising a second phthalocyanine dye conjugated to an immunomodulator capable of binding to immunosuppressive cells or tumor-promoting cells. In some embodiments, the first and second phthalocyanine dyes are the same or different.

[0074] In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulator specifically binds to a molecule selected from the group consisting of 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.

[0075] In some embodiments, the immunomodulator is an antibody or antibody fragment, a small molecule, or a polypeptide.

[0076] In some embodiments, the immunomodulator is not an anti-CTLA4 antibody.

[0077] In some embodiments, the immunomodulator is selected from 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 one of the foregoing.

[0078] 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 molecule on the surface of cells present in the tumor microenvironment; b) irradiating the tumor more than 5 minutes after administration of the conjugate at a wavelength that renders the conjugate cytotoxic, wherein treatment of the tumor by the conjugate and subsequent light irradiation initiates activation of immune cells; and c) administering to the subject a therapeutically effective amount of an immunomodulator capable of enhancing immune cell activity.

[0079] 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 immunomodulator 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), α-galactosylceramide, RC-529, MDF2β, loxoribine, anti-CD40 agonist, SIRPα antagonist, AS04, AS03, flagellin, resiquimod, DAP (diaminopimelate), MDP (muramyl dipeptide) and CAF01 (cationic adjuvant preparation-01). In some embodiments, the immunomodulator is a Toll-like receptor (TLR) agonist, adjuvant or cytokine. In some embodiments, the immunomodulator 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 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 ribosome elongation and initiation factor 4a (LeIF), MEDI9197, SD-101, and imidazoquinoline TLR agonists.

[0080] In some embodiments, the immunomodulator is a cytokine, and the cytokine is IL-4, TNF-α, GM-CSF, or IL-2. In some embodiments, the immunomodulator is administered before, simultaneously with, or after the irradiation. In some embodiments, the immunomodulator is administered no more than 5 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, or 24 hours after the irradiation. In some embodiments, the immunomodulator is administered no more than 5 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, or 24 hours before the irradiation.

[0081] 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 that is capable of binding to the molecule or protein. In some embodiments, the targeting molecule is a second antibody.

[0082] In some embodiments, the phthalocyanine dye has a maximum absorption wavelength of about 600 nm to about 850 nm.

[0083] 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 comprising a reactive group for linking the dye to the targeting molecule.

[0084] In some embodiments, the phthalocyanine dye comprises a compound of the formula:

[0085] in:

[0086] L is the connector;

[0087] Q is a reactive group used to link the dye to the targeting molecule;

[0088] R 2 、R 3 、R 7 and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl;

[0089] 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, wherein R 4 、R 5 、R 6 、R 9 、R 10 and R 11 At least one of the compounds comprises a water-soluble group;

[0090] 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

[0091] X 2 and X 3 Each independently is C1~C 10 Alkylene groups are optionally interrupted by heteroatoms.

[0092] In some embodiments, the phthalocyanine dye comprises a compound of the formula:

[0093]

[0094] in:

[0095] X 1 and X 4 Each independently is C1~C 10 alkylene, optionally interrupted by heteroatoms;

[0096] R 2 、R 3 、R 7 and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl;

[0097] 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, wherein R 4 、R 5 、R 6 、R 9 、R 10 and R 11 At least one of comprises a water-soluble group; and

[0098] R 16 、R 17 、R 18 and R 19 Each is independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino and optionally substituted alkoxy.

[0099] In some embodiments, the phthalocyanine dye comprises IRDye 700DX (IR700).

[0100] In some embodiments, the conjugate is administered at about 50 mg / m 2 ~about 5000 mg / m 2 , about 250mg / m 2 ~about 2500 mg / m 2 , about 750mg / m 2 ~about 1250 mg / m 2 or about 100 mg / m 2 ~about 1000 mg / m 2 Dosage administration.

[0101] 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, 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 carcinoma that is a carcinoma of the bladder, pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, esophagus, or head and neck.

[0102] In some embodiments, the radiation dose is at least 1 J cm at a wavelength of 600 nm to 850 nm. -2 In some embodiments, the tumor is irradiated at a wavelength of 690 nm ± 50 nm or about 690 ± 20 nm.

[0103] 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 after irradiation, compared to the size or volume of the tumor before dosing and irradiation.

[0104] In some embodiments, the PIT treatment method using a phthalocyanine dye conjugate, such as any of the methods described above or provided herein, results in an improvement in a parameter associated with a disorder or cancer in a treated subject population compared to a similarly treated subject population treated with an unconjugated antibody or antigen-binding antibody fragment. In some embodiments, the parameter is one or more selected from the group consisting of: a) objective response rate (ORR); b) progression-free survival (PFS); c) overall survival (OS); d) reduction in toxicity; e) tumor response; or f) quality of life.

[0105] 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.

[0106] In some embodiments, the PIT treatment method using a phthalocyanine dye conjugate, such as any of the methods described above or provided herein, results in an objective 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 the treated subject population.

[0107] In some embodiments, the conjugate contains a phthalocyanine dye as a first dye and further contains a second fluorescent dye that is different from the first dye and is linked to a targeting molecule. In some embodiments, the second fluorescent dye exhibits one or more spectral properties selected from the group consisting of: fluorescence quantum yield in water, extinction coefficient, Stokes shift, absorption and emission at long wavelengths, and photostability that are better than the corresponding spectral properties 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 conjugate at the lesion or tumor present in the object. In some embodiments, the provided method further comprises imaging the lesion or tumor in the object by irradiating or illuminating the tumor with a wavelength that can be absorbed by the second dye.

[0108] In some embodiments, the first dye is IR700. In some embodiments, the second dye is IR700. In some embodiments, the second dye is selected from 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, BODIPYTMR-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, Dylight800, IRDye800, 525, 565, 605, 655, 705 and 800.

[0109] In some embodiments, the first dye is IR700, and the conjugate contains 1 to 10 or 1 to 5 second dye molecules per targeting molecule.

[0110] In some embodiments, the second dye exhibits a Stokes shift of 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.

[0111] 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 greater.

[0112] In some embodiments, the second dye has absorption and emission wavelengths in the spectrum of about 650 nm to 950 nm, about 700 nm to 1000 nm, or about 1000 nm to 1700 nm.

[0113] 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 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.

[0114] In some embodiments, the method further comprises irradiating or illuminating the tumor at a wavelength that is absorbed by the second dye, thereby imaging the subject. In some embodiments, the irradiating or illuminating the tumor is performed using a device selected from a handheld UV 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.

[0115] In some embodiments, a composition containing a conjugate is provided, the conjugate containing a phthalocyanine dye attached to an antibody or antigen-binding antibody fragment that binds to a molecule on the surface of a cell present in a damaged microenvironment, such as a tumor microenvironment. In some embodiments, the composition is formulated for a single dose administration of about 100 mg to 1200 mg of the conjugate. In some embodiments, the composition is formulated for a single dose administration of about 100 mg to 500 mg or about 200 mg to 400 mg. In some embodiments, the composition is formulated for a single dose administration of about 500 mg to 1500 mg, 800 mg to 1200 mg, or 1000 mg to 1500 mg.

[0116] In some embodiments, the volume of the composition is between about 10 mL and 500 mL or between 50 mL and 250 mL. In some embodiments, 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, or 500 mL. In some embodiments, the volume of the composition is between about 10 mL and 1000 mL or between 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.

[0117] In some embodiments, a product is provided, comprising a plurality of sealable containers, each independently 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 a damaged microenvironment, such as a tumor microenvironment. In some embodiments, the total amount of the conjugate in the plurality of sealable containers is about 100 mg to 1500 mg or 100 mg to 1200 mg. In some embodiments, the product comprises packaging material and a label or package insert comprising instructions for combining the contents of a plurality of vials to form a single dose formulation of the composition.

[0118] In some embodiments, the total amount of the conjugate in the plurality of sealable containers is about 100 mg to 1200 mg. In some embodiments, the total amount of the conjugate in the plurality of sealable containers is about 100 mg to 500 mg, about 200 mg to 400 mg, about 500 mg to 1500 mg, about 800 mg to 1200 mg, or about 1000 mg to 1500 mg.

[0119] In some embodiments, the lesion is a tumor.

[0120] In some embodiments, a conjugate is provided, comprising a phthalocyanine dye linked to an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor.

[0121] In some embodiments, the immunomodulator is an antibody or antigen binding fragment that binds to the surface of a tumor, tumor cell, or cancer cell. In some embodiments, the immunomodulator specifically binds to a molecule selected from the group consisting of 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 immunomodulator is selected from 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 one of the foregoing. In some embodiments, the immunomodulator is an antibody or antibody fragment that binds to PD-L1.

[0122] In some embodiments, the immunomodulator is an antibody selected from BMS-935559, MEDI4736, MPDL3280A, and MSB0010718C, or an antigen-binding fragment thereof.

[0123] In some embodiments, a conjugate is provided, comprising 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 that exhibits phototoxicity.

[0124] In some embodiments, the conjugate has the formula:

[0125] [D1-(L1) n ] p -A-[(L2) m -D2] o ,in:

[0126] A is a targeting molecule that can bind to a molecule on the cell surface;

[0127] L1 and L2 are independently selected linkers that can be the same or different;

[0128] n and m are independently 1 or 2;

[0129] D1 is a first dye, which is a phthalocyanine dye that can show phototoxicity;

[0130] D2 is a second dye that is a fluorescent dye, wherein D2 is different from D1;

[0131] p is 1 to 10; and

[0132] o is 1 to 10.

[0133] In some embodiments, the targeting molecule is an antibody or an antigen-binding antibody fragment.

[0134] In some embodiments, the cell surface molecule comprises an antigen, a polypeptide, a lipid or a carbohydrate, or a combination of these molecules.

[0135] In some embodiments, the cell surface molecule is selected from ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, α-4-integrin, α-5-β-3-integrin, α-5-β-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, Cripto-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 receptors), GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, group A (rhodopsin-like) G-protein-coupled receptors, group B (secretin receptor-like) G-protein-coupled receptors, group C (metabotropic glutamate receptor-like) G-protein-coupled receptors, GD2, GP100, GP120, glypican-3, hemagglutinin, heparan sulfate, HER1, HER2, HER3, HER4, HMFG, HPV 16 / 18 and E6 / E7 antigens, hTERT, IL-2R, 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, proteolytic cleavage of collagen IV, proteolytic enzyme 3, inhibin, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (such as P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 patch, RET receptor, plexin , smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEMs, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (such as 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.

[0136] In some embodiments, the cell surface molecule is selected from 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), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate-binding protein, gangliosides (such as 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.

[0137] In some embodiments, the cell surface molecule is selected from 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, 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, sialic acid-binding immunoglobulin-like lectin (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, ILTs, LIRs), SIRPA (signal regulatory protein alpha), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR and VEGF.

[0138] In some embodiments, the targeting molecule is an antibody or antigen-binding fragment selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, tositumomab, Rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab 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, MED I6383, MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, Emactuzumab, CC-90002 and MNRP1685A, or antigen-binding fragments thereof.

[0139] 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.

[0140] In some embodiments, the first dye is a phthalocyanine dye having a maximum absorption wavelength of about 600 nm to about 850 nm.

[0141] In some embodiments, the first dye is a phthalocyanine dye comprising a compound of the formula:

[0142] in:

[0143] L is the connector;

[0144] Q is a reactive group used to link the dye to the targeting molecule;

[0145] R 2 、R 3 、R 7 and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl;

[0146] 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, wherein R 4 、R 5 、R 6 、R 9 、R 10 and R 11 At least one of the compounds comprises a water-soluble group;

[0147] 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

[0148] X 2 and X 3 Each independently is C1~C 10 Alkylene groups are optionally interrupted by heteroatoms.

[0149] In some embodiments, the first dye is a phthalocyanine dye comprising a compound of the formula:

[0150] in:

[0151] X 1 and X 4 Each independently is C1~C 10 alkylene, optionally interrupted by heteroatoms;

[0152] R 2 、R 3 、R 7 and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl;

[0153] R 4 、R 5 、R 6 、R 9 、R 10 and R 11are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl and a chelating ligand, wherein R 4 、R 5 、R 6 、R 9 、R 10 and R 11 At least one of comprises a water-soluble group; and

[0154] R 16 、R 17 、R 18 and R 19 Each is independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino and optionally substituted alkoxy.

[0155] In some embodiments, the first dye is a phthalocyanine dye, comprising IRDye 700DX (IR700).

[0156] In some embodiments, the second fluorescent dye exhibits one or more spectral properties selected from the group consisting of fluorescence quantum yield in water, extinction coefficient, Stokes shift, absorption and emission at long wavelengths, and photostability that are better than the corresponding spectral properties of the first dye.

[0157] In some embodiments, the second dye is IR700. In some embodiments, the second dye is selected from the group consisting of 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, Chromeo642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700, Cy5.5, ICG, Alexa Fluor 750, Dylight755, IRDye 750, Cy7, Cy7.5, Alexa Fluor790, Dylight 800, IRDye 800, 525, 565, 605, 655, 705 and 800.

[0158] In some embodiments, the first dye is IR700, and the conjugate contains 1 to 10 or 1 to 5 second dye molecules per targeting molecule.

[0159] In some embodiments, the second dye exhibits a Stokes shift of 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.

[0160] 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 greater.

[0161] In some embodiments, the absorption and emission wavelengths in the spectrum of the second dye are between about 650 nm and 950 nm, between about 700 nm and 1000 nm, or between about 1000 nm and 1700 nm.

[0162] In some embodiments, the first dye and the second dye do not exhibit overlapping emission and absorption spectra.

[0163] In some embodiments, the second dye is selected from 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.

[0164] In some embodiments, a composition is provided, comprising any of the conjugates described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0165] In some embodiments, methods of treating a disease or condition in a subject are 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) following administration of the conjugate, irradiating the lesion with one or more wavelengths to induce phototoxic activity of the conjugate, thereby treating the disease or condition.

[0166] In some embodiments, a method of using photoimmunotherapy (PIT) to treat a disease or condition such as a tumor in a subject is provided, comprising administering to the subject a therapeutically effective amount of any of the conjugates or compositions described herein. In some embodiments, the method comprises administering a photoimmunotherapy (PIT) to the subject at a wavelength of 660 nm to 740 nm at a dose of at least 1 J cm -2 The tumor is irradiated at a dose of 1 J / cm fiber length or 1 J / cm fiber length, thereby treating the disease or condition in the subject.

[0167] In some embodiments, methods of treating a disease or condition in a subject are 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 administration of the conjugate, irradiating the lesion 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.

[0168] In some embodiments, provided methods include administering radiation at a wavelength of about 400 to about 900 nm at a rate of at least 1 J cm -2 In some embodiments, the methods include irradiating the lesion with a single wavelength. In some embodiments, the methods include irradiating the lesion with two different wavelengths, either simultaneously or sequentially, wherein one wavelength induces phototoxic activity and the other wavelength induces a fluorescent signal.

[0169] In some embodiments, the disease or condition is a tumor.

[0170] In some embodiments, the provided methods include using a wavelength of 660 nm to 740 nm at a rate of at least 1 J cm -2 The tumor is irradiated at a dose of irradiation to treat the tumor in the subject.

[0171] 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, 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 carcinoma that is a carcinoma of the bladder, pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, esophagus, or head and neck.

[0172] 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 dose of about 10 mg / m 2 ~about 500 mg / m 2 In some embodiments, the targeting molecule is an antibody or antigen-binding fragment, such as cetuximab. In some embodiments, cetuximab is administered to the subject prior to administration of the conjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figure 1 Dose-response curves showing inhibition of EGFR phosphorylation in A431 cells by cetuximab and cetuximab-IRDye 700DX conjugate.

[0174] Figure 2 A431 cells were treated with 8 J / cm 2 Dose-response curve of cetuximab-IRDye700DX conjugate for photoimmunotherapy (PIT) under 690 nm light.

[0175] Figure 3 showed that 16 J / cm 2 and 32 J / cm 2 Comparison of the dose-response curves of cetuximab-IRDye700DX conjugate for photoimmunotherapy (PIT) under 690 nm light.

[0176] Figure 4 The schematic structure of the dual-labeled panitumumab-IR700DX-Alexa-488 conjugate is shown.

[0177] Figure 5A and 5B The results showed that different conjugates were used to treat BxPC3 pancreatic cancer cells at different light doses (0-16 J / cm 2 )-induced cell death after photoimmunotherapy (PIT). Figure 5A Shown are cell death following PIT using a single-labeled panitumumab-IR700 conjugate. Figure 5B Shown are cell death after PIT using dual-labeled panitumumab-IR700-Alexa-488 conjugate.

[0178] Figure 6A Shown are the duration of exposure of cetuximab-IRDye 700DX to 500 lux white fluorescent or green LED light and its effect on the formation of soluble aggregates.

[0179] Figure 6B Shown are the effects of pre-exposure of cetuximab-IRDye 700DX to white fluorescent or green LED light on BxPC3PIT activity.

[0180] Figure 6C Shown is the effect of the percentage of cetuximab-IRDye 700DX soluble aggregate formation on PIT activity.

[0181] Figure 7 Shown are PIT activities in the context of sequential staining with cetuximab and donkey anti-human IRDye 700DX (DxHu IR700) secondary antibody.

[0182] Figure 8A Shown is light-dependent killing of BxPC3 cells by biotinylated cetuximab pre-complexed with monomeric streptavidin-IRDye 700DX (mSA IR700).

[0183] Figure 8B Specificity of biotinylated cetuximab pre-complexed with monomeric streptavidin-IRDye 700DX (mSA IR700) for PIT is shown.

[0184] Figure 8C Shown is the effect of pre-exposure of monomeric streptavidin-IRDye 700DX to white light on the PIT-killing activity of biotinylated cetuximab in BxPC3 cells.

[0185] Figure 9A Antibody dose-dependent killing of 4T1 cells by directly conjugated anti-EpCAM-IRDye 700DX is shown.

[0186] Figure 9B The specificity of the anti-EpCAM-IRDye 700DX PIT killing activity was demonstrated.

[0187] Figure 10 Cetuximab-IRDye 700DX demonstrates Fc receptor-specific killing of THP1 cells.

[0188] Figure 11A Demonstrating the specificity of EGF-IRDye 700DX light-dependent killing in A431 cells.

[0189] Figure 11B Shown are the effects of EGF-IRDye 700DX pre-exposure to different types of light on light-dependent killing in A431 cells.

[0190] Figure 12A Shown is light-dependent killing of BxPC3 cells using cholera toxin B-IRDye 700DX.

[0191] Figure 12B Demonstrating the specificity of photoactivated killing by cholera toxin B-IRDye 700DX.

[0192] Figure 12C Shown are the effects of pre-exposure of cholera toxin B-IRDye 700DX to light of different wavelengths on the photoactivated killing of BxPC3 cells.

[0193] Figure 13A Showing light-dependent killing of Vero cells by influenza virus (X-31)-IRDye 700DX.

[0194] Figure 13B Shown is a comparison of the effects of pre-exposure of influenza virus (X-31)-IRDye 700DX to white and green light on photoactivated cell killing.

[0195] Figure 14A Shown is the effect of light dose on the killing activity of SNA-IRDye 700DX in BxPC3 cells.

[0196] Figure 14BShown is the effect of sialidase treatment on the specificity of SNA-IRDye 700DX binding to cells.

[0197] Figure 15 PIT killing of Staphylococcus aureus (S. aureus) by the combination of Cetuximab-IRDye 700DX and laser irradiation is shown.

[0198] Figure 16 Shown is PIT using influenza virus particles pre-complexed with GtxMs Fab-IRDye 700DX against mouse influenza virus (H3N2).

[0199] Figure 17 Shown are light-dependent killing of influenza virus-infected cells by mouse anti-influenza virus (H3N2) and goat anti-mouse IRDye 700DX (GtxMs-IR700).

[0200] Figure 18 Shown is PIT killing of rat embryonic dorsal root ganglion (DRG) neurons using cholera toxin B-IRDye 700DX.

[0201] Figure 19A Shown is the effect of IFNγ treatment on the percentage of cell death in BxPC3 cells.

[0202] Figure 19B Shown are the effects of IFNγ treatment on PD-L1 expression in BxPC3 cells.

[0203] Figure 19C Shown is the effect of IFNγ treatment on the killing activity of anti-PD-L1 IRDye 700DX PIT in BxPC3 cells.

[0204] Figure 20A Shown are the effects of PIT treatment with cetuximab-IRDye 700DX in A431 and FaDu cells on the amount of HMGB1 detected in the extracellular solution.

[0205] Figure 20B Shown is the upregulation of dendritic cell (DC) maturation markers on immature dendritic cells (iDCs) co-cultured with tumors subjected to PIT with cetuximab-IRDye 700DX.

[0206] Figure 20CShown are the effects of activating antigen-presenting cells by PIT-treatment of A431 or FaDu cells (using cetuximab-IRDye 700DX and in the presence of light irradiation) or non-PIT-treatment of A431 or FaDu cells (using cetuximab-IRDye 700DX but in the absence of light irradiation), as evaluated by the expression of the exemplary activation marker CD86.

[0207] Figure 21 Shown are the effects of activating dendritic cells by priming them 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 then stimulating them with an immunomodulator (poly I:C), as evaluated by the expression of exemplary activation markers.

[0208] Figure 22A Shown is the UV-Vis spectrum of Cetuximab-IRDye 700DX-Alexa Flour 488 (CTX700-ALX488).

[0209] Figure 22B Shown is the UV-Vis spectrum of the CTX-700-IRDye 800CW.

[0210] Figure 22C The UV-Vis spectrum of CTX700-Dylight 755 is shown.

[0211] Figure 23A Figure 3. PIT killing activity of cetuximab-IRDye 700DX conjugated or not to DyLight 755 in BxPC3 cells at different concentrations and light doses.

[0212] Figure 23B The PIT killing activity of cetuximab-IRDye800CW, cetuximab-Alexafluor 488, or cetuximab-IRDye 700DX in BxPC3 cells, either conjugated or not, to IRDye 800CW or Alexafluor 488 is shown. The results are expressed as a percentage of the PIT activity relative to that of cetuximab-IRDye 700DX (control).

[0213] Figure 24A Shown is the effect of pre-exposure to white or green light on the formation of soluble aggregates for cetuximab-IRDye 700DX conjugate, cetuximab-IRDye 680RD conjugate, and cetuximab-IRDye 700 + IRDye 680RD dual conjugate.

[0214] Figure 24B Shown are the effects of pre-exposure of Cetuximab-IRDye 700DX conjugate, Cetuximab-IRDye 680RD conjugate, and Cetuximab-IRDye 700 + IRDye 680RD dual conjugate to white or green light on fluorescence normalized to monomer content. DETAILED DESCRIPTION

[0215] I. Photoimmunotherapy

[0216] The present invention provides conjugates, compositions, combinations and methods related to photoimmunotherapy (PIT). Photoimmunotherapy is a molecular targeted therapy using a phthalocyanine-based target-specific photosensitizer, such as a near-infrared (NIR) phthalocyanine dye (such as IR700) coupled to a targeting molecule that targets a protein (such as a cell surface protein on a cell in a disease or condition, such as a cell in a tumor). For example, in some cases, the phthalocyanine dye conjugate used in photoimmunotherapy can include coupling with a monoclonal antibody (mAb), which targets a cell surface protein receptor or a receptor expressed on a microenvironment of disease damage, such as a cell in a tumor microenvironment (which may include tumor cells and other infiltrating cells). In some embodiments, the dye conjugate is activated by irradiation with absorptive light, such as NIR light, which activates the photosensitizer and causes cell killing, thereby reducing or eliminating the damage (such as a tumor) and treating the disease or condition. In some cases, the use of light in the NIR range has resulted in deep tissue penetration, leading to successful eradication of tumors after only a single dose of external NIR light irradiation.

