Phthalocyanine pigment conjugate and its preservation method

Abstract

To provide methods for producing phthalocyanine dye-containing conjugates.SOLUTION: A method of the invention comprises one or more steps of preparing or producing a conjugate, formulating the conjugate and packaging the conjugate. In some aspects, the production method brings about generation of a stable conjugate. Also provided are stable phthalocyanine dye conjugates, compositions and products comprising the stable conjugates as well as administration methods thereof to a subject for photoimmunotherapy. In some embodiments, the phthalocyanine conjugate is conjugated to a targeting molecule such as an antibody that targets the conjugate to a cell or pathogen by binding to a cell surface protein.SELECTED DRAWING: Figure 2A

Description

[Technical Field]

[0001] field This application claims priority to U.S. Provisional Patent Application No. 62 / 206,774, filed on 18 August 2015, entitled "Methods for Manufacturing Phthalocyanine Dye-Macromolecule Conjugates and Stable Conjugates," the contents of which are incorporated in their entirety by reference.

[0002] Inclusion by referencing sequence listings This application is filed together with an electronically formatted sequence listing. The sequence listing is provided as a 9,861-byte file titled 751702000140seqlist.txt, created on August 17, 2016. The electronically formatted information of the sequence listing is incorporated in its entirety by reference.

[0003] field In some aspects, this disclosure relates to a method for producing a phthalocyanine dye-containing conjugate, comprising one or more steps of preparing or producing the conjugate, formulating the conjugate, and packaging the conjugate for storage. In some aspects, the production method results in the production of a stable conjugate. In some aspects, this disclosure further relates to stable phthalocyanine dye conjugates, compositions and products containing the stable conjugate, and methods for administering them to subjects for photoimmunotherapy. In some embodiments, the phthalocyanine dye conjugate is conjugated to a targeting molecule, such as an antibody, which causes the conjugate to target cells or pathogens (target-directed), for example by binding to a cell surface protein. [Background technology]

[0004] background Various treatment methods are available to treat diseases such as cancer. For example, photoimmunotherapy (PIT) is a method that uses a photosensitizer conjugated to a cell surface target molecule, such as an antibody or other targeting molecule that targets a cell surface receptor, to enable targeted killing of specific cells. In some cases, PIT can selectively target diseased cells, such as tumor cells, thereby selectively killing such cells without damaging healthy cells. For use in such methods, there is a need for improved strategies to enhance phthalocyanine dye conjugates, for example, strategies that minimize or avoid photodegradation of the conjugate when used in PIT and improve the activity of the conjugate. Methods and conjugates that satisfy such needs are provided. [Overview of the Initiative]

[0005] overview In some embodiments, a method for producing a phthalocyanine dye conjugate is provided. In some embodiments, the method includes contacting a targeted molecule, e.g., a macromolecule, with a phthalocyanine dye (in some cases, containing a reactive chemical group) under conditions that produce a conjugate containing a phthalocyanine dye linked, e.g., covalently bonded, to the targeted molecule. In some embodiments, the method includes formulating the conjugate in a pharmaceutically acceptable buffer. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and / or after the preparation of the conjugate has a wavelength in the range of about 400 nm to about 650 nm. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and / or after the preparation of the conjugate, e.g., during the contact step and / or the formulation step, has an intensity of less than 500 lux.

[0006] In some embodiments, the method includes contacting a targeting molecule with a phthalocyanine dye in a molar ratio of dye:targeting molecule of 1:1 to 1000:1 or about 1:1 to 1000:1. In some embodiments, the dye contains a reactive chemical group under conditions that produce a conjugate containing the phthalocyanine dye covalently attached to the adherent group of the targeting molecule. In some embodiments, the method includes formulating the conjugate in a pharmaceutically acceptable buffer to a concentration of 0.01 mg / mL to 1000.0 mg / mL or about 0.01 mg / mL to 1000.0 mg / mL. In some embodiments, before, during, and / or after the preparation of the conjugate, for example, during the contact step and / or formulation step, the only light to which the dye and conjugate are exposed has an intensity of less than 500 lux.

[0007] In some embodiments, the conjugate is formulated to a concentration of 0.01 mg / mL to approximately 200.0 mg / mL, or approximately 0.01 mg / mL to approximately 200.0 mg / mL, or approximately 0.5 mg / mL to approximately 10.0 mg / mL. In some embodiments, the conjugate is formulated to a concentration of 0.5 mg / mL to approximately 5.0 mg / mL, or approximately 0.5 mg / mL to approximately 5.0 mg / mL.

[0008] In some embodiments, prior to the contact step, the phthalocyanine dye is dissolved in a solvent under conditions that the only light to which the dye is exposed has a wavelength in the range of about 400 nm to about 650 nm. In some embodiments, the only light to which the dye is exposed while or after being dissolved in the solvent has an intensity of less than 500 lux. In some embodiments, the dye is dissolved in the solvent at a concentration in the range of 0.1 mg / mL to about 100 mg / mL or about 0.1 mg / mL to about 100 mg / mL. In some embodiments, the dye is dissolved in the solvent at a concentration of 1 mg / mL to about 50 mg / mL or about 1 mg / mL to about 50 mg / mL. In some embodiments, the concentration of the phthalocyanine dye in the solvent is about 10 mg / mL. In some embodiments, the solvent is dimethyl sulfoxide (DMSO) or DMF and an aqueous solvent.

[0009] In some embodiments, the formulation step includes a step of conjugating the conjugate.

[0010] In some embodiments, the contact process is performed for at least 5 minutes, at least 15 minutes, at least 30 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, at least 240 minutes, at least 360 minutes, at least 24 hours, at least 72 hours, or at least 120 hours. In some embodiments, the contact process is performed for 5 minutes to 150 hours, 5 minutes to 100 hours, 5 minutes to 48 hours, 5 minutes to 24 hours, 5 minutes to 6 hours, 5 minutes to 2 hours, 5 minutes to 90 minutes, 5 minutes to 60 minutes, 5 minutes to 30 minutes, 30 minutes to 150 hours, 30 minutes to 100 hours, 30 minutes to 48 hours, 30 minutes to 24 hours, 30 minutes to 6 hours, 30 minutes to 2 hours, 30 minutes to 90 minutes, The sessions are conducted for 30-60 minutes, 60 minutes-150 hours, 60 minutes-100 hours, 60 minutes-48 hours, 60 minutes-24 hours, 60 minutes-6 hours, 60 minutes-2 hours, 60 minutes-90 minutes, 90 minutes-150 hours, 90 minutes-100 hours, 90 minutes-48 hours, 90 minutes-24 hours, 90 minutes-6 hours, 90 minutes-2 hours, 2 hours-150 hours, 2 hours-100 hours, 2 hours-48 hours, 2 hours-24 hours, 2 hours-6 hours, 6 hours-150 hours, 6 hours-100 hours, 6 hours-48 hours, 6 hours-24 hours, 24 hours-150 hours, 24 hours-100 hours, 24 hours-48 hours, 48 ​​hours-150 hours, 48 ​​hours-100 hours, or 100 hours-150 hours. In some embodiments, the contact process is carried out at a temperature of 4°C to about 37°C or about 4°C to about 37°C. In some embodiments, the contact process is carried out at a temperature of about 25°C ± 2.0°C, 25°C ± 1.0°C, or 25°C ± 0.3°C.

[0011] In some embodiments, the phthalocyanine dye is covalently or noncovalently linked to the targeting molecule. In some embodiments, the phthalocyanine dye contains a reactive chemical group, and the step of contacting the phthalocyanine dye with the targeting molecule produces a conjugate containing the phthalocyanine dye covalently linked to the attachment group of the targeting molecule.

[0012] In some embodiments, the method further includes a step of quenching the conjugate to remove, for example, an unconjugated dye. In some embodiments, the only light to which the conjugate is exposed during the quenching step has a wavelength in the range of about 400 nm to about 650 nm. In some embodiments, the only light to which the conjugate is exposed during the quenching step has an intensity of less than 500 lux.

[0013] In some embodiments, the formulation step includes ultrafiltration, diafiltration, or dialysis. In some embodiments, the method further includes sterile filtration of the conjugate.

[0014] In some embodiments, the method includes the step of packaging the conjugate in, for example, one or more light-protective containers. In some embodiments, during the packaging step, the only light to which the conjugate is exposed has a wavelength in the range of about 400 nm to about 650 nm. In some embodiments, during the packaging step, the only light to which the dye and conjugate are exposed has an intensity of less than 500 lux.

[0015] In some embodiments, the method comprises the step of dissolving a phthalocyanine dye (in some cases containing a reactive chemical group) in a solvent to a concentration of about 0.1 to 100 mg / mL. In some embodiments, the method further comprises the step of contacting a targeting molecule and a phthalocyanine dye under conditions that produce a conjugate containing a covalently bonded phthalocyanine dye linked to the targeting molecule, for example, in a dye:targeting molecule molar ratio of 1:1 to 1000:1. In some embodiments, the method further comprises the step of formulating the conjugate in a pharmaceutically acceptable buffer to a concentration of 0.01 to 1000.0 mg / mL or about 0.01 to 1000.0 mg / mL. In some embodiments, the method further comprises the step of packaging the conjugate in one or more photoprotective containers. In some embodiments, before, during, and / or after the steps of the Method, such as dissolution, contact, formulation, and packaging, the only light to which the dye and conjugate are exposed has a wavelength in the range of about 400 nm to about 650 nm, or the only light to which the dye and conjugate are exposed has an intensity of less than 500 lux.

[0016] In some embodiments, a method is provided for producing a phthalocyanine dye-targeting molecule conjugate, comprising the steps of: a) dissolving a phthalocyanine dye in a solvent to a concentration of about 0.1 to 100 mg / mL; b) contacting a targeting molecule and a phthalocyanine dye under conditions that produce a conjugate containing a phthalocyanine dye linked to a targeting molecule, with a molar ratio of dye to targeting molecule of 1:1 to 1000:1; c) formulating the conjugate in a pharmaceutically acceptable buffer to a concentration of 0.01 to about 200.0 mg / mL or about 0.01 to about 200.0 mg / mL; and d) packaging the conjugate in one or more photoprotective containers, wherein in each of steps a) to d), the only light to which the dye and conjugate are exposed has a wavelength in the range of about 400 nm to about 650 nm, or the only light to which the dye and conjugate are exposed has an intensity of less than 500 lux.

[0017] In some of these embodiments, the phthalocyanine dye is covalently or noncovalently linked to the targeting molecule. In some of these embodiments, the phthalocyanine dye comprises a reactive chemical group, and the step of contacting the phthalocyanine dye with the targeting molecule produces a conjugate comprising the phthalocyanine dye covalently linked to the attachment group of the targeting molecule.

[0018] In some embodiments, during the steps of the provided method, the total exposure of the dye and conjugate to any light is 5000 lux hours or less, 2500 lux hours or less, 1000 lux hours or less, 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, or 80 lux hours or less. In some embodiments, during the packaging step of the provided method, the total exposure of the conjugate to any light is 5000 lux hours or less, 2500 lux hours or less, 1000 lux hours or less, 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, or 80 lux hours or less.

[0019] In some embodiments, the dye has a maximum absorption wavelength of 600 nm to about 850 nm or about 600 nm to about 850 nm. In some embodiments, the dye has a maximum absorption wavelength of 650 nm to about 850 nm or about 650 nm to about 850 nm. In some embodiments, the dye has a maximum absorption wavelength of 680 nm to about 850 nm or about 680 nm to about 850 nm.

[0020] In some embodiments, the phthalocyanine dye contains the following formula: TIFF0007901958000001.tif92128In formula, L is the linker; Q is a reactive group for the attachment of the dye to the target molecule; R 2 , R 3 , R 7 , and R 8 Each is independently selected from among optionally substituted alkyls and optionally substituted aryls; R4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of which contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; and X 2 and X 3 are each independently C1-C 10 alkylene which may have a heteroatom intervening.

[0021] In some embodiments, the phthalocyanine dye contains the following formula: TIFF0007901958000002.tif90150 where X 1 and X 4 are each independently C1-C 10 alkylene which may have a heteroatom intervening; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 Each is independently selected from hydrogen, an optionally substituted alkyl, an optionally substituted alkanoyl, an optionally substituted alkoxycarbonyl, an optionally substituted alkylcarbamoyl, and a chelate ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of them contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 Each of these is independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy.

[0022] In some embodiments, the reactive group is selected from amine-reactive chemical groups, sulfhydryl-reactive chemical groups, activated esters, acyl halides, alkyl halides, anhydrides, carboxylic acids, carbodiimides, carbonates, carbamates, haloacetamides, isocyanates, isothiocyanates, maleimides, NHS esters, phosphoramidites, platinum complexes, sulfonic acid esters, and thiocyanates. In some embodiments, the reactive chemical group is a sulfhydryl-reactive chemical group selected from maleimides, haloacetyls, and pyridyl disulfides. In some embodiments, the phthalocyanine dye is covalently bonded to a lysine residue of the targeting molecule. In some embodiments, the reactive group is an amine-reactive chemical group. In some embodiments, the reactive chemical group is an amine-reactive chemical group that is an N-hydroxysuccinimide (NHS) ester.

[0023] In some embodiments, the phthalocyanine dye is IRDye 700DX-NHS (IR700-NHS).

[0024] In some embodiments, the targeting molecule binds directly or indirectly to an antigen or protein. For example, in some embodiments, the targeting molecule is a second binding molecule that binds to a first binding molecule, and the first binding molecule is capable of binding to an antigen or protein. In some embodiments, the targeting molecule is a secondary antibody.

[0025] In some embodiments, the targeted molecule binds to a cell surface target molecule on the surface of a cell or pathogen, such as a stem cell, proliferating cell, cancer cell, hyperplastic cell, tumor cell, inflammatory cell, nerve cell, pathogen, or pathogen-infected cell. In some embodiments, the pathogen is selected from viruses, bacteria, fungi, biofilms, and other prokaryotic cell lines. In some embodiments, the cell is a cancer cell, tumor cell, inflammatory cell, or nerve cell. In some embodiments, the cell is present in the microenvironment of a lesion associated with a disease or pathological condition. In some embodiments, the lesion is a tumor, and the cell is a cancer cell or tumor cell. In some embodiments, the cell is a cancer stem cell or circulating tumor cell.

[0026] In some such embodiments, the inflammatory cells are leukocytes such as neutrophils, eosinophils, basophils, lymphocytes, or monocytes. In some embodiments, the targeting molecule is a nerve cell such as a peripheral nervous system nerve cell or a central nervous system nerve cell. In some embodiments, the nerve cell is a nociceptor such as a nociceptor, mechanonociceptor, cheminonociceptor, or multimode nociceptor. In some embodiments, the targeting molecule binds to a pathogen such as a virus, bacteria, fungi, biofilm, or other prokaryotic cell lineage. In some embodiments, the pathogen is a Gram-negative or Gram-positive bacterium.

[0027] In some embodiments, the cell surface target molecule includes an antigen, polypeptide, lipid or carbohydrate, or a combination thereof.

[0028] In some embodiments, cell surface target molecules include cell membrane phospholipids, prokaryotic peptidoglycans, bacterial cell envelope proteins, viral capsid proteins, ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptors BB1, BB2, BB4, calcitonin receptor, and cancer antigen 125 (CA). 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endothialin, EP2, EP4 EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, Family A G protein-coupled receptor (rhodopsin-like), Family B G protein-coupled receptor (secretin-like), Family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV16 / 18 and E6 / E7 antigens, hTERT, interleukin receptors (e.g., IL-2R, IL11-R, IL-13R), ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE1, MAGE2, MAGE3, MAGE4, ​​MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Not ch-1, NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease-cleaved collagen IV, proteinase 3, inhibitor, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 The following are selected from patched, RET receptor, plexin, smoothed, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptor (VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, and VEGF-3 or FLT-4), voltage-gated ion channels, VPAC1, VPAC2, Wilms tumor 1, Y1, Y2, Y4, and Y5.

[0029] In some embodiments, cell surface target molecules include HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, and cancer antigen 125 (CA). 125), alpha-fetoprotein (AFP), Lewis Y, TAG72, caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate-binding protein, ganglioside (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), VEGFR2, VEGF-A, integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAI The following are selected from LR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinase, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, SK-1 antigen, Bcr-abl, RET, MET, TRKB, TIE2, ALK, ROS, EML4-ALK, ROS1, BRAFV600E, SRC, c-KIT, PDGFR, mTOR, TSC1, TSC2, BTK, KIT, BRCA, CDK 4 / 6, JAK1, JAK2, BRAF, FLT-3, MEK1, MEK2, and SMO. In some embodiments, the cell surface target molecules are HER1 / EGFR, HER2, PD-L1, CD25, EpCAM, EphA2, CD206, CD20, CD44, CD133, mesothelin, glypican-3, or carcinoembryonic antigen (CEA).

[0030] In some embodiments, at least a portion of the targeted molecules are proteins, glycoproteins, antibodies, antibody fragments, antigens, antigen-binding fragments, peptides, polypeptides, tissue-homing peptides, small molecules, synthetic polymers, polymer 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, deoxyribonucleotides, DNA fragments, RNA fragments, aptamers, nucleotide triphosphates, acycloterminator triphosphates, or PNAs, or combinations thereof.

[0031] In some embodiments, the targeting molecule is a tissue-specific homing peptide. In some embodiments, the homing peptide has an amino acid sequence as shown in any of SEQ ID NO: 1 to 52.

[0032] In some embodiments, the targeting molecule is an activatable cell-permeable peptide (ACPP) consisting of RGD polypeptides, iRGD polypeptides, Lyp-1 polypeptides, crypto-1 conjugated polypeptides, somatostatin receptor-conjugated polypeptides, inhibitor-conjugated polypeptides, NGR polypeptides, iNGR polypeptides, or polycationic cell-permeable peptides (CPPs) linked to a neutralizing polyanion via a cleavable linker.

[0033] In some embodiments, ACPP comprises the structure A-X1-B-, where B is a peptide moiety of about 5 to about 20 basic amino acid residues suitable for intracellular uptake; A is a peptide moiety of about 2 to about 20 acidic amino acid residues that, when linked with moiety B, are effective in inhibiting or preventing intracellular uptake of moiety B; X1 is a cleavable linker of about 2 to about 100 atoms; and one or more LYs are linked to the C-terminus of peptide moiety B.

[0034] In some embodiments, the targeted molecules are adrenocorticotropic hormone (ACTH), angiotensin II, atrial natriuretic factor (ANF), bombesin, bradykinin, brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), calcitonin, cardiotrophin 1 (BMP-2), CD22, CD40, cholecystokinin (CCK), ciliary neurotrophic factor (CNTF), CCL1-CCL28, CXCL1-CXCL17, XCL1, XCL2, CX 3CL1, Crypt-1 binding peptide, Vascular endothelial growth factor (VEGF), Epidermal growth factor (EGF), Endothelin 1, Endothelin 1 / 3, FAS-ligand, Fibroblast growth factor 1 (FGF-1), Fibroblast growth factor 2 (FGF-2), Fibroblast growth factor 4 (FGF-4), Fibroblast growth factor 5 (FGF-5), Fibroblast growth factor 6 (FGF-6), Fibroblast growth factor 1 (FGF-7), Fibroblast growth factor 1 (FGF-10), Flt-3, Gastrin, Gastrin-releasing peptide (GRP), Granulocyte colony-stimulating factor (G-CS) F) Granulocyte-macrophage-stimulating factor (GM-CSF), glucagon-like peptide (GLP-1), hepatocyte growth factor (HGF), interferon-alpha (IFN-a), interferon-beta (IFN-b), interferon-gamma (IFNg), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6) Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-9 (IL-9), Interleukin-10 (IL-10), Interleukin-11 (IL-11), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-15 (IL-15), Interleukin-17 (IL-17), Interleukin-19 (IL-19), Luteinizing hormone (LH), Luteinization-releasing hormone (LHRH), Macrophage colony-stimulating factor (M-CSF), Monocyte chemotactic protein 1 (MCP-1),Macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neuromedin B, neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), neurotensin, neuropeptide Y, oxytocin, pituitary adenylate cyclase activating peptide (PACAP), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB (PDGF-AB), platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor CC (PDGF-CC), platelet-derived growth factor Growth factor DD (PDGF-DD), Netrin-1 (NTN1), Netrin-2 (NTN2), Netrin-4 (NTN4), Netrin-G1 (NTNG1) and Netrin-G2 (NTNG2), Ephrin A1 (EFNA1), Ephrin A2 (EFNA2), Ephrin A3 (EFNA3), Ephrin A4 (EFNA4), Ephrin A5 (EFNA5), Semaphorin 3A (SEMA3A), Semaphorin 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 Family Members Bar 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), stroma cell-derived factor 1 (SDF-1),It is selected from substance P, thyroid stimulating hormone (TSH), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), 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.

[0035] In some embodiments, the targeting molecule is an antibody or an antibody fragment.

[0036] In some embodiments, the antibody is cetuximab, panitumumab, zalutumumab, nimotuzumab, trastuzumab, Ado-trastuzumab emtansine, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), afatinib, axitinib, bosutinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, olaparib, palbociclib, pazopanib, pertuzumab, ramucirumab, regorafenib, luxitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, vismodegib, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab (CT-011), AMP-224, MSB001078C or MEDI4736, or an antigen-binding fragment thereof. In some embodiments, the antibody binds to a cell surface target molecule such as HER1 / EGFR, HER2, PD-L1, CD25, EpCAM, EphA2, CD206, CD20, CD44, CD133, mesothelin, glypican-3, or carcinoembryonic antigen (CEA). In some embodiments, the antibody is cetuximab, panitumumab, trastuzumab, BMS-935559, MEDI4736, MPDL3280A or MSB0010718C, or an antigen-binding fragment thereof.

[0037] In some embodiments, the dye-targeting molecule conjugate is cetuximab-IR700, panitumumab-IR700, trastuzumab-IR700, BMS-935559-IR700, MEDI4736-IR700, MPDL3280A-IR700 or MSB0010718C-IR700.

[0038] In some embodiments, the targeting molecule is brought into contact with a phthalocyanine dye in a dye:targeting molecule molar ratio of 1:1 to 100:1 or 1:1 to 10:1. In some embodiments, the dye:targeting molecule molar ratio is at least 4:1 or at least about 4:1, or at least 10:1 or at least about 10:1. In some embodiments, the conjugate contains 1 to about 1000 or about 1 to about 1000 phthalocyanine dye molecules per targeting molecule, 1 to about 10 or about 1 to about 10 phthalocyanine dye molecules per targeting molecule, or 2 to about 5 or about 2 to about 5 phthalocyanine dye molecules per targeting molecule.

[0039] In some embodiments, the conjugate is formulated to a concentration of 1.0 to approximately 5.0 mg / mL or approximately 1.0 to approximately 5.0 mg / mL in, for example, a pharmaceutically acceptable buffer. In some embodiments, the pharmaceutically acceptable buffer is phosphate-buffered saline. In some embodiments, the pharmacoacceptable buffer has a pH of 6.0 to approximately 8.0 or approximately 6.0 to approximately 8.0. In some embodiments, the conjugate is stable for more than 3 months, with more than 90% of the conjugate present as the major monomer component. In some embodiments, the conjugate is stable if it retains more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or approximately 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of its potency, activity, or purity for more than 3 months compared to the conjugate before storage. In some embodiments, a conjugate is stable if more than 90% of it is present as the major monomer component. In some embodiments, the pharmacologically acceptable buffer has a pH of 6.8 to approximately 7.4 or approximately 6.8 to approximately 7.4.

[0040] In some embodiments, the only light to which the dye and conjugate are exposed has a wavelength in the range of about 425 nm to about 575 nm. In some embodiments, the only light to which the dye and conjugate are exposed has an intensity of less than 200 lux.

[0041] In some embodiments, the container containing the conjugate is protected from the transmission of light having wavelengths of 250 nm to approximately 800 nm, or approximately 250 nm to approximately 800 nm, approximately 250 nm to approximately 450 nm, approximately 400 nm to approximately 800 nm, approximately 450 nm to approximately 650 nm, or approximately 600 nm to approximately 720 nm. In some embodiments, the container is protected from the transmission of light such that the light transmittance through the container is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the container is covered with green, blue, amber, translucent, opaque, or opaque foil. In some embodiments, the container is covered with green, blue, amber, translucent, opaque, or a material having a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments of the methods provided herein, the container is selected from vials, tubes, syringes, bags, pouches, and boxes.

[0042] In some embodiments, the light-protective container is a first light-protective container, and the method further includes the step of packaging the first light-protective container inside a second light-protective container. In some embodiments, the second container protects from the transmission of light having wavelengths of 250 nm to about 800 nm, or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm. In some embodiments, the second container protects from the transmission of light such that the light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the second container is covered with a material that is green, blue, amber, translucent, opaque, or has a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the second container is selected from vials, tubes, syringes, bags, pouches, and boxes.

[0043] In some embodiments, the method provided herein further includes the step of packaging a second container in a third light-protective container. In some embodiments, the third container protects from the transmission of light having wavelengths of 250 nm to about 800 nm, or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm. In some embodiments, the third container protects from the transmission of light such that its light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the third container is covered with a material that is green, blue, amber, translucent, opaque, or has a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the third container is selected from vials, tubes, syringes, bags, pouches, and boxes.

[0044] In some embodiments, the amount of conjugate produced by the method is more than 1 gram or about 1 gram, more than 2 grams or about 2 grams, more than 3 grams or about 3 grams, more than 4 grams or about 4 grams, more than 5 grams or about 5 grams, or more than 10 grams or about 10 grams. In some embodiments, the conjugate is produced using Good Manufacturing Practices (GMP).

[0045] In some embodiments, conjugates produced, formulated, or packaged by the methods described herein are provided. In some embodiments, the conjugates are stable for more than three months, and for example, more than 90% of the conjugate is present as the major monomer component.

[0046] In some embodiments, a stable conjugate containing a phthalocyanine dye linked to a targeting molecule is provided. In some embodiments, the stable conjugate is stable for more than 3 months, and for example, more than 90% of the conjugate is present as the major monomer component.

[0047] In some embodiments, a conjugate is stable if it retains more than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of its potency, activity, or purity compared to the conjugate before storage for more than three months. In some embodiments, a conjugate is stable if more than 90% of the conjugate or stable conjugate is present as the major monomer component. In some embodiments, a conjugate is stable if more than 95% of the conjugate or stable conjugate is present as the major monomer component. In some embodiments, a conjugate or stable conjugate is stable for more than six months or more than twelve months. In some embodiments, a conjugate or stable conjugate is stable at temperatures below 30°C.

[0048] In some embodiments, the phthalocyanine pigment contained in the stable conjugate comprises the following formula: TIFF0007901958000003.tif92128In formula, L is the linker; Q is a reactive group for the attachment of the dye to the target molecule; R 2 , R 3 , R 7 , and R 8 Each is independently selected from among optionally substituted alkyls and optionally substituted aryls; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 Each is independently selected from hydrogen, an optionally substituted alkyl, an optionally substituted alkanoyl, an optionally substituted alkoxycarbonyl, an optionally substituted alkylcarbamoyl, and a chelate ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of them contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 Each is independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino and optionally substituted alkoxy; and X 2 and X 3 Each of these C1-C atoms may have a heteroatom interposed between them, independently of each other. 10 It is alkylene.

[0049] In some embodiments, a stable conjugate containing a phthalocyanine dye comprises the following formula: TIFF0007901958000004.tif89150 wherein, X 1 and X 4 are each independently C1-C 10 alkylene which may be intervened by heteroatoms; R 2 , R 3 , R 7 and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and chelating ligands, wherein at least one of R 4 , R 5 , R 6 , R 9 , R 10 and R 11 contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino and optionally substituted alkoxy.

[0050] In some embodiments, the dye contained in the stable conjugate has a maximum absorption wavelength of 600 nm to about 850 nm or about 600 nm to about 850 nm, 650 nm to about 850 nm or about 650 nm to about 850 nm, or 680 nm to about 850 nm or about 680 nm to about 850 nm.

[0051] In some embodiments, the dye contained in the stable conjugate is IRDye 700DX (IR700).

[0052] In some embodiments, the targeting molecule contained in the stable conjugate binds to a cell surface target molecule on the surface of a cell or pathogen, such as a proliferating cell, cancer cell, hyperplastic cell, tumor cell, inflammatory cell, nerve cell, or pathogen. In some embodiments, the cell is a stem cell, proliferating cell, hyperplastic cell, or cell infected with a pathogen. In some embodiments, the pathogen is selected from viruses, bacteria, fungi, biofilms, and other prokaryotic cell lines. In some embodiments, the inflammatory cell is a white blood cell such as a neutrophil, eosinophil, basophil, lymphocyte, or monocyte. In some embodiments, the targeting molecule is a nerve cell such as a peripheral nervous system nerve cell or a central nervous system nerve cell. In some embodiments, the nerve cell is a nociceptor such as a mechanical nociceptor, chemical nociceptor, or polymodal nociceptor. In some embodiments, the targeting molecule binds to a pathogen such as a virus, bacteria, fungi, biofilm, or other prokaryotic cell line. In some embodiments, the pathogen is a gram-negative bacterium or a gram-positive bacterium.

[0053] In some embodiments, the cell surface target molecule bound by the targeting molecule contained in the stable conjugate includes an antigen, polypeptide, lipid or carbohydrate, or a combination thereof.

[0054] In some embodiments, cell surface target molecules include cell membrane phospholipids, prokaryotic peptidoglycans, bacterial cell envelope proteins, viral capsid proteins, ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptors BB1, BB2, BB4, calcitonin receptor, and cancer antigen 125 (CA). 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endothialin, EP2, EP4 EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, Family A G protein-coupled receptor (rhodopsin-like), Family B G protein-coupled receptor (secretin-like), Family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV16 / 18 and E6 / E7 antigens, hTERT, interleukin receptors (e.g., IL-2R, IL11-R, IL-13R), ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE1, MAGE2, MAGE3, MAGE4, ​​MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Not ch-1, NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease-cleaved collagen IV, proteinase 3, inhibitor, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 A selection is made from patched, RET receptor, plexin, smoothed, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptor (VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, and VEGF-3 or FLT-4), voltage-gated ion channels, VPAC1, VPAC2, Wilms tumor 1, Y1, Y2, Y4, and Y5.