[0217] Typically, targeted phototoxicity relies primarily on the binding of the dye conjugate to the cell membrane via a specific targeting molecule (e.g., a macromolecule such as an antibody). For example, studies using the exemplary antibody IR700 molecule have shown that the conjugate must bind to the cell membrane for activation and that effective intracellular localization is not required for cell killing (see, e.g., U.S. Patent No. 8,524,239 and U.S. Published Application No. US20140120119). Photoactivation of cells bound to the conjugate results in rapid cell death and necrosis.

[0218] Generally, PIT primarily induces cell death in cells bound to the phthalocyanine dye conjugate, such as an IR700-antibody conjugate, upon exposure to NIR radiation. Cells that do not express the cell surface protein recognized by the targeting molecule (e.g., an antibody) are not significantly killed. Therefore, because this therapy specifically targets diseased cells, such as those in tumors, its effects are more selective for diseased tissues than for healthy tissues or cells. For example, while the targeted photosensitizer can be distributed throughout the body, it is activated only at the site of intense light application, minimizing the potential for off-target effects. This differs from non-PIT-based approaches, in which the activity of similar therapeutic targeting molecules (e.g., therapeutic antibodies) not conjugated to a photosensitizer (e.g., IR700) remains unlocalized, resulting in a 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.

[0219] In some embodiments, the methods provided involve administering a phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate) at a dose as described below, which achieves an exposure that is much lower than the exposure required for a targeting molecule (e.g., antibody) not conjugated to the phthalocyanine dye to achieve a therapeutic effect. Because PIT requires the conjugated targeting molecule (e.g., antibody) to bind to a cell surface protein to mediate cell killing, receptor occupancy is believed to be a factor that is related to the extent of PIT activity. Therefore, it is believed that the conjugate must have a similar or even higher systemic exposure to ensure exposure to the site of damage (e.g., tumor) to achieve sufficient cell killing compared to the systemic exposure required for a targeting molecule (e.g., antibody) that is not so conjugated (i.e., not conjugated to a photosensitizer such as IR700) to achieve a clinically acceptable therapeutic effect. For example, the Food and Drug Administration (FDA)-approved The dose of cetuximab for the treatment of head and neck cancer is: initial dose 400 mg / m 2 , then 250 mg / m2 weekly 2 In the sample patient population, a single dose of Erbitux at 400 mg / m 2 The average systemic exposure (AUC, such as AUC 0-inf or AUC[0-inf]) was approximately 24,620 μg*h / mL, and one month of treatment (initial dose of 400 mg / m 2 , then 250 mg / m2 weekly 2) was estimated to have a total systemic exposure of approximately 60,056 μg*h / mL (cumulative systemic exposure for four weeks of dosing within 1 month, 1×24,620+3×11,812 μg*h / mL) (Fracasso et al. (2007) Clin. Cancer. Res., 13:986).

[0220] However, it was found herein that PIT cell killing could be observed in a sample patient population following a single dose of cetuximab-IR700 conjugate in an amount that only reached approximately 1810 μg / mL*h (AUC 0-inf ) or 770+ / -47.5 (AUC 0-24 ) average systemic exposure. This amount is up to or about 400 mg / m 2 The clinical therapeutic dose is given In addition, as shown in Example 2, the results show that even at 640 mg / m 2 At higher doses, the mean systemic exposure (AUC 0-24 ) is also 400 mg / m 2 Next The results also showed a 100% objective response rate (ORR) (all patients showed a complete or partial response to treatment) to a single dose of dapoxetine in patients with head and neck cancer, including those who had failed other treatments, demonstrating an unexpectedly rapid and robust response given the low systemic exposures described above. In comparison, even with multiple doses required for continuous exposure, dapoxetine was not significantly associated with the response to a single dose of dapoxetine. The ORR for subjects treated with monotherapy is also generally about 15% or less.

[0221] In addition, most of the use Treatment involves combination therapy with one or more chemotherapy or other anti-cancer treatments. As an example, in one reported study, the improvement in ORR when cetuximab was combined with chemotherapy compared to chemotherapy alone was only about 16%, which is similar to the ORR provided by cetuximab alone (see, e.g., Specenier and Vermorken (2013) Biologics, 7:77-90).

[0222] Thus, the provided methods are based on the discovery that even at low systemic exposures of phthalocyanine dye-targeting molecule conjugates (e.g., IR700-antibody conjugates), the conjugates are sufficiently potent in mediating PIT and cell killing. Thus, the provided methods comprise administering the conjugate at a dose that achieves a systemic exposure that is a fraction of the clinical exposure in humans of a corresponding therapeutic targeting molecule (e.g., a therapeutic antibody) when not conjugated to a phthalocyanine dye (e.g., IR700). In some embodiments, the clinical dose of the therapeutic molecule in humans is a dose described on a label approved for commercialization by a regulatory agency (e.g., FDA, EMA, PDMA). In some embodiments, the low systemic exposure achieved by such a phthalocyanine dye-targeting molecule dose means that the systemic exposure of the targeting molecule is not high enough to exhibit pharmacological activity unless PIT is induced by exposing the conjugate to light. Because a dose of light irradiation can be localized to a disease lesion (eg, a tumor), the provided methods can achieve selective cell killing of only the disease lesion (eg, a tumor) while avoiding unwanted or undesirable off-target activity.

[0223] In addition to the potency of the responses provided by the PIT methods of the present invention, the provided methods are particularly advantageous in the treatment of diseases or conditions (e.g., tumors) where existing therapies, such as existing antibody therapies, are prone to adverse off-target side effects. For example, in some cases, it is difficult to achieve strong anti-cancer activity within a safe range using therapies involving immunomodulators. In some cases, this is because such immunomodulators inhibit (or promote) the same mechanisms that our bodies use to combat autoimmunity. As a result, in many cases, administration of immunosuppressants can result in significant therapeutic side effects. Due to the safety of many immunosuppressants (and other therapeutic antibodies), acceptable doses compromise efficacy, necessitating dosing cycles to achieve or maintain a continuous threshold systemic exposure, and / or requiring co-administration with other chemotherapeutic or anticancer agents that carry a higher risk of adverse side effects. The provided methods address these issues because 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 also achieving selective cell killing at the site of disease or lesion. Therefore, the benefits of low dose and / or low systemic exposure are important to achieve safety.

[0224] In some embodiments, the fluorescent properties of phthalocyanine dyes also enable us to monitor PIT therapy using any of a variety of imaging methods or other methods that can detect fluorescent signals. In some embodiments, the assessment of fluorescence, for example by imaging, can be used to monitor the uptake or presence of the conjugate at a lesion (e.g., a tumor) prior to PIT. In some embodiments, the assessment of fluorescence, for example by imaging, can be used to irradiate the lesion (e.g., a tumor) to ensure that PIT is directed to the site of the lesion (e.g., a tumor). In some embodiments, the assessment of fluorescence can be used in surgery, where the edge of the lesion (e.g., a tumor) can be observed by fluorescence, and then residual tumor cells at the edge can be killed by PIT.

[0225] In some embodiments, the conjugate contains another fluorescent dye in addition to a phthalocyanine dye (e.g., IR700). For example, in some cases, IR700 is not an ideal dye for imaging because, for example, it exhibits one or more spectral properties that may not be suitable for ensuring specific and effective fluorescent labeling. In some embodiments, other fluorophores commonly used to label proteins may exhibit higher fluorescence quantum yields, larger Stokes shifts, larger extinction coefficients at the excitation wavelength, and / or longer wavelengths for deeper tissue penetration. Therefore, in some embodiments, a dual-labeled conjugate is provided, wherein a targeting molecule (e.g., an antibody or antigen-binding antibody fragment) is conjugated to a first dye and also to a second fluorescent dye, wherein the first dye is a phthalocyanine dye (e.g., IR700) and the second fluorescent dye exhibits one or more properties that are improved or superior to the first dye and are selected from the group consisting of: higher quantum yield, larger Stokes shift, larger extinction coefficient, and / or longer wavelength. In some embodiments, the dual-labeled conjugate can be used to monitor PIT therapy as described above, or to treat PIT by activating the phthalocyanine dye, e.g., IR700. In some embodiments, the first and second dyes are selected to minimize energy transfer between them, eg, the first and second dyes are selected to avoid or minimize overlap of emission and absorption spectra.

[0226] In some embodiments, the present invention also provides combination therapies for use in conjunction with photoimmunotherapy to treat a disease or condition in a subject. In some embodiments, the combination therapy can be used in a method for treating a tumor or cancer. In some embodiments, the combination of photoimmunotherapy and administration of other therapeutic agents (such as immunomodulators, anticancer agents, or other agents) can improve the efficacy of treating tumors, which in some cases can improve the efficacy of treatment or increase the survival of the subject receiving treatment.

[0227] In some aspects, provided combination therapies utilize the cytotoxic killing and / or lytic effects induced by PIT, synergizing with tumor therapy to enhance therapeutic efficacy. In specific aspects, one or more additional therapeutic agents can be administered to a subject with a tumor prior to completing PIT, wherein PIT activates a phthalocyanine dye conjugate via light irradiation. In some embodiments, the prior administration of the additional therapeutic agent can render the tumor microenvironment more responsive to PIT or the additional therapeutic agent following subsequent tumor irradiation.

[0228] 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 the immune response to the PIT-induced tumor-associated agent released by lysed cells. In another example, the additional therapeutic agent can be an anticancer agent, which in some aspects is administered prior to irradiation to increase the systemic availability of the anticancer agent and promote its delivery or uptake into the tumor area in response to changes in tumor permeability induced by PIT. In some embodiments, the enhanced therapeutic effect of the combination therapy can result in a greater reduction in tumor size (e.g., tumor volume or weight) or a greater or longer survival of the subject compared to a treatment involving only one of the therapies. In some embodiments, the therapeutic effect of the combination therapy can be synergistic compared to a treatment involving only the phthalocyanine dye conjugate / PIT or a treatment involving only the additional therapeutic agent, such as only the immunomodulator or only the anticancer agent.

[0229] A. Conjugates containing phthalocyanine dyes and targeting molecules

[0230] The methods, compositions, and combinations provided herein include conjugates comprising a photosensitizer, such as a phthalocyanine dye, such as IR700, and a targeting molecule (such as 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 the photosensitizer, such as IR700, to the cell surface protein enables the conjugate to target cells implicated in a disease or condition, such as a tumor or cancer, an infection, an inflammatory disease or condition, a neuronal disease or condition, or other diseases or conditions. In some embodiments, the target cells (such as cells expressing a cell surface protein capable of binding to the targeting molecule) are present in the microenvironment of a lesion associated with the disease or condition, such as a tumor microenvironment. In some embodiments, targeting of cells enhances the efficacy of PIT induced by localized irradiation of a lesion (such as a tumor) with a wavelength absorbed by the phthalocyanine dye (such as a near-infrared (NIR) wavelength), thereby rendering cell killing selective for cells to which the dye-targeting molecule conjugate binds.

[0231] In some embodiments, the phthalocyanine dye conjugate for combined therapy provided by the present invention comprises a dye molecule coupled to a targeting molecule via a linker group. In one aspect, the conjugate is represented by Formula I:

[0232] A-[(L) n -D] p

[0233] (I)

[0234] in:

[0235] A is a targeting molecule capable of binding to cells or tissues;

[0236] L is a linker selected independently for each p;

[0237] n is 1 or 2;

[0238] D is a hydrophilic phthalocyanine selected independently for each p; and

[0239] p is independently 1, 2, 3, 4, 5 or greater than 5, for example up to 1000. For example, p may be 1-1000, such as typically 1-10 or 2-5.

[0240] In some embodiments, the phthalocyanine dye conjugate is made by a method or process as described below, wherein the phthalocyanine dye-targeting molecule conjugate, such as IR700-targeting molecule (such as IR700-antibody) conjugate, is prepared under light-protected conditions. In some embodiments, the method comprises: 1) preparing or providing a phthalocyanine dye and a targeting molecule; 2) contacting the targeting molecule with the phthalocyanine dye under conditions that minimize exposure of the dye to produce the conjugate; and 3) formulating, purifying and / or molecule conjugate to produce a composition containing a drug substance, wherein one or more steps, such as, in some cases, all steps, are performed under conditions that minimize exposure of the dye or the conjugate containing the dye to ambient light. In some embodiments, the phthalocyanine dye-targeting molecule conjugate, such as the IR700-targeting molecule (such as the IR700-antibody) conjugate, is a conjugate described in U.S. Patent No. 62 / 206,774, or is prepared using the method for making a conjugate described in U.S. Patent No. 62 / 206,774, which is incorporated herein by reference.

[0241] 1. Phthalocyanine dyes

[0242] Phthalocyanine is a group of photosensitizer compounds with a phthalocyanine ring system. Phthalocyanine is an azaporphyrin containing four benzindole groups (i.e., C 32 H 16N8), which form stable chelates with metal and metalloid cations. In these compounds, the center of the ring is occupied by a metal ion (diamagnetic or paramagnetic), which can carry one or two ligands, depending on the ion. Furthermore, the ring periphery can be unsubstituted or substituted. International Publication No. WO 2005 / 099689 and U.S. Patent No. 7,005,518 describe the synthesis of various phthalocyanines and their use in photodynamic therapy.

[0243] In some embodiments, phthalocyanines strongly absorb red or near-infrared radiation, with an absorption peak between approximately 600 nm and 810 nm, which in some cases enables deep tissue penetration of light. Phthalocyanines are generally stable to light. This photostability is generally advantageous in pigments and dyes, as well as in many other uses of phthalocyanines.

[0244] In some embodiments, the phthalocyanine dye is water-soluble and contains a luminescent fluorophore portion with at least one water-solubilizing portion. In some embodiments, the water-solubilizing portion contains silicon. In some embodiments, the phthalocyanine dye has a core atom of Si, Ge, Sn or Al or the like. In some embodiments, the phthalocyanine dye exists in the form of a single core isomer, substantially free of other isomers. In some embodiments, the phthalocyanine dye contains a joint with a reactive group or an activatable group that can form a bond between the joint and the targeting molecule. In some embodiments, the phthalocyanine dye can be customized to emit fluorescence at a specific wavelength.

[0245] 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 represented by Formula Ia:

[0246]

[0247] in

[0248] L is selected from direct attachment or covalent attachment;

[0249] Q is a reactive or activatable group that may be part of the linker L and is any group that can react to form a bond between L and the targeting molecule A;

[0250] R 2 、R 3 、R 7 and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl;

[0251] R 4 、R 5 、R6 、R 9 、R 10 and R 11 If present, each is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, or a chelating ligand, wherein R 4 、R 5 、R 6 、R 9 、R 10 and R 11 At least one of the compounds comprises a water-soluble group;

[0252] 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 Each is a functional group and may be independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino or optionally substituted alkoxy;

[0253] Or in another embodiment, i) R 13 and R 14 and the carbon to which they are attached, or ii) R 17 and R 18 and the carbon to which they are attached, or iii) R 21 and R 22 and at least one of the carbon atoms to which they are attached is linked to form a fused ring; and

[0254] X 2 and X 3 Each independently is C1~C 10 Alkylene groups are optionally interrupted by heteroatoms.

[0255] In some embodiments, L is a covalent link. In some embodiments, the covalent link is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1 to 60 atoms, such as 1 to 45 atoms or 1 to 25 atoms. In some cases, the atom may be selected from C, N, P, O and S. In some embodiments, L may have additional hydrogen atoms to balance the valence (in addition to the 1 to 60 atoms mentioned above). Typically, the link comprises an ether, thioether, amine, ester, carbamate, urea, thiourea, oxygen or amide bond; or a single bond, double bond, triple bond or aromatic carbon-carbon bond; or a phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen or nitrogen-platinum bond; or a combination of aromatic or heteroaromatic bonds.

[0256] In some embodiments, L is of the formula -R 1 -YX 1 -Y 1 - indicates that, where R 1 is a divalent free radical or directly connected; Y and Y 1 are each independently selected from a direct link, oxygen, optionally substituted nitrogen or sulfur; and X 1 Select from direct connection and C1-C 10 Alkylene is optionally separated by atoms. Divalent radicals include, but are not limited to, optionally substituted alkylene, optionally substituted alkyleneoxycarbonyl, optionally substituted alkylenecarbamoyl, optionally substituted alkylenesulfonyl, and optionally substituted arylene.

[0257] Exemplary 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.

[0258] In some embodiments, Q contains a reactive group for optionally connecting to a material such as a targeting molecule. As used herein, the term "reactive group" refers to a portion of a compound that can chemically react with a functional group on other substances (such as a targeting molecule) to form a connection such as a covalent connection. In general, the reactive group is an electrophile or a nucleophile, which can be covalently connected by being exposed to the corresponding functional group as a nucleophile or an electrophile. Alternatively, the reactive group is a photoactivated group and only becomes chemically reactive after being irradiated with light of a suitable wavelength. In general, the coupling reaction between the reactive dye to be coupled and the targeting molecule causes one or more atoms of the reactive group Q to participate in a new connection, which couples the dye to the targeting molecule.

[0259] In some embodiments, Q contains a reactive group that is reactive with a carboxyl, amine or 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 (such as iodoacetamide), isocyanates, isothiocyanates, maleimides, NHS esters, phosphoramidites, platinum complexes, sulfonates and thiocyanates for optionally being connected with the targeting molecule. In some embodiments, the reactive group is reactive with a carboxyl, amine or thiol group on the targeting molecule. In some embodiments, the reactive chemical group is a sulfhydryl reactive chemical group, such as maleimide, haloacetyl and pyridyl disulfide. In some embodiments, the reactive group is amine reactive. In some embodiments, the reactive group is an NHS ester.

[0260] In some embodiments, R 2 、R 3 、R 7 and R 8 Each is an optionally substituted alkyl group, such as an optionally substituted methyl group, an ethyl group or an isopropyl group.

[0261] In some embodiments, R 4 、R 5 、R 6 、R 9 、R 10 and R 11 At least one of the R 4 、R 5 、R 6 、R 9 、R 10 and R 11As used herein, "water-soluble group" refers to a group containing one or more polar and / or ionic substituents that increase the solubility of the entire molecule in aqueous media. In some cases, R 4 、R 5 、R 6 、R 9 、R 10 and R 11 At least two of the alkyl groups contain water-soluble groups. In other embodiments, three or more of the alkyl groups contain water-soluble groups. Water-soluble groups include, but are not limited to, carboxylic acid groups (—CO2 - ), sulfonic acid group (—SO3 - ), sulfonyl (—SO2 - ), sulfate (—SO4 -2 ), hydroxyl (—OH), phosphate (—OPO3 -2 ), phosphonic acid group (—PO3 -2 ), amino groups (-NH2) and optionally substituted quaternary ammonium nitrogens, each with an optional counterion.

[0262] 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.

[0263] In some embodiments, R 4 、R 5 、R 6 、R 9 、R 10 and R 11 The attached nitrogen atom may be trivalent or tetravalent.

[0264] 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 Each is hydrogen.

[0265] In some embodiments, X 2 and X 3 Each independently selected from C1 to C 10 In some embodiments, the alkylene group is optionally separated by atoms. 2 and / or X 3 The nitrogen on the α-Hydroxy-L-pyrrolidone may optionally be quaternized.

[0266] In some embodiments, the phthalocyanine dye is represented by Formula Ib:

[0267]

[0268] in

[0269] X 1 and X 4 Each independently is C1~C 10 alkylene, optionally interrupted by heteroatoms; and

[0270] 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 As defined herein.

[0271] In some embodiments, the reactive group is an NHS ester. In some embodiments, the reactivity of the NHS ester can be modified by varying the alkylene X between the NHS ester and the carbamate functional group. 4 In some embodiments, the alkylene group X between the NHS ester and the carbamate functional group is 4 The length of X is inversely proportional to the reactivity of the NHS ester. 4 In other embodiments, X 4 In some embodiments, X 1 is C6-alkylene. In other embodiments, X 1 It is a C3-alkylene group.

[0272] In some embodiments, the phthalocyanine dye has an overall electronic charge of zero. In some cases, this charge neutrality can be achieved by one or more optional counterions or quaternary ammonium nitrogens.

[0273] In some embodiments, the phthalocyanine dye has sufficient solubility in aqueous solutions so that when it is attached to a soluble targeting molecule, the targeting molecule maintains its solubility. In some embodiments, the dye is also soluble in organic media (such as DMSO or DMF).

[0274] In some embodiments, the phthalocyanine dye has a maximum light absorption in the near infrared range (NIR range). In some embodiments, the phthalocyanine dye has a maximum light absorption wavelength of 400 nm to 900 nm, such as 600 nm to 850 nm, such as 680 nm to 850 nm, for example, about 690 nm ± 50 nm to 690 ± 20 nm. In some embodiments, the phthalocyanine dye can be effectively excited by commercially available laser diodes that emit light of the above wavelengths.

[0275] In some embodiments, the phthalocyanine dye containing a reactive group is IR700 NHS ester, such as IRDye700DX NHS ester (Li-Ccor 929-70010, 929-70011). Therefore, in some embodiments, the dye is a compound having the following structure:

[0276]

[0277] Chemical formula: C 74 H 96 N 12 Na4O 27 S6Si3

[0278] Exact mass: 1952.37

[0279] Molecular weight: 1954.22

[0280] IRDye 700DX NHS Ester

[0281] For the purposes of the present invention, 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. Generally, IR700 has several advantageous chemical properties. The amino-reactive IR700 is a relatively hydrophilic dye that can be covalently coupled to antibodies using the NHS ester of IR700. In general, IR700 also has better photosensitive properties than common photosensitizers such as hematoporphyrin derivatives. (1.2×10 at 630nm 3 M -1 cm -1 ), tetrakis-m-hydroxyphenylchlorin; (2.2×10 at 652nm 4 M -1 cm -1 ) and mono-L-aspartyl chlorin e6; (4.0×10 at 654 nm 4 M -1 cm -1) is more than 5 times higher than the extinction coefficient (2.1×10 5 M -1 cm -1 ).

[0282] Phthalocyanine dyes as herein described can be made with commercially available starting materials. The core structure is synthesized by the condensation of two or more different diiminoisoindolines. The synthetic strategy using different dinitrile or diiminoisoindoline can result in the distribution of substitution and / or regioisomers of phthalocyanine in varying degrees. An exemplary synthetic scheme for generating the dye has been described in U.S. Patent No. 7,005,518.

[0283] In some embodiments, in any method provided by the present invention, targeting molecule (such as antibody) is directly or indirectly connected with phthalocyanine dye (such as IR700). In some embodiments, targeting molecule (such as antibody) provides covalent bond or non-covalent interaction to be directly or indirectly connected with phthalocyanine dye (such as IR700). In some embodiments, the covalent or non-covalent interaction or connection are direct or indirect. In some embodiments, the connection includes indirect connection, such as by the connection of a joint (such as any exemplary joint mentioned above), binding part or domain or reactive group. In some embodiments, the connection includes the direct interaction between targeting molecule and phthalocyanine dye (such as IR700). In other embodiments, one or both of targeting molecule and phthalocyanine dye are connected with one or more joints, and the interaction is indirect, such as the interaction between the joint connected with a molecule and another molecule or the interaction between two joints connected with targeting molecule or phthalocyanine dye respectively.

[0284] In some embodiments, phthalocyanine dye is non-covalently linked or is connected to the targeting molecule.For example, phthalocyanine dye forms complex with the targeting molecule by non-covalent interaction.In some embodiments, phthalocyanine dye (such as IR700) contains the part or domain that can produce non-covalent interaction with the linking group of the targeting molecule.In some embodiments, described method comprises phthalocyanine dye (such as IR700) and targeting molecule (such as antibody) are hatched or its combination is made, to form non-covalent interaction between dyestuff and targeting molecule.In certain embodiments, the non-covalent interaction between targeting molecule and phthalocyanine dye comprises for example electrostatic interaction, van der Waals force, hydrophobic interaction, π effect, ionic interaction, hydrogen bond, halogen bond and / or their combination or based on any interaction of above-mentioned one or more powers. In some embodiments, the targeting molecule and the phthalocyanine dye are linked using interactions that mimic non-covalent molecular interactions, such as ligand-receptor interactions, antibody-antigen interactions, avidin-biotin interactions, streptavidin-biotin interactions, histidine-divalent metal ion interactions (e.g., Ni, Co, Cu, Fe), interactions between multimerization (e.g., dimerization) domains, glutathione-S-transferase (GST)-glutathione interactions, and / or any combination thereof.

[0285] In some embodiments, the non-covalent interaction portion or domain is attached to or is part of a targeting molecule and forms a non-covalent interaction, such as a 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 a phthalocyanine dye molecule and forms a non-covalent interaction, such as a complex, with the targeting molecule. In some embodiments, the method comprises incubating or contacting a targeting molecule conjugated to biotin (e.g., antibody-biotin, e.g., cetuximab-biotin) and a phthalocyanine dye conjugated to avidin or its analog or streptavidin or its analog, including monomeric forms thereof (e.g., monomeric avidin-IR700 or monomeric streptavidin-IR700). In some embodiments, the phthalocyanine dye (e.g., IR700) forms a non-covalent complex with the targeting molecule by virtue of the non-covalent interaction between avidin, streptavidin, or its analog and biotin.

[0286] 2. Targeting molecules

[0287] In some embodiments, the phthalocyanine dye is coupled to a targeting molecule through the reactive group of the dye molecule. In some embodiments, the targeting molecule can, for example, be combined with the cell surface molecules (such as cell surface receptors) on the cell or pathogen to target the conjugate to the cell or pathogen. In some embodiments, the targeting molecule, such as macromolecule, can be selectively combined with the cell of the desired cell type, the cell with a specific phenotype, or the cell displaying one or more cell surface markers or antigens. In some cases, the targeting molecule is combined with the cell in cancer cells, tumor cells, inflammatory cells, immune cells, neurons, stem cells, proliferative cells or hyperplasia. In some cases, the targeting molecule is combined with a pathogen or a cell infected by a pathogen. In some embodiments, the cell is an inflammatory cell such as a leukocyte, such as a neutrophil, eosinophil, basophil, lymphocyte or 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, which is a peripheral nervous system neuron or a central nervous system neuron, such as a nociceptor, such as a thermal nociceptor, a mechanical nociceptor, a chemical nociceptor, or a polymorphic nociceptor. In some embodiments, the targeting molecule is bound to a pathogen or a pathogenic cell, such as a virus, bacteria, fungus, biofilm, or other prokaryotic cell system. In some embodiments, the targeting molecule is bound to a pathogen, which is a gram-negative or gram-positive bacteria.

[0288] In some embodiments, the targeting molecule (e.g., an antibody) of the phthalocyanine dye conjugate binds to a protein on the surface of a cell in the microenvironment of a lesion associated with or as a result of a disease or condition. For example, in some embodiments, the conjugate binds to a protein on the surface of a cell in a tumor microenvironment associated with a tumor. In some embodiments, the conjugate binds to a protein in the extracellular matrix of the tumor microenvironment.

[0289] As used herein, "cell present in the microenvironment of an injury" refers to any cell present in a cellular environment associated with the injury, disease, or disorder, for example, any cell present in or directly adjacent to a tumor, such as a cell present in the tumor microenvironment or in the extracellular matrix within the tumor microenvironment.

[0290] As used herein, "cells present in the tumor microenvironment" refers to any cell present in the cellular environment in which a tumor occurs, for example, any cell present in a tumor or directly adjacent to a tumor, including proliferative tumor cells (such as cancer cells), tumor stroma, blood vessels, infiltrating inflammatory cells (such as immune cells) and various related tissue cells (such as fibroblasts). It should therefore be understood that when referring to a tumor, it refers not only to tumor cells that may include malignant cells or cancer cells, but also to other cells present in the tumor microenvironment that regulate tumor growth, including immune cells. 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). It should be recognized that in some aspects, many non-cancerous cells present in or around a tumor can regulate the proliferation, angiogenesis, invasion and / or metastasis of tumor cells, thereby promoting tumor growth. Therefore, in some cases, targeting such non-cancerous cells present in tumors, such as immune cells (such as T cells, e.g., regulatory T cells) can serve as an effective therapy for killing tumors through PIT.

[0291] Typically, cancer cells containing tumor-associated antigens should be recognized by the immune system. Generally speaking, in an active immune system, immune cells such as cytotoxic T cells attack and eradicate these cancer cells. Under normal physiological conditions, the T cell-mediated immune response begins by the recognition of antigens by the T cell receptor (TCR) and is regulated by the balance of co-stimulatory signals and inhibitory signals (such as immune checkpoint proteins). In particular, CD4+ and CD8+ T cells expressing TCR can be activated by the recognition of antigenic peptides expressed 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 the antigen, and the presence of CD4+ T cells can assist this phenomenon.

[0292] However, in the case of tumor, the tumor microenvironment has a mechanism for suppressing the immune system, thereby escaping immune recognition and preventing or reducing the killing of tumor cells. For example, in some cases, as a mechanism for escaping the immune system, immune checkpoint proteins can be dysregulated in tumors, thereby leading to the suppression of the immune response in the tumor microenvironment. In some cases, tumor infiltrating lymphocytes may include Tregs (such as CD4+CD25+T cells), which are cells (Whiteside, TL (2008) Oncogene, 27:5904-5912) that can suppress other T cell proliferation in the microenvironment. In some cases, other mechanisms can play a role in preventing immune cells from reaching tumor antigens, thereby also contributing to the ability of tumors to escape the immune system.

[0293] In some embodiments, the targeting molecule is a targeting molecule that is bound to a cell surface protein on a tumor cell or cancer cell. In some embodiments, the targeting molecule is bound to a cell surface protein on an immune cell or other non-cancerous cell present in the tumor microenvironment. In some embodiments, the targeting molecule is bound to a T lymphocyte, such as a Treg, a dendritic cell, a natural killer (NK) cell, a B cell, a macrophage or other immune cells present in the tumor microenvironment. In some cases, the tumor or 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.

[0294] Exemplary targeting molecules, such as tumor or cancer targeting molecules, include, but are not limited to, any targeting molecules described in published PCT International Application Nos. WO2014120974, WO2014176284, WO2015042325, U.S. Patent No. 8,524,239, or U.S. Patent Publication No. US20140120119.

[0295] Exemplary targeting molecules include, but are not limited to, proteins, glycoproteins, antibodies, antibody fragments, antigens, antigen-binding fragments, peptides, polypeptides, small molecules, polymeric synthetic molecules, polymeric nanoparticles, liposomes, enzyme substrates, hormones, neurotransmitters, cellular metabolites, viral particles, viral capsids, viral nanoparticles, bacterial particles, markers, cells, haptens, avidin, streptavidin, monomeric streptavidin, biotin, carbohydrates, oligosaccharides, polysaccharides, nucleic acids, deoxynucleic acids, DNA fragments, RNA fragments, nucleotide triphosphates, acyclovir terminator triphosphates, or PNAs.

[0296] In some embodiments, the targeting molecule is an amino acid, peptide, protein, tyramine, polysaccharide, ion complex moiety, nucleoside, nucleotide, oligonucleotide, psoralen, drug, hormone, lipid, lipid assembly, polymer, polymer microparticle, biological cell or virus. In some embodiments, the targeting molecule is an antigen, steroid, vitamin, drug, metabolite, toxin, environmental pollutant, nucleic acid polymer, carbohydrate, lipid or glass, plastic or other non-biological polymer. In some embodiments, the targeting molecule is a cell, cell system, cell fragment or subcellular particle such as virus particle, bacterial particle, viral component, biological cell (such as animal cell, plant cell, bacterium, yeast or protozoa) or cellular component. In some embodiments, reactive dye can mark the functional group on the cell surface, in the cell membrane, in the organelle or in the cytoplasm.

[0297] In some embodiments, the targeting molecule targets an antigen, for example, any structural substance that is a target that can be bound by the targeting molecule, or binds thereto. In some embodiments, the antigen is a cell surface molecule such as a receptor that is expressed on the surface of a cell, or constitutes a part thereof. In some embodiments, for example, the antigen is a molecule expressed on the surface of a cell present in a tumor, including any cell present in the tumor microenvironment, or constitutes a part thereof. Examples of cell surface molecules include, but are not limited to, antigens, peptides, lipids, polysaccharides, carbohydrates, or nucleic acids containing antigenic determinants that are recognized, for example, by immune cells. In some embodiments, antigens include tumor-specific peptides (such as peptides visible on the surface of cancer cells) or immunogenic fragments thereof. In some embodiments, the targeting molecule is an antibody or an antigen-binding antibody fragment thereof.

[0298] In some embodiments, the cell surface molecule can be ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, α-4-integrin, α-5-β-3-integrin, α-5-β-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, Cripto-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 receptors), GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, group A (rhodopsin-like) G-protein-coupled receptors, group B (secretin receptor-like) G-protein-coupled receptors, group C (metabotropic glutamate receptor-like) G-protein-coupled receptors, GD2, GP100, GP120, glypican-3, hemagglutinin, heparan sulfate, HER1, HER2, HER3, HER4, HMFG, HPV 16 / 18 and E6 / E7 antigens, hTERT, IL-2R, 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, proteolytic cleavage of collagen IV, proteolytic enzyme 3, inhibin, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (such as P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 patch, RET receptor, plexin , smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEMs, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (such as 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.

[0299] In some embodiments, the targeting molecule is a binding partner such as a ligand that can bind to a cell surface protein, such as a cell surface receptor. In some embodiments, the targeting molecule is selected from 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 ganglion neurotrophic factor (CNTF), CCL1-CCL28, CXCL1-CXCL17, XCL1, XCL2, CX3CL1, cripto 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 7 (FGF-7), fibroblast growth factor 10 (FGF-1 0), Flt-3, gastrin, gastrin-releasing peptide (GRP), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), glucagon-like peptide-1 (GLP-1), hepatocyte growth factor (HGF), alpha interferon (IFN-a), beta interferon (IFN-b), gamma interferon (IFNg), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1), interleukin-3 (IL-3), 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), leukocytes Interleukin-17 (IL-17), interleukin-19 (IL-19), luteinizing hormone (LH), luteinizing hormone-releasing hormone (LHRH), macrophage colony-stimulating factor (M-CSF), monocyte chemoattractant 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), efenatin A1 (EFNA1), efenatin A2 (EFNA2), efenatin A3 (EFNA3), efenatin A4 (EFNA4), efenatin A5 (EFNA5), Semaphorin 3A (SEMA3A), 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 (SEMA6B), 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), Wnt1, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15 or Wnt16,Sonic hedgehog, Desert hedgehog, and Indian hedgehog.

[0300] In some embodiments, the targeting molecule can be an immunomodulator, which can bind to a cell surface molecule or protein on an immune cell that inhibits or activates the body's immune response. In some embodiments, the binding of the immunomodulator to a cell surface molecule or protein can stimulate an immune response against tumors and / or pathogens, for example, by inhibiting immunosuppression or by enhancing immunostimulation. In some embodiments, the cell surface molecule or protein can be 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, 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, sialic acid-binding immunoglobulin-like lectin (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, ILTs, LIRs), SIRPA (signal regulatory protein alpha), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR or VEGF. In some embodiments, the targeting molecule is an antibody or antigen-binding fragment, which is an immunomodulator. In some embodiments, the immunomodulator is an immune checkpoint inhibitor.

[0301] In some embodiments, the cell surface molecule can be 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), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate-binding protein, gangliosides (such as 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 or SK-1 antigen.

[0302] In some embodiments, the targeting molecule is an antibody or antigen-binding antibody fragment that specifically binds to an antigen as described below, wherein the antigen is a cell surface molecule expressed on the surface of a cell or a portion thereof. Such antibodies include antibodies or antigen-binding antibody fragments that can bind to cell surface proteins described herein, such as cell surface receptors, and other cell surface molecules. In some cases, the antibody can bind to an antigen of a protein, including a tumor-specific protein, expressed on cells in a tumor.

[0303] In some embodiments, the targeting molecule is directly or indirectly bound to an antigen or protein. For example, in some embodiments, the targeting molecule is a second binding molecule that is bound to a first binding molecule, and the first binding molecule is capable of binding to the antigen or protein. For example, the targeting molecule is a second antibody that is bound to a first binding molecule such as a first antibody, and the first binding molecule is capable of binding to a protein or antigen, such as a cell surface protein or cell surface receptor. Therefore, in some embodiments, the dye is coupled to a second antibody.

[0304] An "antibody" is a polypeptide ligand comprising at least a light chain and / or a heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen epitope. Typically, an antibody is composed of a heavy chain and a light chain, each of which has a variable region, designated as a heavy chain variable region (V H ) region and light chain variable (V L ) area. The V H Area and V L The two regions are collectively responsible for binding to the antigen recognized by the antibody. The term antibody includes intact antibodies and antigen-binding antibody fragments that have antigen binding, such as Fab fragments, Fab' fragments, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). ScFv proteins are fusion proteins in which the light chain variable region of an immunoglobulin and the heavy chain variable region of an immunoglobulin are connected by a linker, while in dsFvs, the chains are mutated to introduce disulfide bonds to stabilize the association of the chains. The term also includes conventional engineered forms, such as modified forms of immunoglobulins, chimeric antibodies such as humanized mouse antibodies, and heteroconjugate antibodies such as bispecific antibodies. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J. Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0305] In general, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain types, or isotypes, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0306] The heavy and light chains each contain a constant region and a variable region, also referred to as a "domain." When combined, the heavy and light chain variable regions typically bind specifically to an antigen. The light and heavy chain variable regions may contain a "framework" region, also referred to as a "complementarity determining region" or "CDR," separated by three hypervariable regions. The extent of framework regions and CDRs has been established (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is incorporated herein by reference). The Kabat database is currently maintained online. The sequences of the framework regions of different light and heavy chains are relatively conserved within species such as humans. The antibody framework region, formed by the combination of the framework regions of the light and heavy chain components, serves to position and align the CDRs in three-dimensional space.

[0307] CDRs are generally responsible for binding to the epitope of the antigen. The CDRs of each chain are generally referred to as CDR1, CDR2 and CDR3, which are numbered sequentially starting from the N-terminus and are usually also identified by the chain in which the specific CDR is located. H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is located, while V L CDR1 is the CDR1 of the variable domain of the light chain of the antibody in which it is located. Antibodies with different specificities, such as having different binding sites for different antigens, have different CDRs. Although CDRs vary between different antibodies, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. These positions in the CDRs are called specificity determining residues (SDRs).

[0308] Mention "V H ” or “VH” refers to the variable region of an immunoglobulin heavy chain, including the variable region of Fv, scFv, dsFv or Fab. L ” or “VL” refers to the variable region of an immunoglobulin light chain, including the variable region of Fv, scFv, dsFv or Fab.

[0309] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule that is different from an intact antibody, comprises a portion of an intact antibody, and binds to the antigen bound by the intact antibody. 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 (such as scFv); and multispecific antibodies formed from antibody fragments. Other antibody fragments or multispecific antibodies formed from antibody fragments include multivalent scFv, bispecific scFv, or scFv-CH3 dimers. Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells.

[0310] "Monoclonal antibodies" are antibodies produced by a single clone of B lymphocytes or cells transfected with the light and heavy chain genes of an antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by making hybrid antibody-forming cells from hybridoma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0311] "Chimeric antibodies" have framework residues from one species, such as human, and the CDRs that typically confer antibody binding ability from another species, such as mouse, that specifically bind to mesothelin.

[0312] A "humanized" immunoglobulin is an immunoglobulin that includes a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is called the "donor," and the human immunoglobulin that provides the framework is called the "acceptor." In some embodiments, the CDRs are from the donor immunoglobulin in the humanized immunoglobulin. The constant region need not be present, but if present, it can be substantially identical to a human immunoglobulin constant region, for example, at least about 85-90%, such as about 95% or more identical. Thus, a portion of the humanized immunoglobulin, but not the CDRs, is substantially identical to the corresponding portion of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody that comprises 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 be substituted with a limited number of amino acids taken from the donor framework. Humanized or other monoclonal antibodies may have other conservative amino acid substitutions that have little effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by genetic engineering methods (see, for example, US Pat. No. 5,585,089).

[0313] "Human" antibodies (also called "fully human" antibodies) are antibodies that include human framework regions and CDRs from human immunoglobulins. 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 engineered to include CDRs from a different human antibody. A portion of a human immunoglobulin can be substantially identical to a corresponding portion of a natural human immunoglobulin sequence.

[0314] "Specific binding" refers to the ability of a molecule such as an antibody or antigen-binding fragment to bind specifically to an antigen such as a tumor-specific antigen, as opposed to binding to unrelated proteins such as β-actin, such as non-tumor proteins. In some embodiments, a molecule such as an antibody or fragment, including a molecule such as an antibody or fragment linked to a phthalocyanine dye molecule, specifically binds to a target such as a cell surface protein with a binding constant that is at least 10 greater than the binding constant for other molecules in the sample or subject. 3 M -1 , 10 4 M -1 or 10 5 M -1 In some embodiments, a molecule such as an antibody or fragment thereof has a molecular weight greater than or equal to about 10 6 M -1 , greater than or equal to about 10 7 M -1 , greater than or equal to about 10 8 M -1 , or greater than or equal to about 10 9 M -1 , 10 10 M -1 , 10 11 M -1 or 10 12 M -1 The equilibrium binding constant (K A ). Antibodies can also be used with 10 -6 M, 10 -7 M, 10 -8 M, 10 - 10 M, 10 -11 M or 10 -12 The equilibrium dissociation constant (K D ) is characterized by. In some embodiments, the equilibrium dissociation constant (K D ) can be 1 nM or lower. K D or K AAffinity constants such as can be estimated empirically, or affinity can be determined comparatively by, for example, comparing the affinity of one antibody to another antibody for a particular antigen. For example, affinity can be readily determined using techniques known in the art, such as by competitive ELISA (enzyme-linked immunosorbent assay), or using a surface plasmon resonance apparatus such as the Biacore T100 (available from Biacore, Inc., Piscataway, NJ), radioimmunoassay using a radiolabeled target antigen, or by other methods known to those skilled in the art.

[0315] 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 that can be conjugated to the phthalocyanine dye (e.g., IR700) include, but are not limited to, cetuximab, panitumumab, zalutumumab, nimotuzumab, trastuzumab, Ado-trastuzumab emtansine, tositumomab, and trastuzumab. Rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab 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, MSB0010718C, MEDI4736, PDR001, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, Emactuzumab, CC-90002 and MNRP1685A, or antibody binding fragments thereof.

[0316] In some embodiments, the targeting molecule is a tissue-specific homing peptide. For example, in some embodiments, the homing peptide may comprise an amino acid sequence as set forth in any one 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 Cripto-1 binding polypeptide, a somatostatin receptor binding polypeptide or an inhibin binding polypeptide, an NGR polypeptide, or an iNGR polypeptide.

[0317] In some embodiments, the targeting molecule is a viral particle, such as a virus-like particle, a virus-like nanoparticle, or a viral 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 and L2 capsid proteins. In some embodiments, the targeting molecule binds to infected cells. In some embodiments, the targeting molecule is as described in WO2015042325.

[0318] In some embodiments, virus-like particles (VLPs) refer to organized capsid-like structures, for example, roughly spherical or cylindrical in shape, which contain a self-assembled ordered array of L1 or L1 and L2 capsid particles and do not include a viral genome. In some embodiments, virus-like particles are similar to real virus particles in morphology and antigenicity, but they lack viral genetic material such as viral nucleic acids, making the particles non-infectious. VLPs can be used to deliver prophylactics, therapeutics, or diagnostics, or agents such as closed-loop or linear DNA or RNA molecules to recipient cells.

[0319] In some embodiments, VLPs may have altered immunogenicity and / or antigenicity relative to wild-type VLPs. For example, VLPs may be assembled from capsid particles having variant capsid proteins that have altered immunogenicity and / or antigenicity. In some embodiments, variant capsid proteins that have altered immunogenicity and / or antigenicity refer to natural or synthetic changes, such as mutations, substitutions, deletions, PEGylations, or insertions of amino acids, thereby reducing or avoiding recognition of the capsid protein by pre-existing, for example, endogenous, viral serotype-specific antibodies. The variant capsid protein may be a human papillomavirus (HPV) L1 variant, a non-human papillomavirus L1 variant, or a papillomavirus L1 variant based on a combination of amino acids from different HPV serotypes.

[0320] In some embodiments, the VLP is a papillomavirus VLP. The VLP can be, for example, a human papillomavirus VLP derived from a virus that infects 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, VLPs derived from bovine papillomavirus, mouse papillomavirus, cottontail rabbit papillomavirus, and macaque or rhesus monkey papillomavirus particles. In some embodiments, the VLP is a bovine papillomavirus virus-like nanoparticle, such as a type 1 virus-like nanoparticle, for example, assembled from the BPV L1 capsid protein or a combination of BPV L1 and BPV L2 capsid proteins.

[0321] In some embodiments, capsid protein refers to a protein monomer, multiple of which form a capsomere oligomer. In some embodiments, capsomere refers to the basic oligomeric structural unit of the viral capsid, i.e., the protein shell that protects the viral genetic material. In some embodiments, the capsid protein can include the papillomavirus L1 major capsid protein and the papillomavirus L2 minor capsid protein. In some embodiments, the VLP contains only the L1 capsid protein, while in other embodiments, the VLP contains a mixture or combination of the L1 and L2 capsid proteins.

[0322] 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 capsid protein in the virus-like particle. In some embodiments, the percentage of L1 capsid protein in the virus-like particle is at least 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 capsid protein in the virus-like particle. For example, in some embodiments, the percentage of L2 capsid protein in the virus-like particle is at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0323] In some embodiments, the virus-like particle comprises 12 to 72 L2 proteins. In some embodiments, the virus-like particle comprises 360 L1 proteins and 12 to 72 L2 proteins. In some embodiments, the capsid proteins assemble into virus-like nanoparticles having a diameter of 20 to 60 nm. For example, the capsid proteins can assemble into virus-like nanoparticles having a diameter of at least or about 20, 25, 30, 35, 40, 45, 50, 55, or 60 nm.

[0324] 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.

[0325] In some embodiments, the targeting molecule is a DARPin (designed ankyrin repeat protein). Generally, 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, which are incorporated herein by reference in their entireties). In some embodiments, DARPins are genetically engineered antibody-mimicking proteins that possess high specificity and high binding affinity for a target protein. In some embodiments, DARPins have a structure comprising at least two ankyrin repeat motifs, for example, at least three, four, or five ankyrin repeat motifs. A DARPin can have any suitable molecular weight, depending on the number of repeat motifs. For example, a DARPin comprising three, four, or five ankyrin repeat motifs can have a molecular weight of approximately 10 kDa, approximately 14 kDa, or approximately 18 kDa, respectively.

[0326] In some embodiments, a DARPin comprises a core portion that provides structure and a target-binding portion that resides 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 varies depending on the target.

[0327] In some embodiments, the conjugate contains multiple dye residues per targeting molecule, the number of which is about 1 to about 1000, such as about 1 to about 100, about 1 to about 50, about 1 to about 25, about 1 to about 10, or about 1 to about 5. In some embodiments, the ratio of dye molecules to targeting molecules is 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 any two of the foregoing values. In some embodiments, the targeting molecule can 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 can contain more than 1000 dye molecules or less than 10 dye molecules.