[0055] In some embodiments, cell surface target molecules include HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, and cancer antigen 125 (CA). 125), alpha-fetoprotein (AFP), Lewis Y, TAG72, caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate-binding protein, ganglioside (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), VEGFR2, VEGF-A, integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAI The following are selected from LR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinase, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, SK-1 antigen, Bcr-abl, RET, MET, TRKB, TIE2, ALK, ROS, EML4-ALK, ROS1, BRAFV600E, SRC, c-KIT, PDGFR, mTOR, TSC1, TSC2, BTK, KIT, BRCA, CDK 4 / 6, JAK1, JAK2, BRAF, FLT-3, MEK1, MEK2, and SMO. In some embodiments, the cell surface target molecules are HER1 / EGFR, HER2, PD-L1, CD25, EpCAM, EphA2, CD206, CD20, CD44, CD133, mesothelin, glypican-3, or carcinoembryonic antigen (CEA).

[0056] In some embodiments, at least a portion of the targeted molecules are proteins, glycoproteins, antibodies, antibody fragments, antigens, antigen-binding fragments, peptides, polypeptides, tissue-homing peptides, small molecules, synthetic polymers, polymer 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, deoxyribonucleotides, DNA fragments, RNA fragments, aptamers, nucleotide triphosphates, acycloterminator triphosphates or PNAs, or combinations thereof.

[0057] In some embodiments, the targeting molecule is a tissue-specific homing peptide. In some embodiments, the homing peptide has an amino acid sequence as shown in any of SEQ ID NO: 1 to 52.

[0058] In some embodiments, the targeting molecule is an activatable cell-permeable peptide (ACPP) consisting of RGD polypeptides, iRGD polypeptides, Lyp-1 polypeptides, crypto-1 conjugated polypeptides, somatostatin receptor-conjugated polypeptides, inhibitor-conjugated polypeptides, NGR polypeptides, iNGR polypeptides, or polycationic cell-permeable peptides (CPPs) linked to a neutralizing polyanion via a cleavable linker.

[0059] In some embodiments, ACPP comprises the structure A-X1-B-, where B is a peptide moiety of about 5 to about 20 basic amino acid residues suitable for intracellular uptake; A is a peptide moiety of about 2 to about 20 acidic amino acid residues that, when linked with moiety B, are effective in inhibiting or preventing intracellular uptake of moiety B; X1 is a cleavable linker of about 2 to about 100 atoms; and one or more LYs are linked to the C-terminus of peptide moiety B.

[0060] In some embodiments, the targeted molecules are adrenocorticotropic hormone (ACTH), angiotensin II, atrial natriuretic factor (ANF), bombesin, bradykinin, brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), calcitonin, cardiotrophin 1 (BMP-2), CD22, CD40, cholecystokinin (CCK), ciliary neurotrophic factor (CNTF), CCL1-CCL28, CXCL1-CXCL17, XCL1, XCL2, CX 3CL1, Crypt-1 binding peptide, Vascular endothelial growth factor (VEGF), Epidermal growth factor (EGF), Endothelin 1, Endothelin 1 / 3, FAS-ligand, Fibroblast growth factor 1 (FGF-1), Fibroblast growth factor 2 (FGF-2), Fibroblast growth factor 4 (FGF-4), Fibroblast growth factor 5 (FGF-5), Fibroblast growth factor 6 (FGF-6), Fibroblast growth factor 1 (FGF-7), Fibroblast growth factor 1 (FGF-10), Flt-3, Gastrin, Gastrin-releasing peptide (GRP), Granulocyte colony-stimulating factor (G-CS) F) Granulocyte-macrophage-stimulating factor (GM-CSF), glucagon-like peptide (GLP-1), hepatocyte growth factor (HGF), interferon-alpha (IFN-a), interferon-beta (IFN-b), interferon-gamma (IFNg), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6) Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-9 (IL-9), Interleukin-10 (IL-10), Interleukin-11 (IL-11), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-15 (IL-15), Interleukin-17 (IL-17), Interleukin-19 (IL-19), Luteinizing hormone (LH), Luteinization-releasing hormone (LHRH), Macrophage colony-stimulating factor (M-CSF), Monocyte chemotactic protein 1 (MCP-1),Macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neuromedin B, neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), neurotensin, neuropeptide Y, oxytocin, pituitary adenylate cyclase activating peptide (PACAP), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB (PDGF-AB), platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor CC (PDGF-CC), platelet-derived growth factor Growth factor DD (PDGF-DD), Netrin-1 (NTN1), Netrin-2 (NTN2), Netrin-4 (NTN4), Netrin-G1 (NTNG1) and Netrin-G2 (NTNG2), Ephrin A1 (EFNA1), Ephrin A2 (EFNA2), Ephrin A3 (EFNA3), Ephrin A4 (EFNA4), Ephrin A5 (EFNA5), Semaphorin 3A (SEMA3A), Semaphorin 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 Family Members Bar 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), stroma cell-derived factor 1 (SDF-1),Selected from Substance P, Thyroid-Stimulating Hormone (TSH), Transforming Growth Factor Alpha (TGF-α), Transforming Growth Factor Beta (TGF-β), Tumor Necrosis Factor Alpha (TNF-α), Thrombin, Vasoactive Intestinal Peptide (VIP), Wntl, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wntl0a, Wntl0b, Wnt11, Wnt14, Wnt15, or Wnt16, Sonic Hedgehog, Desert Hedgehog, and Indian Hedgehog.

[0061] In some embodiments, the targeting molecule is an antibody or an antibody fragment.

[0062] In some embodiments, the antibodies include cetuximab, panitumumab, zaltumumab, nimotuzumab, trastuzumab, Ado-trastuzumab emtansine, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, and CEA-scan. Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), afatinib, axitinib, bosutinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, olaparib, palbociclib, pazopanib, pertuzumab, ramucirumab The antibody is either regorafenib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, bismodegib, basiliximab, ipilimumab, nivolumab, pembrolizumab, lambrolizumab, MPDL3280A, pizilizumab (CT-011), MSB001078C, BMS-935559 or MEDI4736, AMP-224, or an antigen-binding fragment thereof. In some embodiments, the antibody binds to cell surface target molecules such as HER1 / EGFR, HER2, PD-L1, CD25, EpCAM, EphA2, CD206, CD20, CD44, CD133, mesothelin, glypican-3, or carcinoembryonic antigen (CEA). In some embodiments, the antibody is cetuximab, panitumumab, trastuzumab, BMS-935559, MEDI4736, MPDL3280A, or MSB0010718C, or an antigen-binding fragment thereof.

[0063] In some embodiments, stable dye-targeting molecule conjugates include cetuximab-IR700, panitumumab-IR700, trastuzumab-IR700, BMS-935559-IR700, MEDI4736-IR700, MPDL3280A-IR700, or MSB0010718C-IR700.

[0064] In some embodiments, the stable conjugate contains 1 to about 1000 or about 1 to about 1000 phthalocyanine dye molecules per targeted molecule, 1 to about 10 or about 1 to about 10 phthalocyanine dye molecules per targeted molecule, or 2 to about 5 or about 2 to about 5 phthalocyanine dye molecules per targeted molecule.

[0065] In some embodiments, compositions containing a conjugate or a stable conjugate are provided.

[0066] In some embodiments, pharmaceutical compositions are provided that contain a conjugate or a stable conjugate and a pharmaceutically acceptable excipient.

[0067] In some embodiments, the composition is formulated in phosphate-buffered saline. In some embodiments, the composition has a pH greater than 6.0.

[0068] In some embodiments, pharmaceutical compositions are provided that contain a phthalocyanine dye linked to a targeted molecule and a pharmaceutically acceptable excipient. In some such embodiments, the composition has a pH greater than 6.0, and the conjugate in the composition is stable for more than 3 months, for example, with more than 90% of the conjugate present as the major monomer component. In some such embodiments, the conjugate in the composition is stable if it retains more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of its potency, activity, or purity for more than 3 months compared to the conjugate before storage. In some such embodiments, the conjugate in the composition is stable if more than 90% of the conjugate present as the major monomer component. In some embodiments, the pH of the composition is greater than 6.0, or is between pH 6.0 and about 8.0, or about pH 6.0 and about 8.0, including values ​​at both ends.

[0069] In some embodiments, the concentration of the conjugate in the composition is 0.01 mg / mL to about 200 mg / mL or about 0.01 mg / mL to about 200 mg / mL. In some embodiments, the concentration of the conjugate in the composition is 0.5 mg / mL to about 10 mg / mL or about 0.5 mg / mL to about 10 mg / mL. In some embodiments, the concentration of the conjugate in the composition is 1.0 to about 5.0 mg / mL or about 1.0 to about 5.0 mg / mL. In some embodiments, the concentration of the conjugate in the composition is 1.8 to about 2.1 mg / mL or about 1.8 to about 2.1 mg / mL.

[0070] In some embodiments, the volume of the composition is 0.5 mL to about 100 mL or about 0.5 mL to about 100 mL, 1 mL to about 50 mL or about 1 mL to about 50 mL, or 1 mL to about 10 mL or about 1 mL to about 10 mL.

[0071] In some embodiments, a container containing a conjugate or a stable conjugate is provided. In some embodiments, the container is protected from the transmission of light having wavelengths of 500 nm to 725 nm or about 500 nm to 725 nm, or 650 nm to 725 nm or about 650 nm to 725 nm. In some embodiments, the container is protected from the transmission of light such that the light transmittance through the container is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the container is covered with a material that is green, blue, amber, translucent, opaque, or has a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the container is covered with an opaque foil.

[0072] In some embodiments, a packaging system is provided for protecting phthalocyanine dye-targeted molecule conjugates from light. In some embodiments, the packaging system includes an internal packaging material comprising a container as described herein. In some embodiments, the internal packaging material has a light transmittance of 5% or less. In some embodiments, the external packaging material contains the internal packaging material. In some embodiments, the external packaging material has a light transmittance of 5% or less. In some embodiments, the internal packaging material comprises an opaque foil.

[0073] In some embodiments, a packaging system is provided for protecting a phthalocyanine dye-targeted molecule conjugate from light, comprising a first container (e.g., any container provided herein) and a second container containing the first container, wherein the second container protects from the transmission of light having wavelengths of 250 nm to about 800 nm, or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm. In some embodiments, the second container is protected from light transmission such that its light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the second container is covered with a material that is green, blue, amber, translucent, opaque, or has a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the first and second containers are independently selected from vials, tubes, syringes, bags, pouches, and boxes.

[0074] In some embodiments, any of the provided packaging systems further comprises a third container containing a second container, wherein the third container protects from the transmission of light having wavelengths of 250 nm to about 800 nm, or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm. In some embodiments, the third container protects from the transmission of light such that its light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the third container is green, blue, amber, translucent, opaque, or coated with a material having a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the third container is selected from vials, tubes, syringes, bags, pouches, and boxes.

[0075] In some embodiments, a kit is provided comprising one of the containers or packaging systems described herein; a photoprotective cover capable of covering a device capable of administering a composition comprising a phthalocyanine dye-targeted molecule conjugate; and optionally, instructions for use. In some embodiments, the administration device is an intravenous bag. In some embodiments, the photoprotective cover protects from the transmission of light having wavelengths of 250 nm to about 800 nm or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm. In some embodiments, the photoprotective cover protects from the transmission of light such that the light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the light-protective cover is covered with a material that is green, blue, amber, translucent, opaque, or has a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.

[0076] In some embodiments, a method is provided for preparing a composition comprising a phthalocyanine dye conjugate for administration, comprising the steps of: depackaging one or more of the containers described herein, or one or more of the packaging systems described herein, including any of the containers described herein; and transferring the composition present in one or more containers to a device capable of administering the composition to a subject, wherein the sole light to which the composition is exposed has a wavelength in the range of about 400 nm to about 650 nm, or the sole light to which the composition is exposed has an intensity of less than 500 lux. In some embodiments, the sole light to which the composition is exposed has an intensity of less than 200 lux or less than 100 lux. In some embodiments, the method provided is carried out in a biosafety cabinet, biosafety hood, or sterile environment. In some embodiments, one or more containers together contain a therapeutically effective dose of phthalocyanine dye conjugate. In some embodiments, one or more containers comprise at least 2, 4, 6, 8, 10, 12, 18, or 24, or about at least 2, 4, 6, 8, 10, 12, 18, or 24, or 2, 4, 6, 8, 10, 12, 18, or 24 containers. In some embodiments, the method provided is carried out for 1 hour or less, 30 minutes or less, or 15 minutes or less; or the total exposure of the composition to any light during the method is 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, 50 lux hours or less, or 25 lux hours or less.

[0077] In some embodiments, the administration device is an intravenous bag. In some embodiments, the administration device includes a photoprotective cover that can cover the device. In some embodiments, the photoprotective cover protects from the transmission of light having wavelengths of 250 nm to about 800 nm, or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm.

[0078] In some embodiments, the light-protective cover protects from light transmission such that the light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the light-protective cover is covered with a material that is green, blue, amber, translucent, opaque, or has a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.

[0079] In some embodiments, a photoprotective device is provided that includes a composition prepared using the method provided herein.

[0080] In some embodiments, a method for removing unwanted cells or pathogens from a subject, comprising: (a) administering a composition comprising a phthalocyanine dye conjugate to the subject from one of the photoprotective devices provided herein, wherein the composition is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step; and (b) irradiating the unwanted cells or pathogens with at least 1 J cm at a wavelength of 660–740 nm. -2 Alternatively, a method is provided which includes the step of irradiating with a dose of 1 J / cm fiber length, thereby removing unwanted cells from the target.

[0081] In some embodiments, a method for removing unwanted cells or pathogens from a subject, comprising the steps of: a) administering a therapeutically effective amount of any of the conjugates or compositions described herein to the subject, wherein the conjugate is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step; and b) irradiating the unwanted cells or pathogens with at least 1 J cm at a wavelength of 660–740 nm. -2 Alternatively, a method is provided which includes the step of irradiating with a dose of 1 J / cm fiber length, thereby removing unwanted cells from the target.

[0082] In some embodiments, a method for removing unwanted cells or pathogens from a subject, comprising the steps of: a) administering a therapeutically effective dose of a conjugate containing IRDye 700DX (IR700) ligated to a targeting molecule capable of binding to the unwanted cells or pathogens, wherein the conjugate is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step; and b) irradiating the unwanted cells or pathogens with at least 1 J cm at a wavelength of 600-800 nm. -2 Alternatively, a method is provided which includes the step of irradiating with a dose of 1 J / cm fiber length, thereby removing unwanted cells or pathogens from the target.

[0083] In some embodiments, a method for removing unwanted cells or pathogens from a subject, comprising: a) administering a therapeutically effective amount of a first binding molecule capable of binding to the unwanted cells or pathogens to the subject; b) administering a conjugate molecule containing IRDye 700DX (IR700) linked to a targeting molecule, wherein the targeting molecule is a second binding molecule capable of binding to the first binding molecule; and c) injecting the unwanted cells or pathogens with at least 1 J cm at a wavelength of 600-800 nm. -2 Alternatively, a method is provided which includes the step of irradiating with a dose of 1 J / cm fiber length, thereby removing unwanted cells or pathogens from the target.

[0084] In some embodiments, a method for removing unwanted cells or pathogens from a sample, comprising: (a) administering a composition comprising a phthalocyanine dye conjugate to the sample from one of the photoprotective devices provided herein, wherein the composition is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step; and (b) irradiating the unwanted cells or pathogens with at least 1 J cm at a wavelength of 660–740 nm. -2 Alternatively, a method is provided which includes the step of irradiating the sample with a dose of 1 J / fiber length cm, thereby removing unwanted cells from the sample.

[0085] In some embodiments, a method for removing unwanted cells or pathogens from a sample, comprising the steps of: a) administering a therapeutically effective amount of any of the conjugates or compositions described herein to the sample, wherein the conjugate is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step; and b) irradiating the unwanted cells or pathogens with at least 1 J cm at a wavelength of 660–740 nm. -2 Alternatively, a method is provided which includes the step of irradiating the sample with a dose of 1 J / fiber length cm, thereby removing unwanted cells from the sample.

[0086] In some embodiments, a method for removing unwanted cells or pathogens from a sample, comprising the steps of: a) administering a therapeutically effective amount of a conjugate containing IRDye 700DX (IR700) linked to a targeting molecule capable of binding to the unwanted cells or pathogens to the sample, wherein the conjugate is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step; and b) irradiating the unwanted cells or pathogens with at least 1 J cm at a wavelength of 600-800 nm. -2 Alternatively, a method is provided which includes the step of irradiating the sample with a dose of 1 J / cm fiber length, thereby removing unwanted cells or pathogens from the sample.

[0087] In some embodiments, a method for removing unwanted cells or pathogens from a sample, comprising: a) administering a therapeutically effective amount of a first binding molecule capable of binding to the unwanted cells or pathogens to the sample; b) administering a conjugate molecule containing IRDye 700DX (IR700) linked to a targeting molecule to the sample, wherein the targeting molecule is a second binding molecule capable of binding to the first binding molecule; and c) injecting the unwanted cells or pathogens with at least 1 J cm at a wavelength of 600-800 nm. -2 Alternatively, a method is provided which includes the step of irradiating the sample with a dose of 1 J / cm fiber length, thereby removing unwanted cells or pathogens from the sample.

[0088] In some embodiments, the method is performed in vitro or ex vivo. In some embodiments, the method is performed using an extracorporeal device.

[0089] In some embodiments of the methods provided herein, the first conjugating molecule is administered to the subject before the conjugate, or the first conjugating molecule and the conjugate are administered to the subject simultaneously. In some embodiments, the targeting molecule is a secondary antibody. In some embodiments, before and during administration of the conjugate, the conjugate is not exposed to ambient light of an intensity greater than 500 lux.

[0090] In some embodiments, cells are stem cells, proliferative cells, cells in a hyperplastic state, inflammatory cells, negatively regulatory immune cells (which may be T cells), pathogen-infected cells, nerve cells, adipocytes, or adipocytes. In some embodiments, cells are cancer cells or tumor cells. In some embodiments, cells are associated with, cause, or contribute to the pathogenesis of a disease or condition. In some embodiments, the disease or condition is a tumor or cancer, an infection, an inflammatory disease or condition, or a neurological disease or condition. In some embodiments, the cell is a nerve cell and the disease or condition is a neurological disorder (optionally pain); the cell is a fat cell or adipocyte and the disease or condition is associated with excess fat; the cell is a cell infected with a pathogen and the disease or condition is an infection; the cell is a pathogen and the disease or condition is an infection; the cell is an inflammatory cell and the disease or condition is an inflammatory disease; the cell is an immune cell (optionally a regulatory T cell) and the disease or condition is a tumor or cancer; or the cell is a tumor or cancer cell and the disease or condition is a tumor or cancer.

[0091] In some embodiments, cells are present in the microenvironment of a lesion associated with a disease or pathological condition, or are in a hyperplastic state. In some embodiments, the lesion is a tumor, and the disease or pathological condition is a tumor or cancer. In some embodiments, the method treats the disease or pathological condition.

[0092] In some embodiments, a method for removing pathogen-infected cells in a subject, comprising the steps of: a) administering a therapeutically effective amount of a conjugate molecule containing IRDye 700DX (IR700) linked to a targeting molecule to the subject, wherein the targeting molecule is capable of directly or indirectly binding to the pathogen-infected cells; and b) irradiating the pathogen-infected cells with at least 1 J cm at a wavelength of 600-800 nm. -2 Alternatively, a method is provided which includes the step of irradiating with a dose of 1 J / cm fiber length to remove pathogen-infected cells in a target. In some embodiments, the pathogen is a virus, bacteria, fungi, biofilm, or other prokaryotic cell lineage. In some embodiments, before and during administration of the conjugate, the conjugate is not exposed to ambient light of an intensity greater than 500 lux.

[0093] In some embodiments, a method for removing pathogen-infected cells from a sample, comprising the steps of: a) administering a therapeutically effective amount of a conjugate molecule containing IRDye 700DX (IR700) linked to a targeting molecule to the sample, wherein the targeting molecule is capable of directly or indirectly binding to the pathogen-infected cells; and b) irradiating the pathogen-infected cells with at least 1 J cm at a wavelength of 600-800 nm. -2 Alternatively, a method is provided which includes the step of irradiating the sample with a dose of 1 J / cm fiber length to remove pathogen-infected cells. In some embodiments, the pathogen is a virus, bacteria, fungi, biofilm, or other prokaryotic cell lineage. In some embodiments, before and during administration of the conjugate, the conjugate is not exposed to ambient light of an intensity greater than 500 lux.

[0094] In some embodiments, the method is performed in vitro or ex vivo. In some embodiments, ex vivo irradiation is a method performed using an extracorporeal device. In some embodiments, a method is provided for treating hyperplasia or tumors in a subject. In some embodiments, the method includes a step of administering a therapeutically effective amount of a conjugate or stable conjugate or composition to a subject, wherein the conjugate is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step. In some embodiments, the method involves irradiating the hyperplasia or tumor with at least 1 J cm at a wavelength of 660–740 nm. -2 The method further includes irradiating with a dose of 1 J / cm of fiber length, thereby treating a tumor in the target.

[0095] In some embodiments, methods are provided for treating hyperplasia or tumors in a subject. In some such embodiments, the method includes administering a therapeutically effective dose of a conjugate containing IRDye 700DX (IR700) linked to a targeting molecule to the subject. In some embodiments, the conjugate is targeted to the hyperplasia or tumor, and before and during the administration step, the conjugate is not exposed to ambient light of an intensity greater than 500 lux. In some embodiments, the method involves applying at least 1 J cm to the tumor at a wavelength of 600-800 nm. -2 The method further includes irradiating with a dose of 1 J / cm of fiber length, thereby treating a tumor in the target.

[0096] In some embodiments, methods are provided for treating hyperplasia or tumors in a sample. In some embodiments, the method includes the step of administering a therapeutically effective amount of a conjugate or stable conjugate or composition to a sample, wherein the conjugate is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step. In some embodiments, the method involves applying at least 1 J cm of wavelength 660-740 nm to the hyperplasia or tumor. -2 The method further includes irradiating the sample with a dose of 1 J / cm of fiber length, thereby treating the tumor in the sample.

[0097] In some embodiments, a method for treating hyperplasia or tumors in a sample is provided. In some such embodiments, the method comprises administering a therapeutically effective amount of a conjugate containing IRDye 700DX (IR700) linked to a targeting molecule to a sample. In some embodiments, the conjugate is targeted to the hyperplasia or tumor, and before and during the administration step, the conjugate is not exposed to ambient light of an intensity greater than 500 lux. In some embodiments, the method involves applying at least 1 J cm to the tumor at a wavelength of 600-800 nm. -2 The method further includes irradiating the sample with a dose of 1 J / cm of fiber length, thereby treating the tumor in the sample.

[0098] In some embodiments, the method is a method for treating a tumor, wherein a targeting molecule of the conjugate causes the conjugate to target the tumor or the tumor microenvironment. In some embodiments, the irradiation of the tumor is at a wavelength of 600-800 nm and at least 1 J cm -2 Alternatively, irradiation at a dose of 1 J / cm of fiber length is used to treat a tumor, for example, a tumor in a subject or sample.

[0099] In some embodiments, the targeting molecule is an antibody, antigen-binding fragment, protein, glycoprotein, peptide, polypeptide, virus, viral capsid, or viral particle. In some embodiments, the targeting molecule is an antibody or antibody fragment.

[0100] In some embodiments, the administration is carried out under fluorescent or LED lighting, and in the absence of direct or indirect sunlight.

[0101] In some embodiments, any exposure of the conjugate to light below 500 lux is an exposure of less than 20 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. In some embodiments, any exposure of the conjugate to light is light with an intensity of 50 lux or less.

[0102] In some embodiments, a tumor is cancer. In some embodiments, cancer is cancer located in the head and neck, breast, liver, colon, ovaries, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung. In some embodiments, cancer is cancer of the blood.

[0103] In some embodiments, the conjugate is targeted to proteins expressed in the tumor. In some embodiments, the conjugate is targeted to proteins expressed on the surface of cells present in the tumor microenvironment. In some embodiments, the cells are tumor cells, immune cells, or cancer stem cells.

[0104] In some embodiments, the proteins expressed in tumors include ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptors BB1, BB2, BB4, calcitonin receptor, and cancer antigen 125 (CA). 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endothialin, EP2, EP4 EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, Family A G protein-coupled receptor (rhodopsin-like), Family B G protein-coupled receptor (secretin-like), Family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV16 / 18 and E6 / E7 antigens, hTERT, IL11-R, IL-13R, ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE1, MAGE2, MAGE3, MAGE4, ​​MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-ESO -1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease-cleaved collagen IV, proteinase 3, inhibitor, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 Patched, RET receptor, Plexin, Smoothed, sst1, sst2A, sst2B, sst3, sst4, sst5, Substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptor (VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, and VEGF-3 or FLT-4), voltage-gated ion channels, VPAC1, VPAC2, Wilms tumor 1, Y1, Y2, Y4, or Y5.

[0105] In some embodiments, the proteins expressed in tumors include HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, and cancer antigen 125 (CA). 125), alpha-fetoprotein (AFP), Lewis Y, TAG72, caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate-binding protein, ganglioside (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), VEGFR2, VEGF-A, integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAI These are LR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinase, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, SK-1 antigen, Bcr-abl, RET, MET, TRKB, TIE2, ALK, ROS, EML4-ALK, ROS1, BRAFV600E, SRC, c-KIT, PDGFR, mTOR, TSC1, TSC2, BTK, KIT, BRCA, CDK 4 / 6, JAK1, JAK2, BRAF, FLT-3, MEK1, MEK2, or SMO.

[0106] In some embodiments, the conjugate is targeted to proteins expressed in the tumor. In some embodiments, cells, hyperplasia, or tumors are irradiated at wavelengths of 600 nm to approximately 850 nm or approximately 600 nm to approximately 850 nm. In some embodiments, tumors are irradiated at wavelengths of 690 ± 50 nm or 690 ± 20 nm.

[0107] In some embodiments, cells, hyperplasia, or tumors are 2J cm -2 ~about 400J cm-2 Or approximately 2J cm -2 ~about 400J cm -2 The cells, hyperplasia, or tumors are irradiated with a dose of at least 2 J / cm fiber length to approximately 500 J / cm fiber length or approximately 2 J / cm fiber length to approximately 500 J / cm fiber length. In some embodiments, cells, hyperplasia, or tumors are irradiated with at least 2 J / cm -2 , 5J cm -2 , 10J cm -2 , 25J cm -2 , 50J cm -2 , 75J cm -2 , 100J cm -2 , 150J cm -2 , 200J cm -2 , 300J cm -2 , 400J cm -2 Alternatively, 500 J cm -2 , or at least about 2J cm -2 , 5J cm -2 , 10J cm -2 , 25J cm -2 , 50J cm -2 , 75J cm -2 , 100J cm -2 , 150J cm -2 , 200J cm -2 , 300J cm -2 , 400J cm -2 Alternatively, 500 J cm -2Irradiated with a dose of at least 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 / The fiber is irradiated with a dose of at least approximately 2 J / cm, 5 J / cm, 10 J / cm, 25 J / cm, 50 J / cm, 75 J / cm, 100 J / cm, 150 J / cm, 200 J / cm, 250 J / cm, 300 J / cm, 400 J / cm, or 500 J / cm.

[0108] In some embodiments, the disease or condition is a tumor, and the tumor is a superficial tumor. In some embodiments, the tumor is at least 10 J / cm 2 , 25J / cm 2 50 J / cm 2 , 150 J / cm 2 Alternatively, 250 J / cm² 2 , or at least about 10 J / cm² 2 , 25J / cm 2 50 J / cm 2 , 150 J / cm 2 Alternatively, 250 J / cm² 2 , or approximately 10 J / cm 2 , 25J / cm 2 50 J / cm 2 , 150 J / cm 2 Alternatively, 250 J / cm² 2 It is irradiated with this dose.

[0109] In some embodiments, the disease or condition is a tumor, and the tumor is a stromal tumor. In some embodiments, the tumor is irradiated with a dose of at least 50 J / cm fiber length, 100 J / cm fiber length, 200 J / cm fiber length or 300 J / cm fiber length, or about at least 50 J / cm fiber length, 100 J / cm fiber length, 200 J / cm fiber length or 300 J / cm fiber length, or about 50 J / cm fiber length, 100 J / cm fiber length, 200 J / cm fiber length or 300 J / cm fiber length.

[0110] In some embodiments, cells, hyperplasia, or tumors are irradiated within 12, 24, 36, 72, or 96 hours after administration of the conjugate, or within approximately 12, 24, 36, 72, or 96 hours, or within approximately 12, 24, 36, 72, or 96 hours. In some embodiments, the targeted molecule is administered up to 96 hours prior to administration of the conjugate. In some embodiments, the conjugate is administered at a dose of 0.5 mg / kg to approximately 100 mg / kg, or approximately 0.5 mg / kg to approximately 100 mg / kg, or 20 mg / m². 2 ~about 4000mg / m 2 Alternatively, approximately 20 mg / m² 2 ~about 4000mg / m 2 It is administered in a certain amount.

[0111] In some embodiments, the conjugate is administered in amounts of at least 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 8.0 mg / kg, 16.0 mg / kg, 32.0 mg / kg or 64 mg / kg, or approximately at least 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 8.0 mg / kg, 16.0 mg / kg, 32.0 mg / kg or 64 mg / kg, or approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 8.0 mg / kg, 16.0 mg / kg, 32.0 mg / kg or 64 mg / kg; or the conjugate is administered in amounts of at least 20 mg / m² 2 , 40 mg / m² 2 , 160 mg / m² 2 , 320 mg / m² 2 , 640 mg / m² 2 , 1280 mg / m² 2 Alternatively, 2560 mg / m² 2 , or at least 20 mg / m² 2 , 40 mg / m² 2 , 160 mg / m² 2 , 320 mg / m² 2 , 640 mg / m² 2 , 1280 mg / m² 2 Alternatively, 2560 mg / m² 2 , or 20 mg / m² 2 , 40 mg / m² 2 , 160 mg / m² 2 , 320 mg / m² 2 , 640 mg / m² 2 , 1280 mg / m² 2 Alternatively, 2560 mg / m² 2 , or approximately 20 mg / m² 2 , 40 mg / m² 2 , 160 mg / m² 2 , 320 mg / m² 2 , 640 mg / m² 2 , 1280 mg / m² 2 Alternatively, 2560 mg / m² 2 It is administered in a certain amount.