[0328] 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 about 2 to about 5, such as about 2 to about 4, such as 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 per targeting molecule can be about 10 to about 1000, 10 to about 500, 50 to about 500, or 50 to about 1000. Therefore, in some embodiments, the targeting molecule can contain about 10 to about 1000 dye molecules.

[0329] In some embodiments, for example, when the targeting molecule is a VLP, a capsid protein can be coupled to more than one dye molecule. For example, a capsid protein, such as the LI or L2 capsid protein, can be coupled to 1 to 5, such as 1, 2, 3, 4, or 5, dye molecules. Thus, a capsid protein can have more than one amino acid coupled to a dye molecule. In some embodiments, a capsid protein can be coupled to 1 to 2, 1 to 3, or 2 to 3 dye molecules. Thus, a capsid protein can have 1, 2, 3, 4, or 5 different amino acids, such as lysine, arginine, and / or histidine, or other amino acids coupled to a dye molecule.

[0330] 3. Other imaging dyes

[0331] In some embodiments, the conjugate may optionally contain other dyes, such that the targeting molecule can be coupled to two or more different fluorescent dyes. For example, a conjugate containing a targeting molecule is provided, wherein the targeting molecule is coupled to a first dye and a second fluorescent dye, wherein the first dye is a phthalocyanine dye, such as any of the above-mentioned phthalocyanine dyes (e.g., IR700), and the second fluorescent dye is different from the first dye. In one aspect, the conjugate is represented by formula (II):

[0332] [D1-(L1) n ] p -A-[(L2) m -D2] o

[0333] (II)

[0334] in:

[0335] A is a targeting molecule capable of binding to cells or tissues;

[0336] L1 and L2 are linkers independently selected relative to each o or p, wherein each L1 and L2 is as defined above for L;

[0337] n and m are independently 1 or 2;

[0338] D1 is a hydrophilic phthalocyanine dye independently selected with respect to each p, wherein D1 is as defined above for D;

[0339] D2 is a fluorescent dye independently selected for each o; and

[0340] p and o are independently 1, 2, 3, 4, 5 or greater than 5, for example up to 1000. For example, p and o can each independently be 1-1000, for example typically 1-10 or 2-5.

[0341] In some embodiments, the first dye D1 is a phthalocyanine dye, such as a near-infrared (NIR) phthalocyanine dye, such as any of the dyes described above. In some embodiments, the phthalocyanine dye is or comprises a photosensitizer compound, such that it can exhibit phototoxic activity upon exposure to near-infrared light. In some embodiments, D1 is IR700.

[0342] In some embodiments, the second dye D2 is selected to provide a stronger fluorescence than the first dye D1 (such as IR700) for observation. Therefore, in some aspects, the compound of formula II is used for fluorescence imaging and photoimmunotherapy simultaneously. For example, the damage or tumor irradiated sends a fluorescent signal from the second fluorescent dye to achieve the detection of the conjugate at the damage or tumor present in the object. In some embodiments, the conjugate can be used to monitor the combination of dye and target (such as tumor) by fluorescence imaging of D2, or to eradicate cells related to disease or illness, such as tumor cells, by adopting photoimmunotherapy based on activation of D1 (such as IR700). In some embodiments, the second dye D2 shows one or more spectral properties selected from the group consisting of: (such as in water) fluorescence quantum yield, extinction coefficient, Stokes shift, absorption and emission at long wavelengths, and photostability. In some embodiments, D2 is not IR700.

[0343] In some embodiments, the additional dye D2 is a fluorescent dye. In some embodiments, D2 can be, but is 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, Cy 5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye800, 525, 565, 605, 655, 705 or 800.

[0344] In some embodiments, D2 absorbs light and emits fluorescence at a longer wavelength, while the tissue's own fluorescence is weak, substantially absent or eliminated, or absent or eliminated. Therefore, in some aspects, D2 contributes to deep tissue penetration of imaging. In some embodiments, D2 is a near-infrared (NIR) dye having absorption and emission wavelengths in the NIR spectrum of about 650 to 1450 nm, for example, about 650 to 900 nm, about 700 to 1000 nm, or about 750 to 950 nm. In some embodiments, D2 is a second near-infrared (NIR-II) dye having absorption and emission wavelengths in the NIR-II spectrum of about 1000 to 1700 nm, for example, about 1000 to 1400 nm. In some embodiments, D2 is a dye having absorption and emission wavelengths in the visible spectrum of about 300 to 750 nm, for example, about 400 to 600 nm, or about 400 to 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 long wavelength excitation and emission properties.In some embodiments, any of the provided methods can further comprise imaging the lesion or tumor in the subject by irradiating or illuminating the tumor with a wavelength that is absorbed by the second dye.

[0345] In some embodiments, the other dye has a large extinction coefficient at the excitation wavelength. In some embodiments, the extinction coefficient of D2 in water is greater than about 10,000 MoI. -1 cm -1 , for example greater than about 25,000 Mol -1 cm -1 , greater than about 50,000 Mol -1 cm -1 , greater than about 75,000 Mol -1 cm -1 , greater than about 100,000 Mol -1 cm -1 , greater than about 150,000 Mol -1 cm -1 , greater than about 200,000 Mol -1 cm -1 , greater than about 250,000 Mol -1 cm -1 or greater than about 300,000 Mol -1 cm -1 .

[0346] In some embodiments, when coupled to a protein, the second dye D2 has a higher fluorescence quantum yield than the first dye D1. In some embodiments, the additional dye has a high fluorescence quantum yield in water. In some embodiments, the second dye D2 has a quantum yield in water greater than 5%, such as greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 40%, greater than 50%, or greater.

[0347] In some embodiments, the second dye D2 has a large Stokes shift (EX max and EM max In some embodiments, the further dye D2 exhibits a Stokes shift greater than 15 nm, 20 nm, 25 nm, e.g., 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.

[0348] In some embodiments, the other dye D2 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 other dye D2 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 other dye D2 can be Alexa Fluor 488, IRDye 680, IRDye 800, or Dylight 755.

[0349] In some embodiments, the conjugate contains multiple second dye D2 residues per targeting molecule, the number of which is about 1 to about 1000, such as about 1 to about 100, about 1 to about 50, about 1 to about 25, about 1 to about 10, or about 1 to about 5. In some embodiments, the ratio of the second dye molecules to the targeting molecule is 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 any two of the foregoing values. In some embodiments, the ratio of the second dye molecules to the targeting molecule is 1 to 10 or 1 to 5 second dye molecules per targeting molecule.

[0350] II. Pharmaceutical Compositions and Products

[0351] The present invention provides a pharmaceutical composition containing a phthalocyanine dye-targeting molecule conjugate (such as an IR700-antibody conjugate). In some embodiments, the composition can be used in the PIT method described herein. The phthalocyanine dye-targeting molecule conjugate is, for example, an IR700-antibody conjugate. In some embodiments, the composition can be provided in combination with other therapeutic agents (such as immunomodulators or anticancer agents). In some embodiments, the phthalocyanine dye-targeting molecule conjugate and one or both of the other therapeutic agents, such as immunomodulators or anticancer agents, can be packaged into a product in the form of separate compositions for administration together, sequentially, or intermittently. The combination can be packaged into a medicine kit.

[0352] 1. Compositions, preparations and dosage forms

[0353] In some embodiments, the compound such as conjugate can be formulated in a pharmaceutically acceptable buffer containing, for example, a pharmaceutically acceptable carrier or carrier. Typically, a pharmaceutically acceptable carrier or carrier, such as a carrier or carrier present in a pharmaceutically acceptable buffer, can be known in the art. Remington Pharmaceutical Sciences, E.W. Martin, Mack Publishing Co., Easton, Pennsylvania, 19th edition (1995) describes compositions and preparations suitable for drug delivery of one or more therapeutic compounds. Pharmaceutically acceptable compositions are typically prepared based on the approval of regulatory agencies or other agencies, according to the pharmacopoeias generally accepted for animals and humans.

[0354] Pharmaceutical compositions may include a carrier, such as a diluent, adjuvant, excipient, or vehicle, with which the compound is administered. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, by E.W. Martin. Such compositions contain a therapeutically effective amount of the compound, typically in purified form, and an amount of a carrier to provide a form suitable for 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 for intravenous administration of the pharmaceutical composition. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The composition may contain, along with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant such as magnesium stearate, calcium stearate, and talc; and a binder such as starch, natural gums such as gum arabic, gelatin, glucose, molasses, polyvinylpyrrolidone, cellulose and its derivatives, povidone, cross-linked povidone, and other 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, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water and ethanol. If desired, the composition may also contain small amounts of wetting agents or emulsifiers or pH buffers, such as acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate and other such agents.

[0355] In some embodiments, the pharmaceutical preparation can be in liquid form, such as a solution, syrup, or suspension. Such liquid preparations can be prepared in a conventional manner using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl parabens, or sorbic acid). In some embodiments, the pharmaceutical preparation can be in lyophilized form and reconstituted with water or other suitable carriers before use.

[0356] In some embodiments, the properties of the pharmaceutically acceptable buffer or carrier depend on the specific form of administration adopted. For example, in some embodiments, parenteral formulations can include an injectable liquid as a carrier, which includes a pharmaceutically and physiologically acceptable liquid, such as water, normal saline, balanced salt solution, aqueous dextrose or glycerol. In some embodiments, for solid compositions, such as powders, pills, tablets or capsule forms, non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, in some embodiments, the pharmaceutical composition administered can contain a minimum amount of non-toxic adjuvants, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0357] The compound can be formulated into a suitable pharmaceutical formulation, such as a solution, suspension, tablet, dispersible tablet, pill, capsule, powder, sustained-release formulation or elixir for oral administration, as well as a transdermal patch formulation and a dry powder inhaler. In general, the compound is formulated into a pharmaceutical composition using techniques and processes well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, 4th edition, 1985, 126). Typically, the mode of preparation is a function of the route of administration.

[0358] The compositions 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, topical, local, otic, inhalation, buccal (e.g., sublingual), and transdermal administration or any route. Other modes of administration are also contemplated. Administration can be local, topical, or systemic, depending on the treatment site. Local administration to the area in need of treatment can be achieved, for example, but not limited to, by local infusion during surgery, topical application in conjunction with, for example, a wound dressing after surgery, by injection, by catheter, by suppository, or by implant.

[0359] Parenteral administration, typically characterized by injection, can be considered herein, and the injection can be subcutaneous, intramuscular, intratumoral, intravenous, or intradermal. Injections can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for preparing solutions or suspensions in liquids before injection, or 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 an activator in the form of a solvent, such as a pH buffer, a metal ion salt, or other such buffer. The pharmaceutical composition can also contain a small amount of other non-toxic excipient substances, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins. Also contemplated herein are extended-release or sustained-release systems that maintain the dose at a constant level (see, for example, U.S. Patent No. 3,710,795). The percentage of active compound contained in such parenteral compositions is highly dependent upon the specific nature thereof, as well as the activity of the compound and the needs of the subject.

[0360] Injection is designed to be used for local and systemic administration. The preparation of parenteral administration comprises sterile solution for injection, sterile dry soluble product such as lyophilized powder, for being combined with solvent before use, comprises subcutaneous injection tablet, sterile suspension for injection, for being combined with sterile dry insoluble product and sterile emulsion of carrier before use.Solution can be aqueous or non-aqueous.If it is intravenous administration, then suitable carrier comprises physiological saline or phosphate buffered saline (PBS) and the solution of thickening agent and solubilizing agent containing glucose, polyethylene glycol and polypropylene glycol and their mixture etc.

[0361] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobials, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, metal chelators or chelating agents, and other pharmaceutically 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. Antimicrobials can be added to parenteral formulations packaged in multidose containers at bacteriostatic or fungistatic concentrations and include phenol or cresols, mercury, benzyl alcohol, chlorobutanol, methyl and propyl parahydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates.

[0362] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0363] The composition can be formulated for single or multiple dose administration. The medicament can be formulated for direct administration. The composition can be provided as a liquid or a lyophilized formulation. When the composition is provided in lyophilized form, it can be reconstituted with a suitable buffer, such as sterile saline solution, immediately prior to use.

[0364] The composition can also be administered sequentially, intermittently or in the same composition as other biologically active agents. Administration can also include controlled release systems, including controlled release formulations and controlled release devices, such as administration by pumps.

[0365] The most appropriate route in any given case depends on a variety of factors, such as the nature of the disease, the progression of the disease, the severity of the disease, and the specific composition used. For example, the composition is administered systemically, such as by intravenous administration. Subcutaneous administration can also be used, although this requires more absorption time to ensure equivalent bioavailability as intravenous administration.

[0366] Pharmaceutical compositions can be formulated into dosage forms suitable for various routes of administration. Pharmaceutically and therapeutically active compounds and their derivatives are generally formulated and administered in unit dosage forms or multi-dose dosage forms. Each unit dose contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect, as well as the necessary pharmaceutical carriers, vehicles, or diluents. Unit dosage forms include, but are not limited to, tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, and oil-in-water emulsions containing an appropriate amount of the compound or its pharmaceutically acceptable derivatives. Unit dosage forms can be contained in ampoules and syringes, or individually packaged tablets or capsules. Unit dosage forms can be administered in partial form or in multiple forms. Multi-dose dosage forms are multiple identical unit dosage forms packaged in a single container that are administered in separate unit dosage forms. Examples of multi-dose dosage forms include medicine bottles, vials of tablets or capsules, or bottles containing pints or gallons. Thus, multi-dose dosage forms are multiple unit dosage forms that are not packaged separately. Typically, a dosage form or composition can be prepared containing the active ingredient in an amount ranging from 0.005% to 100%, with the balance consisting of non-toxic carriers. The pharmaceutical composition can be formulated into dosage forms suitable for various administration routes.

[0367] The concentration of the pharmaceutically active compound is adjusted so that a single injection can provide an effective amount to produce the desired pharmacological effect. As known in the art, the exact dosage depends on the age, weight and condition of the patient or animal. Unit dose parenteral formulations are packaged in ampoules, medicine bottles or syringes with needles. The volume of the pharmaceutically active compound liquid solution or reconstituted powder formulation is a function of the disease to be treated and the specific product selected for packaging. As known and practiced in the art, all parenteral formulations must be sterile. In some embodiments, the composition can be provided as a lyophilized powder, which can be reconstituted into solutions, emulsions and other mixtures for administration. It can also be reconstituted and formulated as a solid or gel. Lyophilized powder can be prepared from any of the above solutions.

[0368] The sterile lyophilized powder can be prepared by dissolving the phthalocyanine dye-targeting molecule conjugate in a buffer solution. The buffer solution may contain excipients that enhance the stability of the powder or other pharmacological components of a reconstituted solution prepared from the powder.

[0369] In some embodiments, subsequent sterile filtration of the solution and subsequent freeze-drying under standard conditions known to those skilled in the art can provide the desired formulation. In short, the lyophilized powder is prepared by the following method: excipients such as dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other suitable agents are dissolved in a suitable buffer known to those skilled in the art, such as citrate, sodium phosphate or potassium phosphate, or other such buffers. Next, the selected enzyme is added to the resulting mixture and stirred until it dissolves. The resulting mixture is sterile filtered or treated to remove particles and ensure sterility, and is dispensed into vials for freeze-drying. Each vial can contain a single dose (1 mg to 1 g, typically 1 to 100 mg, such as 1 to 5 mg) or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as about 4°C to room temperature. Reconstitution of the lyophilized powder with a buffer solution provides a formulation for parenteral administration. The exact amount depends on the indication being treated and the compound selected. The amount can be determined empirically.

[0370] In some embodiments, the pH of the composition is about 6 to 10, such as about 6 to 8, about 6.9 to 7.3, such as about pH 7.1. In some embodiments, the pH of the pharmaceutically acceptable buffer is at least or about 5, at least or about 6, at least or about 7, at least or about 8, at least or about 9, or at least or about 10, or is 7.1.

[0371] The composition can be formulated for single dose administration or multiple dose administration.The medicament can be formulated for direct administration.

[0372] In some embodiments, the composition provided by the present invention is formulated to be about 0.01 mg to about 3000 mg, 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 100 mg, about 0.1 mg to about 50 The dosage range is preferably from 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 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 can be 0.5mL to 1000mL, such as 0.5mL to 100mL, 0.5mL to 10mL, 1mL to 500mL, 1mL to 10mL, such as at least or about 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 a single dose administration of about 100mg to 500mg or about 200mg to 400mg. In some embodiments, the composition is formulated for a single dose administration of about 500mg to 1500mg, 800mg to 1200mg or 1000mg to 1500mg. In some embodiments, the volume of the composition is about 10 mL to 1000 mL or 50 mL to 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.

[0373] In some embodiments, the entire vial contents of the formulation can be removed for administration, or can be divided into multiple doses for multiple administrations. After removing a certain amount of the drug for administration, the formulation can be further diluted as needed, for example in water, saline (e.g., 0.9%), or other physiological solutions.

[0374] In some embodiments, compositions containing immunomodulators or anticancer agents are also provided, which can be prepared according to the known or standard formulation guidelines described above. In some embodiments, the immunomodulator, anticancer agent and / or phthalocyanine dye-targeting molecule conjugate (such as IR700-targeting molecule, for example IR700-antibody conjugate) are formulated into separate compositions. In some embodiments, the immunomodulator is provided as a composition separate from the phthalocyanine dye-targeting molecule conjugate, and the two compositions are administered separately. In some embodiments, the anticancer agent is provided as a composition separate 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 phthalocyanine dye-targeting molecule conjugates, immunomodulators and / or anticancer agents can be administered by different routes of administration.

[0375] 3Packaging and products

[0376] The present invention also provides an article of manufacture comprising packaging material, any pharmaceutical composition or combination provided herein, and labeling indicating that the composition or combination is used to treat cancer. Exemplary articles of manufacture include single-chamber containers and dual-chamber containers. Such containers include, but are not limited to, tubes, bottles, and syringes. The container may also include a needle for subcutaneous administration.

[0377] In some embodiments, the pharmaceutical agents can be provided separately for packaging into a product. In some embodiments, the product comprises a pharmaceutical composition comprising a phthalocyanine dye-targeting molecule conjugate, such as an IR700-antibody conjugate, and an immunomodulator. In some embodiments, the product comprises a pharmaceutical composition comprising a phthalocyanine dye-targeting molecule conjugate (such as an IR700-antibody conjugate) and an anticancer agent. In some embodiments, the product comprises a pharmaceutical composition comprising a phthalocyanine dye-targeting molecule conjugate (such as an IR700-antibody conjugate), an immunomodulator, and an anticancer agent.

[0378] Goods provided by the present invention comprise packaging materials. The packaging materials used for packaging pharmaceutical products are well known to those skilled in the art. Referring to, for example, U.S. Patents 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein by reference in its entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, medicine bottles, containers, syringes, bottles and any packaging materials suitable for selected preparations and the desired mode of administration and treatment. The selection of packaging depends on the medicament. Generally speaking, the packaging does not react with the composition contained therein.

[0379] The ingredients can be packaged in the same or different containers. For example, in some embodiments, the ingredients are packaged separately in the same container. Generally, examples of such containers include: a container having a closed, confined space containing a phthalocyanine dye-targeting molecule conjugate, and a container having a closed, confined space separately containing other ingredients, so that subsequent areas are separated and the ingredients can be administered separately. Any container or other product can be considered, as long as the agent is separated from the other ingredients before administration. As a suitable embodiment, see, for example, the containers described in U.S. Patents 3,539,794 and 5,171,081. In some embodiments, a plurality of containers are provided, each container containing a phthalocyanine dye-targeting molecule conjugate, an immunomodulator or an anticancer agent. In this example, a plurality of containers can be packaged together into a medicine box.

[0380] In some embodiments, the container containing the phthalocyanine dye-targeting molecule conjugate is contained within a light-proof container. In some embodiments, the container is a medicine vial, such as a depyrogenated glass medicine vial. In some embodiments, the container, such as a medicine vial, blocks specific wavelengths of light, such as wavelengths 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 light of certain wavelengths or intensities. For example, in some embodiments, the container has a light transmittance of no greater than 50%, no greater than 40%, no greater than 30%, no greater than 20%, no greater than 10%, no greater than 5%, or no greater than 1%. In some embodiments, the container prevents the transmission of light having a wavelength of about 500 nm to 725 nm, such as about 650 nm to 725 nm, or prevents the transmission of light having an intensity exceeding 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 wrapped in an opaque material such as foil, such as aluminum foil. In some embodiments, the container is sterile or depyrogenated.

[0381] In some embodiments, the conjugate is provided in multiple sealable containers. For example, the containers can each independently 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 total amount of the conjugate in the multiple sealable containers is about 100 mg to 1500 mg or 100 mg to 1200 mg. In some embodiments, the total amount of the conjugate in the multiple sealable containers is about 100 mg to 500 mg, about 200 mg to 400 mg, about 500 mg to 1500 mg, about 800 mg to 1200 mg, or about 1000 mg to 1500 mg.

[0382] In some embodiments, the article of manufacture comprises packaging material and a label or package insert comprising instructions for combining the contents of multiple vials to create a single-dose formulation of the composition.

[0383] In some embodiments, the container is also packaged to protect the contents from light. In some embodiments, a packaging system is provided, comprising an inner packaging material comprising a container containing a phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-targeting molecule conjugate, such as an IR700-antibody conjugate), and optionally comprising a container containing an immunomodulatory agent or an anticancer agent. In some embodiments, the inner packaging material has a light transmittance of less than 20%, such as less than 15%, less than 10%, less than 5%, or less than 1%. In some embodiments, the packaging system comprises an outer packaging material comprising the inner packaging material. In some embodiments, the outer packaging material has a light transmittance of less than 20%, such as less than 15%, less than 10%, less than 5%, or less than 1%. In some embodiments, the inner or outer packaging material comprises an opaque foil, such as aluminum foil. In some embodiments, the second packaging material is an aluminum bag. In some embodiments, the outer packaging material comprises cardboard.

[0384] In some embodiments, the inner and / or outer packaging material is suitable for storage of the conjugate. In some embodiments, the inner and / or outer packaging material is suitable for shipping of the conjugate.

[0385] The selected compositions (including their preparations) can also be provided as kits. The kits can include the pharmaceutical compositions described herein and a device for administration provided as the preparations. The kits can optionally include instructions for use containing dosage, dosing regimen, and mode of administration. The kits can also include the pharmaceutical compositions described herein and a diagnostic device.

[0386] In some embodiments, the composition for administering pharmaceutical agents such as phthalocyanine dye-targeting molecule conjugates contains an effective amount of each pharmaceutical agent, along with conventional pharmaceutical carriers and excipients suitable for the type of administration contemplated.

[0387] In some embodiments, a single dose of a medicament such as a phthalocyanine dye-targeting molecule conjugate is contained in a container, for example, a container for storing the medicament. In some embodiments, a single dose of the medicament is contained in multiple containers. Therefore, in some embodiments, multiple containers, such as medicine bottles, are combined in a container for administration of the medicament, such as an intravenous (IV) injection bag. In some embodiments, the container for administration, such as an IV injection bag, is prepared as follows: one or more containers containing the medicament are opened and the contents are placed in the bag, for example, until the dose of the medicament required for administration such as an infusion is reached. During the preparation of the administration container such as the IV bag, light protection measures, such as the various light protection measures described herein, are used to avoid exposure of the medicament to light.