[0112] In some embodiments of the methods provided herein, the targeting molecule is administered, for example, to a subject or sample, before the administration of the conjugate. In some embodiments, the targeting molecule is 10 mg / m² 2 ~about 500mg / m 2 Or approximately 10 mg / m² 2 ~about 500mg / m 2 It is administered in a dose within the specified range.

[0113] In some embodiments, the targeting molecule is an antibody or an antigen-binding fragment. In some embodiments, the antibody is cetuximab.

[0114] In some embodiments, a conjugate containing a phthalocyanine dye and a targeting molecule is provided. In some such embodiments, the targeting molecule is an activatable cell-permeable peptide (ACPP) consisting of tissue-specific homing peptides, RGD polypeptides, iRGD polypeptides, Lyp-1 polypeptides, crypto-1 binding polypeptides, somatostatin receptor-binding polypeptides, inhibitor-binding polypeptides, NGR polypeptides, iNGR polypeptides, polycationic cell-permeable peptides (CPPs) linked to a neutralizing polyanion via a cleavable linker, or an antibody, such as Ado-trastuzumab emtansine, afatinib, axitinib, or bosomite. The antigens are tinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, everolimus, ibrutinib, imatinib, lenvatinib, nilotinib, olaparib, palbociclib, pazopanib, ramucirumab, regorafenib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, bismodegib, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pizilizumab (CT-011), AMP-224, MSB001078C, or MEDI4736, or their antigen-binding fragments.

[0115] In some embodiments, ACPP comprises the structure A-X1-B-, where B is a peptide moiety of about 5 to about 20 basic amino acid residues suitable for intracellular uptake; A is a peptide moiety of about 2 to about 20 acidic amino acid residues that, when linked with moiety B, are effective in inhibiting or preventing intracellular uptake of moiety B; X1 is a cleavable linker of about 2 to about 100 atoms; and one or more LYs are linked to the C-terminus of peptide moiety B.

[0116] In some embodiments, the homing peptide has the sequence shown in any of SEQ ID NO: 1 to 52. In some embodiments, the dye is IR700. [Brief explanation of the drawing]

[0117] [Figure 1A] This shows the relative amounts of cetuximab-IRDye 700DX in high molecular weight (aggregate) and monomeric forms, as assessed by high-performance liquid chromatography (HPLC-SEC) using a size exclusion column for samples in a clear vial before 24 hours of light exposure. [Figure 1B] This shows the relative amounts of cetuximab-IRDye 700DX in high molecular weight (aggregate) and monomeric forms, as assessed by high-performance liquid chromatography (HPLC-SEC) analysis using a size exclusion column for samples in clear vials after 24 hours of light exposure. [Figure 2A] This study shows the duration of exposure to 500 lux of white fluorescence or green LED light for cetuximab-IRDye 700DX and its effect on soluble aggregate formation. [Figure 2B] This study demonstrates the effect of pre-exposure to white fluorescence or green LED light with cetuximab-IRDye 700DX on BxPC3 PIT activity at various light doses and durations. [Figure 2C] The effect of cetuximab-IRDye 700DX on the percentage of PIT activity that causes soluble aggregate formation is shown. [Figure 3]The PIT-killing activity was demonstrated using cetuximab and serial staining with donkey anti-human-IRDye 700DX (DxHu IR700) secondary antibody. [Figure 4A] This demonstrates photodependent killing of BxPC3 cells using biotinylated cetuximab pre-conjugated with monomeric streptavidin-IRDye 700DX (mSA IR700). [Figure 4B] This demonstrates the specificity of PIT using biotinylated cetuximab pre-conjugated with monomeric streptavidin-IRDye 700DX (mSA IR700). [Figure 4C] This study demonstrates the effect of biotinylated cetuximab on the PIT-killing activity of monomeric streptavidin-IRDye 700DX pre-exposed to white light in BxPC3 cells. [Figure 5] A shows antibody dose-dependent killing of 4T1 cells using directly conjugated anti-EpCAM-IRDye 700DX. B shows the specificity of the PIT-killing activity of anti-EpCAM-IRDye 700DX. [Figure 6] This demonstrates Fc receptor-specific killing of THP1 cells by cetuximab-IRDye 700DX. [Figure 7A] This demonstrates the photodependent toxicity of EGF-IRDye 700DX in A431 cells. [Figure 7B] This study demonstrates the effect of EGF-IRDye 700DX, pre-exposed to different types of light, on photodependent cell toxicity in A431 cells. [Figure 8A] This demonstrates the photodependent killing of BxPC3 cells using cholera toxin B-IRDye 700DX. [Figure 8B] This demonstrates the photoactivatability and specificity of cholera toxin B-IRDye 700DX. [Figure 8C] This study demonstrates the effect of prior exposure to cholera toxin B-IRDye 700DX at different wavelengths of light on photoactivatable cytosis in BxPC3 cells. [Figure 9]A shows the photodependent toxicity of Vero cells using influenza virus (X-31)-IRDye 700DX. B shows the effect of influenza virus (X-31)-IRDye 700DX on photoactivated cell toxicity after prior exposure to white light versus green light. [Figure 10] A shows the effect of light dose on the killing activity of SNA-IRDye 700DX in BxPC3 cells. B shows the effect of sialidase treatment on the specificity of SNA-IRDye 700DX that binds to cells. [Figure 11] This shows PIT killing of Staphylococcus aureus (S. aureus) by cetuximab-IRDye 700DX combined with laser irradiation. [Figure 12] This shows the PIT (Plant Identification Time) of influenza virus particles using mouse anti-influenza virus (H3N2) pre-complexed with GtxMs Fab-IRDye 700DX. [Figure 13] This study demonstrates photodependent toxicity of influenza virus-infected cells using mouse anti-influenza virus (H3N2) and goat anti-mouse IRDye 700DX (GtxMs-IR700). [Figure 14] This shows PIT-induced necrotizing of rat embryonic dorsal root ganglion (DRG) neurons using cholera toxin B-IRDye 700DX. [Figure 15] A shows the effect of prior exposure to white or green light on soluble aggregate formation of cetuximab-IRDye 700DX conjugate, cetuximab-IRDye 680RD conjugate, and cetuximab-IRDye 700+IRDye 680RD double conjugate. B shows the effect of prior exposure to white or green light on fluorescence normalized to monomer content of cetuximab-IRDye 700DX conjugate, cetuximab-IRDye 680RD conjugate, and cetuximab-IRDye 700+IRDye 680RD double conjugate. [Modes for carrying out the invention]

[0118] Detailed explanation I. Method for producing phthalocyanine dye-targeted molecule conjugates In some embodiments, a method is provided for preparing or manufacturing phthalocyanine dye-targeted molecule conjugates, such as IRDye 700DX (IR700)-targeted molecule (e.g., antibody) conjugates, comprising steps of producing, formulating, and / or packaging the conjugates. In some embodiments, the method is carried out under conditions that reduce or prevent the aggregation or degradation of the dye by protecting the conjugate from light that may photoactivate the dye in the conjugate, for example, during the manufacturing process. In some embodiments, the method provided also includes a step of protecting the conjugate from exposure to acidic pH, such as an acidic pH of less than 6.0. The method provided produces dye conjugates that are stable for more than 3 months, generally more than 6 months or more than 12 months, including dye conjugates that are stable under storage conditions.

[0119] In some embodiments, methods are employed to produce phthalocyanine dye-targeting molecule conjugates for use in photoimmunotherapy. Photoimmunotherapy is a molecularly targeted therapy that utilizes phthalocyanine dye-based target-specific photosensitizers, such as near-infrared (NIR) phthalocyanine dyes (e.g., IR700), conjugated to targeting molecules (e.g., targeting cell surface proteins on tumor cells). For example, in some cases, the phthalocyanine dye conjugates used in photoimmunotherapy may involve conjugation to monoclonal antibodies (mAbs) that target tumor-specific cell surface proteins, e.g., tumor-specific cell surface receptors. In some embodiments, activation of the dye conjugate by irradiation with absorbent light, such as NIR light, excites the photosensitizer, resulting in cell killing. In some cases, the use of light in the NIR range leads to deeper tissue penetration, resulting in successful tumor eradication with only a single dose of external NIR light irradiation.

[0120] Typically, PIT primarily kills cells that are conjugated with phthalocyanine dyes, such as IR700-antibody conjugates, after they are irradiated with NIR, while only a small number of cells that do not express cell surface proteins recognized by the targeting molecule (e.g., antibody) are killed. Therefore, because this therapy is specifically targeted to diseased cells such as tumor cells, its effects are highly selective for diseased tissue compared to healthy tissue or cells. For example, while targeted photosensitizers can be distributed throughout the body, photosensitizers are only active when strong light is applied, reducing the possibility of off-target effects.

[0121] Generally, targeted phototoxicity appears to rely primarily on the binding of dye conjugates to the cell membrane via specific targeting molecules (e.g., macromolecules such as antibodies). For example, studies using the exemplary antibody-IR700 molecule have shown that the conjugate must bind to the cell membrane to be active, and that cell killing does not require intracellular localization to be effective (see, e.g., U.S. Patent No. 8,524,239 and U.S. Patent Application Publication No. US20140120119). Photoactivation of cells bound to the conjugate results in rapid cell death and necrosis.

[0122] In general, phthalocyanine dyes, particularly IRDye 700DX (IR700), are extremely photostable. For example, IR700 has been reported to be 45 to 128 times more photostable than other near-infrared dyes and does not aggregate (see Peng et al. (2006) Proc. SPIE 6097, 60970E; also see www.licor.com / bio / products / reagents / irdye / 700dx / photostability.html). IR700 dyes have also been reported not to exhibit the same aggregation problems as other dyes when formulated at acidic pH. Similarly, Peng et al. reported that when IR700 is conjugated to an antibody, it exhibits substantially the same fluorescence excitation and absorption spectra as the unconjugated dye, thereby demonstrating that the conjugate retains its fluorescence properties. In several aspects, the photostability of IR700 allows for its use in applications where the dye is exposed to continuous excitation by light for extended periods without the need for protection from light. This is in contrast to other fluorophore dyes, which are not photostably stable and cannot maintain their fluorescence when exposed to light for extended periods.

[0123] However, it is found herein that conjugating a dye necessary for PIT activity to a targeting molecule reduces the stability of the dye, resulting in a greater tendency for the conjugate to aggregate and a decrease in activity (e.g., PIT activity). This effect occurs even if the monomeric dye retains its photostability and fluorescence properties. In some cases, particularly for therapeutic applications, this can reduce activity and thereby limit the effectiveness of the conjugate as a PIT agent. This result has not been shown with other dye conjugates (e.g., IRDye 680 conjugate), which exhibit lower photostability but are less prone to aggregation when conjugated to other molecules. Therefore, it is found herein that phthalocyanine dye conjugates, such as IR700 used in PIT, are particularly sensitive to soluble aggregate formation when exposed to light compared to conjugates of other dyes (including other 700 nm dyes). In some aspects, this is problematic because monomeric purity and pharmacological activity (e.g., PIT activity) are essential for the therapeutic use of phthalocyanine dye conjugates (e.g., IR700 conjugates), as changes in purity or activity can significantly affect photoactivatable toxic activity.

[0124] These observations are partly based on HPLC-SEC analysis of dye conjugates prepared or exposed under different conditions. For example, the examples provided herein demonstrate that aggregation of the bound dye portion of antibody-dye conjugates can occur when the conjugate is exposed to light for longer periods, as is evident from the increase in high molecular weight species, which are larger in size than the major monomer peak (compare Figures 1A and 1B). The results also show that substantial aggregation of IR700 conjugates occurs in the presence of white light, which is not observed in IRDye 680 conjugates conjugated to the same molecule (see, for example, Figure 15A). In some cases, reduced dye stability may mean that the dye conjugate is more susceptible to photo-induced aggregation, which may minimize the use of dye conjugates after prolonged exposure to light. It is also found that the overall instability of dye conjugates is evident when the conjugate is formulated at an acidic pH. For example, as shown in the examples, the dye conjugate exhibits increased aggregation at pH levels below 6.0.

[0125] The observations provided establish that, in some cases, photoprotection of phthalocyanine dye conjugates is necessary to minimize aggregation and maintain activity, particularly to ensure consistency in product manufacturing (e.g., when used in accordance with Good Manufacturing Practice (GMP) methods). Therefore, methods for improving the stability, e.g., integrity, purity, activity, or potency of dye conjugates are provided herein. In some embodiments, the method includes steps of protecting the dye or dye conjugate from excitation light during one or more steps of handling or preparing the dye, carrying out conjugation of the dye with a targeting molecule (e.g., an antibody), formulating the dye, and / or packaging the dye. In some embodiments, if light is required, for example, to visualize the process of making or producing the conjugate, the photoprotection includes carrying out one or more, in some cases all, of the above steps only in the presence of green light of a wavelength not absorbed by the dye, e.g., 400 nm to about 650 nm or about 400 nm to about 650 nm. In some embodiments, any light present during one or more of the above steps is light with an intensity of less than 500 lux, for example, less than 200 lux. In some embodiments, the total light exposure of the dye and conjugate to any light during the process of fabricating, manufacturing or producing the conjugate is 5000 lux hours or less, for example, 80 lux hours or less. In some embodiments, the total light exposure of the dye and conjugate to any light during the process of packaging the conjugate is 5000 lux hours or less, for example, 80 lux hours or less.

[0126] In some embodiments, the method includes the step of formulating a dye in a pharmaceutically acceptable buffer with a pH greater than 6.0, for example, generally pH 6.0 to about 8.0 or about pH 6.0 to about 8.0.

[0127] In some embodiments, a method is also provided for protecting a dye conjugate from excitation light by packaging a drug product in a container or other product that protects against light transmission, such that the container or other product exhibits a transmittance of 40% or less of light having wavelengths of 250 nm to about 800 nm, or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm. In some embodiments, the container or other product has a transmittance of less than 5% or 5% or less of any light. In some embodiments, such a container or product containing a dye conjugate product is an internal packaging material, and one or more other external containers or products are provided that wrap or enclose the internal packaging material, for example, to provide further light protection.

[0128] In some embodiments, stable dye conjugates are provided. In some embodiments, by practicing the method provided, the purity, impurities, integrity, composition, and potency of the conjugate do not change beyond acceptable specifications for manufacturing purposes supporting clinical or commercial use. In some embodiments, the conjugate is stable, exhibits minimal aggregation, and retains potency and activity after, for example, processing, manufacturing, or storage of the dye. In some embodiments, the dye conjugate is stable for more than 3 months, 4 months, 5 months, for example more than 6 months, more than 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. In some embodiments, such stability exists when stored for that time at a temperature below 30°C, for example more than 2-8°C.

[0129] With respect to dye conjugates, the term “stable” means that, after being stored for longer than the required time, for example, more than 3 months, for example, more than 6, 12, or 24 months, or more than approximately 6, 12, or 24 months, more than 90% or approximately more than 90% of the total molecular weight of the conjugates present in the sample are present as major monomer components, or less than 10.0% of the total molecular weight of the conjugates present in the sample are present as high molecular weight components, or that the conjugate retains at least 20% to a maximum of 100% of its integrity, such as its physical and functional qualities, including one or more of its purity (e.g., aggregates, e.g., percentage of monomer content relative to content of higher molecular weight components), identity (e.g., chemical composition, e.g., structural features), potency (e.g., concentration or amount required to produce a pharmacological response), or activity (e.g., PIT killing), compared to the conjugate before being stored for the required time (e.g., t=0).

[0130] In some embodiments, the conjugate is stable when, after being stored for longer than required time, for example, more than 3 months, for example, more than 6 months, 12 months, or 24 months, more than 90% of the total molecular weight of the conjugate present in the sample is the major monomer component, for example, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more of the total molecular weight of the conjugate present in the sample is the major monomer component. In some embodiments, dye conjugates are stable after storage for more than 3 months, for example, 6 months, 12 months, or 24 months, when 10.0% or less of the conjugate is present as a high molecular weight component as a percentage of the total molecular weight of the conjugates present in the sample, generally when 9.0% or less, 8.0% or less, 7.0% or less, 6.0% or less, 5.0% or less, 4.0% or less, or 3.0% or less of the high molecular weight component as a percentage of the total molecular weight of the conjugates present in the sample. In some embodiments, the presence of high molecular weight components or major monomer components can be identified using any method that can separate molecules based on size, for example by performing HPLC-SEC.

[0131] In some embodiments, a conjugate is stable if, after more than three months, for example, more than six, twelve, or twenty-four months, or approximately more than six, twelve, or twenty-four months, its integrity, purity, identity, potency, or activity is retained at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the integrity, purity, identity, potency, or activity of the conjugate before storage for that time (e.g., the conjugate at t=0), or approximately at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the potency of a conjugate may be related to the affinity of the conjugate for binding to its targeting molecule. In some embodiments, potency is related to its ED 50In other words, it can be evaluated by a measure of the dose or amount of the conjugate that is pharmacologically effective or that exhibits the desired effect in 50% of subjects exposed to the conjugate. In some embodiments, activity relates to biological activity, including the therapeutic effect and pharmacological activity of the conjugate resulting from in vivo administration, such as the activity of the conjugate to induce PIT killing. In some embodiments, biological activity can be observed in an in vitro system designed to test such activity. In some embodiments, the purity of the conjugate relates to the presence of monomers of the conjugate compared to aggregates (e.g., high molecular weight components). In some embodiments, purity can be evaluated based on the percentage of monomers (e.g., major monomer peak) to aggregates (e.g., high molecular weight components) in the composition. In some embodiments, the presence of high molecular weight components or major monomer components can be identified using any method that can separate molecules based on size, for example by performing HPLC-SEC.

[0132] In some embodiments, the major monomeric component of a dye conjugate generally refers to the molecular weight species of the dye conjugate, representing the combined molecular weight of the dye and targeting molecule present in the conjugate. Generally, the major monomeric component is the species present in the largest amount in a sample of dye conjugate. For example, by HPLC-SEC, the major monomeric component is generally the species of dye conjugate present as the largest peak in the preparation of the dye conjugate. The exact molecular weight range in a sample of dye conjugate depends on the specific sample (e.g., specific dye and targeting molecule) and the preparation method (e.g., the ratio of dye to targeting molecule). Those skilled in the art will recognize such species. For example, for a conjugate containing IR700 dye (with a molecular weight of approximately 1954.22 Da) and antibody (with an average molecular weight of approximately 150,000 Da for full-length antibodies), the molecular weight range of the major monomer component is typically about 151,000 Da to 165,000 Da, e.g., 154,000 Da to 158,000 Da. In the exemplary experiments depicted in Figures 1A and 1B, the major monomer component of the exemplary antibody-dye conjugate includes components that elute by HPLC-SEC between 8 and 9 minutes.

[0133] In some embodiments, the high molecular weight component of a dye conjugate generally refers to a molecular weight species of the dye conjugate that exhibits a molecular weight greater than that of the major monomer component. In some embodiments, the increased or greater molecular weight may be due to the aggregation of the dye. In some embodiments, the aggregation may be due to the formation of dimers, trimers, or higher-order oligomers. The precise molecular weight range of the high molecular weight component in a dye conjugate sample will depend on the specific sample (e.g., the specific dye and targeting molecule), the preparation method (e.g., the ratio of dye to targeting molecule), and, in some cases, the degree or range of aggregation. Those skilled in the art will recognize such species. In some embodiments, for dye conjugates of full-length antibodies and dyes, such as IR700 dyes, the high molecular weight component may be due to the presence of dimers, trimers, or higher-order oligomers, generally having molecular weights greater than 200,000 Da, e.g., 300,000 Da, 350,000 Da, 400,000 Da, 450,000 Da, 500,000 Da or larger. In the exemplary experiments depicted in Figures 1A and 1B, the high molecular weight component of the exemplary antibody-dye conjugate includes components that elute by HPLC-SEC for 8 minutes, e.g., between 6 and 8 minutes.

[0134] A. Conjugate containing phthalocyanine dye and targeting molecule The methods provided herein include the step of producing a conjugate containing a photosensitizer, e.g., a phthalocyanine dye, e.g., IR700, and a targeting molecule (e.g., an antibody), e.g., an antibody that binds to a cell surface protein. In some embodiments, the targeting molecule conjugated to the photosensitizer, e.g., a phthalocyanine dye (e.g., IR700), enables the targeting of the conjugate to cell surface molecules, e.g., cell surface receptors, of cells involved in diseases or pathologies such as tumors or cancer, infections, inflammatory diseases or conditions, neurological diseases or conditions, or other diseases or conditions. In some embodiments, cell targeting increases the effectiveness of PIT induced by local irradiation of a target, e.g., irradiation of a tumor in a target, at wavelengths absorbed by the phthalocyanine dye, e.g., near-infrared (NIR) wavelengths.

[0135] The phthalocyanine dye conjugates provided herein for use in combination therapies comprise a dye molecule conjugated to a target molecule via a linker group. In one aspect, the conjugate is the conjugate represented by formula I: A-[(L) n -D] p (I) During the ceremony, A is a targeting molecule that can bind to cells or tissues; L is a linker that is independently selected for each p; n is either 1 or 2; D is a hydrophilic phthalocyanine dye independently selected for each p; and p can be independently 1, 2, 3, 4, 5, or greater than 5, for example, up to 1000. For example, p can be 1 to 1000, for example, generally 1 to 10 or 2 to 5.

[0136] Phthalocyanines are a group of photosensitizer compounds that have a phthalocyanine ring system. Phthalocyanines are azaporphyrins (i.e., C) containing four benzoindole groups connected by nitrogen bridges in a 16-membered ring in which carbon and nitrogen atoms are arranged alternately. 32 H 16 Phthalocyanines (N8) form stable chelates with metallic and nonmetallic cations. In these compounds, the ring center is occupied by a metallic ion (either diamagnetic or paramagnetic) which may harbor one or two ligands depending on the ion. In addition, the periring ring may be unsubstituted or substituted. The synthesis and use of a wide variety of phthalocyanines in photodynamic therapy are described in International Publication WO 2005 / 099689 and U.S. Patent No. 7,005,518.

[0137] In some embodiments, phthalocyanines strongly absorb red or near-infrared light, with absorption peaks between approximately 600 and 810 nm, allowing for deep penetration of tissue by light in some cases. Phthalocyanines are generally photostable. This photostability is typically advantageous in pigments and dyes, as well as in many other applications of phthalocyanines.

[0138] In some embodiments, the phthalocyanine dye is water-soluble and contains a luminescent fluorophore moiety having at least one aqueous solubilizing moiety. In some embodiments, the aqueous solubilizing moiety contains silicon. In some embodiments, the phthalocyanine dye has a core atom such as Si, Ge, Sn, or Al. In some embodiments, the phthalocyanine dye exists as a single core isomer that is essentially free of other isomers. In some embodiments, the phthalocyanine dye contains a linker having a reactive or activatable group that can form a bond between the linker and the targeting molecule. In some embodiments, the phthalocyanine dye can be conditioned to fluoresce at a specific wavelength.

[0139] In some embodiments, the phthalocyanine dye contains a linker, i.e., a linker-phthalocyanine dye moiety (LD). In some embodiments, the linker contains a reactive group. In some embodiments, the phthalocyanine dye is a phthalocyanine dye represented by formula II: TIFF0007901958000005.tif100128In formula, L is selected from direct or covalent linkage; Q is a reactive or activatable group, which can be part of linker L and, by reaction, can form a bond between L and the target molecule A; R 2 , R 3 , R 7 , and R 8 Each is independently selected from an optionally substituted alkyl and an optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 If present, each is independently selected from hydrogen, an optionally substituted alkyl, an optionally substituted alkanoyl, an optionally substituted alkoxycarbonyl, an optionally substituted alkylcarbamoyl, or a chelate ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of them contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 Each of these is a functional group that can be independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted alkoxy; Alternatively, in an alternative manner, i)R 13 and R 14 and the carbon to which these are attached, or ii)R 17 and R 18 and the carbon to which these are attached, or iii) R 21 and R 22 These and at least one carbon to which they are attached bond together to form a fused ring; and X 2 and X 3 Each of these C1-C atoms may have a heteroatom interposed between them, independently of each other. 10 It is alkylene.

[0140] In some embodiments, L is a covalent bond. In some embodiments, the covalent bond is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1 to 60 atoms, e.g., 1 to 45 atoms or 1 to 25 atoms. In some cases, such atoms can be selected from C, N, P, O, and S. In some embodiments, L can have additional hydrogen atoms (in addition to the 1 to 60 atoms) that satisfy the valence. Generally, the bonds include ether, thioether, amine, ester, carbamate, urea, thiourea, oxy or amide bonds; or single, double, triple or aromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen or nitrogen-platinum bonds; or any combination of aromatic or heteroaromatic bonds.

[0141] In some forms, L is given by formula -R 1 -YX 1 -Y 1 - is L, and in the formula, R 1 is a divalent radical or directly linked; Y and Y 1 Each is independently selected from directly linked, oxygen, nitrogen which may be substituted, or sulfur; and X 1 This includes direct linkages and C1-C atoms that may have an intervening atom. 10 Selected from alkylenes. The divalent radicals include, non-limitingly, substituted alkylenes, substituted alkyleneoxycarbonyls, substituted alkylenecarbamoyls, substituted alkylenesulfonyls, and substituted arylenes.

[0142] Exemplary R 1The 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.

[0143] In some embodiments, Q contains a reactive group for any attachment to a material such as a targeted molecule. As used herein, the term “reactive group” or “reactive chemical group” means a portion of a compound that is capable of chemically reacting with a functional group on a different material (e.g., a targeted molecule) to form a linkage such as a covalent linkage. Typically, a reactive group is an electrophile or nucleophile that can form a covalent linkage through exposure to a corresponding functional group, which is a nucleophile or electrophile, respectively. Alternatively, a reactive group is a photoactivatable group that becomes chemically reactive only after irradiation with light of an appropriate wavelength. Typically, a conjugation reaction between a conjugated reactive dye and a targeted molecule incorporates one or more atoms of the reactive group Q into a new linkage that attaches the dye to the conjugated targeted molecule.

[0144] In some embodiments, Q contains a reactive group that is reactive with a carboxyl group, amine, or thiol group on the targeting molecule. Preferred reactive groups include, non-limitingly, amine-reactive chemical groups, sulfhydryl-reactive chemical groups, activated esters, acyl halides, alkyl halides, anhydrides, carboxylic acids, carbodiimides, carbonates, carbamates, haloacetamides (e.g., iodoacetamide), isocyanates, isothiocyanates, maleimides, NHS esters, phosphoramidites, platinum complexes, sulfonic acid esters, and thiocyanates for any attachment to the targeting molecule. In some embodiments, the reactive group is reactive with a carboxyl group, amine, or thiol group on the targeting molecule. In some embodiments, the reactive group is a sulfhydryl-reactive chemical group, e.g., maleimide, haloacetyl, and pyridyl disulfide. In some embodiments, the reactive group is amine-reactive. In some embodiments, the reactive group is an NHS ester.

[0145] In some embodiments, R 2 , R 3 , R 7 , and R 8 These are, respectively, substituted alkyl groups, such as substituted methyl, ethyl, or isopropyl groups.

[0146] In some embodiments, R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of them contains a water-soluble group. For example, R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The alkyl portion is substituted with a water-soluble substituent. As used herein, “water-soluble group” refers to a group comprising one or more polar and / or ionic substituents that improve the overall solubility of the molecule in an aqueous medium. In some cases, R 4 , R5 , R 6 , R 9 , R 10 , and R 11 At least two of them contain water-soluble groups. In other embodiments, three or more contain water-soluble groups. The water-soluble groups are, non-limitingly, carboxylates (-CO2 - ) group, sulfonate (-SO3 - ) group, sulfonyl (-SO2 - ) base, sulfate (-SO4 -2 ) group, hydroxyl (-OH) group, phosphate (-OPO3 -2 ) group, phosphonate (-PO3 -2 It comprises a ) group, an amine (-NH2) group, and an optionally substituted quaternary nitrogen (each having any counterion).

[0147] 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 bioacceptable counterion.

[0148] In some embodiments, R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The nitrogen atom to which the compound is attached can be trivalent or tetravalent.

[0149] 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 of them is hydrogen.

[0150] In some embodiments, X 2 and X 3Each of these C1-C atoms may have an intervening atom. 10 Selected from alkylenes. In some embodiments, X 2 and / or X 3 The nitrogen added may, in some cases, be quaternary.

[0151] In some embodiments, the phthalocyanine dye is a phthalocyanine dye represented by formula III: TIFF0007901958000006.tif97150 formula, X 1 and X 4 Each of these C1-C atoms may have a heteroatom interposed between them, independently of each other. 10 It is alkylene; and R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 16 , R 17 , R 18 , R 19 , X 2 , and X 3 This is as defined herein.

[0152] In some embodiments, the reactive group is an NHS ester. In some embodiments, the reactivity of the NHS ester is due to the X between the NHS ester and the carbamate functional group. 4 This can be adjusted by changing the length of the alkylene group. In some embodiments, the X between the NHS ester and the carbamate functional group 4 The length of the alkylene group is inversely proportional to the reactivity of the NHS ester. In some embodiments, X 4 is C5-alkylene. In another embodiment, X 4 is C3-alkylene. In some embodiments, X 1 is C6-alkylene. In other embodiments, X1 It is a C3-alkylene.

[0153] In some embodiments, phthalocyanine dyes have a total charge of zero. This neutral charge can, in certain cases, be obtained with one or more arbitrary counterions or quaternary nitrogen.

[0154] In some embodiments, phthalocyanine dyes have sufficient solubility in aqueous solutions, as the targeted molecules retain their solubility when attached to soluble targeting molecules. In some embodiments, the dyes are also soluble in organic media (e.g., DMSO or DMF).

[0155] In some embodiments, phthalocyanine dyes have maximum light absorption in the near-infrared (NIR) region. In some embodiments, phthalocyanine dyes have maximum light absorption wavelengths between 600 nm and 850 nm, for example between 680 nm and 850 nm, for example, approximately 690 nm ± 50 nm or 690 ± 20 nm. In some embodiments, phthalocyanine dyes can be efficiently excited by commercially available laser diodes that emit light at these wavelengths.

[0156] In some embodiments, the phthalocyanine dye containing the reactive group is IR700 NHS ester, for example, IRDye 700DX NHS ester (LiCor 929-70010, 929-70011). Therefore, in some embodiments, the dye is a compound having the following formula: TIFF0007901958000007.tif106160

[0157] For the purposes of this specification, the terms “IR700,” “IRDye 700DX,” or their variations refer to the above formula when the dye is conjugated to a target molecule, for example, via a reactive group. Generally, IR700 has several desirable chemical properties. Amino-reactive IR700 is a relatively hydrophilic dye and can be covalently conjugated with antibodies using the NHS ester of IR700. Typically, IR700 is also a hematoporphyrin derivative, Photofrin® (1.2 × 10⁻¹⁶ at 630 nm). 3 M -1 cm -1 ), meta-tetrahydroxyphenylchlorine; Foscan® (2.2 × 10 at 652 nm) 4 M -1 cm -1 ), and mono-L-aspartylchlorin e6;NPe6 / Laserphyrin(registered trademark) (4.0 × 10 at 654nm) 4 M -1 cm -1 ) has an absorption coefficient more than 5 times higher than conventional photosensitizers (maximum absorption at 689nm is 2.1 × 10⁻¹⁰). 5 M -1 cm -1 ) has.

[0158] The phthalocyanine dyes described herein can be prepared using commercially available starting materials. Their core structure is synthesized by the condensation of two or more different diiminoisoindolines. Synthetic strategies using different dinitriles or diiminoisoindolines can lead to phthalocyanines with varying degrees of substitution and / or positional isomers with varying degrees of distribution. An exemplary synthetic scheme for producing the dyes is described in U.S. Patent No. 7,005,518.

[0159] In some embodiments, phthalocyanine dyes are conjugated to targeting molecules via reactive groups of the dye molecule. In some embodiments, the targeting molecule is a molecule whose conjugate can be directed to target cells or pathogens, for example, by binding to cell surface molecules (e.g., cell surface receptors) on cells or pathogens. In some embodiments, the targeting molecule, e.g., a macromolecule, can selectively bind to a desired cell type, a cell having a specific phenotype, or a cell presenting one or more cell surface markers or antigens. In some cases, the targeting molecule binds to cells that are cancer cells, tumor cells, inflammatory cells, immune cells, nerve cells, stem cells, proliferative cells, or cells in a hyperplastic state. In some embodiments, the targeting molecule binds to pathogens or pathogen-infected cells. In some embodiments, the cells are inflammatory cells such as leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, or monocytes. In some embodiments, the cells are immune cells such as T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils. In some embodiments, the cells are nerve cells, such as peripheral nervous system nerve cells or central nervous system nerve cells, such as nociceptors (e.g., thermal nociceptors, mechanonociceptors, chemonociceptors, or multimode nociceptors). In some cases, the targeting molecule binds to pathogens or pathogenic cells, such as viruses, bacteria, fungi, biofilms, or other prokaryotic cell lines. In some embodiments, the targeting molecule binds to pathogens, such as Gram-negative bacteria or Gram-positive bacteria.

[0160] In some embodiments, a targeting molecule (e.g., an antibody) of a phthalocyanine dye conjugate binds to a protein on the surface of one or more cells present in the lesion microenvironment that is associated with or present as a result of a disease or pathological condition. For example, in some embodiments, the conjugate binds to a protein on the surface of one or more cells present in the tumor microenvironment that is associated with or present within a tumor. In some embodiments, the conjugate binds to a protein present in the extracellular matrix of the tumor microenvironment.

[0161] As used herein, “cells present in the lesion microenvironment” means any cells present in the cellular environment associated with a lesion, disease, or disorder, for example, any cells present in or directly adjacent to a tumor, for example, the tumor microenvironment, or cells present in the extracellular matrix within the tumor microenvironment.

[0162] As used herein, “cells present in the tumor microenvironment” refers to any cells present in the cellular environment in which a tumor resides, including proliferating tumor cells (e.g., cancer cells), tumor stroma, blood vessels, infiltrating inflammatory cells (e.g., immune cells), and various related tissue cells (e.g., fibroblasts), for example, any cells present in or directly adjacent to the tumor. Thus, reference to a tumor is understood to refer not only to tumor cells, which may include malignant or cancer cells, but also to other cells present in the tumor microenvironment (including immune cells) that regulate tumor growth. In some cases, immune cells present in the tumor microenvironment may include T lymphocytes (including regulatory T lymphocytes (Treg)), dendritic cells, natural killer (NK) cells, B cells, macrophages, and other immune cells (Whiteside (2008) Oncogene, 27:5904-5912). In some aspects, it is recognized that many non-cancerous cells present in and around a tumor can regulate 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 the tumor, such as immune cells (e.g., T cells like regulatory T cells), can be an effective treatment for killing tumors with PIT.

[0163] Generally, cancer cells contain tumor-specific antigens that should be recognized by the immune system. Typically, in an active immune system, immune cells such as cytotoxic T cells attack and eliminate these cancer cells. Under normal physiological conditions, the T cell-mediated immune response is initiated by antigen recognition by the T cell receptor (TCR) and regulated by a balance of co-stimulatory and inhibitory signals (e.g., immune checkpoint proteins). In particular, CD4+ and CD8+ T cells expressing TCRs can be activated by the recognition of antigenic peptides presented on antigen-presenting cells on major histocompatibility complex (MHC) class I or class II molecules, respectively. In some aspects, activated CD8+ cells, or cytotoxic T cells, can kill antigen-expressing tumor cells, which may be assisted by the presence of CD4+ T cells.

[0164] However, in the case of tumors, the tumor microenvironment has mechanisms to evade immune recognition by suppressing the immune system, thereby preventing or reducing the killing of tumor cells. For example, in some cases, immune checkpoint proteins may be disregulated in tumors, thereby leading to the suppression of the immune response in the tumor microenvironment as a mechanism for evading the immune system. In some cases, tumor-infiltrating lymphocytes may include Tregs (e.g., CD4+CD25+ T cells), which are cells capable of suppressing the proliferation of other T cells in the microenvironment (Whiteside, TL (2008) Oncogene, 27:5904-5912). In some cases, other mechanisms may act to inhibit the approach of immune cells to tumor antigens, thereby contributing to the tumor's ability to evade the immune system.

[0165] In some embodiments, the targeted molecule is a molecule that binds to cell surface proteins on tumor or cancer cells. In some embodiments, the targeted molecule binds to cell surface proteins on the surface of T lymphocytes, e.g., Tregs, dendritic cells, natural killer (NK) cells, B cells, macrophages, or other immune cells present in the tumor microenvironment. For example, the tumor or cancer cells may be associated with cancer located in the head and neck, breast, liver, colon, ovaries, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung. In some embodiments, the targeted molecule binds to cells that are cancer stem cells or circulating tumor cells.

[0166] Exemplary targeted molecules, such as macromolecules (including those targeting tumors or cancer), include, but are not limited to, any of those described in Published International PCT Applications WO2014120974, WO2014176284, WO2015042325, U.S. Patent No. 8,524,239, or U.S. Patent Publication US20140120119.

[0167] Exemplary targeting molecules include, but are not limited to, proteins, glycoproteins, antibodies, antibody fragments, antigens, antigen-binding fragments, peptides, polypeptides, small molecules, synthetic polymers, polymer nanoparticles, liposomes, enzyme substrates, hormones, neurotransmitters, cellular metabolites, viral particles, viral capsids, viral nanoparticles, bacterial particles, markers, cells, haptens, avidins, streptavidin, monomeric streptavidin, biotin, carbohydrates, oligosaccharides, polysaccharides, nucleic acids, deoxyribonucleotides, DNA fragments, RNA fragments, nucleotide triphosphates, acycloterminator triphosphates, or PNAs.

[0168] In some embodiments, the targeted molecule is an amino acid, peptide, protein, tyramine, polysaccharide, ionic complex moiety, nucleoside, nucleotide, oligonucleotide, psoralen, drug, hormone, lipid, lipid aggregate, polymer, polymer microparticle, living cell, or virus. In some embodiments, the targeted molecule is an antigen, steroid, vitamin, drug, metabolite, toxin, environmental pollutant, nucleic acid polymer, carbohydrate, lipid, or glass, plastic, or other non-biopolymer. In some embodiments, the targeted molecule is a cell, cell line, cell fragment, or particle smaller than a cell, such as a viral particle, bacterial particle, viral component, living cell (e.g., animal cell, plant cell, bacterium, yeast, or protist), or cellular component. In some embodiments, the reactive dye can label functional groups on the cell surface, in the cell membrane, organelle, or in the cytoplasm.

[0169] In some embodiments, the targeting molecule can selectively bind to a desired cell type, a cell having a specific phenotype, or a cell presenting one or more cell surface markers or antigens. In some embodiments, the targeting molecule is a tumor targeting molecule. In some embodiments, the targeting molecule can bind to tumor or cancer cells. In some embodiments, the targeting molecule targets or binds to a cell surface, for example, a marker or antigen on the cell surface of a tumor cell.

[0170] In some embodiments, the targeting molecule targets or binds to an antigen (e.g., any structural substance that can be targeted by the molecule). In some embodiments, the antigen is a cell surface molecule expressed on the cell surface, such as a protein (e.g., a receptor), or is included as part thereof. In some embodiments, for example, the antigen is a molecule expressed on the surface of cells present in a tumor (including any cells present in the tumor microenvironment), or is included as part thereof. Examples of cell surface molecules to which the targeting molecule can bind include, non-limitingly, antigens, peptides, lipids, polysaccharides, carbohydrates, or nucleic acids containing antigenic determinants (such as those recognized by immune cells). In some examples, the antigen includes a tumor-specific peptide (e.g., one found on the surface of cancer cells) or an immunogenic fragment thereof.

[0171] In some embodiments, the targeting molecule is a binding partner, such as a ligand, that can bind to cell surface molecules, such as cell surface proteins (e.g., cell surface receptors). In some embodiments, the targeting molecule is adrenocorticotropic hormone (ACTH), angiotensin II, atrial natriuretic factor (ANF), bombesin, bradykinin, brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), calcitonin, cardiotrophin 1 (BMP-2), CD22, CD40, cholecystokinin (CCK), ciliary neurotrophic factor (CNTF), CCL1-CCL28, CXCL1-C XCL17, XCL1, XCL2, CX3CL1, Crypt-1 binding peptide, Vascular endothelial growth factor (VEGF), Epidermal growth factor (EGF), Endothelin 1, Endothelin 1 / 3, FAS-ligand, Fibroblast growth factor 1 (FGF-1), Fibroblast growth factor 2 (FGF-2), Fibroblast growth factor 4 (FGF-4), Fibroblast growth factor 5 (FGF-5), Fibroblast growth factor 6 (FGF-6), Fibroblast growth factor 1 (FGF-7), Fibroblast growth factor 1 (FGF-10), Flt-3, Gust Phosphorus, gastrin-releasing peptide (GRP), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-stimulating factor (GM-CSF), glucagon-like peptide (GLP-1), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN-b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 11 (IL-11), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17 (IL-17), Interleukin 19 (IL-19),Luteinizing hormone (LH), luteinizing-releasing hormone (LHRH), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neuromedin B, neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), neurotensin, neuropeptide Y, oxytocin, pituitary adenylate cyclase-activating peptide (PACAP), platelet-derived growth factor AA (PDGF- AA), platelet-derived growth factor AB (PDGF-AB), platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor CC (PDGF-CC), platelet-derived growth factor DD (PDGF-DD), netrin-1 (NTN1), netrin-2 (NTN2), netrin-4 (NTN4), netrin-G1 (NTNG1) and netrin-G2 (NTNG2), ephrin A1 (EFNA1), ephrin A2 (EFNA2), ephrin A3 (EFNA3), ephrin A4 (EFNA4), ephrin A5 (EFNA5), semaphorin 3A (SEMA3A), semaphorin 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 (SEM A6B), Semaphorin 6D (SEMA6D), Semaphorin 7A (SEMA7A), SLIT1, SLIT2, SLIT3, SLIT and NTRK Family Member 1 (SLITRK1), SLIT and NTRK Family Member 2 (SLITRK2), SLIT and NTRK Family Member 3 (SLITRK3), SLIT and NTRK Family Member 4 (SLITRK4), SLIT and NTRK Family Member 5 (SLITRK5), SLIT and NTRK Family Member 6 (SLITRK6),Prostaglandin E2 (PGE2), RANTES, Somatostatin-14, Somatostatin-28, Stem cell factor (SCF), Stroma cell-derived factor 1 (SDF-1), Substance P, Thyroid-stimulating hormone (TSH), Transforming growth factor alpha (TGF-α), Transforming growth factor beta (TGF-β), Tumor necrosis factor alpha (TNF-α), Thrombin, Vasoactive intestinal peptide (VIP), Wntl, Wnt2, Wnt2b / 13, Wnt3 , selected from Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wntl0a, Wntl0b, Wnt11, Wnt14, Wnt15 or Wnt16, Sonic Hedgehog, Desert Hedgehog, and Indian Hedgehog, or its binding fragment capable of binding to its congeneral cell surface molecules such as cell surface proteins (e.g., cell surface receptors).

[0172] In some embodiments, the targeted molecule may be an immunomodulator that can bind to cell surface molecules or proteins on immune cells to suppress or activate the body's immune response. In some embodiments, the binding of the immunomodulator to cell surface molecules or proteins may stimulate the immune response against tumors and / or pathogens, for example, by inhibiting immunosuppression or by enhancing immune stimulation. In some embodiments, the cell surface molecules or proteins may 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, C D155, CD226, TIGIT, CD96, Galectin 3, CD40, CD40L, CD70, LIGHT (TNFSF14), HVEM (TNFRSF14), B7-H3 (CD276) These may include Ox40L (TNFSF4), CD137L (TNFSF9, GITRL), B7RP1, ICOS (CD278), ICOSL, KIR, GAL9, NKG2A (CD94), GARP, TL1A, TNFRSF25, TMIGG2, BTNL2, butyrophyllin family, CD48, CD244, Siglec family, CD30, CSF1R, MICA (MHC class I polypeptide-related sequence A), MICB (MHC class I polypeptide-related sequence B), NKG2D, KIR family (killer cell immunoglobulin-like receptor), LILR family (leukocyte immunoglobulin-like receptor, CD85, ILT, LIR), SIRPA (signal regulatory protein alpha), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR, or VEGF.

[0173] In some embodiments, cell surface molecules include cell membrane phospholipids, prokaryotic peptidoglycans, bacterial cell envelope proteins, viral capsid proteins, ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptors BB1, BB2, BB4, calcitonin receptor, and cancer antigen 125 (CA). 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endothialin, EP2, EP4 EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, Family A G protein-coupled receptor (rhodopsin-like), Family B G protein-coupled receptor (secretin-like), Family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV16 / 18 and E6 / E7 antigens, hTERT, interleukin receptors (e.g., IL-2R, IL11-R, IL-13R), ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE1, MAGE2, MAGE3, MAGE4, ​​MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Not ch-1, NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease-cleaved collagen IV, proteinase 3, inhibitor, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 This could be patched, RET receptor, plexin, smoothed, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptor (VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, and VEGF-3 or FLT-4), voltage-gated ion channel, VPAC1, VPAC2, Wilms tumor 1, Y1, Y2, Y4, or Y5.

[0174] In some embodiments, cell surface molecules include HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72, caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate-binding protein, ganglioside (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), integrin αVβ3, and integrin. It may be Grin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, Tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinase, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, or SK-1 antigen.

[0175] In some embodiments, the targeting molecule is an antibody or antibody fragment that specifically binds to an antigen, such as a cell surface molecule on a tumor cell. Such antibodies include antibodies or antigen-binding antibody fragments capable of binding to cell surface molecules, such as cell surface proteins (e.g., cell surface receptors) as described herein. In some cases, the antibody can bind to an antigen of a protein expressed on cells in the tumor, including a tumor-specific protein.

[0176] In some embodiments, the targeting molecule binds directly or indirectly to an antigen or protein. For example, in some embodiments, the targeting molecule is a second binding molecule that binds to a first binding molecule capable of binding to an antigen or protein. For example, the targeting molecule is a secondary antibody that binds to a first binding molecule capable of binding to a protein or antigen, such as a cell surface protein or cell surface receptor, such as a primary antibody. Thus, in some embodiments, the dye is conjugated to the secondary antibody.

[0177] An antibody is a polypeptide ligand that specifically recognizes and binds to an antigen epitope, such as a tumor-specific protein, and contains at least one light chain or heavy chain immunoglobulin variable region. Generally, antibodies are characterized by their variable heavy chain (V H ) region and variable light chain (V L It is composed of heavy and light chains, each having a variable region called the ) region. H Region and V L The region as a whole is involved in the binding of antigens that are recognized by antibodies.

[0178] Antibodies include intact immunoglobulins and antibody fragments exhibiting antigen binding, such as Fab fragments, Fab' fragments, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). The scFv protein is a fusion protein in which the light chain variable region and the heavy chain variable region of immunoglobulin are linked by a linker, while in dsFv, the chain is mutated to introduce a disulfide bond that stabilizes the chain association. This term also includes genetically modified forms, such as chimeric antibodies, e.g., humanized mouse antibodies, and heteroconjugate antibodies, e.g., bispecific antibodies. (See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J. Immunology, 3) rd See also Ed., WH Freeman & Co., New York, 1997.

[0179] Typically, naturally occurring immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (k). There are five major heavy chain classes, or isotypes, that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0180] Each heavy and light chain contains a constant region and a variable region, also known as a “domain.” In combination, the variable regions of the heavy and light chains generally bind specifically to the antigen. The variable regions of the light and heavy chains may contain a “framework” region interposed by three hypervariable regions, also called “complementarity-determining regions” or “CDRs.” The scope of the framework region and CDRs is defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, incorporated herein by reference). The Kabat database is currently maintained online. Sequences of different light or heavy chain framework regions are relatively conserved within species such as humans. The framework region of an antibody, which is the combined framework region of the constituent light and heavy chains, helps to position and align the CDRs in three-dimensional space.

[0181] CDRs are typically involved in the binding of an antigen to an epitope. The CDRs on each chain are typically referred to as CDR1, CDR2, and CDR3, sequentially numbered starting from the N-terminus, and are also generally identified by the chain on which a particular CDR is located. Therefore, V H CDR3 is located in the variable domain of the antibody heavy chain where it is found, on the other hand, V LCDR1 is a CDR1 derived from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities, such as different binding sites for different antigens, have different CDRs. While CDRs vary by antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. These positions within a CDR are called specificity-determining residues (SDRs).

[0182] "V H References to "VH" or "VH" refer to the variable region of the immunoglobulin heavy chain, including those of Fv, scFv, dsFv, or Fab. L References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including those of Fv, scFv, dsFv, or Fab.

[0183] In particular, the antibodies offered are antibody fragments. An "antibody fragment" refers to a molecule other than the intact antibody, including a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Other antibody fragments or multispecific antibodies formed from antibody fragments include polyvalent scFv, bispecific scFv, or scFv-CH3 dimers. Antibody fragments can be produced by a variety of techniques, but are not limited to, proteolysis of intact antibodies and production by recombinant host cells.

[0184] A "monoclonal antibody" is an antibody produced by a single clone of a B lymphocyte or by cells transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells derived from a fusion of myeloma cells and immunosplenic cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0185] A "chimeric antibody" has a framework residue derived from one species, such as a human, and a CDR (generally antigen-binding-constituting) derived from another species, such as a mouse antibody, which specifically binds to mesothelin.

[0186] A “humanized” immunoglobulin is an immunoglobulin comprising a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDR is referred to as the “donor,” and the human immunoglobulin providing the framework is referred to as the “acceptor.” In some embodiments, the CDR in the humanized immunoglobulin is derived from the donor immunoglobulin. A constant region is not required, but if one is present, it may be substantially identical to the human immunoglobulin constant region, e.g., at least about 85–90%, e.g., about 95% or more. Therefore, a portion of the humanized immunoglobulin (perhaps excluding the CDR) is substantially identical to a portion of the corresponding native human immunoglobulin sequence. A “humanized antibody” is an antibody containing immunoglobulins with humanized light chains and humanized heavy chains. The humanized antibody binds to the same antigen as the donor antibody providing the CDR. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids derived from the donor framework. Humanized or other monoclonal antibodies may have additional conserved amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by genetic modification (see, for example, U.S. Patent No. 5,585,089).

[0187] A “human” antibody (also called a “fully human” antibody) is an antibody that contains a human framework region and a CDR derived from human immunoglobulin. In some embodiments, the framework and CDR are derived from human heavy and / or light chain amino acid sequences of the same origin. However, a framework derived from one human antibody can be modified to contain CDRs derived from different human antibodies. A portion of a human immunoglobulin may be substantially identical to a portion of the corresponding native human immunoglobulin sequence.

[0188] "Specifically binding" refers to the ability of a molecule, such as an antibody or antigen-binding fragment, to specifically bind to an antigen, such as a tumor-specific antigen, compared to binding to unrelated proteins, such as non-tumor proteins (e.g., β-actin). In some embodiments, a molecule such as an antibody or fragment (including a molecule attached to a phthalocyanine dye molecule) binds to a target, such as a cell surface protein, at least 10 times more than its binding constant for other molecules in the sample or subject. 3 M -1 Big, 10 4 M -1 Large or 10 5 M -1 It binds specifically with a large binding constant. In some embodiments, molecules such as antibodies or fragments of antibodies bind specifically with a large binding constant of about 10 6 M -1 The above, approximately 10 7 M -1 The above, approximately 10 8 M -1 The above, or about 10 9 M -1 , 10 10 M -1 , 10 11 M -1 Or 10 12 M -1 The equilibrium association constant (K) A ) has. The antibody also has 10 -6 M, 10 -7 M, 10 -8 M, 10 -10 M, 10 -11 M or 10 -12 M or a lower equilibrium dissociation constant (KD It can be characterized by the equilibrium dissociation constant (K D ) can be less than 1 nM. K D or K A Such affinity constants can be estimated empirically or determined by comparing affinities, for example, by comparing the affinity of one antibody against a particular antigen with that of another antibody. For example, such affinities can be readily determined using techniques known in the art, such as by competitive ELISA (enzyme-linked immunosorbent assay) or by radioimmunoassay using a radiolabeled target antigen with a surface plasmon resonance spectrometer such as the Biacore T100 (available from Biacore, Inc., Piscataway, NJ), or by other methods known to those skilled in the art.

[0189] In some embodiments, phthalocyanine dyes (e.g., IR700) are conjugated to antibodies or antigen-binding antibody fragments. For example, in some aspects, the phthalocyanine dye-targeting molecule conjugate is the IR700-antibody conjugate. Exemplary antibodies that can be conjugated with phthalocyanine dyes (e.g., IR700) include, but are not limited to, cetuximab, panitumumab, zaltumumab, nimotuzumab, trastuzumab, Ado-trastuzumab emtansine, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, and CEA-scan. Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), afatinib, axitinib, bosutinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, olaparri B, palbociclib, pazopanib, pertuzumab, ramucirumab, regorafenib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, bismodegib, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pizilizumab (CT-011), MK-3475, BMS- 936559, MPDL3280A, Tremelimumab, IMP321, BMS-986016, LAG525, Urelumab, PF-05082566, TRX518, MK-4166, Dasetuzumab, Lucatumumab, SEQ-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR0916, AMP-224, MSB00107 This product contains 18C, MEDI4736, PDR001, rHIgM12B7, urocuplumab, BKT140, valrirumab (CDX-1127), ARGX-110, MGA271, lirirumab (BMS-986015, IPH2101), IPH2201, AGX-115, emactuzumab, CC-90002, and MNRP1685A or its antibody-conjugated fragments.

[0190] In some embodiments, the targeted molecule is a tissue-specific homing peptide. For example, in some embodiments, the homing peripeptide may contain an amino acid sequence shown in any of SEQ ID NO: 1 to 52. In some embodiments, the targeted molecule is an RGD polypeptide, e.g., iRGD polypeptide, Lyp-1 polypeptide, crypto-1 binding polypeptide, somatostatin receptor binding polypeptide, or inhibitor-binding polypeptide, NGR polypeptide, or iNGR polypeptide.

[0191] In some embodiments, the targeting molecule is an activatable cell-permeable peptide (ACPP) composed of a polycationic cell-permeable peptide (CPP) linked to a neutralizing polyanion via a cleavable linker. For example, in some embodiments, the ACPP comprises the structure A-X1-B-, where B is a peptide moiety of about 5 to about 20 basic amino acid residues suitable for intracellular uptake; A is a peptide moiety of about 2 to about 20 acidic amino acid residues that, when linked with moiety B, is effective in inhibiting or preventing the intracellular uptake of moiety B; X1 is a cleavable linker of about 2 to about 100 atoms; and one or more LYs are linked to the C-terminus of peptide moiety B.

[0192] In some embodiments, the targeting molecule is a viral particle, such as a virus-like particle, virus-like nanoparticle, or viral capsid. In some embodiments, the targeting molecule is a virus-like nanoparticle. In some embodiments, the virus-like nanoparticle is assembled from an L1 capsid protein. In some embodiments, the virus-like nanoparticle is assembled from a combination of an L1 capsid protein and an L2 capsid protein. In some embodiments, the targeting molecule binds to and infects a cell. In some embodiments, the targeting molecule is the targeting molecule described in WO2015042325.

[0193] In some embodiments, virus-like particles (VLPs) refer to organized capsid-like structures, such as those roughly spherical or cylindrical, that contain a self-assembled, ordered array of L1 or L1 and L2 capsomers and do not contain a viral genome. In some embodiments, virus-like particles are morphologically and antigenically similar to true virions, but they lack viral genetic material such as viral nucleic acids, which makes the particles non-infectious. VLPs can be used to deliver drugs, such as prophylactic, therapeutic, or diagnostic agents, or encapsulated circular or linear DNA or RNA molecules to recipient cells.

[0194] In some embodiments, VLPs may have modified immunogenicity and / or antigenicity compared to wild-type VLPs. VLPs may be assembled, for example, from capsomers having immunogenic and / or antigenically modified mutant capsid proteins. In some embodiments, immunogenic and / or antigenically modified mutant capsid proteins are those in which amino acids are modified spontaneously or synthetically, for example by mutation, substitution, deletion, pegylation, or insertion, thereby reducing or preventing recognition of the capsid protein by existing (e.g., endogenous) viral serotype-specific antibodies. Mutant capsid proteins may be human papillomavirus (HPV) L1 mutants, non-human papillomavirus L1 mutants, or papillomavirus L1 mutants based on amino acid combinations derived from different HPV serotypes.

[0195] In some embodiments, the VLP is a papillomavirus VLP. The VLP may be a human papillomavirus VLP, such as one derived from a virus capable of infecting humans, while in other embodiments, the VLP may be a non-human papillomavirus VLP. Examples of non-human VLPs include, but are not limited to, those derived from bovine papillomavirus, murine papillomavirus, cotton rabbit papillomavirus, and macaque or rhesus monkey papillomavirus particles. In some embodiments, the VLP is a bovine papillomavirus-like nanoparticle, such as type 1 virus-like nanoparticles (e.g., assembled from BPV L1 capsid protein or a combination of BPV L1 and BPV L2 capsid proteins).

[0196] In some embodiments, capsid proteins refer to protein monomers, some of which form capsomer oligomers. In some embodiments, capsomers refer to the basic oligomeric structural units of the viral capsid, which is the protein outer covering protecting the viral genetic material. In some embodiments, capsid proteins may include papillomavirus L1 major capsid proteins and papillomavirus L2 minor capsid proteins. In some embodiments, VLPs contain only L1 capsid proteins, while in other embodiments, VLPs contain a mixture of L1 and L2 capsid proteins, or a combination thereof.

[0197] In some embodiments, the percentage of L1 capsid protein in virus-like particles is greater than the percentage of L2 capsid protein in virus-like particles. For example, in some embodiments, the percentage of L1 capsid protein in virus-like particles is 80% to 100% of the total number of capsid proteins in virus-like particles. In some embodiments, the percentage of L1 capsid protein in virus-like particles is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the percentage of L2 capsid protein in virus-like particles is between 1% and 25% of the total number of capsid proteins in the virus-like particles. For example, in some embodiments, the percentage of L2 capsid protein in virus-like particles is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0198] In some embodiments, the virus-like particles contain 12 to 72 L2 proteins. In some embodiments, the virus-like particles contain 360 L1 proteins and 12 to 72 L2 proteins. In some embodiments, the capsid protein is assembled into virus-like nanoparticles having a diameter of 20 to 60 nm. For example, the capsid protein can be assembled into virus-like nanoparticles having a diameter of at least 20, 25, 30, 35, 40, 45, 50, 55 or 60 nm, or approximately 20, 25, 30, 35, 40, 45, 50, 55 or 60 nm.

[0199] In some embodiments, the target molecule is a DARPin (designed ankyrin repeat protein). Typically, DARPins are derived from natural ankyrin repeat proteins and bind to proteins including, for example, human receptors, cytokines, kinases, human proteases, viruses, and membrane proteins (see Molecular Partners AG Zurich Switzerland; Chapter 5. “Designed Ankyrin Repeat Proteins (DARPins): From Research to Therapy”, Methods in Enzymology, vol 503: 101-134 (2012); and “Efficient Selection of DARPins with Sub-nanomolar Affinities using SRP Phage Display”, J. Mol. Biol. (2008) 382, ​​1211-1227, the entire disclosure of which is incorporated herein by reference). In some embodiments, DARPins are antibody-mimicking proteins prepared via genetic modification that have high specificity and high binding affinity to the target protein. In some embodiments, DARPin has a structure comprising at least two ankyrin repeat motifs, for example, at least three, four, or five ankyrin repeat motifs. DARPin can have any suitable molecular weight depending on the number of repeat motifs. For example, DARPin comprising three, four, or five ankyrin repeat motifs may have molecular weights of about 10 kDa, about 14 kDa, or about 18 kDa, respectively.