[0388] III. Treatment Methods

[0389] In some embodiments, methods of using compositions containing phthalocyanine dye-targeting molecule conjugates (e.g., IR700-targeting molecule conjugates, such as IR700-antibody conjugates) and their use in PIT are provided. In some embodiments, the phthalocyanine dye-targeting molecule conjugate targets cells or pathogens associated with a disease or condition, for example, by binding to cell surface proteins or cell surface receptors expressed on the cells. Such methods and uses include therapeutic methods and uses, for example, involving administering a molecule to a subject suffering from a disease, condition, or disorder, followed by irradiation for photoimmunotherapy (PIT), thereby causing photolysis of such cells or pathogens to achieve treatment of the disease or disorder. In some embodiments, the methods can be used to treat tumors or cancer, wherein the administered phthalocyanine dye-targeting molecule conjugate (IR700-targeting molecule conjugate, such as IR700-antibody conjugate) targets cells associated with the tumor, thereby causing photolysis of such cells and, in some cases, achieving treatment of the tumor. Uses include the use of the compositions in such methods and treatments, as well as the use of the compositions in the preparation of medicaments for implementing such treatment methods. In some embodiments, the methods and uses can thereby treat a disease or condition or disorder, such as a tumor or cancer, in a subject.

[0390] In some embodiments, the method comprises administering a phthalocyanine dye-targeting molecule conjugate (such as an IR700-antibody conjugate) to a subject under conditions in which the target cells to be killed are usually in contact with the conjugate. In some embodiments, the method results in the binding of the targeting molecule (such as an antibody) portion of the conjugate to a cell surface protein associated with a tumor or cancer. After contact or administration of the conjugate, the local area of ​​the object containing target cells such as tumor-associated cells is exposed or irradiated with light absorbed by the dye, usually NIR light, to activate the conjugate to achieve specific cell killing. In some embodiments, the conjugate is administered at a wavelength of 600 nm to 850 nm at a rate of at least 1 J cm -2 In some embodiments, the method for administering the phthalocyanine dye-targeting molecule conjugate (such as IR700-antibody conjugate) includes the method described in U.S. Patent No. 8,524,239 or U.S. Patent Publication No. US2014 / 0120119.

[0391] A. Tumors and Subjects to be Treated

[0392] In some embodiments, the lesion is a tumor. In some embodiments, the tumor is 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, cancer may 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 adjacent cells, release of abnormal levels of cytokines or other secretory products, suppression or exacerbation of inflammation or immune response, invasion of surrounding or distant tissues or organs such as lymph nodes, etc. Metastatic disease may refer to cancer cells leaving the original tumor site and migrating to other parts of the body through, for example, the bloodstream or lymphatic system. In some embodiments, the target cell of the method disclosed herein is a cancer cell or an immune cell. In some embodiments, the cancer cell is a cancer stem cell. In some embodiments, the target cell of the method disclosed herein is a cancer cell, a tumor cell, an inflammatory cell, an immune cell, a neuron, a stem cell, a proliferative cell, or a cell in proliferation.

[0393] The target cells can be unwanted cells or cells whose growth is undesirable, such as tumors or cancer cells. In some embodiments, the cells can be grown in culture or present in a mammal to be treated, such as a subject suffering from cancer. Any target cell can be treated using the methods claimed in the present invention. In some embodiments, the target cells express cell surface proteins that are essentially not found on the surface of other normal cells. In some embodiments, antibodies that specifically bind to such proteins can be selected, and phthalocyanine dye-antibody conjugates can be produced 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.

[0394] In some embodiments, the tumor cell is a cancer cell, such as a cell in a subject suffering from cancer. Exemplary cells that can be targeted in the methods disclosed herein include cells of the following tumors: liquid tumors, such as leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia and myeloblasts, promyelocytic cells, myelomonocytic cells, monocytic and erythroid leukemias), chronic leukemias (e.g., chronic myeloid (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 cell is a solid tumor cell, such as a sarcoma or carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic and other sarcomas, synovial tumor, 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, cervix), In some embodiments, the cancer is squamous cell carcinoma of the head and neck.

[0395] Exemplary tumors such as cancers that can be treated using the methods claimed herein include solid tumors, such as breast cancer, such as lobular carcinoma and ductal carcinoma, sarcomas, lung cancer, such as non-small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma, lung mesothelioma, colorectal adenocarcinoma, gastric cancer, prostate cancer, ovarian cancer, such as serous cystadenocarcinoma and mucinous cystadenocarcinoma, ovarian germ cell tumor, testicular cancer, and germ cell tumor, pancreatic cancer, bile duct adenocarcinoma, hepatocellular carcinoma, bladder cancer, such as transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma, renal cell adenocarcinoma, endometrial cancer, such as adenocarcinoma and mixed paramectomycin tumor (carcinosarcoma), Endocervical, ectocervical and vaginal cancers such as adenocarcinomas and squamous cell carcinomas thereof, skin tumors such as squamous cell carcinomas, basal cell carcinomas, malignant melanomas, skin adnexal tumors, Kaposi's sarcoma, cutaneous lymphocytic tumors, skin adnexal tumors and various sarcomas and Merkel cell carcinomas, esophageal cancer, nasopharyngeal and oropharyngeal cancers including squamous cell carcinomas and adenocarcinomas thereof, salivary gland cancers, brain and central nervous system tumors such as tumors of glial, neuronal and meningeal origin, peripheral nerve tumors, soft tissue sarcomas and bone and chondrosarcomas, lymphatic system tumors including B-cell and T-cell malignant lymphomas. In some embodiments, the tumor is an adenocarcinoma.

[0396] The method can also be used to treat liquid tumors, such as lymphocytic, leukocytic or other types of leukemia. In some embodiments, the tumor to be treated is a blood tumor, for example, a leukemia such as acute lymphocytic 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, lymphomas such as Hodgkin lymphoma and non-Hodgkin lymphoma, and myeloma.

[0397] In some embodiments, the conjugate targets a protein expressed on the surface of a lesion or on the surface of cells present in the lesion microenvironment. For example, in some embodiments, the conjugate targets a protein expressed on the surface of cells in a tumor or on the surface of cells present in the tumor microenvironment. Exemplary such cell surface proteins are any of the proteins described herein, including those described above.

[0398] In some embodiments, the protein on the cell surface of the target cell being targeted is not present in significant amounts on other cells. For example, the cell surface protein may be a receptor that is only seen on the target cell type.

[0399] In some embodiments, proteins expressed in tumors, such as tumor-specific proteins, can be HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, cancer antigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate-binding protein, gangliosides (such as 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, 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 the proteins described above.

[0400] In some embodiments, the cell surface protein is associated with a tumor, for example, a tumor-specific protein or tumor-specific antigen, such as members of the EGF receptor family (such as HER1, 2, 3, and 4) and cytokine receptors (such as CD20, CD25, IL-13R, CD5, CD52, etc.). In some embodiments, the tumor-specific protein is a protein that is unique to or more abundant in cancer cells compared to other cells such as normal cells. For example, HER2 is common in breast cancer, while HER1 is common in adenocarcinomas and can be found in many organs such as the pancreas, breast, prostate, and colon.

[0401] Targeting molecules, such as antibodies or antibody fragments, that are visible in target cells and specific for the protein can be used to formulate exemplary tumor-associated proteins of phthalocyanine dye-antibody conjugates, including but not limited to: any MAGE (melanoma-associated antigen E), including MAGE 1, MAGE 2, MAGE 3 and MAGE 4, any tyrosinase, mutant ras, mutant p53, p97 melanoma antigen, human milk fat globule (HMFG) that may be associated with breast tumors, any BAGE (human B melanoma-associated antigen E), including BAGE1 and BAGE2, any GAGE ​​(G antigen), including GAGE1, GAGE2-6, various gangliosides, and CD25.

[0402] Other tumor-associated proteins include HPV 16 / 18 and E6 / E7 antigens associated with cervical cancer, mucin (MUC 1)-KLH antigen, which may be associated with breast cancer, CEA (carcinoembryonic antigen), which may be associated with colorectal cancer, gp100, which may be associated with, for example, 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 or other cancers, alpha-fetoprotein (AFP), which may be associated with liver cancer, Lewis Y antigen, which may be associated with colorectal, bile duct, breast, small cell lung and other cancers, tumor-associated glycoprotein 72 (TAG72), which may be associated with adenocarcinoma, and the PSA antigen, which may be associated with prostate cancer.

[0403] Other exemplary tumor-associated proteins include, but are not limited to, PMSA (prostate-specific membrane antigen), which may be associated with solid tumor neovasculature and prostate cancer; HER-2 (human epidermal growth factor receptor 2), which may be associated with breast, ovarian, gastric, and uterine cancers; HER-1, which may be associated with lung, anal, glioblastoma, and adenocarcinomas; NY-ESO-1, hTERT (aka telomerase), protease 3, and Wilms' tumor 1 (WT-1), which may be associated with melanoma, sarcoma, testicular, and other cancers.

[0404] In some embodiments, the tumor-associated protein is CD52, which may be associated with chronic lymphocytic leukemia, CD33, which may be associated with acute myeloid leukemia, or CD20, which may be associated with non-Hodgkin's lymphoma.

[0405] Therefore, the methods disclosed herein can be used to treat any cancer that expresses a tumor-specific protein. In some embodiments, the tumor therapeutic agent is an antibody, an antigen-binding fragment, a protein, a glycoprotein, a peptide, a polypeptide, a virus, a viral capsid, or a viral particle. In some embodiments, the tumor therapeutic agent is an antibody or an antigen-binding fragment.

[0406] In some embodiments, the subject is a human or non-human mammal. In some embodiments, the subject is a human or veterinary subject, such as a mouse. In some embodiments, the subject is a mammal, such as a human suffering from or seeking treatment for cancer. In some embodiments, the methods disclosed herein are used to treat a subject having a tumor, such as a tumor described herein. In some embodiments, the tumor has been previously treated, such as surgically or chemically removed, and the methods disclosed herein are subsequently used to kill any unwanted residual tumor cells that may remain in the subject.

[0407] The methods disclosed herein can be used to treat any mammalian subject, such as a human with a tumor such as cancer or one that has previously undergone removal or treatment. Subjects requiring the therapies disclosed herein can include human subjects with cancer, wherein the cancer cells express tumor-specific proteins on their surfaces that are capable of specifically binding to the phthalocyanine dye-targeting molecule conjugate. For example, the methods disclosed herein can be used alone or in combination with radiation or other chemotherapy as initial treatment for cancer. The methods disclosed herein can also be used for patients whose previous radiation or chemotherapy has failed. Thus, in some embodiments, the subject is a subject who has received other therapies, but which did not provide the desired therapeutic response. The methods provided herein can also be used for patients with localized and / or metastatic cancer.

[0408] In some embodiments, the method includes selecting a subject who would benefit from a therapy disclosed herein, such as selecting a subject having a tumor that expresses a cell surface protein, such as a tumor-specific protein that can specifically bind to a phthalocyanine dye-targeting molecule conjugate. For example, if the subject is diagnosed with breast cancer that expresses HER1, the subject can be selected for treatment with an anti-HER1-IR700 molecule, such as cetuximab-IR700.

[0409] B. Dosage and Administration

[0410] The compositions provided herein containing phthalocyanine dye-targeting molecule conjugates (e.g., IR700-antibody conjugates) are administered in an amount sufficient to produce a therapeutically useful effect. Generally, 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 alleviates observed side effects compared to the dose and amount required for a single treatment with one of the aforementioned agents.

[0411] For patients who require administration of a phthalocyanine dye-targeting molecule conjugate (such as an IR700-antibody conjugate) alone or in combination with one or more other agents, the optimal dose of the phthalocyanine dye-targeting molecule conjugate (such as an IR700-antibody conjugate) can be determined through standard dose-response and toxicity studies known in the art.

[0412] The amount of therapeutic agents such as phthalocyanine dye-targeting molecule conjugates (such as IR700-antibody conjugates) administered to human or animal subjects depends on a number of factors related to the subject, such as the subject's overall health. 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 by various in vitro, in vivo, or ex vivo immunoassays. In some embodiments, the agents disclosed herein 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 of implementation, the subject to be treated, the severity and type of the symptoms to be treated, and the mode of administration.

[0413] In some embodiments, a therapeutically effective amount is an amount of the composition alone or in combination with other therapeutic agents such as chemotherapeutic agents, sufficient to achieve the desired effect in the subject or cell being treated with the composition. The therapeutically effective amount of a therapeutic agent such as a phthalocyanine dye-targeting molecule conjugate (such as an IR700-antibody conjugate) can depend on a variety of factors, including but not limited to: the subject or cell 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 an amount or concentration sufficient to prevent the progression of metastasis, slow the progression, or cause the regression of the disease, or to alleviate the symptoms caused by the disease, such as cancer. In some embodiments, a therapeutically effective amount or concentration is an amount or concentration sufficient to increase the survival time of a patient suffering from a tumor.

[0414] In some embodiments, the therapeutically effective dose of the conjugate is about 10 mg / m 2 ~5000mg / m 2 , for example, about 10 mg / m 2 ~3000 mg / m 2 , 10mg / m 2 ~1500 mg / m 2 , 10mg / m 2 ~750 mg / m 2 , 10mg / m 2 ~500 mg / m 2 , 10mg / m 2 ~250 mg / m 2 , 10mg / m 2 ~200 mg / m2 、10mg / 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 / m 2 ~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 / m2 , 200mg / m 2 ~5000mg / m 2 , 200mg / m 2 ~3000 mg / m 2 , 200mg / m 2 ~1500 mg / m 2 , 200mg / m 2 ~750 mg / m 2 , 200mg / m 2 ~500 mg / m 2 , 200mg / m 2 ~250 mg / m 2 , 250mg / m 2 ~5000mg / m 2 , 250mg / m 2 ~3000 mg / m 2 , 250mg / m 2 ~1500 mg / m 2 , 250mg / m 2 ~750 mg / m 2 , 250mg / m 2 ~500 mg / m 2 , 500mg / m 2 ~5000mg / m 2 , 500mg / m 2 ~3000 mg / m 2 , 500mg / m 2 ~1500 mg / m 2 , 500mg / m 2 ~750 mg / m 2 , 750mg / m 2 ~5000mg / m 2 , 750mg / m 2 ~3000 mg / m 2 , 750mg / m 2 ~1500 mg / m 2 , 1500mg / m 2 ~5000mg / m 2 , 1500mg / m 2 ~3000 mg / m 2 and 3000 mg / m 2 ~5000mg / m 2 In some embodiments, the therapeutically effective dose of the conjugate is no greater than 10 mg / m 2 , 50mg / m 2 , 75mg / m 2, 100mg / m 2 , 150mg / m 2 , 200mg / m 2 , 225mg / m 2 , 250mg / m 2 , 300mg / m 2 , 400mg / m 2 , 500mg / m 2 , 600mg / m 2 , 700mg / m 2 , 800mg / m 2 , 900mg / m 2 , 1000mg / m 2 , 1250mg / 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 about 50 mg / m 2 ~about 5000 mg / m 2 , about 250mg / m 2 ~about 2500 mg / m 2 , about 750mg / m 2 ~about 1250 mg / m 2 or about 100 mg / m 2 ~about 1000 mg / m 2 In some embodiments, the dose is about 160 mg / m 2 , 320mg / m 2 , 640mg / m 2 or 1280 mg / m 2 .

[0415] In some embodiments, the therapeutically effective dose of the conjugate is about 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 2.5 mg / kg, 2.5 mg / kg to 150 mg / kg, 2.5 mg / kg to 100 mg / kg, 2.5 mg / kg to 75 mg / kg, 2.5 mg / kg to 60 mg / kg, 2.5 mg / kg to 50 mg / kg, 2.5 mg / kg to 25 mg / kg, 2.5 mg / kg to 10 mg / kg, 2.5 mg / kg to 7.5 mg / kg, 2.5 mg / kg to 5.0 mg / kg, 5.0 mg / kg to 150 mg / kg, 5.0 mg / kg to 100 mg / kg, 5.0 mg / kg to 75 mg / kg, 5.0 mg / kg to 60 mg / kg, 5.0 mg / kg to 50 mg / kg, 5.0 mg / kg to 25 mg / kg, 5.0 mg / kg to 10 mg / kg, 5.0 mg / kg to 7.5 mg / kg, 7.5 mg / kg to 150 mg / kg, 7.5 mg / kg to 100 mg / kg, 7.5 mg / kg to 75 mg / kg, 7.5 mg / kg to 60 mg / 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, 2 5mg / kg~60mg / kg, 25mg / kg~50mg / kg, 50mg / kg~150mg / kg, 50mg / kg~100mg / kg, 50mg / kg~75mg / kg, 50mg / kg~60mg / kg, 60 mg / kg~150mg / kg, 60mg / kg~100mg / kg, 60mg / kg~75mg / kg, 75mg / kg~150mg / kg, 75mg / kg~100mg / kg and 100mg / kg~150mg / kg. In some embodiments, the therapeutically effective dose of the conjugate is no greater than 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, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, or 150 mg / kg.

[0416] In some embodiments, the therapeutically effective amount is at least about 0.01 milligram (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.

[0417] In some embodiments, the method comprises administering a therapeutically effective amount of a phthalocyanine dye-targeting molecule conjugate, such as an IR700-antibody conjugate, to a subject suffering from a disease or condition. In some embodiments, the phthalocyanine dye-targeting molecule conjugate targets cells present in the microenvironment of a tumor, lesion, or hyperplasia. In some embodiments, the therapeutically effective amount of the conjugate is administered intravenously. In some embodiments, the therapeutically effective amount of the conjugate is administered intratumorally.

[0418] In some embodiments, the dose of the conjugate is at least 10 μg / kg, such as at least 100 μg / kg, at least 500 μg / kg, or at least 500 μg / kg, for example, at a dose of 10 μg / kg to 1000 μg / kg, such as about 100 μg / kg, about 250 μg / kg, about 500 μg / kg, about 750 μg / kg, or about 1000 μg / kg, for example, when administered intratumorally or intraperitoneally (IP). In some embodiments, for example when administered in a topical solution, the dose is at least 1 μg / ml, such as at least 500 μg / ml, such as 20 μg / ml to 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.

[0419] In some embodiments, the therapeutically effective dose is a dose administered to a human. In some embodiments, the average human body weight is 60-85 kg, such as about 75 kg.

[0420] In some embodiments, a therapeutically effective dose is an amount of a conjugate comprising a phthalocyanine dye conjugated to a targeting molecule (e.g., an antibody or antigen-binding antibody fragment) that is administered to achieve 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, such as the systemic exposure achieved by administering a clinically acceptable dose of the drug targeting molecule alone.

[0421] The term "systemic exposure" refers to the actual body exposure of the drug targeting molecule in the plasma (blood or serum) after administration of the drug targeting molecule, which can be expressed as the area under the plasma drug concentration-time curve (AUC), which is determined by pharmacokinetic analysis after administration of the drug targeting molecule. In some cases, AUC is expressed in mg*h / L or its corresponding units (such as μg*h / L). In some embodiments, AUC is measured as the average AUC in a patient population, such as a sample patient population, for example, the average AUC of one or more patients. In some embodiments, systemic exposure refers to the area under the curve (AUC) in the range of 0 to infinity (inf or ∞). 0-∞ or AUC[0-inf]), including all measured data and data extrapolated from measured pharmacokinetic (PK) parameters, such as the mean AUC from a patient population, such as a sample patient population. In some embodiments, AUC is predicted based on one month of PK information. 0-∞In some embodiments, systemic exposure refers to the AUC (AUC) within the range of 0 to the last experimental determination time point. 0-最后 In some embodiments, the systemic exposure is the exposure (AUC) that occurs during photoirradiation or irradiation, because PIT depends on the dose or amount of the conjugate in the tumor at the time of the photoirradiation or irradiation. In some embodiments, the photoirradiation or irradiation is performed within a range of about 24 hours ± 3 hours, such as 24 hours ± 2 hours after administration of the conjugate. Therefore, in some embodiments, the systemic exposure refers to the area under the curve (AUC) within the range of 0 to 24 hours (AUC). 0-24 In some embodiments, systemic exposure refers to the mean area under the curve (AUC) from a patient population, such as a sample patient population, over the range of 0 to 24 hours. 0-24 or AUC[0-24]).

[0422] In some embodiments, a therapeutically effective dose is an amount of a conjugate comprising a phthalocyanine dye conjugated to a targeting molecule (e.g., an antibody or antigen-binding antibody fragment) that is administered to achieve a systemic exposure based on AUC 0-24 The AUC values ​​were significantly lower than those of clinically acceptable doses of the targeting molecule not so conjugated. 0-∞ In some embodiments, this is because the therapeutically effective systemic exposure of the conjugate achieved during PIT (light irradiation or radiation) is the relevant period for the above-mentioned PIT activity. In contrast, in some cases, for a targeting molecule that is not so conjugated (such as unconjugated cetuximab) to achieve a therapeutically effective systemic exposure, for example, to achieve pharmacological activity, the targeting molecule (such as an antibody) must be present at a much higher exposure. For example, according to the FDA guidance for Erbitux (cetuximab), the antibody is administered at 400 mg / m 2 250 mg / m2 per week 2 In some cases, patients may require continuous treatment with an unconjugated targeting molecule (e.g., antibody) for more than one month.

[0423] The term "therapeutically effective systemic exposure" refers to the systemic exposure that is considered to be clinically acceptable pharmacological activity and / or to achieve a therapeutic effect while having an acceptable safety profile achieved by a drug targeting molecule (such as an antibody). With the level of skill in the art, it is possible to determine or decide the dosage of a drug targeting molecule (such as an antibody) that is clinically acceptable and / or to achieve a therapeutic effect while having an acceptable safety profile. In some embodiments, the clinically acceptable dose of the drug targeting molecule is determined as a result of a clinical trial in animals, especially humans, which is implemented, for example, by the Food and Drug Administration (FDA) or other regulatory agencies (such as EMA, PDMA). In some embodiments, the therapeutically effective systemic exposure includes the systemic exposure resulting from a single dose of a drug targeting molecule (such as an antibody) or from repeated administration of a drug targeting molecule (such as an antibody) in a cycle of administration, for example, daily, weekly, biweekly, or monthly dosing.

[0424] Exemplary FDA-approved, clinically acceptable dosing regimens for exemplary antibody drugs are listed 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 known from pharmacokinetic studies in a subject population that is administered a single initial dose or by repeated dosing over a dosing cycle at a clinically acceptable or approved dose (see, e.g., Fracasso et al. (2007) Clin. Cancer. Res., 13:986 for systemic exposure (AUC) observed following a single dose of cetuximab within the FDA-approved dose range).

[0425]

[0426]

[0427] Retrieved from www.accessdata.fda.gov / scripts / cder / drugsatfda / index.cfm

[0428] In some embodiments, the therapeutically effective dose of the conjugate is a dose of the conjugate comprising a phthalocyanine dye (e.g., IR700) conjugated to a targeting molecule (e.g., an antibody or antigen-binding antibody fragment) that achieves an average systemic exposure (e.g., AUC) in a patient population, such as a sample patient population, that is no greater than 75%, 70%, 60%, 50%, 45%, 40%, 35%, 25%, or 20% of the average therapeutically effective systemic exposure of the corresponding targeting molecule (e.g., an antibody or antigen-binding antibody fragment) that is not so conjugated in the patient population, such as a sample patient population. Generally, the systemic exposure of the conjugate is high enough to demonstrate phototoxicity by PIT.