[0200] In some embodiments, DARPin comprises a core portion that provides structure and a target-binding portion located outside the core and binding to a target. In some embodiments, the structural core comprises a conserved amino acid sequence, and the target-binding portion comprises a different amino acid sequence depending on the target.

[0201] In some embodiments, the conjugate contains multiple pigment residues, each targeting molecule, of 1 to about 1000 or about 1 to about 1000, for example, 1 to about 100 or about 1 to about 100, 1 to about 50 or about 1 to about 50, 1 to about 25 or about 1 to about 25, 1 to about 10 or about 1 to about 10, 1 to about 5 or about 1 to about 5. In some aspects, the ratio of dye molecule to target molecule is 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1 or 1000:1, or approximately 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 these values ​​or approximately between any two of these values. In some embodiments, the targeted molecule may contain up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 dye molecules. In some embodiments, the targeted molecule may contain more than 1000 dye molecules or less than 10 dye molecules.

[0202] In some embodiments, for example, when the targeting molecule is a polypeptide such as an antibody or antigen-binding fragment, the number of dye molecules per targeting molecule can be 2 to 5 or about 2 to 5, e.g., 2 to 4 or about 2 to 4, e.g., about 3 or 3. In some embodiments, for example, when the targeting molecule is a nanoparticle such as a virus-like particle (VLP), the number of dye molecules and targeting molecules can be 10 to 1000, 10 to 500, 50 to 500 or 50 to 1000, or about 10 to 1000, about 10 to 500, about 50 to 500 or about 50 to 1000. Thus, in some embodiments, the targeting molecule may contain about 10 to about 1000 dye molecules.

[0203] In some embodiments, for example, when the targeting molecule is a VLP, more than one pigment molecule may be conjugated to a single capsid protein. For example, a single capsid protein, such as an LI or L2 capsid protein, may be conjugated to 1-5, e.g., 1, 2, 3, 4, or 5 pigment molecules. Thus, more than one amino acid of the capsid protein may be conjugated to pigment molecules. In some embodiments, a single capsid protein may be conjugated to 1-2, 1-3, or 2-3 pigment molecules. Thus, the pigment molecule may be conjugated to 1, 2, 3, 4, or 5 different amino acids of the single capsid protein, e.g., lysine, arginine, and / or histidine, or other amino acids.

[0204] B. Photoprotective preparation of dye-targeted molecule conjugates In some embodiments, methods or processes are provided for preparing phthalocyanine dye-targeting molecule conjugates, such as IR700-targeting molecule (e.g., IR700-antibody) conjugates, under photoprotective conditions. In some embodiments, the method comprises: 1) preparing or providing a phthalocyanine dye and a targeting molecule; 2) contacting the targeting molecule and the phthalocyanine dye under conditions that produce or generate a conjugate with minimal exposure to the dye; and 3) formulating, purifying and / or isolating the conjugate to produce a composition containing the active pharmaceutical ingredient, wherein one or more of the steps, for example all of the steps in some cases, are carried out with minimal exposure of the dye or the dye-containing conjugate to ambient light.

[0205] In some embodiments, before, during, and after the preparation of the dye and / or conjugate, the dye and / or conjugate are not exposed to any ambient light or to light having an intensity greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the dye and / or conjugate are not exposed to light having an intensity greater than 700 lux for more than 20 minutes, more than 10 minutes, or more than 5 minutes. In some embodiments, the dye and / or conjugate are not exposed to light having an intensity greater than 200 lux for more than 20 minutes, more than 10 minutes, or more than 5 minutes.

[0206] In some embodiments, the dye and / or conjugate are protected from ambient light, such as near-infrared light, before, during, or after one or more steps of the Method or all steps of the Method. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and after the preparation of the conjugate has wavelengths that are not absorbed or substantially not absorbed by the dye. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and after the preparation of the API is green light. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and after the preparation of the API has wavelengths in the range of 400 nm to 650 nm or approximately 400 nm to 650 nm, for example, 400 nm to 600 nm, 425 nm to 575 nm, or 450 nm to 550 nm.

[0207] In some embodiments, the only light to which the dye and / or conjugate are exposed between one or more or all of the steps of the Method has a wavelength that is not absorbed or substantially absorbed by the dye and an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the only light to which the dye and / or conjugate are exposed between one or more or all of the steps of the Method is green light having an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the only light to which the dye and conjugate are exposed during one, more or all of the steps of the Method has a wavelength in the range of 400 nm to 650 nm or about 400 nm to 650 nm, for example, 400 nm to 600 nm or about 400 nm to 600 nm, 425 nm to 575 nm or about 425 nm to 575 nm, or 450 nm to 550 nm or about 450 nm to 550 nm, and has an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux.

[0208] In some embodiments, before, during, and after the preparation, production, formulation, and / or packaging of the dyes and / or conjugates, the dyes and / or conjugates are not exposed to any ambient light, or have a total light exposure of 5000 lux or less for 2500 lux or less, 1000 lux or less, 500 lux or less, 250 lux or less, 100 lux or less, or 80 lux or less. In some embodiments, in the methods provided herein, the dyes and / or conjugates are not exposed to any ambient light, or have a total light exposure of 5000 lux or less for 2500 lux or less, 1000 lux or less, 500 lux or less, 250 lux or less, 100 lux or less, or 80 lux or less, during the execution of the entire method. For example, the total light exposure of the dye and / or conjugate during the entire method, or before, during, and after the preparation, production, formulation, and / or packaging of the dye and / or conjugate, is 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, 50 lux hours or less, or 25 lux hours or less. The total light exposure is determined by multiplying the illuminance (lux) by the exposure duration (hours).

[0209] In some embodiments, during one, more or all of the steps of the method provided herein, the total exposure of the dye and / or conjugate to any light is 5000 lux hours or less, 2500 lux hours or less, 1000 lux hours or less, 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, or 80 lux hours or less. For example, the total light exposure of the dye and / or conjugate during the packaging step is 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, 50 lux hours or less, or 25 lux hours or less.

[0210] In some embodiments, the dye and / or conjugate are protected from light by performing a procedure in the Method that uses one or more containers that protect the contents from light or a particular wavelength or intensity of light during one or more or all of the steps of the Method. For example, in some embodiments, one, two or three or more light-protective containers are used in the Method. For example, in some embodiments, the containers have a light transmittance of 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. In some embodiments, the container protects against the transmission of light having wavelengths of 250nm to 800nm ​​or about 250nm to 800nm, for example, 250nm to 450nm or about 250nm to 450nm, 400nm to 800nm ​​or about 400nm to 800nm, 450nm to 650nm or about 450nm to 650nm, 500nm to 725nm or about 500nm to 725nm, 600nm to 720nm or about 600nm to 720nm, or 650nm to 725nm or about 650nm to 725nm, or does not transmit light of an intensity greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the dye and / or conjugate are prepared in a translucent or opaque container. In some embodiments, the container is green, blue, or amber in color. In some embodiments, the container is covered with an opaque material, such as foil, such as aluminum foil. In some embodiments, the container is covered with a material having a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the container is a vial, tube, syringe, bag, pouch, and / or box.

[0211] In some embodiments, a method for producing a conjugate includes the step of preparing or producing the conjugate. In some embodiments, such a method includes the step of providing a phthalocyanine dye. In some embodiments, the phthalocyanine dye is provided in an aqueous form, such as an aqueous solution. In some embodiments, the dye is provided in a lyophilized form, such as a lyophilized powder, and is reconstituted or dissolved in a solvent to form an aqueous solution. For example, in some embodiments, a phthalocyanine dye containing a reactive group, such as an IR700 NHS ester, is dissolved in a solvent. In some embodiments, the method includes the step of dissolving the phthalocyanine dye in a solvent, for example, before conjugation of the dye to a target molecule. In some embodiments, the solvent is an organic solvent such as dimethyl sulfoxide (DMSO) or DMF. In some examples, the solvent is an aqueous solvent. In some embodiments, the dye is dissolved in the solvent at concentrations ranging from 0.1 mg / mL to 100 mg / mL or approximately 0.1 mg / mL to 100 mg / mL, 1 mg / mL to 50 mg / mL or approximately 1 mg / mL to 50 mg / mL, 1 mg / mL to 15 mg / mL or approximately 1 mg / mL to 15 mg / mL, or at a concentration of 10 mg / mL or approximately 10 mg / mL.

[0212] In some embodiments, during the process of preparing the dye for use in the method, the phthalocyanine dye, such as IR700 NHS ester, is protected from exposure to ambient light. In some embodiments, before, during, and after the preparation of the phthalocyanine dye, the dye is not exposed to light such as ambient light, or is exposed only to light having wavelengths in the range of 400 nm to 650 nm or approximately 400 nm to 650 nm, for example, 425 nm to 475 nm or approximately 425 nm to 475 nm. In some embodiments, the phthalocyanine dye is not exposed to light having an intensity greater than 700 lux, or is not exposed to light having an intensity greater than 700 lux for more than 10 minutes or more than 5 minutes. In some embodiments, the phthalocyanine dye is not exposed to light having an intensity greater than 200 lux, or is not exposed to light having an intensity greater than 200 lux for more than 10 minutes or more than 5 minutes.

[0213] In some embodiments, the step of preparing or producing the conjugate includes providing a targeting molecule (e.g., an antibody) for conjugation with a phthalocyanine dye such as IR700. In some embodiments, the targeting molecule is prepared before conjugation with the phthalocyanine dye. In some embodiments, the step of preparing the targeting molecule includes concentrating or diluting the targeting molecule to a specific amount or concentration before the conjugation reaction. In some embodiments, the step of preparing the targeting molecule includes replacing the targeting molecule with a buffer, e.g., a buffer compatible with or suitable for the conjugation reaction. In some embodiments, the step of preparing the targeting molecule includes adjusting the pH to a pH suitable for use in the conjugation reaction. For example, a targeting molecule such as an antibody is prepared at a pH of 6 to 10 or about 6 to 10, e.g., between 8 and 9 or about 8 to 9, e.g., about 8.5, e.g., 8.46.

[0214] In some embodiments, the targeting molecule, such as an antibody, is buffered to a buffer, for example, using tangential flow filtration (TFF), or ultrafiltration / dialysis filtration. In some embodiments, the TFF includes a regenerated membrane, such as a regenerated cellulose membrane. In some embodiments, the buffer to which the targeting molecule is buffered is a sodium phosphate buffer, such as 100 mM sodium phosphate, with a pH of 8.5 or 8.65. In some embodiments, tangential flow filtration is carried out until the desired pH of the filtrate is reached. In some embodiments, the desired pH is between 6 and 10 or about 6 and 10, for example, between 8 and 9 or about 8 and 9, for example, about 8.5, for example, 8.46.

[0215] In some embodiments, the targeted molecule is provided in an amount between 0.01g and 100g or about 0.01g and 100g, between 1g and 50g or about 1g and 50g, between 1g and 25g or about 1g and 25g, between 5g and 15g or about 5g and 15g, or 12g or about 12g. In some embodiments, the volume of the targeted molecule preparation is between 0.01L and 100L or about 0.01L and 100L, between 1L and 50L or about 1L and 50L, between 1L and 15L or about 1L and 15L, or 6L or about 6L. In some embodiments, the concentration of the targeting molecule, such as an antibody, is less than 0.01 mg / mL, or between 0.1 mg / mL and 100.0 mg / mL or approximately between 0.1 mg / mL and 100.0 mg / mL, between 0.1 mg / mL and 50 mg / mL or approximately between 0.1 mg / mL and 50 mg / mL, between 0.1 mg / mL and 10 mg / mL or approximately between 0.1 mg / mL and 10 mg / mL, or between 1 mg / mL and 5 mg / mL or approximately between 1 mg / mL and 5 mg / mL, or 5 mg / mL or approximately 5 mg / mL, or 4.5 mg / mL or approximately 4.5 mg / mL, or 2 mg / mL or approximately 2 mg / mL. In some embodiments, the targeting molecule, such as an antibody, is diluted to a concentration between 0.1 mg / mL and 100.0 mg / mL or approximately 0.1 mg / mL and 100.0 mg / mL, between 0.1 mg / mL and 50 mg / mL or approximately 0.1 mg / mL and 50 mg / mL, between 0.1 mg / mL and 10 mg / mL or approximately 0.1 mg / mL and 10 mg / mL, between 1 mg / mL and 5 mg / mL or approximately 1 mg / mL and 5 mg / mL, or between 1.8 mg / mL and 2.4 mg / mL or approximately 1.8 mg / mL and 2.4 mg / mL, or to a concentration of 2 mg / mL or approximately 2 mg / mL.

[0216] In some embodiments, the targeting molecule, such as an antibody, is filtered through a sterile filter such as a 0.2 μm or 0.22 μm filter. In some embodiments, the prepared targeting molecule is stored at a temperature below 30°C, for example, generally below 26°C, 20°C, 15°C, or 10°C, for example, generally between 2 and 8°C or approximately between 2 and 8°C. In some embodiments, the weight of the targeting molecule is determined.

[0217] In some embodiments, a method for producing a conjugate includes contacting a targeting molecule, such as one of the above (e.g., an antibody), with a phthalocyanine dye (e.g., IR700). In some embodiments, the phthalocyanine dye and the targeting molecule are mixed together in a vessel such as a reactor. In some embodiments, the contact step is carried out in a vessel or container, such as a reactor. In some embodiments, the container is a tube, bottle, or carboy. In some embodiments, the container has a maximum capacity of about 1 L, 2 L, 5 L, 10 L, 15 L, 20 L, 30 L, 40 L, 50 L, or 100 L, or at least 1 L, 2 L, 5 L, 10 L, 15 L, 20 L, 30 L, 40 L, 50 L, or 100 L. In some embodiments, the container is a 40 L carboy. In some embodiments, the container has a maximum capacity of approximately 100 μL, 500 μL, 1 mL, 1.5 mL, 5 mL, 15 mL, 50 mL, 250 mL, or 500 mL, or at least 100 μL, 500 μL, 1 mL, 1.5 mL, 5 mL, 15 mL, 50 mL, 250 mL, or 500 mL. In some embodiments, the container or vessel is translucent or opaque, green or amber in color, and / or covered with an opaque foil, such as aluminum foil.

[0218] In some embodiments, the amount of dye used to contact the targeting molecule is calculated based on the weight of the targeting molecule present in the container or vessel. For example, in some embodiments, the amount of dye is added such that the final molar ratio of dye to targeting molecule is 1:1 to 1000:1 or approximately 1:1 to 1000:1, 1:1 to 100:1 or approximately 1:1 to 100:1, 1:1 to 10:1 or approximately 1:1 to 10:1, 1:1 to 4:1 or approximately 1:1 to 4:1, or approximately 4:1 or 4:1.

[0219] In some embodiments, for example, when the targeting molecule is a virus-like particle (VLP), the ratio of dye molecule to targeting molecule is between 10:1 and 1000:1 or approximately between 10:1 and 1000:1, between 10:1 and 500:1 or approximately between 10:1 and 500:1, between 50:1 and 500:1 or approximately between 50:1 and 500:1, or between 50:1 and 1000:1 or approximately between 50:1 and 1000:1. Thus, in some embodiments, the targeting molecule may contain approximately 10 to approximately 1000 dye molecules. In some aspects, the ratio of dye molecule to target molecule is 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 approximately 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 these values ​​or approximately between any two of these values. In some embodiments, the targeted molecule may contain up to 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 targeted molecule may contain more than 1000 dye molecules or fewer than 10 dye molecules.

[0220] In some embodiments, the ratio of dye to targeting molecule is selected so that a desired number of dye residues are incorporated per targeting molecule. In some embodiments, the desired number of dye residues per targeting molecule is 1 to 50 or about 1 to 50, 1 to 25 or about 1 to 25, 1 to 10 or about 1 to 10, 1 to 5 or about 1 to 5, 2 to 5 or about 2 to 5, 2 to 3 or about 2 to 3, or about 3 or 3.

[0221] In some embodiments, for example, when the targeting molecule is a VLP, more than one pigment molecule can be conjugated to a single capsid protein. For example, a single capsid protein, such as an LI or L2 capsid protein, can be conjugated to 1-5, e.g., 1, 2, 3, 4, or 5 pigment molecules. Thus, more than one amino acid of the capsid protein can be conjugated to pigment molecules. In some embodiments, a single capsid protein can be conjugated to 1-2, 1-3, or 2-3 pigment molecules. Thus, the pigment molecules can be conjugated to 1, 2, 3, 4, or 5 different amino acids of the single capsid protein, e.g., lysine, arginine, and / or histidine, or other amino acids.

[0222] In some embodiments, the contact step is carried out under conditions in which the dye and the targeting molecule associate covalently or non-covalently, for example, to react together or otherwise associate or link together.

[0223] In some embodiments, the phthalocyanine dye comprises a reactive chemical group, and the step of contacting the phthalocyanine dye with a targeting molecule produces a conjugate containing the phthalocyanine dye covalently bonded to the attachment group of the targeting molecule.

[0224] In some embodiments, the dye and the targeting molecule are brought into contact at a controlled temperature or in a temperature-controlled unit such as an incubator or cooler. In some embodiments, the method includes the step of bringing a phthalocyanine dye (e.g., IR700) and a targeting molecule (e.g., an antibody) into contact at a temperature in the range of 4°C to 37°C or about 4°C to 37°C, for example 10°C to 30°C or about 10°C to 30°C, 20°C to 30°C or about 20°C to 30°C, or 23°C to 27°C or about 23°C to 27°C, or at a temperature of about 25°C ± 2.0°C, 25°C ± 1.0°C or 25°C ± 0.3°C, for example 25°C or about 25°C. In some embodiments, the contact step is carried out at room temperature, for example between 21°C and 25°C, for example about 23°C.

[0225] In some embodiments, the contact step includes incubating, for example, reacting, the dye and the targeting molecule. In some embodiments, the contact step can be carried out in a reactor. In some embodiments, the contact step includes, at least in part of the contact step, mixing (for example, by stirring) the combined dye and targeting molecule composition. In some embodiments, the contents are stirred, for example, on a stirring plate. In some embodiments, the contents are stirred for about 5 to 30 minutes or at least 5 to 30 minutes, for example, about 5 to 20 minutes, for example, about 10 to 15 minutes.

[0226] In some embodiments, the contact process is performed for at least 5 minutes, at least 15 minutes, at least 30 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, at least 240 minutes, at least 360 minutes, at least 24 hours, at least 72 hours, or at least 120 hours. In some embodiments, the contact process is performed for 5 minutes to 150 hours, 5 minutes to 100 hours, 5 minutes to 48 hours, 5 minutes to 24 hours, 5 minutes to 6 hours, 5 minutes to 2 hours, 5 minutes to 90 minutes, 5 minutes to 60 minutes, 5 minutes to 30 minutes, 30 minutes to 150 hours, 30 minutes to 100 hours, 30 minutes to 48 hours, 30 minutes to 24 hours, 30 minutes to 6 hours, 30 minutes to 2 hours, 30 minutes to 90 minutes, The sessions are conducted for 30-60 minutes, 60 minutes-150 hours, 60 minutes-100 hours, 60 minutes-48 hours, 60 minutes-24 hours, 60 minutes-6 hours, 60 minutes-2 hours, 60 minutes-90 minutes, 90 minutes-150 hours, 90 minutes-100 hours, 90 minutes-48 hours, 90 minutes-24 hours, 90 minutes-6 hours, 90 minutes-2 hours, 2 hours-150 hours, 2 hours-100 hours, 2 hours-48 hours, 2 hours-24 hours, 2 hours-6 hours, 6 hours-150 hours, 6 hours-100 hours, 6 hours-48 hours, 6 hours-24 hours, 24 hours-150 hours, 24 hours-100 hours, 24 hours-48 hours, 48 ​​hours-150 hours, 48 ​​hours-100 hours, or 100 hours-150 hours. In some embodiments, the contact step is carried out for a duration of 5 minutes to 6 hours, for example, 5 minutes to 4 hours, 5 minutes to 2 hours, 5 minutes to 60 minutes, 5 minutes to 30 minutes, for example, about 5 minutes to 20 minutes, for example, about 10 minutes to 15 minutes. In some embodiments, the method includes a step of incubating, for example, contacting a phthalocyanine dye (e.g., IR700) with a targeting molecule (e.g., an antibody) for at least 15 minutes or about 15 minutes, at least 30 minutes or about 30 minutes, at least 60 minutes or about 60 minutes, at least 90 minutes or about 90 minutes, at least 120 minutes or about 120 minutes, or at least 150 minutes or about 150 minutes. In some embodiments, the method includes a step of reacting, for example, contacting the dye with the targeting molecule for 90 to 150 minutes or about 90 to 150 minutes, for example, 120 minutes.

[0227] In some embodiments, the dye and the targeting molecule are mixed in an aqueous buffer, which may contain an organic solvent such as DMSO or DMF. In some embodiments, the solvent is an aqueous solvent. In some embodiments, the pH of the buffer is between 6 and 10 or about 6 and 10, for example between 7 and 10 or about 7 and 10, between 8 and 10 or about 8 and 10, or between 8 and 9 or about 8 and 9.

[0228] In some embodiments, the contact process is carried out under photoprotective conditions. For example, in some embodiments, during the contact process, the dye and conjugate are not exposed to any ambient light or to light having an intensity greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the dye and conjugate are not exposed to light having an intensity greater than 700 lux for more than 10 minutes or more than 5 minutes. In some embodiments, the dye and / or conjugate are not exposed to light having an intensity greater than 200 lux for more than 10 minutes or more than 5 minutes.

[0229] In some embodiments, during the contact process, the dye and conjugate are protected from ambient light, such as near-infrared light. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and after the contact process has wavelengths that are not absorbed or substantially not absorbed by the dye. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and after the contact process is green light. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and after the contact process has wavelengths in the range of 400 nm to 600 nm or about 400 nm to 600 nm, for example, 425 nm to 575 nm or about 425 nm to 575 nm, or 450 nm to 550 nm or about 450 nm to 550 nm.

[0230] In some embodiments, the only light to which the dye and conjugate are exposed during the contact process has a wavelength that is not absorbed or substantially absorbed by the dye and has an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the only light to which the dye and conjugate are exposed during the contact process has a wavelength in the range of 400 nm to 600 nm or about 400 nm to 600 nm, for example, 425 nm to 575 nm or about 425 nm to 575 nm, or 450 nm to 550 nm or about 450 nm to 550 nm and has an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the only light to which the dye and conjugate are exposed during the contact process is green light, for example, light with a wavelength of 425 nm to 575 nm, having an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the total exposure of the dye and / or conjugate to any light during the contact process is 5000 lux hours or less, 2500 lux hours or less, 1000 lux hours or less, 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, or 80 lux hours or less, 50 lux hours or less, or 25 lux hours or less.

[0231] In some embodiments, the dyes and conjugates are protected from light during the contact process by using a container that protects the contents from light, or from specific wavelengths or intensities of light. For example, in some embodiments, the container has a light transmittance of 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. In some embodiments, the container protects from the transmission of light having wavelengths between 500 nm and 725 nm or about 500 nm and 725 nm, for example between 650 nm and 725 nm or about 650 nm and 725 nm, or does not transmit light with intensities greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the dyes and / or conjugates are prepared in a translucent or opaque container. In some embodiments, the container is green, blue, or amber in color. In some embodiments, the container is covered with an opaque material, such as foil, such as aluminum foil.

[0232] In some embodiments, in any of the methods provided herein, a targeting molecule (e.g., an antibody) is directly or indirectly linked to a phthalocyanine dye (e.g., IR700). In some embodiments, the targeting molecule (e.g., an antibody) is directly or indirectly linked to a phthalocyanine dye (e.g., IR700) via covalent or non-covalent interactions. In some embodiments, the covalent or non-covalent interaction or linkage is direct or indirect. In some embodiments, attachment includes indirect linkage, for example, through a linker (e.g., any of the exemplary linkers described above), a binding site or domain or a reactive group. In some embodiments, linkage includes a direct interaction between the targeting molecule and the phthalocyanine dye (e.g., IR700). In other embodiments, one or both of the targeting molecule and the phthalocyanine dye are linked to one or more linkers, and the interaction is indirect, for example, between a linker attached to one molecule and another molecule, or between two linkers attached to the targeting molecule or the phthalocyanine dye, respectively.

[0233] In some embodiments, phthalocyanine dyes are non-covalently linked to or associated with a targeting molecule. For example, a phthalocyanine dye forms a complex with a targeting molecule via a non-covalent interaction. In some embodiments, a phthalocyanine dye (e.g., IR700) contains a moiety or domain capable of non-covalently interacting with the adherent groups of the targeting molecule. In some embodiments, the method includes the step of incubating or binding a phthalocyanine dye (e.g., IR700) with a targeting molecule (e.g., an antibody) to form a non-covalent interaction between the dye and the targeting molecule. In some examples, the non-covalent interaction between the targeting molecule and the phthalocyanine dye includes, for example, electrostatic interactions, van der Waals forces, hydrophobic interactions, π effects, ionic interactions, hydrogen bonds, halogen bonds and / or combinations thereof, or any interaction that depends on one or more of these forces. In some embodiments, the targeting molecule and the phthalocyanine dye are linked using interactions that mimic non-covalent molecular interactions, such as ligand-receptor interactions, antibody-antigen interactions, avidin-biotin interactions, streptavidin-biotin interactions, histidine-divalent metal ion (e.g., Ni, Co, Cu, Fe) interactions, interactions between multimerization (e.g., dimerization) domains, glutathione S-transferase (GST)-glutathione interactions, and / or any combination thereof.

[0234] In some embodiments, a non-covalent interaction moiety or domain is attached to or is part of the targeting molecule and forms a non-covalent interaction, e.g., a complex, with a phthalocyanine dye (e.g., IR700). In other embodiments, a non-covalent interaction molecule or domain is attached to or is part of the targeting molecule and forms a non-covalent interaction, e.g., a complex, with the targeting molecule. In some embodiments, the method includes the step of incubating or contacting a biotin-conjugated targeting molecule (e.g., antibody-biotin, e.g., cetuximab-biotin) with a phthalocyanine dye (including its monomeric form (e.g., monomeric avidin-IR700 or monomeric streptavidin-IR700)) conjugated to avidin or an analogue thereof or streptavidin or an analogue thereof. Thanks to non-covalent interactions between avidin, streptavidin or its analogues and biotin, in some embodiments, phthalocyanine dyes (e.g., IR700) form non-covalent complexes with targeting molecules.

[0235] In some embodiments, the phthalocyanine dye is covalently linked to the targeting molecule, for example, by covalent bonding. In some embodiments, the phthalocyanine dye (e.g., IR700) contains a reactive group that links it to the targeting molecule. In some embodiments, contact between the targeting molecule (e.g., an antibody) and the phthalocyanine dye (e.g., IR700), for example, a dye containing a reactive chemical group, produces a conjugate containing the dye linked to the attachment group of the targeting molecule. In some embodiments, attachment includes indirect linking, for example, through a linker. An exemplary linker is described above. In some embodiments, attachment includes direct linking or covalent linking, where the covalent linking is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1 to 60 atoms selected from, for example, C, N, P, O, and S. In some embodiments, attachment may contain any combination of ethers, thioethers, amines, esters, carbamates, ureas, thioureas, oxy or amide bonds. In some embodiments, the attachment may include single bonds, double bonds, triple bonds or aromatic carbon-carbon bonds, phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, nitrogen-platinum bonds, or aromatic or heteroaromatic bonds.

[0236] In some embodiments, the method includes the step of reacting a phthalocyanine dye (e.g., IR700) with a targeting molecule (e.g., an antibody) to form a covalent bond between the dye and the targeting molecule. In some embodiments, the bond is, for example, an amide, a secondary or tertiary amine, a carbamate, an ester, an ether, an oxime, a phosphate ester, a sulfonamide, a thioether, a thiourea, or a urea. In some embodiments, the bond is covalent, for example, an amide or carbamate bond. In some embodiments, the covalent bond is a phosphate or other linking group. In some embodiments, the bond is a phosphate diester bond (for example, for DNA or RNA).

[0237] In some embodiments, the reactive groups of the dye react with the attachment groups of the targeting molecule, such as thiol, hydroxyl, carboxyl, or amino groups, to form an adhesion between the dye and the targeting molecule. In some embodiments, the attachment groups of the targeting molecule are lysine residues. Therefore, in some embodiments, a phthalocyanine dye (e.g., IR700) is covalently bonded to the lysine residue of the targeting molecule.

[0238] In some embodiments, after the contact step, the reaction is quenched by adding a quenching agent, such as glycine. The term “quenching” refers to the process of reacting unreacted reactive groups with an excess of a nonspecific quenching agent (also called a quencher) to stop a reaction, for example, between a dye and a targeted molecule. The specific agent or quencher used depends on the specific reactive groups that associate with the dye. For example, an NHS ester crosslinking reaction can be quenched in the presence of a buffer containing an amine, such as Tris or a buffer containing glycine.

[0239] In some embodiments, the quenching step removes any unreacted dye. In some embodiments, the amount of quenching agent added is at least 200 mM or about 200 mM, at least 500 mM or about 500 mM, at least 1 M or about 1 M, at least 2 M or about 2 M, at least 5 M or about 5 M, or at least 10 M or about 10 M. In some embodiments, the quenching reaction involves the addition of 1 M glycine. In some embodiments, the final concentration of the quenching reagent after it has been added to the conjugation reaction is at least 1 mM or about 1 mM, at least 2 mM or about 2 mM, at least 3 mM or about 3 mM, at least 4 mM or about 4 mM, at least 5 mM or about 5 mM, or at least 10 mM or about 10 mM. In some embodiments, the final concentration of the quenching reagent, such as glycine, is 4.2 nM or about 4.2 nM.

[0240] In some embodiments, during the quenching step, the contents of the reactor are mixed, for example, stirred, on a stirring plate or the like. In some embodiments, the contents of the reactor are stirred at a speed of 100 rpm to 1000 rpm or about 100 rpm to 1000 rpm, 200 rpm to 500 rpm or about 200 rpm to 500 rpm, or at 300 ± 50 rpm or 300 rpm. In some embodiments, the quenching reaction is mixed for at least 5 minutes or about 5 minutes, at least 10 minutes or about 10 minutes, or at least 15 minutes or about 15 minutes. In some embodiments, the quenching reaction is mixed for about 10 to 12 minutes.

[0241] In some embodiments, after mixing of the quenching reaction, the vessel, such as a reactor, is returned to a controlled temperature, for example, in an incubator. In some embodiments, the contents of the vessel are incubated at, for example, 21°C to 30°C or about 21°C to 30°C, for example, 23°C to 27°C or about 23°C to 27°C, for example, 25°C or about 25°C. In some embodiments, incubation of the quenching step, for example, additional incubation after mixing of the quenching reagent and the contents of the reactor, is carried out for at least 10 minutes or about 10 minutes, at least 15 minutes or about 15 minutes, at least 20 minutes or about 20 minutes, at least 25 minutes or about 25 minutes, or at least 30 minutes or about 30 minutes. In some embodiments, incubation is carried out for 20 to 25 minutes or about 20 to 25 minutes.

[0242] In some embodiments, the quenching process is carried out under light-protected conditions. For example, in some embodiments, during the quenching process, the conjugate is not exposed to any ambient light or to light having an intensity greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the conjugate is not exposed to light having an intensity greater than 700 lux for more than 10 minutes or more than 5 minutes. In some embodiments, the conjugate is not exposed to light having an intensity greater than 200 lux for more than 10 minutes or more than 5 minutes.

[0243] In some embodiments, during the quenching process, the conjugate is protected from ambient light, such as near-infrared light. In some embodiments, the only light to which the conjugate is exposed during the quenching process has wavelengths that are not absorbed or substantially absorbed by the dye or conjugate. In some embodiments, the only light to which the conjugate is exposed during the quenching process is green light. In some embodiments, the only light to which the conjugate is exposed during the quenching process has wavelengths in the range of 400 nm to 600 nm or about 400 nm to 600 nm, for example, 425 nm to 575 nm or about 425 nm to 575 nm, or 450 nm to 550 nm or about 450 nm to 550 nm.

[0244] In some embodiments, the only light to which the conjugate is exposed during the quenching process has a wavelength that is not absorbed or substantially absorbed by the conjugate and has an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the only light to which the conjugate is exposed during the quenching process has a wavelength in the range of 400 nm to 600 nm or about 400 nm to 600 nm, for example, 425 nm to 575 nm or about 425 nm to 575 nm, or 450 nm to 550 nm or about 450 nm to 550 nm and has an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the only light to which the conjugate is exposed during the quenching process is green light, for example, light with a wavelength of 425 nm to 575 nm, having an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the total exposure of the dye and / or conjugate to any light during the quenching process is 5000 lux hours or less, 2500 lux hours or less, 1000 lux hours or less, 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, or 80 lux hours or less, 50 lux hours or less, or 25 lux hours or less.

[0245] In some embodiments, the conjugate is protected from light during the quenching process by using a container that protects the contents from light, or from specific wavelengths or intensities of light. For example, in some embodiments, the container has a light transmittance of 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. In some embodiments, the container protects from the transmission of light having wavelengths between 500 nm and 725 nm or about 500 nm and 725 nm, for example between 650 nm and 725 nm or about 650 nm and 725 nm, or does not transmit light of intensities greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the conjugate is prepared in a translucent or opaque container. In some embodiments, the container is green, blue, or amber in color. In some embodiments, the container is covered with an opaque material, such as foil, such as aluminum foil.

[0246] In some embodiments, the manufacturing methods provided herein include one or more steps of formulating, purifying, or isolating a conjugate to produce an active pharmaceutical ingredient. In some embodiments, the conjugate is 0.1 mg / mL to about 1000 mg / mL, 0.1 mg / mL to about 500 mg / mL, 0.1 mg / mL to about 200 mg / mL, 0.1 mg / mL to about 100 mg / mL, 0.1 mg / mL to about 50 mg / mL, 0.1 mg / mL to about 10 mg / mL, 0.5 mg / mL to about 10 mg / mL, or 0.5 mg / mL to about 5 mg / mL, and It is formulated to concentrations within the range of approximately 0.1 mg / mL to 1000 mg / mL, approximately 0.1 mg / mL to 500 mg / mL, approximately 0.1 mg / mL to 200 mg / mL, approximately 0.1 mg / mL to 100 mg / mL, approximately 0.1 mg / mL to 50 mg / mL, approximately 0.1 mg / mL to 10 mg / mL, approximately 0.5 mg / mL to 10 mg / mL, or approximately 0.5 mg / mL to 5 mg / mL.

[0247] In some embodiments, a method for formulating a conjugate may include steps of concentrating or diluting the conjugate, replacing the conjugate with a pharmaceutically acceptable buffer, or sterilizing it.

[0248] In some embodiments, the formulation process includes a step of concentrating the conjugate. In some embodiments, the concentration process includes a step of reducing the volume of the conjugate. In some embodiments, the volume reduction is achieved using an ultrafiltration / diafiltration system. In some embodiments, the volume of the conjugate is reduced from 10L, 15L, 20L, 25L, 30L, 40L, or 50L, or from approximately 10L, 15L, 20L, 25L, 30L, 40L, or 50L, to 5L, 8L, 9L, 10L, 12L, or 15L, or from approximately 5L, 8L, 9L, 10L, 12L, or 15L. In some embodiments, the final volume after concentration is between 8L and 10L or approximately between 8L and 10L. In some embodiments, the conjugate is 0.1 mg / mL to approximately 1000 mg / mL, 0.1 mg / mL to approximately 500 mg / mL, 0.1 mg / mL to approximately 200 mg / mL, 0.1 mg / mL to approximately 100 mg / mL, 0.1 mg / mL to approximately 50 mg / mL, 0.1 mg / mL to approximately 10 mg / mL, 0.5 mg / mL to approximately 10 mg / mL, 0.5 mg / mL to approximately 5 mg / mL, or 1.8 mg / mL to approximately 2.1 mg / mL, and The concentrate is concentrated to a concentration within the range of approximately 0.1 mg / mL to 1000 mg / mL, approximately 0.1 mg / mL to 500 mg / mL, approximately 0.1 mg / mL to 200 mg / mL, approximately 0.1 mg / mL to 100 mg / mL, approximately 0.1 mg / mL to 50 mg / mL, approximately 0.1 mg / mL to 10 mg / mL, approximately 0.5 mg / mL to 10 mg / mL, approximately 0.5 mg / mL to 5 mg / mL, or approximately 1.8 mg / mL to 2.1 mg / mL. In some embodiments, the conjugate is concentrated to 2.0 mg / mL or approximately 2.0 mg / mL.

[0249] In some embodiments, the formulation step includes a step of diluting the conjugate. In some embodiments, the dilution of the conjugate includes a step of increasing the volume of the buffer containing the conjugate from, for example, 5 L, 10 L, 15 L, 20 L, 30 L, 40 L, or 50 L, or from about 5 L, 10 L, 15 L, 20 L, 30 L, 40 L, or 50 L, to 20 L, 30 L, 40 L, 50 L, or 75 L, or to about 20 L, 30 L, 40 L, 50 L, or 75 L. In some procedures, the conjugate is 0.1 mg / mL to approximately 1000 mg / mL, 0.1 mg / mL to approximately 500 mg / mL, 0.1 mg / mL to approximately 200 mg / mL, 0.1 mg / mL to approximately 100 mg / mL, 0.1 mg / mL to approximately 50 mg / mL, 0.1 mg / mL to approximately 10 mg / mL, 0.5 mg / mL to approximately 10 mg / mL, or 0.5 mg / mL to approximately 5 mg / mL. It is diluted to a concentration within the range of approximately 0.1 mg / mL to 1000 mg / mL, approximately 0.1 mg / mL to 500 mg / mL, approximately 0.1 mg / mL to 200 mg / mL, approximately 0.1 mg / mL to 100 mg / mL, approximately 0.1 mg / mL to 50 mg / mL, approximately 0.1 mg / mL to 10 mg / mL, approximately 0.5 mg / mL to 10 mg / mL, or approximately 0.5 mg / mL to 5 mg / mL.

[0250] In some embodiments, the formulation step includes a step of purifying the conjugate. In some embodiments, the conjugate is purified by gel permeation chromatography using an instrument such as a SEPHADEX G-50 column, or by dialysis, to remove unconjugated dyes. In some embodiments, the conjugate is ultrafiltered or dialyzed, for example, by using tangential flow filtration (TFF). In some embodiments, ultrafiltration / dialysis is carried out in the dark or under photoprotective conditions to avoid exposure of the conjugate to ambient light.

[0251] In some embodiments, the formulation step includes replacing the phthalocyanine dye-targeted molecule conjugate (e.g., IR700-targeted molecule conjugate, e.g., IR700-antibody conjugate) from the reaction buffer to a pharmaceutically acceptable buffer. In some embodiments, the buffer exchange may be performed by ultrafiltration / diafiltration.

[0252] In some embodiments, the conjugate is formulated in a pharmaceutically acceptable buffer containing, for example, a pharmaceutically acceptable carrier or vehicle. Generally, any pharmaceutically acceptable carrier or vehicle, such as those present in a pharmaceutically acceptable buffer, may be known in the art. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995) describes compositions and formulations suitable for the pharmaceutically acceptable delivery of one or more therapeutic compounds.

[0253] In some embodiments, the pH of the composition is between 6 and 10 or about 6 and 10, for example between 6 and 8 or about 6 and 8, between 6.9 and 7.3 or about 6.9 and 7.3, for example about pH 7.1. In some embodiments, the pH of the pharmaceutically acceptable buffer is at least 5 or about 5, at least 6 or about 6, at least 7 or about 7, at least 8 or about 8, at least 9 or about 9, or at least 10 or about 10, or 7.1.

[0254] In some embodiments, the properties of the pharmaceutically acceptable buffer or carrier depend on the specific mode of administration employed. For example, in some embodiments, parenteral formulations may include an injection solution containing a pharmaceutically and physiologically acceptable fluid, such as water, saline, equilibrium salt solution, aqueous dextrose, or glycerol as the vehicle. In some embodiments, for solid compositions, such as powders, pills, tablets, or capsules, the non-toxic solid carrier may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutically acceptable composition may, in some embodiments, contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0255] In some embodiments, the pharmaceutically acceptable buffer is phosphate-buffered saline (PBS). In some embodiments, the PBS has a pH of 7.1 or approximately 7.1.

[0256] In some embodiments, the formulation step includes filtering the conjugate, for example, by sterile filtration. In some embodiments, the conjugate is filtered by passing it through a sterile filter, for example, a filter of about 0.2 μm, for example, a filter of 0.22 μm.

[0257] In some embodiments, the conjugate is protected from light of wavelengths strongly absorbed by the conjugate during the formulation process. For example, in some embodiments, the concentration process is carried out in a photoprotective cooling device.

[0258] In some embodiments, the formulation process is carried out under photoprotective conditions. For example, in some embodiments, during the formulation process, the conjugate is not exposed to any ambient light or to light having an intensity greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the conjugate is not exposed to light having an intensity greater than 700 lux for more than 10 minutes or more than 5 minutes. In some embodiments, the conjugate is not exposed to light having an intensity greater than 200 lux for more than 10 minutes or more than 5 minutes.

[0259] In some embodiments, during the formulation process, the conjugate is protected from ambient light, such as near-infrared light. In some embodiments, the only light to which the conjugate is exposed during the formulation process has wavelengths that are not absorbed or substantially not absorbed by the conjugate. In some embodiments, the only light to which the conjugate is exposed during the formulation process is green light. In some embodiments, the only light to which the conjugate is exposed during the formulation process has wavelengths in the range of 400 nm to 600 nm or about 400 nm to 600 nm, for example, 425 nm to 575 nm or about 425 nm to 575 nm, or 450 nm to 550 nm or about 425 nm to 550 nm.

[0260] In some embodiments, the only light to which the conjugate is exposed during the formulation process has a wavelength that is not absorbed or substantially absorbed by the conjugate and has an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the only light to which the conjugate is exposed during the formulation process has a wavelength in the range of 400 nm to 600 nm or about 400 nm to 600 nm, for example, 425 nm to 575 nm or about 425 nm to 575 nm, or 450 nm to 550 nm or about 425 nm to 550 nm and has an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the only light to which the conjugate is exposed during the formulation process is green light, for example, light with a wavelength of 425 nm to 575 nm, having an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the total exposure of the dye and / or conjugate to any light during the formulation process is a total exposure of 5000 lux hours or less, 2500 lux hours or less, 1000 lux hours or less, 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, or 80 lux hours or less, 50 lux hours or less, or 25 lux hours or less.

[0261] In some embodiments, the conjugate is protected from light during the formulation process by placing the container in which the reaction takes place under light protection, such as a dark area. For example, in some embodiments, ultrafiltration / diafiltration is carried out in the dark by placing an ultrafiltration / diafiltration device, such as a TFF, in a dark area, such as a cooling device. In some embodiments, the conjugate is protected from light during the formulation process by using a container that protects the contents from light, or from a specific wavelength or intensity of light. For example, in some embodiments, the container has a light transmittance of 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. In some embodiments, the container protects against the transmission of light having wavelengths between 500 nm and 725 nm or approximately between 500 nm and 725 nm, for example between 650 nm and 725 nm or approximately between 650 nm and 725 nm, or does not transmit light of intensity greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the conjugate is prepared in a translucent or opaque container. In some embodiments, the container is green, blue, or amber in color. In some embodiments, the container is covered with an opaque material, for example, foil, for example, aluminum foil. In some embodiments, the container is covered with a material having a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.

[0262] In some embodiments, the formulated active pharmaceutical ingredient is stored before packaging the drug product, e.g., vial filling. In some embodiments, the formulated conjugate is stored in the dark and / or in an opaque or translucent container, e.g., a green or amber container, or in a container covered with opaque foil, e.g., aluminum foil. In some embodiments, the formulated active pharmaceutical ingredient is stored in a cooling device at a temperature between 2 and 8°C or approximately 2 and 8°C, e.g., 4°C or approximately 4°C.

[0263] Packaging of active pharmaceutical ingredients for the production of drug products In some embodiments, the method includes the step of packaging a phthalocyanine dye-targeted molecule conjugate (e.g., IR700-targeted molecule conjugate, e.g., IR700-antibody conjugate) to package a drug prepared, for example, as described above, to obtain a packaged drug product. In some embodiments, the active pharmaceutical ingredient is packaged, for example, in one or more containers, within four weeks of preparation, for example, within one, two, or three weeks of preparation. In some embodiments, the containers are vials, tubes, syringes, bags, pouches or boxes or a combination thereof.

[0264] Furthermore, containers, such as photoprotective containers, and / or devices, such as photoprotective devices, containing any of the conjugates or compositions described herein, or any conjugate or composition produced or generated using any of the methods described herein. Also provided herein are packaging systems for protecting any of the conjugates or compositions described herein, or any conjugate or composition produced or generated using any of the methods described herein. In some embodiments, such a packaging system includes one or more of the containers described herein.

[0265] Furthermore, kits or products are provided that contain a container, device, and / or packaging system for the protection of the conjugate or composition, and for storage and / or administration. The kit may include a container and / or packaging system, a photoprotective cover capable of covering a device capable of administering a composition containing a phthalocyanine dye-targeted molecule conjugate; and optionally, instructions for use. The kit may also include a label or accompanying information sheet on or accompanying the contents of the kit. The kit or product may further include an accompanying information sheet describing instructions for the use, storage, or administration of the conjugate or composition contained in the container and / or packaging system.

[0266] In some embodiments, the conjugate is packaged in one or more containers, such as photoprotective containers. In some embodiments, the container is a vial, such as a pyrogen-removed glass vial. In some embodiments, the container, such as a vial, blocks light of a specific wavelength, such as a dye or dye-targeted molecule conjugate. Thus, in some embodiments, the container protects the conjugate contained therein from light having wavelengths less than 250 nm or about 250 nm, or between 550 nm and 750 nm, or about 550 nm and 750 nm. In some embodiments, the container protects from the transmission of light having wavelengths between 500 nm and 725 nm, such as between 650 nm and 725 nm, or about 650 nm and 725 nm. In some embodiments, the container allows transmission of light of specific wavelengths, such as 400nm to 600nm or approximately 400nm to 600nm, for example 425nm to 575nm or approximately 425nm to 575nm, or 450nm to 550nm or approximately 450nm to 550nm. In some embodiments, the container is green, blue, amber, translucent, opaque, or covered with an opaque material, such as foil, for example aluminum foil. In some embodiments, the container is sterilized or pyrogen-removed. In some embodiments, the container is protected from light transmission such that the light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.

[0267] In some embodiments, the container has a maximum capacity of at least 5 mL or about 5 mL, at least 10 mL or about 10 mL, at least 25 mL or about 25 mL, at least 50 mL or about 50 mL, for example 51 ± 1 mL, at least 100 mL or about 100 mL, at least 250 mL or about 250 mL, at least 500 mL or about 500 mL, or at least 1 L or about 1 L.

[0268] In some embodiments, for example, if the container is a vial, the vial is stoppered and crimped before filling. In some embodiments, the average weight of empty vials is determined and used to determine the weight range of vials to be filled.

[0269] In some embodiments, packaging includes semi-automated aseptic filling. For example, in some embodiments, the conjugate is filled into containers, e.g., vials, using a peristaltic pump and filling needle assembly. In some embodiments, the conjugate is aseptically filtered before filling, for example, through a filter of about 0.2 μm, e.g., a filter of 0.22 μm. In some embodiments, the sterile filtrate is weighed to determine the approximate number of containers to be filled, e.g., vials.

[0270] In some embodiments, the method includes filling vials with a dye conjugate active pharmaceutical ingredient in volumes of at least 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 1 mL, 1.5 mL, 2.0 mL, 3.0 mL, 5.0 mL, 10.0 mL, 20.0 mL, 30.0 mL, 40.0 mL, 50.0 mL, or more, for example, generally 0.5 mL to 50 mL or 1 mL to 10 mL. In some embodiments, all vials to be filled are filled to contain the same volume and amount of dye conjugate. In some embodiments, the manufacturing method results in the filling of multiple vials, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more vials.

[0271] In some embodiments, a single dose of the conjugate is contained in a single container. In some embodiments, a single dose is provided in multiple containers, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more containers.

[0272] In some embodiments, after the filling process, the container, such as a vial, is stoppered, sealed, and crimped. In some embodiments, the container is stored in, for example, an opaque bottle at a temperature of, for example, 26°C or less or about 26°C, such as 20°C, 15°C, 8°C, 0°C, -20°C, or -80°C or less or about 20°C, 15°C, 8°C, 0°C, -20°C, or -80°C, and is protected from light. In some embodiments, the temperature is 20-26°C or about 20-26°C, such as 23±3°C, or 2-8°C or about 2-8°C, such as 5±3°C, such as 4°C or about 4°C or 5°C or about 5°C, or below 0°C, such as about -20 or -80°C.

[0273] In some embodiments, the container, such as a vial, is labeled. In some embodiments, the labeling is carried out at room temperature, taking care to avoid the conjugate being exposed to room temperature for any given time. For example, in some embodiments, the container is exposed to ambient temperature for less than 30 minutes or about 30 minutes, for example, less than 20 minutes or about 20 minutes, less than 10 minutes or about 10 minutes, less than 2 minutes or about 2 minutes, or less than 1 minute or about 1 minute.

[0274] In some embodiments, the container is further packaged so that the contents are protected from light. In some embodiments, a packaging system is provided which includes an internal packaging material that includes a container containing a phthalocyanine dye-targeted molecule conjugate (e.g., IR700-targeted molecule conjugate, e.g., IR700-antibody conjugate). In some embodiments, the internal packaging material has a light transmittance of less than 20%, e.g., less than 15%, less than 10%, less than 5%, or less than 1%. In some embodiments, the packaging system includes an external packaging material that includes the internal packaging material. In some embodiments, the external packaging material has a light transmittance of less than 20%, e.g., less than 15%, less than 10%, less than 5%, or less than 1%. In some embodiments, the internal or external packaging material includes an opaque foil, e.g., aluminum foil. In some embodiments, the container is covered with a material having a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the second packaging material is an aluminum pouch. In some embodiments, the external packaging material includes cardboard.

[0275] In some embodiments, the internal and / or external packaging material is suitable for storing the conjugate. In some embodiments, the internal and / or external packaging material is suitable for transporting the conjugate.

[0276] Furthermore, a packaging system is provided for protecting phthalocyanine dye-targeted molecule conjugates from light, comprising one or more containers, for example, one, two, three or more containers. Each or all of the containers in the packaging system provided herein may be photoprotective containers, for example, any photoprotective containers described herein.

[0277] In some embodiments, the packaging system comprises two containers: a first container comprising one of the containers described herein, a second container comprising the first container, and the second container protecting from the transmission of light having wavelengths of 250 nm to about 800 nm or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm.

[0278] In some embodiments, the second container is protected from light transmission such that its light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the second container is green, blue, amber, translucent, opaque, or coated with a material having a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the first and second containers are independently selected from vials, tubes, syringes, bags, pouches, and boxes.

[0279] In some embodiments, any of the provided packaging systems further comprises a third container containing a second container, wherein the third container protects from the transmission of light having wavelengths of 250 nm to about 800 nm, or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm. In some embodiments, the third container protects from the transmission of light such that its light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the third container is green, blue, amber, translucent, opaque, or coated with a material having a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the third container is selected from vials, tubes, syringes, bags, pouches, and boxes.

[0280] In some embodiments, a container or container or packaging system containing a conjugate, for example, a single container or multiple containers containing a single dose, is packaged in a kit. Thus, in some embodiments, the kit contains one or more single doses. In some embodiments, the kit includes instructions for administering the conjugate, for example, under photoprotective conditions. In some embodiments, the kit contains materials used for photoprotection of the conjugate, such as opaque foil, opaque containers, opaque intravenous (IV) bags, or opaque sleeves (for example, for covering IV bags).

[0281] For example, in some embodiments, the kit includes one of the containers described herein or one of the packaging systems described herein; a photoprotective cover capable of covering a device capable of administering a composition comprising a phthalocyanine dye-targeted molecule conjugate; and optionally, instructions for use. In some embodiments, the administration device is an intravenous bag or a syringe. In some embodiments, the photoprotective cover protects from the transmission of light having wavelengths of 250 nm to about 800 nm or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm. In some embodiments, the photoprotective cover protects from the transmission of light such that the light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the light-protective cover is covered with a material that is green, blue, amber, translucent, opaque, or has a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.

[0282] In some embodiments, the conjugate is protected from ambient light, such as near-infrared light, before, during, and after packaging. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and after preparation of the active pharmaceutical ingredient is green light. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and after preparation of the active pharmaceutical ingredient has wavelengths in the range of 400 nm to 600 nm or about 400 nm to 600 nm, for example, 425 nm to 575 nm or about 425 nm to 575 nm, or 450 nm to 550 nm or about 450 nm to 550 nm. In some embodiments, the only light to which the dye and conjugate are exposed before, during, and after preparation of the active pharmaceutical ingredient has wavelengths that are not absorbed by the dye or are substantially not absorbed by the dye.

[0283] In some embodiments, before, during, and after the preparation of the active pharmaceutical ingredient, the dye and conjugate are not exposed to any ambient light or to light having an intensity greater than 700 lux, greater than 600 lux, greater than 500 lux, greater than 400 lux, greater than 300 lux, greater than 200 lux or greater than 100 lux. In some embodiments, the total exposure of the dye and / or conjugate to any light during the packaging process is less than or equal to 5000 lux hours, less than or equal to 2500 lux hours, less than or equal to 1000 lux hours, less than or equal to 500 lux hours, less than or equal to 25 lux hours.

[0284] Characteristics of phthalocyanine pigment-targeting molecule conjugates In some embodiments, stable phthalocyanine dye-targeting molecule conjugates, e.g., IR700 dye-targeting molecule conjugates, e.g., IR700-antibody conjugates, are provided. Also provided are conjugates produced, formulated, or packaged according to any of the manufacturing methods described herein, e.g., stable conjugates.

[0285] In some embodiments, the conjugate has a molar ratio of dye:targeting molecule (e.g., IR700:targeting molecule) of 1:1 to 1000:1 or approximately 1:1 to 1000:1, 1:1 to 100:1 or approximately 1:1 to 100:1, 1:1 to 10:1 or approximately 1:1 to 10:1, 1:1 to 4:1 or approximately 1:1 to 4:1, or approximately 4:1 or 4:1.

[0286] In some embodiments, the conjugate (e.g., IR700-targeting molecule, e.g., IR700-antibody) is 0.1 mg / mL to 1000 mg / mL, 0.1 mg / mL to 500 mg / mL, 0.1 mg / mL to 200 mg / mL, 0.1 mg / mL to 100 mg / mL, 0.1 mg / mL to 50 mg / mL, 0.1 mg / mL to 10 mg / mL, 0.5 mg / mL to 10 mg / mL, 0.5 mg / mL to 5 mg / mL or The concentration is within the range of 1.8 mg / mL to 2.1 mg / mL, or approximately 0.1 mg / mL to 1000 mg / mL, 0.1 mg / mL to 500 mg / mL, 0.1 mg / mL to 200 mg / mL, 0.1 mg / mL to 100 mg / mL, 0.1 mg / mL to 50 mg / mL, 0.1 mg / mL to 10 mg / mL, 0.5 mg / mL to 10 mg / mL, 0.5 mg / mL to 5 mg / mL, or 1.8 mg / mL to 2.1 mg / mL. In some embodiments, the conjugate has a concentration of approximately 2.0 mg / mL or a concentration of 2.0 mg / mL.

[0287] In some embodiments, the amount of conjugate produced by the method is more than 1 gram or about 1 gram, more than 2 grams or about 2 grams, more than 3 grams or about 3 grams, more than 4 grams or about 4 grams, more than 5 grams or about 5 grams, or more than 10 grams or about 10 grams. In some embodiments, the conjugate is produced using Good Manufacturing Practices (GMP).

[0288] In some embodiments, the phthalocyanine dye-targeted molecule conjugates provided (e.g., IR700-targeted molecule conjugates, e.g., IR700-antibody conjugates) are stable for or after storage for, for example, three months or more, and exhibit minimal aggregation, e.g., photo-induced aggregation. Thus, in some embodiments, the conjugates are stable for at least three, four, five, six, seven, eight, or nine months, or for about one year or at least one year or more. In some embodiments, the conjugates are stable for more than one year.

[0289] In some embodiments, the stability of the conjugate can be measured by evaluating the percentage of monomer content of the conjugate. In some embodiments, the conjugate exhibits a monomer content of more than 90%, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% for 3 months or more after preparation or storage. In some embodiments, the stability of the conjugate exists if the conjugate exhibits less than 10% high molecular weight (HMW) species, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% HMW for 3 months or more after preparation or storage.

[0290] In some embodiments, the potency or activity of a conjugate can be measured by evaluating the ED50 of the conjugate or by evaluating the ability of the conjugate to induce or mediate PIT killing. In some embodiments, the conjugate exhibits a potency or activity of more than 30% or about 30% after storage, for example, more than 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or about 40%, 50%, 60%, 70%, 80%, 90%, or 95%, compared to the conjugate before storage, for example, compared to the conjugate at t=0, for 3 months or more.

[0291] In some embodiments, stable phthalocyanine dye-targeting molecule conjugates, such as IR700 dye-targeting molecule conjugates (e.g., IR700-antibody conjugates), are stable as a result of preparing, producing, or storing the dye or dye conjugate under the photoprotective conditions described above.

[0292] In some embodiments, the factor affecting the stability of the conjugate is protection of the conjugate from light, and consequently, from photo-induced aggregation. Therefore, in some embodiments, the stability of the conjugate is conferred by protecting the conjugate from light, or from specific wavelengths or intensities of light. For example, in some embodiments, the conjugate is not exposed to any ambient light or light having an intensity greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the conjugate is not exposed to light having an intensity greater than 700 lux for more than 10 minutes or more than 5 minutes. In some embodiments, the conjugate is not exposed to light having an intensity greater than 200 lux for more than 10 minutes or more than 5 minutes. In some embodiments, during the steps of the provided method, the total exposure of the dye and conjugate to any light is less than or equal to 5000 lux hours, less than or equal to 2500 lux hours, less than or equal to 1000 lux hours, less than or equal to 500 lux hours, less than or equal to 250 lux hours, less than or equal to 100 lux hours, or less than or equal to 80 lux hours.

[0293] In some embodiments, the conjugate is protected from ambient light, such as near-infrared light. In some embodiments, the only light to which the conjugate is exposed has wavelengths that are not absorbed or substantially not absorbed by the conjugate. In some embodiments, the only light to which the conjugate is exposed is green light. In some embodiments, the only light to which the conjugate is exposed has wavelengths in the range of 400 nm to 600 nm or about 400 nm to 600 nm, for example, 425 nm to 575 nm or about 425 nm to 575 nm, or 450 nm to 550 nm or about 450 nm to 550 nm.

[0294] In some aspects, the only light to which the conjugate is exposed has a wavelength that is not absorbed or substantially absorbed by the conjugate, and an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some aspects, the only light to which the conjugate is exposed has an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux. In some embodiments, the only light to which the conjugate is exposed has a wavelength in the range of 400 nm to 600 nm or approximately 400 nm to 600 nm, for example, 425 nm to 575 nm or approximately 425 nm to 575 nm, or 450 nm to 550 nm or approximately 450 nm to 550 nm, and has an intensity of less than 700 lux, less than 600 lux, less than 500 lux, less than 400 lux, less than 300 lux, less than 200 lux, or less than 100 lux.