[0429] In some embodiments, the conjugate is administered so that the mean systemic exposure (AUC[0-inf] or AUC 0-∞ ) to about 250 μg / mL*h (used interchangeably with μg*h / mL) to 100,000 μg / mL*h, about 500 μg / mL*h to 50,000 μg / mL*h, about 500 μg / mL*h to 25,000 μg / mL*h, about 500 μg / mL*h to 18,000 μg / mL*h, about 500 μg / mL*h to 10,000 μg / mL*h, about 500 μg / mL*h to 5,000 μg / mL*h, or about 500 μg / mL*h to 2,500 μg / mL*h. In some embodiments, the conjugate is administered so that the systemic exposure (AUC[0-inf] or AUC 0-∞ ) no greater than 100,000 μg / mL*h, no greater than 75,000 μg / mL*h, no greater than 50,000 μg / mL*h, no greater than 40,000 μg / mL*h, no greater than 30,000 μg / mL*h, no greater than 20,000 μg / mL*h, no greater than 10,000 μg / mL*h, no greater than 5,000 μg / mL*h, or no greater than 2,500 μg / mL*h.

[0430] In some embodiments, the conjugate is administered so that the mean systemic exposure (AUC[0-24] or AUC 0-24) to about 100 μg / mL*h to 25,000 μg / mL*h, about 200 μg / mL*h to 10,000 μg / mL*h, about 500 μg / mL*h to 5,000 μg / mL*h; or by the AUC of a patient population, such as a sample patient population, after administration of the conjugate. 0-24 The measured mean systemic exposure is no greater than 25,000 μg / mL*h, no greater than 15,000 μg / mL*h, no greater than 10,000 μg / mL*h, no greater than 5,000 μg / mL*h, no greater than 2,500 μg / mL*h, no greater than 1,000 μg / mL*h, or no greater than 500 μg / mL*h. In some embodiments, the plasma conjugate AUC after administration of the conjugate is no greater than 25,000 μg / mL*h, no greater than 15,000 μg / mL*h, no greater than 10,000 μg / mL*h, or no greater than 500 μg / mL*h. 0-24 About 500 μg / mL*h to 8,000 μg / mL*h, about 500 μg / mL*h to 5,000 μg / mL*h, about 500 μg / mL*h to 2,000 μg / mL*h, or about 1000 μg / mL*h to 4,000 μg / mL*h.

[0431] In some embodiments, the therapeutically effective dose of the phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate) is less than the single-administered therapeutically effective dose of the corresponding targeting molecule (e.g., antibody or antigen-binding antibody fragment) that is not so conjugated, for example, no more than 75%, 70%, 60%, 50%, 45%, 40%, 35%, 25%, or 20% of the single-administered therapeutically effective dose of the corresponding targeting molecule that is not so conjugated.

[0432] In some embodiments, the therapeutically effective dose of the phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate) is less than the initial dose in a repeated dosing regimen of the therapeutically effective dose of the corresponding targeting molecule (e.g., antibody or antigen-binding antibody fragment) that is not so conjugated, for example, no more than 75%, 70%, 60%, 50%, 45%, 40%, 35%, 25%, or 20% of the initial dose in a repeated dosing regimen of the therapeutically effective dose of the corresponding targeting molecule that is not so conjugated.

[0433] In some embodiments, the therapeutically effective dose of the phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibody conjugate) is less than the average dose in a repeated dosing regimen of the therapeutically effective dose of the corresponding targeting molecule (e.g., antibody or antigen-binding antibody fragment) that is not so conjugated, for example, no more than 75%, 70%, 60%, 50%, 45%, 40%, 35%, 25%, or 20% of the average dose in a repeated dosing regimen of the therapeutically effective dose of the corresponding targeting molecule that is not so conjugated.

[0434] In some embodiments, the therapeutically effective dose of the conjugate is less than 400 mg / m 2 Less than 300mg / m 2 Less than 250mg / m 2 Less than 225 mg / m 2 Less than 200mg / m 2 Less than 180mg / m 2 Less than 100mg / m 2 or less than 50 mg / m 2 In some embodiments, the therapeutically effective dose of the conjugate is about 50 mg / m 2 ~400 mg / m 2 , 100mg / m 2 ~300 mg / m 2 , 100mg / m 2 ~250 mg / m 2 or 100 mg / m 2 ~160 mg / m 2 In some embodiments, the therapeutically effective dose of the conjugate is about 80 mg / m 2 ~240 mg / m 2 , 80mg / m 2 ~220 mg / m 2 , 80mg / m 2 ~200 mg / m 2 , 80mg / m 2 ~180 mg / m 2 , 80mg / m 2 ~160 mg / m 2 , 80mg / m 2 ~140 mg / m 2 , 80mg / m 2 ~120 mg / m 2 , 80mg / m 2 ~100 mg / m 2 , 100mg / m 2 ~240 mg / m 2 , 100mg / m 2 ~220 mg / m 2 , 100mg / m 2 ~200 mg / m 2 , 100mg / m 2 ~180 mg / m 2 , 100mg / m 2 ~160 mg / m 2 , 100mg / m 2 ~140 mg / m 2, 100 mg / m 2 ~120 mg / m 2 , 120 mg / m 2 ~240 mg / m 2 , 120 mg / m 2 ~220 mg / m 2 , 120 mg / m 2 ~200 mg / m 2 , 120 mg / m 2 ~180 mg / m 2 , 120 mg / m 2 ~160 mg / m 2 , 120 mg / m 2 ~140 mg / m 2 , 140 mg / m 2 ~240 mg / m 2 , 140 mg / m 2 ~220 mg / m 2 , 140 mg / m 2 ~200 mg / m 2 , 140 mg / m 2 ~180 mg / m 2 , 140 mg / m 2 ~160 mg / m 2 , 160 mg / m 2 ~240 mg / m 2 , 160 mg / m 2 ~220 mg / m 2 , 160 mg / m 2 ~200 mg / m 2 , 160 mg / m 2 ~180 mg / m 2 , 180 mg / m 2 ~240 mg / m 2 , 180 mg / m 2 ~220 mg / m 2 , 180 mg / m 2 ~200 mg / m 2 , 200 mg / m 2 ~220 mg / m 2 or 200 mg / m 2 ~240 mg / m 2 .

[0435] 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 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 about 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 6.5 mg / kg.

[0436] In some embodiments, the therapeutically effective amount is 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.

[0437] In some embodiments, the conjugate is IR700-cetuximab. In some embodiments, the therapeutically effective amount of the IR700-cetuximab conjugate is at least or about 160 mg / m 2 , 320mg / 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.

[0438] In some embodiments, a therapeutically effective dose of the conjugate is administered in a single dose. In some embodiments, a therapeutically effective dose is administered by only one injection or one infusion in a dosing regimen or cycle, for example, only once in a dosing regimen or cycle. For example, in one dosing regimen or cycle, no subsequent dose of the conjugate is administered. In some embodiments, the dosing regimen can be repeated. In some embodiments, repeated doses, such as repeated single doses, are administered at a time when the first dose has been cleared from the subject, which in some cases is a time when there is no detectable systemic exposure of the conjugate. Therefore, in some embodiments, the administration of the conjugate is not performed to achieve continuous systemic exposure of the conjugate, which is different from many existing therapies, including antibody therapies, in which repeated administration is required in a dosing regimen or cycle to maintain continuous systemic exposure. In some embodiments, the dosing regimen or cycle is repeated once a week, once every two weeks, once a month, twice a year, once a year, or at a lower frequency as needed.

[0439] In some embodiments, in any of the methods provided herein, if residual damage remains after the previous treatment with the conjugate, the dosing regimen is repeated. In some embodiments, the method further comprises evaluating the presence of residual damage in the subject, and if residual damage remains, the dosing regimen is repeated. In some embodiments, if residual damage remains more than or about 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months, or 1 year after the previous conjugate administration begins, the dosing regimen is repeated. In some embodiments, if residual damage remains about 4 weeks after the previous conjugate administration begins, the dosing regimen is repeated.

[0440] In some embodiments, in a dosing regimen or cycle, no subsequent dose of a targeting molecule, such as a therapeutic targeting molecule (such as a therapeutic antibody), that is not conjugated to a photosensitizer (such as IR700) is administered. For example, in some embodiments, no separate targeting molecule is administered after administration of a phthalocyanine dye-targeting molecule conjugate.

[0441] Those skilled in the art will recognize that higher or lower doses of the phthalocyanine dye-targeting molecule conjugate may also be used, for example, depending on the specific pharmaceutical agent. In some embodiments, a dose such as a daily dose is administered in one or multiple doses, such as 2, 3, or 4 doses or a single formulation. The phthalocyanine dye-targeting molecule conjugate may be administered alone, in the presence of a pharmaceutically acceptable carrier, or in the presence of other therapeutic agents such as immunomodulators, anticancer agents, or other anti-tumor agents.

[0442] In some embodiments, the phthalocyanine dye-targeting molecule conjugate can be administered systemically or topically 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, vaginal, 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 by topical application.

[0443] Compositions comprising the phthalocyanine dye-targeting molecule conjugates can be administered locally or systemically using any method known in the art, for example, to a subject suffering from a tumor such as cancer or a subject who has previously had a tumor removed, for example, by surgery. While specific examples are provided, those skilled in the art will appreciate that other methods of administering the agents disclosed herein may also be employed. Such methods may include, for example, providing continuous infusion to a subject in need of treatment using a catheter or implantable pump over a period of hours to days.

[0444] In some embodiments, the phthalocyanine dye-targeting molecule conjugate is administered parenterally, including by direct injection or infusion into the tumor, such as intratumoral administration. In some embodiments, the phthalocyanine dye-targeting molecule conjugate is administered to the tumor by applying the agent to the tumor, such as by bathing the tumor in a solution containing the agent, such as the phthalocyanine dye-targeting molecule conjugate, or by pouring the agent onto the tumor.

[0445] Additionally or alternatively, the compositions disclosed herein can be administered systemically, for example, intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, subcutaneously, or orally, to a subject having a tumor, such as cancer.

[0446] There is no absolute limit to the dosage of the phthalocyanine dye-targeting molecule conjugate administered to a subject, but it depends on the nature of the composition, its active ingredient, its undesirable side effects such as immune response to the agent, the subject to be treated, the type of symptoms to be treated, and the mode of administration. Generally, the dosage is a therapeutically effective amount, for example, an amount sufficient to achieve the desired biological effect, such as an amount that effectively reduces the size of a tumor, such as volume and / or weight, or slows further growth of a tumor, or alleviates undesirable symptoms of a tumor.

[0447] In some embodiments, compositions for administering pharmaceutical agents such as phthalocyanine dye-targeting molecule conjugates contain, in addition to an effective amount of the pharmaceutical agent, conventional pharmaceutical carriers and excipients suitable for the intended type of administration. For example, in some embodiments, parenteral formulations may contain a sterile aqueous solution or suspension of the conjugate. In some embodiments, compositions for enteral administration may contain an effective amount of the phthalocyanine dye-targeting molecule conjugate in an aqueous solution or suspension, which may optionally include a buffer, a surfactant, a thixotropic agent, and a fragrance.

[0448] C. Dosing Regimens and Photoimmunotherapy

[0449] PIT comprises administration of a composition comprising a phthalocyanine dye-targeting molecule conjugate (eg, IR700-antibody conjugate) followed by irradiation. In some embodiments, the method comprises irradiating the tumor.

[0450] In some embodiments, after contacting cells with the phthalocyanine dye-targeting molecule conjugate, the cells are irradiated. Irradiation methods are known in the art. Because only cells expressing the cell surface protein are typically recognized by the targeting molecule, sufficient amounts of the conjugate are typically bound to these cells. Because irradiation only kills cells that have bound the conjugate and generally spares other cells, the possibility of unwanted side effects, such as killing normal cells, is reduced.

[0451] In some embodiments, cells are irradiated in vivo, for example, in a subject that has previously been administered a phthalocyanine dye-targeting molecule conjugate. In some embodiments, the subject is irradiated, for example, in a tumor in the subject.

[0452] In some embodiments, irradiation is performed after administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, irradiation or exposure is performed or implemented about 30 minutes to 96 hours after administration of the phthalocyanine dye-targeting molecule conjugate (such as IR700-antibody conjugate), for example, about 30 minutes to 48 hours, 30 minutes to 24 hours, or 12 hours to 48 hours after administration of the conjugate, such as usually 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. For example, the irradiation can be implemented within about 24 hours after administration of the conjugate. In some embodiments, the conjugate comprising a targeting molecule is administered to the subject more than 6 hours prior to irradiation or exposure of the tumor, wherein the conjugate is attached to the tumor. In some embodiments, the conjugate is administered to the subject more than about 12 hours, 24 hours, 26 hours, 48 ​​hours, 72 hours, or 96 hours prior to irradiation or exposure of the tumor.

[0453] In some embodiments, the subject may receive one or more other therapies described herein (such as immunomodulators or anticancer agents) at or after irradiation. In some cases, the one or more other therapies are also administered after the administration of the phthalocyanine dye-targeting molecule conjugate (such as IR700-antibody conjugate). In some embodiments, the other therapy is administered within about 0 to 24 hours of irradiation, for example, within about 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours of irradiation.

[0454] In some embodiments, prior to irradiation, the subject may receive one or more other therapies described herein. In some embodiments, the one or more other therapies may be administered before, during, or after the administration of the phthalocyanine dye-targeting molecule conjugate (such as an IR700-antibody conjugate), and are typically administered before irradiation of the subject. In some embodiments, the other therapeutic agent (such as an immunomodulator or anticancer agent) may be administered during or simultaneously with the administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the other therapeutic agent (such as an immunomodulator or anticancer agent) may be administered after or subsequently to the administration of the phthalocyanine dye-targeting molecule conjugate. For example, in some embodiments, the conjugate is administered before the one or more other therapies, and the conjugate and the one or more other therapies are administered separately before irradiating the tumor. In some embodiments, the conjugate is administered after the one or more other therapies, and the conjugate and the one or more other therapies are administered separately before irradiating the tumor. In some embodiments, irradiation is performed after administration of the other therapy (eg, an immunomodulatory agent or an anticancer agent) and the phthalocyanine dye-targeting molecule conjugate.

[0455] In some embodiments, the other therapeutic agent is an immunomodulator, and the immunomodulator is administered 6 hours to 4 weeks before irradiation, such as typically more than about 12 hours, 24 hours, 36 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 4 weeks before irradiation. In some embodiments, the phthalocyanine dye-targeting molecule (such as IR700-antibody conjugate) is administered 6 hours to 96 hours before irradiation, such as typically about 6 hours, 12 hours, 24 hours, 36 hours, 72 hours, or 96 hours before irradiation.

[0456] In some embodiments, before irradiation, the subject may receive an immunomodulator, such as an immune checkpoint inhibitor. In some embodiments, the immunomodulator is typically administered before the subject is irradiated. In some embodiments, the immunomodulator is administered about 12 hours to 2 months, such as 12 hours to 1 month, 12 hours to 3 weeks, 12 hours to 2 weeks, 12 hours to 1 week, and 1 week to 1 month before irradiation, for example, typically at least 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 1 month before irradiation of the tumor. In some embodiments, the immunomodulator can be administered before, after, or simultaneously with the conjugate, as long as both the immunomodulator and the conjugate are administered before irradiation. In some embodiments, the immunosuppressant can be administered before the administration of the phthalocyanine dye-targeting molecule conjugate (such as IR700-antibody conjugate). In some embodiments, the immunomodulator can be administered during or simultaneously with the administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the immunomodulator can be administered after or subsequent to the administration of the phthalocyanine dye-targeting molecule conjugate, for example, three times a week, twice a week, once a week, once every two weeks, once every three weeks, or once a month after irradiation.

[0457] For example, in some cases, the conjugate is administered at least 12 hours, such as about 12 hours to 48 hours, before irradiation, and the immunosuppressant is administered at least 12 hours, such as about 12 hours to about 1 month before irradiation. In some embodiments, the conjugate is administered no more than about 36 hours, 24 hours, 18 hours, or 12 hours before irradiation, and the immunosuppressant is administered more than 12 hours before irradiation, such as typically more than 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 1 month before irradiation.

[0458] In some embodiments, the immunosuppressant itself is a conjugate containing a phthalocyanine dye, for example, a phthalocyanine dye linked to an antibody or antigen-binding fragment as an immunomodulator. In some embodiments, the immunomodulator is an IR700-antibody conjugate, comprising an immunomodulatory antibody (such as a checkpoint inhibitor) that binds to a checkpoint protein (such as PD-L1) on tumor cells. In some embodiments, the immunomodulator (such as an IR700-antibody conjugate as an immunomodulator) is administered before the administration of the phthalocyanine dye-targeting molecule conjugate, for example, 12 hours to 2 months before the 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 before the administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the immunomodulator (such as an IR700-antibody conjugate as an immunomodulator) is administered during or at the same time as the administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the immunomodulator (such as an IR700-antibody conjugate as an immunomodulator) is administered after the administration of the phthalocyanine dye-targeting molecule conjugate, for example, 12 hours to 2 months after the 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 the administration of the phthalocyanine dye-targeting molecule conjugate.

[0459] In some embodiments, irradiation is performed or implemented after administration of the immunomodulator (e.g., IR700-antibody conjugate as an immunomodulator) and the phthalocyanine dye-targeting molecule conjugate. In some embodiments, irradiation is performed or implemented after administration of the phthalocyanine dye-targeting molecule conjugate.

[0460] In some embodiments, the combination therapy method comprises two irradiations or exposures. In some embodiments, the combination therapy method involves a first tumor irradiation after administration of the immunomodulator (e.g., an IR700-antibody conjugate as an immunomodulator) and a second tumor irradiation after administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, each irradiation is performed within 6 to 48 hours after administration of each conjugate, for example, typically at least about 6 hours, 12 hours, 24 hours, or 36 hours after administration of each conjugate.

[0461] In some embodiments, the combination therapy comprising the immunomodulator (e.g., an IR700-antibody conjugate as an immunomodulator) and the phthalocyanine dye-targeting molecule conjugate comprises only a single irradiation. In some embodiments, the phthalocyanine dye-targeting molecule conjugate is administered at least 12 hours after administration of the immunomodulator (e.g., an IR700-antibody conjugate as an immunomodulator) and within 6 to 48 hours before irradiation of the tumor.

[0462] In some embodiments, the immunomodulator (e.g., an IR700-antibody conjugate as an immunomodulator) and the phthalocyanine dye-targeting molecule conjugate are administered via the same route of administration. In some embodiments, the immunomodulator (e.g., an IR700-antibody conjugate as an immunomodulator) and the phthalocyanine dye-targeting molecule conjugate are both administered systemically. In other embodiments, the conjugates are both administered intravenously.

[0463] In some embodiments, the subject may receive an anticancer agent prior to irradiation. In some embodiments, the anticancer agent is typically administered prior to irradiation. In some embodiments, the irradiation is performed or implemented approximately 5 minutes to 2 weeks after administration of the anticancer agent, such as 5 minutes to 1 week, 5 minutes to 3 days, 5 minutes to 48 hours, 5 minutes to 24 hours, 5 minutes to 12 hours, 5 minutes to 6 hours, and 5 minutes to 1 hour, typically 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 irradiation of the tumor. In some embodiments, the anticancer agent may be administered before, after, or simultaneously with the conjugate, as long as both the anticancer agent and the conjugate are administered prior to irradiation. In some embodiments, the anticancer agent may be administered prior to administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the anticancer agent may be administered during or concurrently with administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the anticancer agent can be administered after the phthalocyanine dye-targeting molecule conjugate is administered.

[0464] For example, in some cases, the conjugate is administered at least 12 hours, such as about 12 hours to 48 hours, before irradiation, and the anticancer agent is administered at least 5 minutes, such as about 5 minutes to about 24 hours, before irradiation. In some embodiments, the conjugate is administered no more than about 36 hours, 24 hours, 18 hours, or 12 hours before irradiation, and the anticancer agent is administered more than 5 minutes before irradiation, and typically no more than 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours before irradiation.

[0465] In some embodiments, cells such as tumors are irradiated with a therapeutic dose of radiation at a wavelength in the range of about 400 nm to about 900 nm, such as about 500 nm to about 900 nm, such as about 600 nm to about 850 nm, such as about 600 nm to about 740 nm, such as 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 such as tumors are irradiated with a therapeutic dose of radiation at a wavelength in the range of 600 nm to 850 nm, such as 660 nm to 740 nm. In some embodiments, cells, such as tumors, are irradiated at a wavelength of at least about 600 nm, 620 nm, 640 nm, 660 nm, 680 nm, 700 nm, 720 nm, or 740 nm, such as 690 ± 50 nm, for example, about 680 nm.

[0466] In some embodiments, the concentration of at least 1 J cm -2 , such as at least 10 J cm -2 , at least 30 J cm -2 , at least 50 J cm -2 , at least 100 J cm -2 or at least 500 J cm -2 In some embodiments, the radiation dose is about 1 to about 1000 J cm -2 , about 1 to about 500 J cm -2 , about 5 to about 200 J cm -2 , about 10 to about 100 J cm -2 or about 10 to about 50 J cm -2 In some embodiments, at a rate of at least about 2 J cm -2 、5J cm -2 、10J cm -2 、25J cm -2 、50Jcm -2 、75J cm -2 、100J cm -2 、150J cm -2 , 200 J cm -2 、300J cm -2 , 400J cm -2 or 500 J cm -2 The dose is used to irradiate cells such as tumors.

[0467] In some embodiments, cells such as tumors are irradiated or irradiated at a dose of at least 1 J / cm fiber length, such as 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 irradiation dose is 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, such as tumors, are irradiated at a dose of 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.

[0468] In some embodiments, the dose of irradiation or exposure in a human subject is from about 1 to about 400 J cm -2 , about 2 to about 400 J cm -2 , about 1 to about 300 J cm -2 , about 10 to about 100 J cm -2 or about ~ about 50J cm -2 , such as at least about or within about or about 10 J cm -2 , at least 30 J cm -2 , at least 50 J cm -2 , at least 100 J cm -2 In some embodiments, the irradiation dose in a human subject is about 1 to 300 J / cm fiber length, 10 to 100 J / cm fiber length, or 10 to 50 J / cm fiber length, such as at least about or falling within about or being 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 was found that the irradiation dose to achieve PIT in a human subject can be less than the dose required for PIT in a mouse. For example, in some cases, 50 J / cm 2 (50J cm -2 ) dose of light radiation could not effectively induce PIT in an in vivo tumor mouse model, whereas we could effectively induce PIT in human patients clinically.

[0469] In some embodiments, the radiation dose after administration of the composition comprising the phthalocyanine dye-targeting molecule conjugate is at least 1 J cm at a wavelength of 660 to 740 nm.-2 or 1 J / cm fiber length, for example at least 10 J cm at a wavelength of 660-740 nm -2 Or 10 J / cm fiber length, at least 50 J cm at a wavelength of 660-740 nm -2 or 50 J / cm fiber length, or at least 100 J cm at a wavelength of 660-740 nm -2 Or 100 J / cm fiber length, for example, 1.0 to 5001.0 J cm at a wavelength of 660 to 740 nm -2 or 1.0 to 500 J / cm fiber length. In some embodiments, the wavelength is 660 to 710 nm. In some embodiments, the radiation dose after administration of the composition comprising the phthalocyanine dye-targeting molecule conjugate is at least 1.0 J cm at a wavelength of 680 nm. -2 or 1 J / cm fiber length, for example at a wavelength of 680 nm it is at least 10 J cm -2 or 10 J / cm fiber length, at least 50 J cm at a wavelength of 680 nm -2 or 50 J / cm fiber length, or at least 100 J cm at a wavelength of 680 nm -2 Or 100 J / cm fiber length, for example, 1.0 to 500 J cm at a wavelength of 680 nm -2 or 1.0 to 500 J / cm fiber length. In some embodiments, multiple irradiations are performed, for example, at least 2, at least 3, or at least 4 irradiations, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 irradiations are performed separately. Exemplary irradiations after administration of the conjugate or composition provided by the present invention include irradiation at a wavelength of 660 nm to 740 nm, at a wavelength of at least 1 J cm -2 Or 1J / cm fiber length dose to irradiate the tumor.

[0470] In some embodiments, light or laser light can be applied to the dye molecule, for example, a cell containing the conjugate for about 5 seconds to about 5 minutes. For example, in some embodiments, the light or laser light is applied for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 seconds, or in a range between any two of the above values, to activate the dye molecule. In some embodiments, light or laser light is applied for about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 minutes or more, or in a range between any two of the above values. In some embodiments, the length of time for applying light or laser light can depend, for example, on the energy of the light or laser light, such as wattage. For example, light or laser light with lower wattage can be applied for a longer time to activate the dye molecule.

[0471] 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 about 30, 35, 40, 45, 50, or 55 minutes after administration of the conjugate, or within a range between any two of the above values. In some embodiments, the light or laser is applied 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 within a range between any two of the above values. In some embodiments, the light or laser is applied about 1 to 24 hours after administration of the conjugate, such as about 1 to 12 hours, 12 to 24 hours, 6 to 12 hours, or more than 24 hours after administration. In some embodiments, the light or laser is applied 36 or 48 hours after administration of the conjugate.

[0472] In some embodiments, cells or subjects can be irradiated once or multiple times. Thus, irradiation can be performed on a single day, or can be repeated over multiple days with the same or different doses, for example, at least 2 different times, 3 different times, 4 different times, 5 different times, or 10 different times. In some embodiments, repeated irradiation can be performed on the same day, on consecutive days, or every 1 to 3 days, every 3 to 7 days, every 1 to 2 weeks, every 2 to 4 weeks, every 1 to 2 months, or at longer intervals.

[0473] In some embodiments, the dose or method of irradiation varies depending on the type or morphology of the tumor.

[0474] In some embodiments, the lesion is a tumor and the tumor is superficial. In another embodiment, the thickness of the tumor is less than 10 millimeters (mm). In some embodiments, the irradiation is performed using a microlens tip fiber for surface irradiation. In some embodiments, the light irradiation dose is about 5 J / cm 2 ~about 200J / cm 2 .

[0475] In some embodiments, provided methods include using a microlens tip fiber for surface irradiation at about 5 J / cm 2 ~about 200J / cm 2 A light dose of about 50 J / cm2 is applied to a superficial tumor in a subject, wherein the tumor is linked to a phototoxic agent comprising a targeting molecule that binds to a cell surface molecule of the tumor. In some embodiments, the light irradiation dose is about 50 J / cm2. 2 .

[0476] In some embodiments, the lesion is a tumor, and the tumor is a mesenchymal tumor. In some embodiments, the tumor is greater than 10 mm in depth or is a subcutaneous tumor. In some embodiments, irradiation is performed using a cylindrical diffuser fiber having a diffusion length of 0.5 centimeters (cm) to 10 cm and a spacing of 1.8 ± 0.2 cm. In some embodiments, the light irradiation dose is about 20 J / cm fiber length or about 500 J / cm fiber length.

[0477] In some embodiments, provided methods comprise irradiating a mesenchymal tumor in a subject using a cylindrical diffusing fiber comprising a diffusion length of 0.5 cm to 10 cm and a spacing of 1.8 ± 0.2 cm at a light dose of about 100 J / cm of fiber length or at a radiation energy fluence rate of about 400 mW / cm, wherein the tumor is attached to a phototoxic agent comprising a targeting molecule that binds to a cell surface molecule of the tumor. In some embodiments, the tumor is greater than 10 mm in depth or is a subcutaneous tumor. In some embodiments, the cylindrical diffusing fiber is placed in a catheter located 1.8 ± 0.2 cm away from the tumor. In some embodiments, the catheter is optically transparent.

[0478] In some embodiments, the methods provided comprise irradiating at one or more wavelengths. In some embodiments, after administration of the conjugate, the lesion or tumor is irradiated at one or more wavelengths to induce phototoxic activity of the first dye of the conjugate and a fluorescent signal of the second dye of the conjugate. For example, in methods employing first and second dyes having different excitation wavelengths coupled to a targeting molecule, two different wavelengths can be used for irradiation. In some embodiments, the methods provided comprise irradiating the lesion with one wavelength. In some embodiments, the methods provided comprise irradiating the lesion with two different wavelengths simultaneously or sequentially, wherein one wavelength induces phototoxic activity and the other wavelength induces a fluorescent signal. For example, in some embodiments, the methods provided comprise irradiating the lesion with one or more wavelengths of about 400 to about 900 nm at a rate of at least 1 J cm -2 The lesions were irradiated with a dose of 1 J / cm fiber length or 1 J / cm fiber length.

[0479] D. Other Therapeutic Agents and Combination Therapies

[0480] In some embodiments, other therapeutic agents, such as immunomodulators or anticancer agents, are administered in combination with photoimmunotherapy agents, such as phthalocyanine dye conjugates, for example, IR700-antibody conjugates. In some embodiments, the combination therapy may include administering a phthalocyanine dye conjugate, such as an IR700-antibody conjugate, in combination with an anticancer agent or immunomodulator.

[0481] In some embodiments, the other agents can be administered at a sufficient time before the implementation of irradiation so that the therapeutic effect of treating tumors is enhanced. In some embodiments, before irradiation in the method of photoimmunotherapy, one or more other therapeutic agents, such as immunomodulators (such as immune checkpoint inhibitors) or anticancer agents (such as antimetabolites) are given to the subject. In one embodiment, the immunomodulator can be administered at a sufficient time before irradiation, for example, usually at least 12 hours before irradiation, so that the immune system responds to the tumor-related agents released by the lysis of tumor cells after photoimmunotherapy. In another embodiment, the anticancer agent can be administered at a sufficient time before irradiation, for example, usually at least 5 minutes before irradiation to achieve systemic utilization of the anticancer agent so that it can be immediately delivered to the tumor by changes in vascular permeability after photoimmunotherapy.

[0482] The one or more other agents, such as immunomodulators or anticancer agents, can be administered before, simultaneously with, after, 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, for example, a certain time after administration of the other therapeutic agent mentioned above. In some embodiments, activation of the phthalocyanine dye photosensitizer of the conjugate by irradiation with light is achieved before administration of the other therapeutic agent mentioned above.

[0483] In some embodiments, the combined effect of the photoimmunotherapy and the one or more other agents can be a synergistic effect compared to treatment involving photoimmunotherapy using only the phthalocyanine dye-targeting molecule conjugate or treatment involving monotherapy using only the other therapeutic agent. In some embodiments, the methods provided by the present invention result in an enhancement or improvement in the desired anti-tumor therapeutic effect, such as an enhancement or improvement in the alleviation or suppression of one or more symptoms associated with cancer, compared to photoimmunotherapy alone or monotherapy alone.

[0484] Treatment with the phthalocyanine dye-targeting molecule conjugate and optionally other therapeutic immunomodulators or anticancer agents can be completed independently on a single day, or can be repeated over several days at the same or different doses. Repeated treatments can be performed on the same day, on consecutive days, or every 1 to 3 days, every 3 to 7 days, every 1 to 2 weeks, every 2 to 4 weeks, every 1 to 2 months, or at longer intervals.

[0485] In some embodiments, the combination therapy includes administering to the subject a therapeutically effective amount of an immunomodulator, such as an immune checkpoint inhibitor. The immunomodulator is administered in an amount of about 0.01 mg to 1000 mg, for example, at least 0.01 mg, 0.1 mg, 1 mg, 10 mg, 1000 mg, 2000 mg, 3000 mg or more. In an exemplary embodiment, an immunomodulator such as an immune checkpoint inhibitor can be administered at about 0.3 mg / kg to 10 mg / kg, or 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, an immunomodulator such as an immune checkpoint inhibitor can be administered by an ascending dosing regimen, including 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 incremental dosing regimen includes administering the first dose of the immunomodulator at about 5 mg / kg, and administering the second dose at about 9 mg / kg. Another step-by-step incremental dosing regimen can include administering the first dose of the immunomodulator at about 3 mg / kg, administering the second dose at about 3 mg / kg, administering the third dose at about 5 mg / kg, administering the fourth dose at about 5 mg / kg, and administering the fifth dose at about 9 mg / kg. On the other hand, a step-by-step incremental dosing regimen can include administering the first dose at 5 mg / kg, administering the second dose at 5 mg / kg, and administering the third dose at 9 mg / kg. In some embodiments, specific administration can be 2 times per week, once per week, once in 2 weeks, once in 3 weeks, or once in 1 month or more. In some cases, administration can be carried out by a cyclic process, and the cyclic process can be repeated, for example, every 1 month, every 2 months, every 3 months, every 6 months, every 1 year or more.

[0486] In some embodiments, the combination therapy comprises administering to a subject a therapeutically effective amount of an anticancer agent, such as any of the anticancer agents described above. In some embodiments, the therapeutically effective dose can be 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 anticancer agent is 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.

[0487] In some embodiments, the administration of the immunomodulator (such as an immune checkpoint inhibitor) or anticancer agent is performed continuously or repeatedly according to its clinical dosing regimen after PIT treatment. Therefore, in some embodiments, in a dosing regimen or dosing cycle according to the method provided by the present invention, the phthalocyanine dye conjugate (such as IR700-antibody conjugate) can be administered only once in, for example, a single dose or a single infusion for PIT, while the administration of the immunomodulator is performed continuously or repeatedly more than once, for example, 3 times a week, 2 times a week, once a week, once every 2 weeks, once every 3 weeks, or once a month during the dosing regimen or dosing cycle. In some embodiments, the dosing regimen or dosing cycle is about 28 days or 4 weeks.

[0488] In some embodiments, the other agent or the administered agent or the other agent in the combination therapy is an unconjugated targeting molecule. In some embodiments, the unconjugated targeting molecule is a targeting molecule that is identical or substantially identical to the targeting molecule of the conjugate. For example, in some embodiments, the targeting molecule, such as an unconjugated antibody targeting a protein or antigen, 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 about 10 mg / m 2 ~about 500 mg / m 2 For example, the targeting molecule is cetuximab, and cetuximab is administered to the subject up to 96 hours prior to administration of the conjugate.

[0489] 1. Immunomodulators

[0490] The present invention provides an immunomodulator that can be co-administered with a PIT method employing a phthalocyanine dye conjugate. Therefore, the combination therapy provided by the present invention (including combinations and methods of use thereof) includes an immunomodulator. In some aspects, an immunomodulator or immunomodulator is a substance that directly or indirectly suppresses or activates the body's immune response. For example, an immunomodulator that stimulates an immune response to tumors and / or pathogens can be used in conjunction with photoimmunotherapy. In some embodiments, the immunomodulator can include a cell-based (e.g., a combined therapy with immune cells such as dendritic cells or T cells) or a non-cell-based immunomodulator.

[0491] Typically, cancer cells containing tumor-specific antigens should be recognized by the immune system. In general, in an active immune system, immune cells such as cytotoxic T cells attack and eradicate these cancer cells. Under normal physiological conditions, the T cell-mediated immune response begins by the recognition of antigens by T cell receptors (TCRs) and is regulated by the balance of co-stimulatory signals and inhibitory signals (such as immune checkpoint proteins). In particular, CD4+ and CD8+ T cells expressing TCRs can be activated by the recognition of antigenic peptides expressed on antigen-presenting cells on major histocompatibility complex (MHC) class I or class II molecules. In some aspects, activated CD8+ cells or cytotoxic T cells can kill tumor cells expressing the antigen, and the presence of CD4+ T cells can assist this phenomenon. In some embodiments, the immune cells are antigen-presenting cells. In some embodiments, the immune cells are dendritic cells.

[0492] However, in the case of tumors, the tumor microenvironment has mechanisms that suppress the immune system, thereby escaping immune recognition and preventing or reducing the killing of tumor cells. For example, in some cases, as a mechanism to evade the immune system, immune checkpoint proteins can be dysregulated in tumors, resulting in the suppression of the immune response in the tumor microenvironment. In some cases, other mechanisms can play a role in preventing immune cells from reaching tumor antigens, thereby also contributing to the ability of tumors to evade the immune system. The combination therapy provided by the present invention simultaneously addresses these two escape mechanisms, so as to provide a stronger immune response against tumors while also killing tumor cells through a photolytic mechanism.

[0493] In some embodiments of the combination therapies provided herein, an immunomodulator is administered to a subject to suppress immunosuppressive signals or enhance immunostimulatory signals. For example, inhibitory checkpoint protein antagonists and / or co-stimulatory receptor agonists can stimulate the host's endogenous anti-tumor immune response by amplifying antigen-specific T cell responses. In some aspects of the provided methods, photoimmunotherapy can also be implemented, which can lead to tumor cell killing, thereby releasing tumor antigens. By combining photoimmunotherapy with the administration of an immunomodulator, the subsequent release of antigens induced by PIT can provide a source of antigen stimulation for T cells, whose responses are amplified or stimulated by the immunomodulator. Thus, in some aspects, the enhanced immune response generated by therapy with an immunomodulator is achieved and prepared to respond to tumor antigens exposed by cell lysis following PIT. Thus, in some aspects, the combination therapies provided herein address natural escape mechanisms that may exist in the tumor microenvironment, thereby providing a more robust immune response against the tumor while also killing tumor cells through photolytic mechanisms.

[0494] Typically, in the provided methods, PIT-mediated cell killing by irradiation and activation of the administered phthalocyanine dye conjugate occurs at a time after the immune response has been stimulated or enhanced following administration of the immunomodulator. In some embodiments, the immunomodulator is administered at a sufficient time prior to irradiation of the administered phthalocyanine dye conjugate to allow amplification of the T cell response to occur prior to PIT-induced cell lysis. Thus, typically, the immunomodulator is administered at a sufficient time prior to irradiation to therapeutically effectively amplify the T cell response, for example, at least 12 hours, such as typically 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 administering irradiation to induce PIT-mediated cell killing by activation of the administered phthalocyanine dye conjugate (e.g., an IR700-targeting molecule conjugate, such as an IR700-antibody dye conjugate).

[0495] In some embodiments, the conjugate is administered before, simultaneously with, or after administration of the immunomodulator. In some embodiments, the conjugate is administered after administration of the immunomodulator and before irradiating the tumor. In some embodiments, the conjugate is administered about 12 hours to 48 hours before irradiating the tumor, and the immunomodulator is administered about 12 hours to about 1 month before irradiating the tumor. In some embodiments, the immunomodulator is administered more than about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 1 month before irradiating the tumor.

[0496] In some embodiments, irradiation of the tumor is performed i) after administration of the immunomodulator and after administration of the conjugate, or ii) only after administration of the conjugate.

[0497] Exemplary dosing regimens and schedules for administering an immunomodulatory agent, a phthalocyanine dye conjugate (eg, an IR700-targeting molecule conjugate, eg, an IR700-antibody dye conjugate), and for irradiation are described elsewhere herein.

[0498] In some embodiments, the combination therapy can be implemented with any immunomodulator that can stimulate, amplify and / or enhance the anti-tumor immune response by, for example, inhibiting immunosuppressive signals or enhancing immunostimulatory signals. In some embodiments, the immunomodulator is a peptide, protein or small molecule. In some embodiments, the protein can be a fusion protein or a recombinant protein. In some embodiments, the immunomodulator binds to a cell surface receptor expressed on an immune target, such as a T cell or B cell or an antigen presenting cell. For example, in some embodiments, the immunomodulator is an antibody or antigen-binding antibody fragment, a fusion protein, a small molecule or a polypeptide.

[0499] In some embodiments, the immunomodulator inhibits the immune checkpoint pathway. The immune system has a variety of inhibitory pathways, which are related to the maintenance of self-tolerance and the regulation of immune responses. It is known that tumors can use certain immune checkpoint pathways as the main mechanism of immune resistance, especially for immune resistance of T cells specific for tumor antigens (Pardoll, 2012, Nature Reviews Cancer 12: 252-264). Because many such immune checkpoints are initiated by ligand-receptor interactions, they can be easily blocked with antibodies against the ligand and / or its receptor.

[0500] Therefore, the use of antagonist molecules that block the immune checkpoint pathway, such as small molecules, nucleic acid inhibitors (such as RNAi) or antibody molecules, has become a promising immunotherapy approach for other diseases. In contrast to the main anticancer agents, checkpoint inhibitors do not necessarily target tumor cells directly, but only need to target lymphocyte receptors or their ligands to enhance the endogenous anti-tumor activity of the immune system. (Pardoll, 2012, Nature Reviews Cancer 12: 252-264).

[0501] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins regulate 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 regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses.

[0502] Immune checkpoint inhibitors include any agent that blocks or inhibits the inhibitory pathways of the immune system in a statistically significant manner. Such inhibitors may include small molecule inhibitors, or may include antibodies or antigen-binding fragments thereof that bind to immune checkpoint receptor ligands and block or inhibit them. Illustrative 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 antigens (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (a molecule belonging to the CD2 family, expressed on all NK, γδ, and memory CD8+ (αβ) T cells), CD160 (also known as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies or their antigen-binding fragments or other binding proteins that bind to and block or inhibit the activity of one or more molecules selected from the group consisting 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. Illustrative 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).

[0503] Programmed cell death 1 (PD1) is an immune checkpoint protein expressed on 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 function of PD1 is to limit the activity of T cells in peripheral tissues during the inflammatory process in response to infection, and to limit autoimmunity (Pardoll, 2012, Nature Reviews Cancer 12:252-264). The expression of PD1 is induced in activated T cells, and the binding of PD1 to one of its endogenous ligands inhibits the activation of T cells by inhibiting stimulatory kinases (Pardoll, 2012, Nature Reviews Cancer 12:252-264). PD1 also inhibits TCR “stop signals” (Pardoll, 2012, Nature Reviews Cancer 12:252-264). PD1 is highly expressed on Treg cells and can enhance their proliferation in the presence of ligands (Pardoll, 2012, Nature Reviews Cancer 12:252-264). Anti-PD-1 antibodies are 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 (BMS's Opdivo), pembrolizumab (Merck's Keytruda), pidilizumab (CureTech's CT-011), lambrolizumab (Merck's MK-3475), and AMP-224 (Merck).

[0504] PD-L1 (also known as CD274 and B7-H1) and PD-L2 (also known as CD273 and B7-DC) are ligands for PD1 and are found on activated T cells, B cells, myeloid cells, macrophages, and some types of tumor cells. Anti-tumor therapy focuses on anti-PD-L1 antibodies. The complex of PD1 and PD-L1 inhibits the proliferation of CD8+ T cells and weakens 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 are 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.

[0505] Cytotoxic T lymphocyte-associated antigen (CTLA-4), also known as CD152, is a co-inhibitory molecule for regulating the activation of T cells. CTLA-4 is a member of the immunoglobulin superfamily and is only expressed on T cells. CTLA-4 inhibits the activation of T cells, and it has been reported that it inhibits helper T cell activity and improves regulatory T cell immunosuppressive activity (Pardoll, 2012, Nature Reviews Cancer 12: 252-264). Although the accurate mechanism of action of CTLA-4 is still under study, it is believed that it inhibits the activation of T cells by competitively combining with CD80 and CD86 with CD28 and delivering the inhibitory signal activity to T cells (Pardoll, 2012, Nature Reviews Cancer 12: 252-264). Anti-CTLA-4 antibodies are 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 notable feature of anti-CTLA-4 is the kinetics of its anti-tumor effect, with a lag period of up to 6 months after initial treatment for a physiological response (Pardoll, 2012, Nature Reviews Cancer 12:252-264). In some cases, tumor size may actually increase after treatment initiation before a decrease is observed (Pardoll, 2012, Nature Reviews Cancer 12:252-264). Exemplary anti-CTLA-4 antibodies include Ipilimumab (Bristol-Myers Squibb) and Tremelimumab (Pfizer). Ipilimumab has recently been approved by the FDA for the treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11:89). In some embodiments, the immunomodulator is not an anti-CTLA-4 antibody.

[0506] 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-infiltrating T cells, including cells that are endowed with potent immunosuppressive activity. An exemplary anti-LAG-3 antibody is BMS-986016. IMP321 is a soluble form of the immune checkpoint molecule LAG-3, which activates dendritic cells and enhances antigen presentation.

[0507] T cell immunoglobulin domain-containing and mucin domain-containing-3 (TIM-3) was originally identified on activated Th1 cells and has been shown to be a negative regulator of the immune response. Blockade of TIM-3 promotes T cell-mediated anti-tumor immunity and has anti-tumor activity in many mouse tumor models. The combination of TIM-3 blockade and other immunotherapeutic agents such as TSR-042, anti-CD137 antibodies and other immunotherapeutic agents can produce additive or synergistic effects in enhancing anti-tumor effects. TIM-3 expression is associated with a variety of different tumor types, including melanoma, NSCLC and renal cancer. In addition, intratumoral TIM-3 expression has been shown to be associated with poor prognosis in many tumor types, including NSCLC, cervical cancer and gastric cancer. Blockade of TIM-3 is also useful in promoting immunity to a variety of chronic viral diseases. TIM-3 has also been shown to interact with multiple ligands, including galectin-9, phosphatidylserine and HMGB1, although at this stage, it is unclear whether these ligands, if any, are involved in the regulation of antitumor responses.

[0508] 4-1BB, also known as CD137, is a transmembrane glycoprotein belonging to the TNFR superfamily. 4-1BB receptors are present on activated T cells and B cells and monocytes. An exemplary anti-4-1BB antibody is urelumab (BMS-663513), which has potential immunostimulatory and anti-tumor activity.

[0509] Glucocorticoid-induced TNFR family-related genes (GITR) are also members of the TNFR superfamily. GITR is upregulated in activated T cells, boosting the immune system. An exemplary anti-GITR antibody is TRX518.

[0510] Cluster of differentiation 40 (CD40) is also a member of the TNFR superfamily. CD40 is a co-stimulatory protein found on antigen-presenting cells that mediates a wide variety of immune and inflammatory responses. CD40 is also expressed on some malignant tumors, where it promotes proliferation. Exemplary anti-CD40 antibodies are dacetuzumab (SGN-40), lucatumumab (Novartis, antagonist), SEA-CD40 (Seattle Genetics), and CP-870,893.

[0511] 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, but rather acts as a second co-stimulatory immune checkpoint molecule. Exemplary anti-OX40 antibodies are MEDI6469 and MOXR0916 (RG7888, Genentech).

[0512] In some embodiments, the immunomodulator is an antibody or an antigen-binding antibody fragment thereof. Exemplary such antibodies include, but are not limited to, daclizumab (Zenapax), bevacizumab, Basiliximab, Ipilimumab, Nivolumab, Pembrolizumab, MPDL3280A, Pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (Atezolizumab), Tremelimumab, IMP321, BMS-986016, LAG525, Urelumab, PF-05082566, TRX518, MK-4166, Dacetuzumab (SGN-40), Lucatumumab (HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR0916, AMP-224, MSB0010718C (Avelumab), MEDI4736, PDR001, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, ARGX-115, Emactuzumab, CC-90002, and MNRP1685A, or antibody-binding fragments thereof.

[0513] CXCR2 is a chemokine receptor expressed on myeloid-derived suppressor cells (MDSCs). CXCR2 contributes to tumor immune evasion. Anti-CXCR2 monoclonal antibody therapy has been shown to enhance anti-PD-1 antibody-induced anti-tumor immune responses and anti-tumor efficacy.

[0514] In some embodiments, the immunomodulator is a cytokine. In some embodiments, the immunomodulator is a cytokine, or an agent that induces enhanced expression of a cytokine in the tumor microenvironment. "Cytokine" is a general term for proteins released by a cell population that act as molecular mediators on other cells. Examples of such cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include 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); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor, such as NGF-β; platelet-derived growth factor; transforming growth factor (TGF), such as TGF-α and TGF-β; Insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and -γ; 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-1a, 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-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins of natural or recombinant cell culture origin, as well as biologically active equivalents of native sequence cytokines. For example, the immunomodulator is a cytokine, and the cytokine is IL-4, TNF-α, GM-CSF or IL-2.