[0295] In some embodiments, the stability of the conjugate exists after storage in a light-protective container. In some embodiments, the conjugate is protected from light by using a container that protects the contents from light, or from specific wavelengths or intensities of light. For example, in some embodiments, the container has a light transmittance of 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. In some embodiments, the container protects from the transmission of light having wavelengths between 500 nm and 725 nm or approximately between 500 nm and 725 nm, for example between 650 nm and 725 nm or approximately between 650 nm and 725 nm, or does not transmit light of intensities greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the conjugate is stored in a translucent or opaque container. In some embodiments, the container is green or amber in color. In some embodiments, the container is covered with an opaque material, such as foil, such as aluminum foil.

[0296] In some embodiments, the stability of the conjugate exists after storage of the conjugate at temperatures below or around 26°C, e.g., 20°C, 15°C, 8°C, 0°C, -20°C, or below or around 20°C, 15°C, 8°C, 0°C, -20°C, or -80°C. In some embodiments, the temperature is 20-26°C or around 20-26°C, e.g., 23±3°C, or 2-8°C or around 2-8°C, e.g., 5±3°C, e.g., 4°C or around 4°C or 5°C or around 5°C, or below 0°C, e.g., around -20°C or -80°C.

[0297] In some embodiments, the pH of the conjugate, for example, the pH of the pharmaceutically acceptable buffer into which the conjugate is formulated, confers stability to the conjugate. In some embodiments, the pH is greater than 6.0, e.g., greater than 7.0 or about 7.0, greater than 8.0 or about 8.0, or greater than 9.0 or about 9.0. In some embodiments, the pH is between 6.0 and 9.0, e.g., 6.0 and 8.0, 6.5 and 7.4, e.g., about 7.1 ± 3.0.

[0298] In some embodiments, stable phthalocyanine dye-targeting molecule conjugates (e.g., IR700-targeting molecule conjugates, e.g., IR700-antibody conjugates) are produced by methods provided herein, for example, the photoprotection method described in subsection C.

[0299] In some embodiments, a stable conjugate is provided that is stable for more than three months when manufactured and stored at a temperature below 26°C, e.g., 2-8°C, under the photoprotection conditions described above, and formulated at a pH greater than 6.0, e.g., 6.0-8.0.

[0300] II. Treatment Methods In some embodiments, methods and uses are provided for using phthalocyanine dye-targeting molecule conjugates (e.g., IR700-targeting molecule conjugate, e.g., IR700-antibody conjugate) to target cells or pathogens associated with a disease or pathological condition, for example, by binding to cell surface molecules, cell surface proteins, or cell surface receptors expressed on cells. Such methods and uses include, for example, therapeutic methods and uses that encompass the step of administering the molecule to a subject having a disease, pathological condition, or disorder, and then irradiating the subject to achieve photoimmunotherapy, thereby causing photodegradation of such cells or pathogens to achieve treatment of the disease or disorder. Uses include the use of the conjugate in such methods and treatments, as well as in the preparation of pharmaceuticals, in order to carry out such therapeutic methods. In some embodiments, methods and uses are provided for using such molecules to treat tumors in a subject using phthalocyanine dye-targeting molecule conjugates (e.g., IR700-targeting molecule conjugate, e.g., IR700-antibody conjugate). In some embodiments, the phthalocyanine dye-targeted molecule conjugate is a stable conjugate, such as any of those described herein. In some embodiments, the phthalocyanine dye-targeted molecule conjugate is produced using the method described herein. In some embodiments, the method thereby treats a disease, condition, or disorder in a subject.

[0301] Furthermore, a method is provided for preparing a composition for administration to a subject, comprising any of the conjugates described herein or a conjugate prepared using the method provided herein, such as a phthalocyanine dye conjugate (e.g., antibody-IR700 conjugate). In some embodiments, the preparation of the conjugate is carried out under photoprotective conditions. In some embodiments, the preparation method comprises the steps of: depackaging one or more of the containers described herein or one or more of the packaging systems described herein that include any of the containers described herein; and transferring the composition present in one or more containers to a device capable of administering the composition to a subject, wherein the only light to which the composition is exposed has a wavelength in the range of about 400 nm to about 650 nm, or the only light to which the composition is exposed has an intensity of less than 500 lux, for example, less than 200 lux or less than 100 lux.

[0302] In some embodiments, the method provided is carried out in a biosafety cabinet, biosafety hood, or sterile environment. In some embodiments, one or more containers together contain a therapeutically effective dose of phthalocyanine dye conjugate. In some embodiments, one or more containers comprise at least 2, 4, 6, 8, 10, 12, 18, or 24, or about 2, 4, 6, 8, 10, 12, 18, or 24, or 2, 4, 6, 8, 10, 12, 18, or 24 containers.

[0303] In some embodiments, the provided method for preparing a phthalocyanine dye conjugate (e.g., antibody-IR700 conjugate) is carried out for 1 hour or less, 30 minutes or less, or 15 minutes or less; or the total exposure of the composition to any light during the method is 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, 50 lux hours or less, or 25 lux hours or less.

[0304] Furthermore, a photoprotective device comprising a composition prepared by the method provided herein is also provided. In some embodiments, the photoprotective device is used for administering the composition or conjugate described herein.

[0305] In some embodiments, the administration device is an intravenous bag or a syringe. In some embodiments, the administration device includes a photoprotective cover that can cover the device. In some embodiments, the photoprotective cover protects from the transmission of light having wavelengths of 250 nm to about 800 nm, or about 250 nm to about 800 nm, about 250 nm to about 450 nm, about 400 nm to about 800 nm, about 450 nm to about 650 nm, or about 600 nm to about 720 nm. In some embodiments, the photoprotective cover protects from the transmission of light such that the light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%. In some embodiments, the photoprotective cover is green, blue, amber, translucent, opaque, or covered with a material having a light transmittance of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.

[0306] In some embodiments, the method generally involves administering a phthalocyanine dye-targeting molecule conjugate to a subject under conditions in which cells targeted for killing are brought into contact with the conjugate. In some embodiments, the method results in the binding of the targeting molecule (e.g., antibody) portion of the conjugate to cell surface proteins associated with tumors or cancer. After contact with the conjugate or administration thereof, a local area of ​​the subject containing target cells, e.g., tumors, is exposed to or irradiated with light absorbed by the dye, generally NIR light, thereby activating the conjugate and achieving specific cell killing. For example, in some embodiments, the method further includes local irradiation of the diseased area in the subject, e.g., local irradiation of tumors. In some embodiments, the irradiation is at a wavelength of 600–850 nm and at least 1 J cm -2The procedure is carried out at a dose of [specify dose]. In some embodiments, the conjugate is targeted to diseased cells, such as tumors, and the irradiation results in cell killing, for example, by photoimmunotherapy (PIT). In some embodiments, the method includes the method described in U.S. Patent No. 8,524,239 or U.S. Publication No. US2014 / 0120119.

[0307] In some embodiments, the administration of the conjugate is carried out under photoprotective conditions. In some embodiments, the administration is carried out under fluorescent or LED lighting. In some embodiments, the administration is carried out in the absence of direct or indirect sunlight.

[0308] In some aspects, before and during administration, the conjugate is not exposed to ambient light or to ambient light having an intensity greater than 700 lux, greater than 600 lux, greater than 500 lux, greater than 400 lux, greater than 300 lux, greater than 200 lux, greater than 100 lux, or greater than 50 lux.

[0309] In some embodiments, the conjugate is not exposed to light having an intensity greater than 700 lux, greater than 600 lux, greater than 500 lux, greater than 400 lux, greater than 300 lux, greater than 200 lux, or greater than 100 lux or greater than 50 lux for more than 20 minutes, more than 10 minutes, or more than 5 minutes. In some embodiments, the dye and / or conjugate is not exposed to light having an intensity greater than 200 lux for more than 10 minutes, or more than 5 minutes. In some embodiments, any exposure of the conjugate to light before and during administration is less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute. In some embodiments, the total exposure of the dye and / or conjugate to any light before or during administration is 5000 lux hours or less, 2500 lux hours or less, 1000 lux hours or less, 500 lux hours or less, 250 lux hours or less, 100 lux hours or less, or 80 lux hours or less, 50 lux hours or less, or 25 lux hours or less.

[0310] In some embodiments, the conjugate is protected from ambient light, such as near-infrared light. In some embodiments, the only light to which the conjugate is exposed has wavelengths that are not absorbed or substantially not absorbed by the conjugate.

[0311] In some embodiments, prior to administration to the subject, the conjugate is protected from light using a container that protects its contents from light, or from specific wavelengths or intensities of light. The container may be a tube, syringe, infusion bag, or other container suitable for injection to transfer the conjugate to the subject. For example, in some embodiments, the container has a light transmittance of 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. In some embodiments, the container protects from the transmission of light having wavelengths between 500 nm and 725 nm or approximately between 500 nm and 725 nm, for example between 650 nm and 725 nm or approximately between 650 nm and 725 nm, or does not transmit light of intensities greater than 700 lux, 600 lux, 500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, the conjugate is administered from or in a translucent or opaque container. In some embodiments, the container is green or amber in color. In some embodiments, the container is covered with an opaque material, such as foil, such as aluminum foil. In some embodiments, the container is an intravenous (IV) bag, and the bag is covered with an opaque sleeve, such as foil, such as aluminum foil.

[0312] The target cells may be undesirable cells, such as tumor cells, or cells whose growth is undesirable. In some embodiments, the cells may be able to grow in culture or may be present in the mammal being treated, e.g., a subject having cancer. Any target cells may be treated in the claimed manner. In some embodiments, the target cells express cell surface proteins that are substantially not found on the surface of other normal cells. In some embodiments, antibodies that specifically bind to such proteins may be selected, and a phthalocyanine dye-antibody conjugate may be generated against those proteins. 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 undesirable transplant rejection.

[0313] Furthermore, methods are provided for removing unwanted cells or pathogens, such as diseased cells or pathogen-infected cells, from a subject using any of the conjugates or compositions described herein, or any conjugates or compositions produced using any of the methods described herein. For example, in some embodiments, unwanted cells may include stem cells, proliferating cells, cells in a hyperplastic state, inflammatory cells, negatively regulatory immune cells (optionally T cells), pathogen-infected cells, nerve cells, adipocytes, or adipocytes. In some embodiments, unwanted cells are cancer cells or tumor cells. In some embodiments, unwanted cells are cancer stem cells or circulating tumor cells. In some embodiments, unwanted pathogens may be viruses, bacterial cells, or fungal cells.

[0314] In some embodiments, methods are provided for removing unwanted cells or pathogens, such as diseased cells or pathogen-infected cells, from a sample using any of the conjugates or compositions described herein, or any conjugates or compositions produced using any of the methods described herein. For example, unwanted cells or pathogens are removed from a biological sample derived from a subject, such as a blood sample, bone marrow sample, or biopsy. In some embodiments, the sample is a blood sample or a tissue sample. In some embodiments, unwanted cells or pathogens are removed from tissue, such as tissue temporarily removed from a subject during surgery or a procedure. In some embodiments, unwanted cells are removed from a sample associated with a device, such as a biofilm on a medical device.

[0315] In some embodiments, irradiation for removal or treatment is achieved in vivo, for example, by direct administration to the subject. In some embodiments, the method is performed in vitro or ex vivo, for example, outside the body of the subject. In some embodiments, the method is performed using an extracorporeal device. Exemplary extracorporeal devices include devices used for hemodialysis, extracorporeal oxygenation, CO2 removal, and apheresis, or devices that contain blood taken from a subject, process the blood (e.g., filter, purify, process, and administer therapeutic agents), and then return the blood to the subject.

[0316] In some embodiments of the methods provided herein, the removal of unwanted cells or pathogens from a sample, such as a blood sample or a tissue sample, includes methods for treatment, such as the treatment of hyperplasia, tumors or infections.

[0317] In some embodiments, unwanted cells are associated with, cause, or contribute to the pathogenesis of a disease or condition. In some embodiments, the condition or disease is a tumor or cancer, an infection, an inflammatory disease or condition, or a neurological disease or condition. In some embodiments, the cells are nerve cells and the disease or condition is a neuropathy (optionally pain); the cells are adipocytes or adipose cells and the disease or condition is associated with excess fat; the cells are pathogen-infected cells and the disease or condition is an infection; the cells are pathogens and the disease or condition is an infection; the cells are inflammatory cells and the disease or condition is an inflammatory disease; the cells are immune cells (optionally regulatory T cells) and the disease or condition is a tumor or cancer; or the cells are tumor or cancer cells and the disease or condition is a tumor or cancer. In some embodiments, the cells are present in the microenvironment of a lesion associated with a disease or condition, or are in a hyperplastic state. In some embodiments, the lesion is a tumor, and the disease or condition is a tumor or cancer. In some embodiments, the method treats the disease or condition.

[0318] The method generally includes the steps of administering a conjugate or composition provided herein, or a conjugate or composition produced using a method described herein, to a target, and irradiating unwanted cells or pathogens to activate the conjugate and thereby remove the cells.

[0319] In some embodiments, a method for removing unwanted cells or pathogens from a subject includes: (a) administering a composition comprising a phthalocyanine dye conjugate to a subject from one of the photoprotective devices provided herein, wherein the composition is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step; and (b) irradiating the unwanted cells or pathogens with at least 1 J cm at a wavelength of 660–740 nm. -2 Alternatively, the process may include irradiating the target with a dose of 1 J / cm fiber length, thereby removing unwanted cells from the target.

[0320] In some embodiments, a method for removing unwanted cells or pathogens from a subject is a) a step of administering a therapeutically effective amount of any of the conjugates or compositions described herein to the subject, wherein the conjugate is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step; and b) irradiating the unwanted cells or pathogens with at least 1 J cm at a wavelength of 660-740 nm. -2 Alternatively, the process may include irradiating the target with a dose of 1 J / cm fiber length, thereby removing unwanted cells from the target.

[0321] In some embodiments, a method for removing unwanted cells or pathogens from a subject includes: a) administering a therapeutically effective dose of a conjugate containing IRDye 700DX (IR700) linked to a targeting molecule capable of binding to the unwanted cells or pathogens, wherein the conjugate is not exposed to ambient light of an intensity greater than 500 lux before and during the administration step; and b) irradiating the unwanted cells or pathogens with at least 1 J cm at a wavelength of 600-800 nm. -2 Alternatively, the procedure includes irradiating the target with a dose of 1 J / cm fiber length, thereby removing unwanted cells or pathogens from the target.

[0322] In some embodiments, a method for removing unwanted cells or pathogens from a subject includes: a) administering a therapeutically effective amount of a first binding molecule capable of binding to the unwanted cells or pathogens to the subject; b) administering a conjugate molecule containing IRDye 700DX (IR700) linked to a targeting molecule, wherein the targeting molecule is a second binding molecule capable of binding to the first binding molecule; and c) injecting the unwanted cells or pathogens with at least 1 J cm at a wavelength of 600-800 nm. -2Alternatively, the method includes irradiating the target with a dose of 1 J / cm fiber length, thereby removing unwanted cells or pathogens from the target. In some embodiments of the methods provided herein, the first conjugating molecule is administered to the target before the conjugate, or the first conjugating molecule and the conjugate are administered to the target simultaneously. In some embodiments, the targeting molecule is a secondary antibody. In some embodiments, before and during the administration of the conjugate, the conjugate is not exposed to ambient light of an intensity greater than 500 lux.

[0323] In some embodiments, a method for removing unwanted cells or pathogens, such as pathogen-infected cells, from a target is a) administering a therapeutically effective amount of a conjugate molecule containing IRDye 700DX (IR700) linked to a targeting molecule to the target, wherein the targeting molecule is capable of directly or indirectly binding to pathogen-infected cells; and b) applying at least 1 J cm to the pathogen-infected cells at a wavelength of 600-800 nm. -2 Alternatively, the method includes irradiating the target with a dose of 1 J / cm fiber length, thereby removing pathogen-infected cells. In some embodiments, the pathogen is a virus, bacteria, fungi, biofilm, or other prokaryotic cell lineage. In some embodiments, before and during administration of the conjugate, the conjugate is not exposed to ambient light of an intensity greater than 500 lux.

[0324] In some embodiments, the object being treated, or the object from which unwanted cells or pathogens are removed, is a tumor, and the phthalocyanine dye-targeting molecule conjugate is targeted to the tumor. In some embodiments, the tumor is cancer. In some embodiments, cancer is 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 malignant tumors characterized by abnormal or uncontrolled cell growth. Other features that may be associated with cancer include metastasis, interference with the normal function of adjacent cells, abnormal levels of release of cytokines or other secretory products, and suppression or exacerbation of inflammatory or immunological responses, and invasion of surrounding or distant tissues or organs such as lymph nodes. Metastatic disease may refer to cancer cells that have left the original tumor site and migrated to other parts of the body, for example, via the bloodstream or lymphatic system. In some embodiments, the cells targeted by the method of disclosure are cancer cells. In some embodiments, the targeted cells are cancer stem cells or circulating tumor cells.

[0325] In some embodiments, tumor cells are cancer cells, e.g., cells in a subject having cancer. Exemplary cells that may be targeted in the manner of disclosure include cells of tumors such as: humoral tumors, e.g., leukemia, including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease). In some aspects, cells are solid tumor cells, such as sarcomas or carcinomas, fibrosarcomas, myxosarcomas, liposarcomas, chondrosarcomas, osteosarcomas, and other sarcomas, synoviomas, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancers, pancreatic cancers, breast cancers, ovarian cancers, prostate cancers, hepatocellular carcinomas, lung cancers, colorectal cancers, squamous cell carcinomas, basal cell carcinomas, adenocarcinomas, such as those of the pancreas, colon, ovaries, lungs, breasts, stomachs, and prostates. Adenocarcinomas of the cervix or esophagus, sweat gland carcinomas, sebaceous gland carcinomas, papillary carcinomas, papillary adenocarcinomas, medullary carcinomas, bronchogenic carcinomas, renal cell carcinomas, hepatocellular carcinomas, cholangiocarcinomas, choriocarcinomas, Wilms' tumors, cervical cancers, testicular cancers, bladder cancers, CNS tumors such as gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal glandomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, and retinoblastomas. In some aspects, the cancer is squamous cell carcinoma of the head and neck.

[0326] Exemplary tumors, e.g., cancers, that can be treated in the claimed manner include solid tumors, e.g., breast carcinomas, e.g., lobular and ductal carcinomas, sarcomas, lung carcinomas, e.g., non-small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma, pulmonary mesothelioma, colorectal adenocarcinoma, gastric cancer, prostate adenocarcinoma, ovarian cancers, e.g., serous cystadenocarcinoma and mucinous cystadenocarcinoma, ovarian germ cell tumors, testicular cancers and germ cell tumors, pancreatic adenocarcinoma, cholangiocarcinoma, hepatocellular carcinoma, bladder cancer (e.g., transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma), renal cell adenocarcinoma, endometrial cancer (e.g., adenocarcinoma and mixed Müllerian duct tumor (carcinosarcoma)), cervical endometrium, cervix, and vagina This includes carcinomas of the body, such as adenocarcinoma and squamous cell carcinoma, tumors of the skin, such as squamous cell carcinoma, basal cell carcinoma, malignant melanoma, appendage tumors, Kaposi's sarcoma, cutaneous lymphoma, adnexal tumors, and various types of sarcomas and Merkel cell carcinoma, esophageal cancer, carcinomas of the nasopharynx and oropharynx (including their squamous cell carcinoma and adenocarcinoma), salivary gland carcinoma, tumors of the brain and central nervous system (including tumors of glial cell, nerve cell, and meningeal origin), tumors of the peripheral nerves, soft tissue sarcomas and bone and cartilage sarcomas, and lymphoid tumors (including B-cell and T-cell malignant lymphoma). In some aspects, the tumor is adenocarcinoma.

[0327] Furthermore, this method can be used to treat humoral tumors, such as lymphoid leukemia, or other types of leukemia. In some embodiments, the tumors treated are hematological tumors, such as leukemias, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prelymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia, lymphomas, such as Hodgkin lymphoma and non-Hodgkin lymphoma, and myeloma.

[0328] In some embodiments, the conjugate is targeted to proteins expressed in tumors.

[0329] In some embodiments, proteins on the cell surface of targeted cells are not present in significant amounts on other cells. For example, a cell surface protein may be a receptor found only on the target cell type.

[0330] In some embodiments, proteins expressed in tumors, such as tumor-specific proteins, include HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, and cancer antigen 125 (CA). 125) These may be alpha-fetoprotein (AFP), Lewis Y, TAG72, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate-binding protein, gangliosides (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, SK-1 antigen, or PD-L1. In some embodiments, the tumor-specific protein is PD-L1, HER1 / EGFR, HER2, CD20, CD25, CD33, CD52, or prostate-specific membrane antigen (PSMA). Other cell surface proteins include any of those described above.

[0331] In some embodiments, cell surface proteins are tumor-specific proteins or tumor-specific antigens, such as members of the EGF receptor family (e.g., HER1, 2, 3, and 4) and members of cytokine receptors (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.). In some embodiments, tumor-specific proteins are proteins that are unique to or highly abundant on cancer cells compared to other cells such as normal cells. For example, HER2 is commonly found in breast cancer, while HER1 is typically found in adenocarcinoma, which can be found in many organs such as the pancreas, breast, prostate, and colon.

[0332] Exemplary tumor-specific proteins that can be found on target cells and which can be formulated into phthalocyanine dye-antibody conjugates using antibodies or antibody fragments specific to those proteins include, but are not limited to: any of the various MAGE (melanoma-associated antigen E) including MAGE1, MAGE2, MAGE3, and MAGE4; any of the various tyrosinases; mutant Ras, mutant p53, p97 melanoma antigens; human milk fat globules (HMFG) that may be associated with breast tumors; any of the various BAGE (human B melanoma-associated antigen E) including BAGE1 and BAGE2; any of the various GAGE ​​(G antigen) including GAGE1, GAGE2-6; various gangliosides; and CD25.

[0333] Other tumor-specific antigens include HPV16 / 18 and E6 / E7 antigens associated with cervical cancer, mucin (MUC 1)-KLH antigen which may be associated with breast carcinoma, CEA (carcinoembryonic antigen) which may be associated with colorectal cancer, gp100 which may be associated with melanoma, MARTI antigen which may be associated with melanoma, cancer antigen 125 (also known as CA125, mucin 16, or MUC16) which may be associated with ovarian and other cancers, alpha-fetoprotein (AFP) which may be associated with liver cancer, Lewis Y antigen which may be associated with colorectal cancer, cholangiocarcinoma, breast cancer, small cell lung cancer, and other cancers, tumor-associated glycoprotein 72 (TAG72) which may be associated with adenocarcinoma, and PSA antigen which may be associated with prostate cancer.

[0334] Other exemplary tumor-specific proteins include, but are not limited to, PMSA (prostatic membrane specific antigen), which may be associated with solid tumor angiogenesis as well as prostate cancer; HER-2 (human epidermal growth factor receptor 2), which may be associated with breast cancer, ovarian cancer, gastric cancer, and uterine cancer; HER-1, which may be associated with lung cancer, anal cancer, and glioblastoma and adenocarcinoma; NY-ESO-1, hTERT (also known as telomerase), proteinase 3, and Wilms tumor 1 (WT-1), which may be associated with melanoma, sarcoma, testicular cancer, and other cancers.

[0335] In some embodiments, the tumor-specific protein may be CD52 and associated with chronic lymphocytic leukemia, CD33 and associated with acute myeloid leukemia, or CD20 and associated with non-Hodgkin lymphoma.

[0336] Therefore, any cancer expressing tumor-specific proteins can be treated using the methods disclosed.

[0337] In some embodiments, the subject is a human or a non-human mammal. In some embodiments, the subject is a human or animal subject, e.g., a mouse. In some embodiments, the subject is a mammal having cancer or whose cancer is being treated, e.g., a human. In some embodiments, the method of disclosure is used to treat a tumor, e.g., a subject having a tumor as described herein. In some embodiments, the tumor has been previously treated and removed, e.g., surgically or chemically, and the method of disclosure is subsequently used to kill any remaining undesirable tumor cells that may remain in the subject.

[0338] The disclosed method can be used to treat any mammalian subject, such as a human, that has a tumor, such as cancer, or whose cancer has been previously removed or treated. Subjects requiring treatment of the disclosed method may include human subjects having cancer, wherein cancer cells express tumor-specific proteins on the cell surface that can specifically bind to phthalocyanine dye-targeting molecule conjugates. For example, the disclosed method can be used as initial treatment for cancer, either alone or in combination with radiotherapy or other chemotherapy. The disclosed method can also be used in patients who have failed with previous radiotherapy or chemotherapy. Thus, in some embodiments, the subject is a subject receiving other treatment, but whose other treatment has not provided the desired therapeutic response. The disclosed method can also be used in patients with localized and / or metastatic cancer.

[0339] In some embodiments, the method includes the step of selecting subjects who will benefit from the treatments of the present disclosure, for example, subjects having tumors that express cell surface proteins, such as tumor-specific proteins, that can specifically bind to phthalocyanine dye-targeting molecule conjugates. For example, if a subject is determined to have breast cancer that expresses HER1, subjects to be treated with an anti-HER1-IR700 molecule such as cetuximab-IR700 may be selected.

[0340] In some embodiments, the composition used for administering the conjugate contains an effective amount of the conjugate together with conventional pharmaceutical carriers and excipients appropriate to the intended type of administration.

[0341] In some embodiments, a single dose of conjugate is contained in a single container, such as a container in which the conjugate is stored. In some embodiments, the container, such as a vial, is a container that is packaged under the photoprotective conditions described above. In some embodiments, a single dose of conjugate is contained in multiple containers. Thus, in some embodiments, multiple containers, such as vials, are combined in a container used for administering the conjugate, such as an intravenous (IV) bag. In some embodiments, the container used for administration, such as an IV bag, is prepared by opening one or more containers containing the conjugate and filling the bag with its contents, for example, until the desired administration of the conjugate for administration, such as an infusion, is achieved. During the preparation of the administration container, such as an IV bag, photoprevention measures are taken to avoid exposure of the conjugate to light, such as the various photoprevention measures described herein.

[0342] In some embodiments, the method includes the step of administering a therapeutically effective amount of a conjugated drug product to a subject. In some embodiments, the method includes the step of administering a therapeutically effective amount of a conjugate containing a dye conjugated to a targeting molecule, for example, IRDye 700DX-Targeting Molecular Conjugate, to a subject. In some embodiments, the IRDye 700DX-Targeting Molecular Conjugate is targeted to a tumor.

[0343] In some embodiments, the therapeutically effective dose is an amount of the composition sufficient to achieve the desired effect in the subject or cells being treated with the composition, either alone or in combination with additional therapeutic agents such as chemotherapeutic agents. The effective dose of a therapeutic agent, such as a phthalocyanine dye-targeted molecule conjugate, may depend on several factors, including, but not limited to, the subject or cells being treated, the specific therapeutic agent, and the mode of administration of the therapeutic composition. In some embodiments, the therapeutically effective dose or concentration is sufficient to prevent disease progression, such as metastasis, or to slow the progression of the disease or to induce disease regression, or to reduce symptoms caused by a disease such as cancer. In some embodiments, the therapeutically effective dose or concentration is sufficient to increase the survival time of a patient with a tumor.

[0344] In some embodiments, the desired response to treatment by the provided method is to reduce or inhibit one or more symptoms associated with cancer. In some embodiments, it is not necessary for one or more symptoms to be completely eliminated for the composition to be effective. For example, administration of a composition containing a phthalocyanine dye-targeted molecule conjugate and subsequent irradiation can reduce tumor size, e.g., tumor volume or weight, or tumor metastasis, by, for example, at least 20%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% compared to the tumor size, volume, weight, or metastasis in the absence of the conjugate.

[0345] In some embodiments, the desired response of treatment by the provided method is to kill a desired amount of a population of cells, for example, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% of the cells compared to cell killing in the absence of conjugate and irradiation.

[0346] In some embodiments, the desired response is to increase the survival time of patients with tumors, or patients whose tumors have recently been removed, by a desired amount, for example, by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% survival compared to survival in the absence of conjugates and irradiation.

[0347] The amount of a drug containing a phthalocyanine dye-targeted molecule conjugate administered to a human or animal subject will vary depending on a number of factors associated with the subject, such as the subject's overall health status. In some embodiments, the effective dose of the drug can be determined by varying the dose of the product and measuring the resulting therapeutic response, such as tumor regression. In some embodiments, the effective dose can be determined through various in vitro, in vivo, or in situ immunoassays. In some embodiments, the disclosed drug can be administered in single or multiple doses as needed to obtain the desired response. In some embodiments, the effective dose depends on the source of application, the subject being treated, the severity and type of the disease being treated, and the mode of administration.

[0348] In some embodiments, the therapeutically effective dose of the conjugate, when administered intravenously, is at least 0.5 milligrams (mg / kg) per 60 kilograms, at least 5 mg / 60 kg, at least 10 mg / 60 kg, at least 20 mg / 60 kg, at least 30 mg / 60 kg, at least 50 mg / 60 kg, for example 0.5 to 50 mg / 60 kg, for example 1 mg / 60 kg, 2 mg / 60 kg, 5 mg / 60 kg, 20 mg / 60 kg, or 50 mg / 60 kg. In some embodiments, the dose of the conjugate is at least 10 μg / kg, for example at least 100 μg / kg, at least 500 μg / kg, or at least 500 μg / kg, for example 10 μg / kg to 1000 μg / kg, for example 100 μg / kg, 250 μg / kg, about 500 μg / kg, 750 μg / kg, or 1000 μg / kg, when administered intratumorally or intraperitoneally (ip). In some embodiments, the dose, when administered, for example in a topical solution, is at least 1 μg / ml, for example at least 500 μg / ml, for example 20 μg / ml to 100 μg / ml, for example 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, or 100 μg / ml.

[0349] In some embodiments, the therapeutically effective dose of the conjugate is 10 mg / m². 2 ~2000mg / m 2 Between or approximately 10 mg / m² 2 ~2000mg / m 2 For example, 10 mg / m² 2 ~1500mg / m 2 Alternatively, approximately 10 mg / m² 2 ~1500mg / m 2 , 25 mg / m² 2 ~2000mg / m 2 Alternatively, approximately 25 mg / m² 2 ~2000mg / m 2 , 200 mg / m² 2 ~1250mg / m 2 Alternatively, approximately 200 mg / m² 2 ~1250mg / m2 500 mg / m² 2 ~1250mg / m 2 Alternatively, approximately 500 mg / m² 2 ~1250mg / m 2 500 mg / m² 2 ~750mg / m 2 Alternatively, approximately 500 mg / m² 2 ~750mg / m 2 , or 750 mg / m² 2 ~1250mg / m 2 Alternatively, approximately 750 mg / m² 2 ~1250mg / m 2 In some embodiments, the therapeutically effective dose is at least 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 2000 mg, 3000 mg or more, or approximately at least 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 2000 mg, 3000 mg or more.