[0515] In some embodiments, the immunomodulator 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), α-galactosylceramide, RC-529, MDF2β, loxoribine, anti-CD40 agonist, SIRPα antagonist, AS04, AS03, flagellin, resiquimod, DAP (diaminopimelate), MDP (muramyl dipeptide) and CAF01 (cationic adjuvant formulation-01). In some embodiments, the immunomodulator is a Toll-like receptor (TLR) agonist, adjuvant or cytokine. In some embodiments, the immunomodulator 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 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 ribosome elongation and initiation factor 4a (LeIF), MEDI9197, SD-101, and imidazoquinoline TLR agonists.

[0516] In some embodiments, the immunomodulator can contain one or more interleukins or other cytokines. For example, the interleukin can include interleukin injection (Multikine), which is a combination of natural cytokines.

[0517] In some embodiments, the immunomodulator is a Toll-like receptor (TLR) agonist. In some embodiments, such agonists may include TLR4 agonists, TLR8 agonists, or TLR9 agonists. Such agonists may be selected from peptidoglycan, poly: C, CpG, 3M003, flagellin, and Leishmania homolog (LeIF) of eukaryotic ribosome elongation and initiation factor 4a.

[0518] In some embodiments, the immunomodulator can be an immunomodulator that enhances the immunogenicity of tumor cells, such as patupirone (epothilone B), epidermal growth factor receptor (EGFR) targeted monoclonal antibody 7A7.27, histone deacetylase inhibitors (such as vorinostat, romidepsin, panobinostat, belinostat and entinostat), n3-polyunsaturated fatty acid docosahexaenoic acid, proteasome inhibitors (such as bortezomib), shikonin (the main component of the root of Lithospermum erythrorhizon) and oncolytic viruses such as TVec (talimogenelaherparepvec). In some embodiments, the immunomodulator activates immunogenic cell death of cancer or tumor, 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. In some embodiments, the immunomodulator can be an epigenetic therapy, such as a DNA methyltransferase inhibitor (such as decitabine, 5-aza-2'-deoxycytidine).

[0519] For example, in some embodiments, the immunomodulator can be a DNA methyltransferase inhibitor, which can regulate the expression of tumor-associated antigens (TAA). TAA is an antigenic substance produced in tumor cells that triggers an immune response. TAA is often downregulated in tumors through DNA methylation to escape the immune system. The inversion of DNA methylation restores the expression of TAA and enhances 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 enhance the immune recognition of cancer cells. Photoimmunotherapy further exposes TAA to the immune system by destroying cells.

[0520] In some embodiments, the immunomodulator itself can be an antibody conjugate containing a phthalocyanine dye connected to an antibody or antigen-binding antibody fragment, and the antibody or antigen-binding antibody fragment is an immunomodulator, such as an immune checkpoint inhibitor. In some embodiments, the immunomodulator is an immunosuppressive molecule on the surface of a targeted tumor cell, such as an immune checkpoint molecule or an agent bound thereto. For example, PD-L1 is an immunosuppressive molecule constitutively expressed or expressed on many tumor cells, which can prevent T cell activation by interacting with its receptor PD-1 expressed on tumor cells. In some aspects, a phthalocyanine dye conjugate containing an immunomodulator bound to an immunosuppressive molecule (such as PD-L1) on a tumor cell can be administered to simultaneously enhance the immune response and specifically kill cancer cells expressing the immunosuppressive molecule, thereby reversing immunosuppression in the tumor microenvironment. In particular, irradiation of tumor cells bound to the conjugate can lead to their activation to mediate PIT-induced cell killing of PD-L1 cancer cells, which will also specifically eliminate cancer cells in the tumor that control T cell suppression in the tumor microenvironment.

[0521] Therefore, the present invention provides a conjugate containing a phthalocyanine dye (such as IR700) connected to an immunomodulator, which binds to an immunosuppressive molecule expressed on tumor cells. For example, in some embodiments, the immunosuppressive molecule expressed on the tumor cells can be an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule expressed on the tumor cells is PD-L1. In some embodiments, the immunomodulator as 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, the present invention provides a conjugate containing a phthalocyanine dye (such as IR700) connected to an antibody or antigen-binding antibody fragment, which binds to PD-L1. Exemplary immune checkpoint inhibitors including antibodies or antigen-binding antibody fragments for PD-L1 are as described above and can all be included in the conjugate provided. 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 include, for example, IR700-BMS-935559, IR700-MEDI4736, IR700-MPDL3280A, and IR700-MSB0010718C. In some embodiments, such conjugates can be applied to photoimmunotherapy methods by, for example, irradiating with light of a wavelength sufficient to activate the dye. Such conjugates can be used in photoimmunotherapy based on monotherapy, or in combination therapy with other phthalocyanine dye conjugates.

[0522] For example, in some embodiments, a combination therapy is provided, wherein a first conjugate is administered to a subject, followed by administration of a second conjugate to the subject, wherein the first conjugate contains a phthalocyanine dye (such as IR700) linked to an immunomodulator, the immunomodulator binds to an immunosuppressive molecule expressed on tumor cells (such as an anti-PD-L1 antibody, such as an IR700-anti-PD-L1 conjugate), and the second conjugate contains a phthalocyanine dye linked to a targeting molecule. Typically, the second conjugate can include any targeting molecule capable of binding to cell surface proteins on cells in the tumor, such as cells present in the tumor microenvironment, such as any of the targeting molecules described above. In some embodiments, the first conjugate and the second conjugate bind to different proteins expressed on cells in the tumor. In some embodiments, the second conjugate can include a phthalocyanine dye (such as IR700) linked to an antibody or antigen-binding antibody fragment, which binds to a cell surface protein expressed on cells in the tumor. Exemplary antibodies or antigen-binding antibody fragments of the second conjugate may include, but are not limited to, bevacizumab, cetuximab, panitumumab, zalutumumab, nimotuzumab, tositumomab, Rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab 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, MED I6383, MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, Emactuzumab, CC-90002 and MNRP1685A, or antibody binding fragments thereof.

[0523] In some embodiments, for example, if the tumor treatment performed by 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, which can reduce the amount or activity of immunosuppressive cells in the tumor, or can block the activity of immunosuppressive markers, or can reduce the activity of tumor-promoting cells in the tumor, or can block the activity of tumor-promoting markers. For example, in some embodiments, a conjugate having a phthalocyanine dye as a first dye is co-administered with an immunomodulatory agent, the immunomodulatory agent comprising a conjugate comprising a second phthalocyanine dye conjugated to the immunomodulatory agent, the immunomodulatory agent being capable of binding to immunosuppressive cells or tumor-promoting cells and modulating the activity of such cells. In some embodiments, the first and second phthalocyanine dyes are the same or different.

[0524] Therefore, in some embodiments, the immunosuppressant itself is a conjugate containing a phthalocyanine dye, for example, a phthalocyanine dye linked to an antibody or antigenic antibody binding fragment as an immunomodulator. In some embodiments, the immunomodulator is an IR700-antibody conjugate, comprising an immunomodulatory antibody (such as a checkpoint inhibitor) that binds to a checkpoint protein (such as PD-L1) on tumor cells. In some embodiments, the immunomodulator (such as an IR700-antibody conjugate as an immunomodulator) is administered before the administration of the phthalocyanine dye-targeting molecule conjugate, for example, 12 hours to 2 months before the 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 before the administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the immunomodulator (such as an IR700-antibody conjugate as an immunomodulator) is administered during or at the same time as the administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the immunomodulator (such as an IR700-antibody conjugate as an immunomodulator) is administered after the administration of the phthalocyanine dye-targeting molecule conjugate, for example, 12 hours to 2 months, 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 the administration of the phthalocyanine dye-targeting molecule conjugate or after irradiation after the administration of the phthalocyanine dye-targeting molecule conjugate.

[0525] In these aspects, the combination therapy typically comprises one or more irradiations with light of a wavelength sufficient to activate the dye of the first and / or second conjugate.

[0526] In some embodiments, at least two irradiations are performed, wherein at least a first irradiation is provided to activate the first conjugate and a second irradiation is provided to activate the second conjugate. In some embodiments, the first irradiation of light is provided to the tumor after administration of the first conjugate. For example, within about 12 hours to 48 hours, such as about 24 hours, after administration of the first conjugate, the tumor can be treated with light to kill cancer cells expressing immunosuppressive molecules, such as killing tumor cells expressing PD-L1. In some embodiments, such cell killing can reactivate or amplify the T cell response in the tumor. In some embodiments, after photoimmunotherapy of the first conjugate by administration and irradiation, the subject can be given the second phthalocyanine dye conjugate, and then, within about 12 hours to 48 hours, such as about 24 hours, after administration of the second conjugate, a second irradiation with light is performed. In some embodiments, the second irradiation activates the second conjugate, which can result in selective cell killing of tumor cells expressing the tumor target molecule recognized by the second conjugate, thereby releasing tumor antigens to induce a strong immunogenic response, just as T cells in the tumor are no longer suppressed by immunosuppressive molecules (such as PD-L1). In some embodiments, the first irradiation is performed before the administration of the second conjugate, for example, at least or about 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours or 24 hours before the administration of the second conjugate.

[0527] In some embodiments, a single irradiation can be performed to simultaneously activate the first and second conjugates, thereby triggering PIT-induced cell killing of tumor cells expressing an immunosuppressive molecule recognized by the first conjugate (such as PD-L1) and tumor cells expressing a tumor target molecule recognized by the second conjugate. Thus, in these aspects, a single photoirradiation of a tumor can simultaneously induce two effects to selectively kill specific tumor cells, thereby releasing tumor antigens and inducing a strong immunogenic response by immunosuppressing the killing of tumor cells, such as those expressing PD-L1. In some embodiments, prior to irradiation, the first conjugate can be administered before, simultaneously with, after, or intermittently with the administration of the second conjugate. In some embodiments, the first conjugate is administered, for example, at least 5 minutes, typically at least 12 hours, or at least 24 hours, before the administration of the second conjugate. In some embodiments, the first and second conjugates are administered simultaneously. In some embodiments, the first and second conjugates are formulated separately. In some embodiments, the first and second conjugates are formulated together in the same composition.

[0528] 2. Anticancer agents

[0529] The present invention also provides an anticancer agent that can be administered in combination with photoimmunotherapy using a phthalocyanine dye-targeting molecule conjugate. Thus, the combination therapies provided herein (including combinations and methods of use thereof) include an anticancer agent, which can include any agent that can be used to reduce, inhibit, or prevent cancer in a subject. Optionally, additional anticancer agents can be used in combination with photoimmunotherapy using a phthalocyanine dye-targeting molecule conjugate and an immunomodulator, for example, to treat various cancers.

[0530] As described herein, PIT-induced cell killing by administering one or more phthalocyanine dye conjugates to a subject with a tumor in combination with irradiation can result in increased tumor permeability, such as increased vascular permeability in the tumor space. It is believed that this increased permeability can lead to a rapid leakage of systemically available molecules into the tumor space, thereby maximizing the tumor's exposure to these molecules. Thus, in some embodiments, in the combination therapies provided herein, the anticancer agent is administered to the subject sufficient time prior to irradiation of the administered phthalocyanine dye-targeting molecule conjugate to render the anticancer agent systemically available, such as typically at least 5 minutes prior to irradiation, such as at least 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours prior to irradiation. In such embodiments, following irradiation and PIT-induced killing of tumor cells, the systemically available anticancer agent can be immediately taken up into the tumor space, where it can provide a therapeutic effect. Therefore, in contrast to methods where the anticancer agent is administered after irradiation and, therefore, after PIT-induced cell killing, there is no lag time for therapeutic effects in this method because the anticancer agent is directly accessible and immediately taken up into the tumor space. This can maximize the therapeutic response to the anticancer agent.

[0531] The appropriate time of administration of a particular anticancer agent prior to irradiation can be determined by one skilled in the art to ensure adequate systemic availability of the anticancer agent. In many cases, the pharmacokinetics of a particular anticancer agent are well known in the art. In some cases, pharmacokinetics can be evaluated by measuring the following parameters after administration: maximum (peak) plasma concentration (C max ), peak time (i.e., the time when the maximum plasma concentration occurs; T max ), minimum plasma concentration (i.e., the minimum plasma concentration between doses of the drug; C min ), elimination half-life (T 1 / 2) and the area under the curve (i.e., the area under the curve generated by plotting time against the plasma concentration of the agent; AUC). The concentration of a particular agent in plasma following subcutaneous administration can be determined by any method known in the art suitable for assessing the concentration of an agent in a blood sample. For example, an immunoassay, such as an ELISA, or a chromatography / mass spectrometry-based assay can be used.

[0532] In some embodiments, the anticancer agent used in the conjoint therapy provided by the present invention may refer to any agent or compound used in anticancer treatment. It is included in the clinical symptoms or diagnostic markers relevant to tumors or cancers or the remission state of the clinical symptoms or diagnostic markers when used alone or in combination with other compounds, and can be used for the combination and composition provided by the present invention. In some embodiments, the therapeutic effect of the anticancer agent is generally related to the penetration or delivery of the anticancer agent to the tumor microenvironment or tumor space. In some embodiments, the anticancer agent is an alkylating agent, a platinum drug, an antimetabolite, an antitumor antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a corticosteroid, a proteasome inhibitor, a kinase inhibitor, a histone deacetylase inhibitor, or an antibody or antigen-binding antibody fragment thereof. In some embodiments, the anticancer agent is a peptide, a protein, or a small molecule drug.

[0533] In some embodiments, the anticancer agent is 5-fluorouracil / leucovorin, oxaliplatin, irinotecan, regorafenib, aflibercept, capecitabine, cisplatin, paclitaxel, topotecan, carboplatin, gemcitabine, docetaxel, 5-FU, ifosfamide, mitomycin, pemetrexed, vinorelbine, carmustine wafers, temozolomide, methotrexate, capecitabine, lapatinib, etoposide, dabrafenib, vemurafenib, liposomal cytarabine, cytarabine, interferon alpha, erlotinib, vincristine, cyclophosphamide, lomustine, procarbazine, sunitinib, somatostatin, doxorubicin, PEGylated liposome encapsulated doxorubicin, epirubicin, eribulin , nab-paclitaxel, ixabepilone, co-trimoxazole, a taxane, vinblastine, temsirolimus, temozolomide, bendamustine, oral etoposide, everolimus, octreotide, lanreotide, dacarbazine, mesna, pazopanib, eribulin, imatinib, regorafenib, sorafenib, nilotinib, dasatinib, celecoxib, tamoxifen, toremifene, dactinomycin D, sirolimus, crizotinib, ceritinib, enzalutamide, abiraterone acetate, mitoxantrone, cabazitaxel, a fluoropyrimidine, oxaliplatin, leucovorin, afatinib, ceritinib, gefitinib, cabozantinib, oxaliplatin, or auroropyrimidine.

[0534] In some embodiments, the anticancer agent is an antibody or antigen-binding antibody fragment. In some embodiments, the anticancer agent can be one or more selected from the group consisting of bevacizumab, cetuximab, panitumumab, ramucirumab, ipilimumab, rituximab, trastuzumab, Ado-trastuzumab emtansine, pertuzumab, nivolumab, lapatinib, dabrafenib, vemurafenib, erlotinib, sunitinib, pazopanib, imatinib, regorafenib, sorafenib, rituximab, rituximab, trastuzumab, trastuzumab, emtansine, pertuzumab, nivolumab, lapatinib, dabrafenib, vemurafenib, erlotinib, sunitinib, pazopanib, imatinib, regorafenib, sorafenib, rituximab ...

[00155] The present invention relates to an anti-cancer drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug, wherein the inhibitor is abacterial drug targeting an inhibitor of abacterial drug,

[0535] In some embodiments, the anticancer agent is an alkylating agent. Alkylating agents are compounds that directly damage DNA and inhibit DNA synthesis by forming covalent bonds with nucleic acids. Exemplary alkylating agents include, but are not limited to, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, and thiotepa, as well as nitrosourea alkylating agents such as carmustine and lomustine.

[0536] In some embodiments, the anticancer agent is a platinum drug. Platinum drugs bind to DNA and cause DNA crosslinking, ultimately triggering apoptosis. Exemplary platinum drugs include, but are not limited to, cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin.

[0537] In some embodiments, the anticancer agent is an antimetabolite. Antimetabolites interfere with DNA and RNA growth by replacing the normal structural modules of RNA and DNA. These agents damage cells during the S phase, when the chromosomes of the cells are replicating. In some cases, antimetabolites can be used to treat leukemia, breast cancer, ovarian cancer and intestinal cancer, as well as other types of cancer. Exemplary antimetabolites include but are not limited to: 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (C13), ... Cytarabine Floxuridine, fludarabine, gemcitabine Hydroxyurea, methotrexate, and pemetrexed

[0538] In some embodiments, the anticancer agent is an antitumor antibiotic. Antitumor antibiotics work by altering the DNA within cancer cells to prevent them from growing and replicating. Anthracyclines are antitumor antibiotics that interfere with enzymes involved in DNA replication. These drugs generally work at all stages of the cell cycle. They can be widely used in various cancers. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin, epirubicin, and idarubicin. Other antitumor antibiotics include actinomycin D, bleomycin, mitomycin C, and mitoxantrone.

[0539] In some embodiments, the anticancer agent is a topoisomerase inhibitor. These drugs interfere with enzymes called topoisomerases, which help separate DNA strands so they can replicate during the S phase. Topoisomerase inhibitors can be used to treat certain leukemias, as well as lung cancer, ovarian cancer, gastrointestinal cancer, and other cancers. Exemplary topoisomerase inhibitors include, but are not limited to, doxorubicin, topotecan, irinotecan (CPT-11), etoposide (VP-16), teniposide, and mitoxantrone.

[0540] In some embodiments, the anticancer agent is a mitotic inhibitor. Mitotic inhibitors are typically plant alkaloids and other compounds derived from natural plant products. They work by stopping mitosis during the M phase of the cell cycle, but in some cases, they can damage cells at all phases by preventing enzymes from producing proteins needed for cell proliferation. Exemplary mitotic inhibitors include, but are not limited to, paclitaxel. Docetaxel Ixapilone Vinblastine vincristine Vinorelbine and estramustine

[0541] In some embodiments, the anticancer agent is a corticosteroid. Corticosteroids, often referred to as st...

Claims

1. Use of a pharmaceutical composition comprising a conjugate comprising a silicon phthalocyanine dye linked to a targeting molecule, wherein the targeting molecule is capable of binding to a protein on the surface of a cell on a tumor or in a tumor microenvironment, for preparing a medicament for treating cancer in a subject, wherein the treatment regimen comprises: a) administering an immune checkpoint inhibitor to a human subject; b) administering the conjugate to a human subject; and c) irradiating the tumor with a cytotoxic wavelength of 600 nm to 850 nm, thereby making the conjugate cytotoxic and treating cancer.

2. Use of a pharmaceutical composition comprising a conjugate comprising a silicon phthalocyanine dye linked to an antibody or antigen-binding antibody fragment for the preparation of a medicament for treating cancer in a subject, wherein the treatment regimen comprises: a) administering an immune checkpoint inhibitor to a human subject; b) administering the conjugate to a human subject; and c) irradiating the tumor with a wavelength of 600 nm to 850 nm, thereby making the conjugate cytotoxic and treating cancer.

3. Use of a pharmaceutical composition comprising a conjugate comprising a silicon phthalocyanine dye linked to an antibody or antigen binding fragment that binds to epidermal growth factor receptor (EGFR) on the surface of a tumor cell, for preparing a drug for treating a tumor, wherein the treatment regimen comprises: a) administering an immune checkpoint inhibitor to a human subject; b) administering the conjugate to a human subject; and c) irradiating the tumor with a wavelength of 600 nm to 850 nm, thereby making the conjugate cytotoxic, thereby treating the tumor.

4. Use of a pharmaceutical composition comprising a conjugate comprising a silicon phthalocyanine dye linked to an antibody or antigen binding fragment that binds to cell surface CD25, for preparing a drug for treating a tumor, wherein the treatment regimen comprises: a) administering an immune checkpoint inhibitor to a human subject; b) administering the conjugate to a human subject; and c) irradiating the tumor with a wavelength of 600 nm to 850 nm, thereby making the conjugate cytotoxic, thereby treating the tumor.

5. Use of a pharmaceutical composition comprising a conjugate comprising a silicon phthalocyanine dye linked to cetuximab or an antigen-binding fragment of cetuximab for the preparation of a medicament for treating a tumor, wherein the treatment regimen comprises: a) administering an anti-PD-1 antibody to a human subject; b) administering the conjugate to a human subject; and c) irradiating the tumor with a wavelength of 600 nm to 850 nm, thereby making the conjugate cytotoxic, thereby treating the tumor.

6. Use of a pharmaceutical composition comprising a conjugate comprising a silicon phthalocyanine dye of formula I linked to a targeting molecule, wherein the targeting molecule is capable of binding to a protein on the surface of a cell on a tumor or in a tumor microenvironment, for the preparation of a medicament for treating cancer in a subject, wherein the treatment regimen comprises: a) administering an immune checkpoint inhibitor to a human subject; b) administering the conjugate to a human subject; and c) irradiating the tumor at a wavelength of 600 nm to 850 nm, thereby making the conjugate cytotoxic, thereby treating the tumor, Wherein formula I is: in: L is the connector; Q is a reactive group used to attach the dye to the targeting molecule; R2, R3, R7 and R8 are each independently selected from: optionally substituted alkyl and optionally substituted aryl; R4, R5, R6, R9, R10 and R11 are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl and a chelating ligand, wherein at least one of R4, R5, R6, R9, R10 and R11 comprises a water-soluble group; R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22 and R23 are each independently selected from the group consisting of hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino and optionally substituted alkoxy; and X2 and X3 are each independently C1-C10 alkylene, optionally interrupted by heteroatoms.

7. Use of a pharmaceutical composition comprising a conjugate comprising an IR700 dye linked to a targeting molecule, wherein the targeting molecule is capable of binding to a protein on the surface of a cell on a tumor or in a tumor microenvironment, for the preparation of a medicament for treating cancer in a subject, wherein the treatment regimen comprises: a) administering an immune checkpoint inhibitor to a human subject; b) administering the conjugate to a human subject; and c) irradiating the tumor with a wavelength of 600 nm to 850 nm, thereby making the conjugate cytotoxic, thereby treating cancer.

8. Use of a pharmaceutical composition comprising a conjugate comprising an IR700 dye linked to an antibody for the preparation of a medicament formulated for intravenous administration to treat a tumor associated with cancer in a subject, wherein prior to use, The pharmaceutical composition comprises the following features: ( a) less than or equal to 3% of free dye, (b) less than or equal to 5% of high molecular weight substances, (c) less than or equal to 5% of low molecular weight species, and (d) greater than or equal to 92% monomer; Treatment options include: i) administering an anti-PD-1 antibody to a human subject; ii) administering the conjugate to a human subject; and iii) irradiating the tumor with a wavelength of 600 nm to 850 nm, thereby making the conjugate cytotoxic, thereby treating the tumor.

9. A phototoxic conjugate comprising at least one silicon phthalocyanine dye conjugated to a targeting molecule that binds to PD-L1.

10. A pharmaceutical composition comprising an IR700 dye linked to cetuximab, wherein the pharmaceutical composition comprises the following features: (a) less than or equal to 3% of free dye, (b) less than or equal to 5% of high molecular weight substances, (c) less than or equal to 5% of low molecular weight species, and (d) Greater than or equal to 92% monomers.

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