[0350] Those skilled in the art will recognize that, for example, depending on a particular conjugate, higher or lower doses may also be used. In some embodiments, the dose, for example, the daily dose, is administered in one or more divided doses, for example, two, three, or four doses, or in a single formulation. The disclosed conjugate may be administered alone, in the presence of a pharmaceutically acceptable carrier, or in the presence of other therapeutic agents, such as other anti-cancer agents.

[0351] In some embodiments, the targeting molecule 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 targeting molecule is an antibody or an antigen-binding fragment.

[0352] In some embodiments, prior to the administration of the conjugate, the subject is administered a targeting molecule, such as an antibody like cetuximab, in its non-conjugate form alone. Generally, the targeting molecule administered before the conjugate is the same targeting molecule administered as part of the conjugate. In some embodiments, the dose of the targeting molecule administered is 10 mg / m². 2 ~2000mg / m 2 Between or approximately 10 mg / m² 2 ~2000mg / m 2 For example, 10 mg / m² 2 ~1500mg / m 2 Alternatively, approximately 10 mg / m² 2 ~1500mg / m 2 , 25 mg / m² 2 ~2000mg / m 2 Alternatively, approximately 25 mg / m² 2 ~2000mg / m 2 , 200 mg / m² 2 ~1250mg / m 2 Alternatively, approximately 200 mg / m² 2 ~1250mg / m 2 500 mg / m² 2 ~1250mg / m 2 Alternatively, approximately 500 mg / m² 2 ~1250mg / m 2 500 mg / m² 2 ~750mg / m 2 Alternatively, approximately 500 mg / m² 2 ~750mg / m 2 , or 25 mg / m² 2 ~100mg / m 2 Alternatively, approximately 25 mg / m² 2 ~100mg / m 2 It is between these two points.

[0353] In some embodiments, the targeted molecule is administered at least one week, at least six days, at least five days, at least 96 hours, at least 72 hours, at least 48 hours, at least 24 hours, or at least 12 hours before administration of the conjugate. In some embodiments, the targeted molecule is administered before administration of the conjugate, within a range of 1 hour to 1 week or approximately 1 hour to 1 week, for example, 1 hour to 96 hours or approximately 1 hour to 96 hours, 1 hour to 48 hours or approximately 1 hour to 48 hours, 1 hour to 24 hours or approximately 1 hour to 24 hours, 24 hours to 96 hours or approximately 24 hours to 96 hours, or 24 hours to 48 hours or approximately 24 hours to 48 hours. In some embodiments, the targeted molecule is administered 96 hours or approximately 96 hours before administration of the conjugate.

[0354] In some embodiments, the conjugate may be administered systemically or topically to the organ or tissue being 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, percutaneous, intranasal, vaginal, and inhalation routes. In some embodiments, the conjugate is administered intravenously. In some embodiments, the conjugate is administered parenterally. In some embodiments, the conjugate is administered intra-enterally. In some embodiments, the conjugate is administered by local injection. In some embodiments, the conjugate is administered as a topical application.

[0355] Compositions containing a conjugate can be administered locally or systemically to subjects having tumors, such as cancer, or to subjects whose tumors have been previously removed, for example, through surgery, using any method known in the art. Specific examples are provided, but those skilled in the art will understand that alternative methods of administration of the disclosed conjugate can be used. Such methods may include, for example, the use of a catheter or implantable pump to provide continuous infusion over several hours to several days to a subject requiring treatment.

[0356] In some embodiments, the conjugate is administered to the tumor by parenteral means, including, for example, direct injection into the tumor, direct injection, or infusion. In some embodiments, the conjugate is administered to the tumor by applying it to the tumor, for example, by immersing the tumor in a solution containing a phthalocyanine dye-targeted molecule conjugate, or by pouring the conjugate onto the tumor.

[0357] Additionally, or alternatively, the disclosed compositions may be administered systemically to subjects having tumors such as cancer, for example, intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, subcutaneously, or orally.

[0358] The dose of the conjugate administered to the subject will not be given up to its absolute limit, but will depend on the properties of the composition and its active ingredients, as well as any unwanted side effects, such as an immune response to the antibody, the subject being treated, the type of disease being treated, and the mode of administration. Generally, the dose will be a therapeutically effective dose, for example, a sufficient amount to achieve the desired biological effect, such as reducing the size of a tumor, e.g., volume and / or weight, or attenuating further tumor growth, or reducing undesirable symptoms of the tumor.

[0359] In some embodiments, for example, for intravenous administration of the conjugate, exemplary doses for administration to a subject for a single treatment may range from 0.5 to 100 mg / 60 kg body weight, 1 to 100 mg / 60 kg body weight, 1 to 50 mg / 60 kg body weight, 1 to 20 mg / 60 kg body weight, for example, about 1 or 2 mg / 60 kg body weight. In some embodiments, the therapeutically effective dose of the conjugate administered intraperitoneally or intratumorally may vary from 10 μg to 5000 μg of conjugate per kg of body weight, for example, 10 μg / kg to 1000 μg / kg, 10 μg / kg to 500 μg / kg, or 100 μg / kg to 1000 μg / kg. In some embodiments, the conjugate may range from 0.5 mg / kg to about 100 mg / kg or about 0.5 mg / kg to about 100 mg / kg, or 20 mg / m³ 2~about 4000mg / m 2 Alternatively, approximately 20 mg / m² 2 ~about 4000mg / m 2 The conjugate is administered in an amount that is at least 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 8.0 mg / kg, 16.0 mg / kg, 32.0 mg / kg or 64 mg / kg, or about at least 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 8.0 mg / kg, 16.0 mg / kg, 32.0 mg / kg or 64 mg / kg, or 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 8.0 mg / kg, 16.0 mg / kg, 32.0 mg / kg or 64 mg / kg, or about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 8.0 mg / kg, 16.0 mg / kg, 32.0 mg / kg or 64 mg / kg; or the conjugate is administered in an amount that is at least 20 mg / m 2 , 40 mg / m² 2 , 160 mg / m² 2 , 320 mg / m² 2 , 640 mg / m² 2 , 1280 mg / m² 2 Alternatively, 2560 mg / m² 2 , or at least 20 mg / m² 2 , 40 mg / m² 2 , 160 mg / m² 2 , 320 mg / m² 2 , 640 mg / m² 2 , 1280 mg / m² 2 Alternatively, 2560 mg / m² 2 , or 20 mg / m² 2 , 40 mg / m² 2 , 160 mg / m² 2 , 320 mg / m² 2 , 640 mg / m² 2 , 1280 mg / m² 2 Alternatively, 2560 mg / m² 2 , or approximately 20 mg / m² 2 , 40 mg / m² 2 , 160 mg / m² 2 , 320 mg / m² 2 , 640 mg / m² 2 , 1280 mg / m²2 Alternatively, 2560 mg / m² 2 It is administered in a certain amount.

[0360] In some embodiments, the dose of the conjugate administered to a human patient is at least 50 mg, for example, at least 100 mg, at least 300 mg, at least 500 mg, at least 750 mg, or even 1 g.

[0361] Treatment using the disclosed conjugate may be completed in one day or repeated over several days with the same or different doses. Repeated treatments may be performed on the same day, daily, or at intervals of 1-3 days, 3-7 days, 1-2 weeks, 2-4 weeks, 1-2 months, or longer intervals.

[0362] In some embodiments, compositions used for administering conjugates contain an effective amount of conjugate together with conventional pharmaceutical carriers and excipients appropriate to 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 intraenteral administration may contain an effective amount of conjugate in an aqueous solution or suspension that may optionally contain buffers, surfactants, thixotropes, and flavoring agents.

[0363] In some embodiments, the method includes the step of irradiating a tumor. In some embodiments, irradiation is achieved between 30 minutes and 96 hours or approximately 30 minutes and 96 hours after administration of the conjugate, for example between 30 minutes and 48 hours, 30 minutes and 24 hours or between 12 hours and 48 hours, for example, generally at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or longer after administration of the conjugate. For example, irradiation can be performed within approximately 24 hours after administration of the conjugate.

[0364] In some embodiments, cells are brought into contact with phthalocyanine dye-targeting molecule conjugates and then irradiated. Methods of irradiation are known in the art. Typically, only cells expressing cell surface proteins are recognized by the targeting molecules, so generally only such cells will have a sufficient amount of conjugate bound to the protein. Irradiation can reduce the possibility of undesirable side effects, such as the killing of normal cells, because it kills only the cells to which the conjugate is bound and generally does not kill other cells.

[0365] In some embodiments, cells are irradiated in vitro, for example, in a tissue culture dish. In some embodiments, cells are irradiated in vivo, for example, to a subject that has been previously administered a phthalocyanine dye-targeted molecule conjugate. In some embodiments, a subject can be irradiated, for example, to a tumor in the subject.

[0366] In some embodiments, light or a laser may be applied to cells containing pigment molecules, e.g., conjugates, for about 5 seconds to about 5 minutes. For example, in some embodiments, light or a laser is applied for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 seconds or for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 seconds, or within the range of any two of these values, to activate the pigment molecules. In some embodiments, light or a laser is applied for 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 minutes or longer, or for about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 minutes or longer, or within the range of any two of these values. In some embodiments, the length of time the light or laser is applied may vary depending, for example, the energy of the light or laser, e.g., wattage. For example, lower wattage light or lasers can be applied for longer periods to activate pigment molecules.

[0367] In some embodiments, light or laser may be applied approximately 30 minutes to 48 hours after administration of the conjugate. For example, in some embodiments, light or laser may be applied 30, 35, 40, 45, 50, or 55 minutes after administration of the conjugate, or approximately 30, 35, 40, 45, 50, or 55 minutes later, or within a range between any two of these values. In some embodiments, light or laser is applied 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 approximately 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, or within a range between any two of these values ​​or approximately between any two of them. In some embodiments, following the administration of the conjugate, light or laser may be applied between 1 and 24 hours or approximately between 1 and 24 hours, for example between 1 and 12 hours or approximately 1 and 12 hours, between 12 and 24 hours or approximately 12 and 24 hours, between 6 and 12 hours or approximately 6 and 12 hours, or more than 24 hours later. In some embodiments, light or laser is applied 36 or 48 hours after the administration of the conjugate. In some embodiments, cells, hyperplasia, or tumors are irradiated within 12, 24, 36, 72, or 96 hours after the administration of the conjugate, or within approximately 12, 24, 36, 72, or 96 hours, or at approximately 12, 24, 36, 72, or 96 hours.

[0368] In some embodiments, the conjugated dye molecules can be activated at preferred wavelengths. Thus, in some embodiments, cells are irradiated with a therapeutic dose of radiation at wavelengths of 660–710 nm or approximately 660–710 nm, e.g., 660–700 nm or 670–690 nm, e.g., 680 nm. In some embodiments, activation of the dye molecules makes them cytotoxic or enables them to produce cytotoxic molecules. Preferred wavelengths include, non-limitingly, ultraviolet, visible, infrared (IR), and near-infrared wavelengths. In some embodiments, the dye molecules are activated at wavelengths of 600–800 nm or approximately 600–800 nm, or 660–740 nm or approximately 660–740 nm, and become cytotoxic. In some embodiments, the dye molecules are approximately 600nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, 770nm, 780nm, 790nm or 800nm, or at least approximately 600nm, 610nm, 62nm The pigment molecules are activated and become cytotoxic at wavelengths of 0 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, or 800 nm, or within a range between any two of these wavelengths or approximately between any two of them. In some embodiments, the pigment molecules are activated at wavelengths below 600 nm or above 800 nm.

[0369] In some embodiments, the tumor is irradiated with wavelengths within the range of 600nm to 800nm ​​or approximately 600nm to 800nm, or 600nm to 740nm or approximately 600nm to 740nm, for example, 640nm to 760nm or approximately 640nm to 760nm, 660nm to 740nm or approximately 660nm to 740nm, 680nm to 720nm or approximately 680nm to 720nm, or 690nm to 710nm or approximately 690nm to 710nm. In some embodiments, the tumor is irradiated with wavelengths of 690±50nm.

[0370] The optimal wavelength for activating dye molecules may depend on the specific dye molecule being used.

[0371] In some embodiments, cells, hyperplasia, or tumors are at least 1 J cm -2 (1J / cm 2 ) or approximately 1 J cm -2 (1J / cm 2 ), for example, at least 10 J cm -2 Alternatively, approximately 10 J cm -2 at least 30 J cm -2 Alternatively, approximately 30 J cm -2 at least 50 J cm -2 Alternatively, approximately 50 J cm -2 at least 100 J cm -2 Alternatively, approximately 100 J cm -2 , or at least 500 J cm -2 Alternatively, approximately 500 J cm -2 For example, at least about 2J cm -2 , 5J cm -2 , 10J cm -2 , 25J cm -2 , 50J cm -2 , 75J cm -2 , 100J cm -2 , 150J cm -2 , 200J cm -2 , 300J cm -2 , 400J cm -2 , or 500J cm -2They are irradiated with a dose of 1-1000 J cm. For example, in some embodiments, cells, hyperplasia, or tumors are irradiated with 1-1000 J cm. -2 Alternatively, approximately 1-1000 J cm -2 , 1-500J cm -2 Alternatively, approximately 1-500 J cm -2 , 10-100J cm -2 Alternatively, approximately 10-100 J cm -2 , or 10-50 J cm -2 Alternatively, approximately 10-50 J cm -2 The tumor is irradiated with at least 0.5 J cm. In some embodiments, the tumor is irradiated with at least 0.5 J cm. -2 at least 1 J cm -2 , at least 2J cm -2 , at least 3J cm -2 , at least 4J cm -2 , or at least 5J cm -2 They are irradiated with a dose of 1 J cm. In some embodiments, cells, hyperplasia, or tumors are irradiated with 1 J cm. -2 It is irradiated with this dose. In some embodiments, cells, hyperplasia, or tumors are irradiated with doses of at least 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, or at least about 2 J / cm fiber length, 5 J / cm fiber length, 10 J / cm fiber length, 25 J / cm fiber length, 50 J / cm fiber length, 75 J / cm fiber length, 100 J / cm fiber length, 150 J / cm fiber length, 200 J / cm fiber length, 250 J / cm fiber length, 300 J / cm fiber length, 400 J / cm fiber length, or 500 J / cm fiber length. In some embodiments, cells are subjected to at least 1 J cm⁻¹ -2 Alternatively, it is irradiated with a dose of 1 J / fiber length cm. In some embodiments, cells, hyperplasia, or tumors are irradiated with 2 J cm -2 ~about 400J cm -2 Or approximately 2J cm-2 ~about 400J cm -2 The radiation is administered at a dose of 2 J / cm fiber length to approximately 500 J / cm, or approximately 2 J / cm to approximately 500 J / cm fiber length.

[0372] In some embodiments, the tumor is a superficial tumor. In some embodiments, the tumor has a load of at least 10 J / cm³. 2 , 25J / cm 2 50 J / cm 2 , 150 J / cm 2 Alternatively, 250 J / cm² 2 , or at least about 10 J / cm² 2 , 25J / cm 2 50 J / cm 2 , 150 J / cm 2 Alternatively, 250 J / cm² 2 , or approximately 10 J / cm 2 , 25J / cm 2 50 J / cm 2 , 150 J / cm 2 Alternatively, 250 J / cm² 2 It is irradiated with this dose.

[0373] In some embodiments, the tumor is a stromal tumor. In some embodiments, the tumor is irradiated with a dose of at least 50 J / cm fiber length, 100 J / cm fiber length, 200 J / cm fiber length or 300 J / cm fiber length, or about at least 50 J / cm fiber length, 100 J / cm fiber length, 200 J / cm fiber length or 300 J / cm fiber length, or about 50 J / cm fiber length, 100 J / cm fiber length, 200 J / cm fiber length or 300 J / cm fiber length.

[0374] In some embodiments, the irradiation dose after administration of a composition containing a phthalocyanine dye-targeted molecule conjugate is at least 1 J cm at a wavelength of 660–740 nm. -2 For example, at a wavelength of 660-740 nm, at least 10 J cm⁻¹ -2 At wavelengths of 660-740 nm, at least 50 J cm⁻¹ -2, or at least 100 J cm at a wavelength of 660-740 nm -2 For example, at wavelengths of 660-740 nm, 1-500 1.0 J cm -2 In some embodiments, the wavelength is 660-710 nm. In some embodiments, the irradiation dose after administration of a composition containing a phthalocyanine dye-targeting molecule conjugate is at least 1.0 J cm at a wavelength of 680 nm. -2 For example, at a wavelength of 680 nm, at least 10 J cm -2 at a wavelength of 680 nm, at least 50 J cm -2 , or at least 100 J cm at a wavelength of 680 nm -2 For example, at a wavelength of 680 nm, 1 to 500 1.0 J cm -2 In some embodiments, multiple irradiations, for example, at least two, at least three, or at least four irradiations, for example, two, three, four, five, six, seven, eight, nine, or ten separate doses are administered.

[0375] In some embodiments, cells or subjects may be irradiated once or multiple times. Thus, irradiation may be completed in one day, or repeated over several days with the same or different doses, for example, at least two, three, four, five, or ten different irradiations. In some embodiments, repeated irradiations may be performed on the same day, consecutively, or at intervals of 1-3 days, 3-7 days, 1-2 weeks, 2-4 weeks, 1-2 months, or longer intervals.

[0376] In some embodiments, the subject may receive one or more other treatments before, during, or after administration of the conjugate. In some embodiments, the subject may receive one or more procedures to remove or reduce the tumor before administration of the conjugate.

[0377] Additional measures Before, during, or after administration of a phthalocyanine dye-targeted molecule conjugate, the subject may receive one or more other treatments. For example, the subject may receive one or more procedures to remove or reduce tumors before administration of the conjugate.

[0378] In some embodiments, one or more other or additional agents administered, or additional agents in combination therapy, are non-conjugate targeting molecules. In some embodiments, the non-conjugate targeting molecule is the same as or substantially the same as the targeting molecule of the conjugate. For example, in some embodiments, a targeting molecule that targets a protein or antigen, e.g., a non-conjugate antibody, is administered to the target prior to the administration of the conjugate. In some embodiments, the targeting molecule is administered up to 96 hours before the administration of the conjugate. In some embodiments, the targeting molecule is administered at a dose of 10 mg / m². 2 ~about 500mg / m 2 Or approximately 10 mg / m² 2 ~about 500mg / m 2 It is administered in doses within a specified range. For example, the targeting molecule is cetuximab, and cetuximab is administered to the subject at least 96 hours before administration of the conjugate.

[0379] Examples of such therapies that can be used in combination with the disclosed PIT method, which may enhance tumor accessibility to additional therapeutic agents for approximately 8 hours after PIT, include, but are not limited to, surgical procedures for tumor removal or reduction, e.g., surgical resection, cryotherapy, or chemoembolization, as well as antitumor pharmaceutical procedures, which may include radiotherapies, anti-cancer chemotherapy agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents. In some examples, the additional therapeutic agents are conjugated to nanoparticles. Specific examples of additional therapeutic agents that may be used include microtubule binders, DNA insertion agents or crosslinking agents, DNA synthesis inhibitors, DNA and / or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators. These agents and procedures, administered in therapeutically effective doses, may be used alone or in combination. Methods and therapeutic doses of such agents are known to those skilled in the art and may be determined by a skilled clinician.

[0380] In some embodiments, microtubule conjugates refer to drugs that interfere with tubulin to stabilize or destabilize microtubule formation, thereby inhibiting cell division. Examples of microtubule conjugates that can be used in combination with the disclosed conjugate therapies include, but are not limited to, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), epothirone, colchicine, dorastatin 15, nocodazole, podophyllotoxin, and rhizoxin. Analogues and derivatives of such compounds can also be used. For example, suitable epothirone and epothirone analogs may be used. Taxoids such as paclitaxel and docetaxel can also be used.

[0381] The following classes of compounds can be used in conjunction with the PIT method disclosed herein: suitable DNA and / or RNA transcription regulators, including, but not limited to, actinomycin D, daunorubicin, doxorubicin and their derivatives and analogues, are also suitable for use in combination with the treatments disclosed herein. DNA insertion agents and crosslinking agents that can be administered to a subject include, but not limited to, cisplatin, carboplatin, oxaliplatin, mitomycins, e.g., mitomycin C, bleomycin, chlorambucil, cyclophosphamide and their derivatives and analogues. Suitable DNA synthesis inhibitors for use as therapeutic agents include, but not limited to, methotrexate, 5-fluoro-5'-deoxyuridine, 5-fluorouracil and their analogues. Examples of suitable enzyme inhibitors include, but not limited to, camptothecin, etoposide, formestan, trichostatin and their derivatives and analogues. Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5-azacitidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone, and their derivatives and analogs. Kinase inhibitors include Gleevac, Iressa, and Tarceva, which prevent the phosphorylation and activation of growth factors.

[0382] Other therapeutic agents that may or may not fall into one or more of the above classifications, such as antitumor agents, are also suitable for administration in combination with the disclosed PIT treatments. Examples of such agents include doxorubicin, apigenin, rapamycin, zebralin, cimetidine, and their derivatives and analogues.

[0383] In some cases, interleukin-2 (IL-2) is also administered to subjects receiving the therapeutic conjugate composition, for example, via intravenous administration. In some embodiments, IL-2 is administered, for example, as an intravenous bolus at a dose of at least 500,000 IU / kg, starting, for example, the day after the administration of the peptide, over 15 minutes every 8 hours for up to 5 days. The dose may be omitted depending on the tolerance of the subject.

[0384] Further examples of treatments that can be used in combination with the administration of the conjugate include, but are not limited to, surgical procedures for tumor removal or reduction, such as surgical resection, cryotherapy, or chemoembolization, as well as antitumor pharmaceutical procedures, which may include radiotherapies, anti-cancer chemotherapy agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents. Specific examples of additional therapeutic agents that can be used include microtubule binders, DNA insertion agents or crosslinking agents, DNA synthesis inhibitors, DNA and / or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and DARPin (engineered ankyrin repeat protein), such as mono-DARPin. These agents and procedures, which may be administered in therapeutically effective doses, can be used alone or in combination. The method and therapeutic dose of such agents may be determined by an experienced clinician.

[0385] In some embodiments, at least a portion of the tumor, such as a metastatic tumor, is surgically removed, irradiated, chemically treated, or a combination thereof, for example via cryotherapy, or chemoembolization, for example, prior to administration of the treatment of the Disclosure, for example, administration of a phthalocyanine dye-targeted molecule conjugate. For example, a subject with a metastatic tumor may have all or part of the tumor surgically resected prior to administration of the treatment of the Disclosure. In some embodiments, one or more chemotherapeutic agents are administered after treatment with the conjugate and irradiation. In some embodiments, a subject has a metastatic tumor and receives radiotherapy, chemoembolization, or both concurrently with administration of the treatment of the Disclosure.

[0386] III. Definition Unless otherwise defined, all technical terms, notes, and other technical and scientific or related terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art in the field to which the claimed subject matter belongs. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be interpreted as differing significantly from the generally understood meaning in the art.

[0387] Where used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly indicates otherwise. For example, “a” or “an” means “at least one” or “one or more.” The aspects and variations described herein are understood to include “consisting of” and / or “essentially being from” the aspects and variations.

[0388] Throughout this disclosure, various aspects of the claimed subject matter are presented in range form. It should be understood that the range form is merely for convenience and conciseness and should not be interpreted as an inflexible limitation on the scope of the claimed subject matter. Therefore, a description of a range should be considered to encompass all possible sub-ranges within that range and all specifically disclo...

Claims

1. (i) a conjugate containing a phthalocyanine dye linked to a targeting molecule, and (ii) Pharmaceutically acceptable excipients A container containing a pharmaceutical composition including, The targeting molecule binds to a cell surface target molecule on the surface of a cell or pathogen. The phthalocyanine dye has the formula: Including, in the formula, L is the linker; Q is a reactive group; R 2 , R 3 , R 7 , and R 8 Each is independently selected from among optionally substituted alkyls and optionally substituted aryls; 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 chelating ligands, where at least one of R 4 、R 5 、R 6 、R 9 、R 10 、and R 11 contains a water-soluble group selected from carboxylate (-CO 2 - ), sulfonate (-SO 3 - ), sulfonyl (-SO 2 - ), sulfate (-SO 4 -2 ), hydroxyl (-OH), phosphate (-OPO 3 -2 ), phosphonate (-PO 3 -2 ), and amine (-NH[[ID= 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 independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino and optionally substituted alkoxy; and X 2 and X 3 Each of these C atoms may have a heteroatom interposed between them, independently of each other. 1 -C 10 It is alkylene, The phthalocyanine dye is linked to the target molecule via an attachment formed by the reaction of the reactive group Q with the attachment group of the target molecule, which is selected from the group consisting of thiol, hydroxyl, carboxyl, and amino reactive groups. More than 90% of the conjugate in the composition exists in monomeric form, and The container is protected from light transmission so that its light transmittance is less than 50%. The container.

2. The container according to claim 1, which is amber, green, or blue.

3. The container according to claim 1, which is covered with a material that is translucent, opaque, or has a light transmittance of less than 50%.

4. (i) a conjugate containing a phthalocyanine dye linked to a targeting molecule, and (ii) Pharmaceutically acceptable excipients A container containing a pharmaceutical composition including, The targeting molecule binds to a cell surface target molecule on the surface of a cell or pathogen. The phthalocyanine dye has the formula: Including, in the formula, L is the linker; Q is a reactive group; R 2 , R 3 , R 7 , and R 8 Each is independently selected from among optionally substituted alkyls and optionally substituted aryls; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 Each is independently selected from hydrogen, an optionally substituted alkyl, an optionally substituted alkanoyl, an optionally substituted alkoxycarbonyl, an optionally substituted alkylcarbamoyl, and a chelate ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of them is carboxylate (-CO 2 - ) group, sulfonate (-SO 3 - ) group, sulfonyl (-SO 2 - ) group, sulfate (-SO 4 -2 ) group, hydroxyl (-OH) group, phosphate (-OPO 3 -2 ) group, phosphonate (-PO 3 -2 ) group, and amine (-NH 2 ) Contains a water-soluble group selected from the groups; 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 independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino and optionally substituted alkoxy; and X 2 and X 3 each independently represents a C 1 -C 10 alkylene which may have a hetero atom intervening therebetween The phthalocyanine dye is linked to the target molecule via an attachment formed by the reaction of the reactive group Q with the attachment group of the target molecule, which is selected from the group consisting of thiol, hydroxyl, carboxyl, and amino reactive groups. More than 90% of the conjugate in the composition exists in monomeric form, and It is amber in color. The container.

5. The container according to any one of claims 1 to 4, wherein the container protects against the transmission of light having wavelengths of 500 nm to 725 nm or 650 nm to 725 nm.

6. The container according to any one of claims 1 to 5, which protects the container from light transmission such that the light transmittance is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.

7. (i) a conjugate containing a phthalocyanine dye linked to a targeting molecule, and (ii) Pharmaceutically acceptable excipients A container containing a pharmaceutical composition including, The targeting molecule binds to a cell surface target molecule on the surface of a cell or pathogen. The phthalocyanine dye has the formula: Including, in the formula, L is the linker; Q is a reactive group; R 2 , R 3 , R 7 , and R 8 Each is independently selected from among optionally substituted alkyls and optionally substituted aryls; 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 chelating ligands, where at least one of R 4 、R 5 、R 6 、R 9 、R 10 、and R 11 contains a water-soluble group selected from carboxylate (-CO 2 - ), sulfonate (-SO 3 - ), sulfonyl (-SO 2 - ), sulfate (-SO 4 -2 ), hydroxyl (-OH), phosphate (-OPO 3 -2 ), phosphonate (-PO 3 -2 ), and amine (-NH 2 ); 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 independently selected from hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino and optionally substituted alkoxy; and X 2 and X 3 Each of these C atoms may have a heteroatom interposed between them, independently of each other. 1 -C 10 It is alkylene, The phthalocyanine dye is linked to the target molecule via an attachment formed by the reaction of the reactive group Q with the attachment group of the target molecule, which is selected from the group consisting of thiol, hydroxyl, carboxyl, and amino reactive groups. More than 90% of the conjugate in the composition exists in monomeric form, and The container protects against the transmission of light having wavelengths of 500 nm to 725 nm. The container.

8. The container according to any one of claims 1 to 7, wherein more than 95% of the conjugate in the composition is present in monomeric form.

9. The container according to any one of claims 1 to 8, wherein more than 90% or more than 95% of the conjugate in the composition is present in monomer form for more than 3 months.

10. The container according to any one of claims 1 to 9, wherein more than 95% of the conjugate in the composition is present in monomer form for more than 3 months.

11. The phthalocyanine dye has the formula: Including, in the formula, X 1 and X 4 Each of these C atoms may have a heteroatom interposed between them, independently of each other. 1 -C 10 It is alkylene; R 2 , R 3 , R 7 , and R 8 Each is independently selected from an orphan alkyl and an orphan aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 Each is independently selected from hydrogen, an optionally substituted alkyl, an optionally substituted alkanoyl, an optionally substituted alkoxycarbonyl, an optionally substituted alkylcarbamoyl, and a chelate ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of them is carboxylate (-CO 2 - ) group, sulfonate (-SO 3 - ) group, sulfonyl (-SO 2 - ) group, sulfate (-SO 4 -2 ) group, hydroxyl (-OH) group, phosphate (-OPO 3 -2 ) group, phosphonate (-PO 3 -2 ) group, and amine (-NH 2 ) comprising a water-soluble group selected from the groups; and 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. A container according to any one of claims 1 to 10.

12. The container according to any one of claims 1 to 11, wherein the phthalocyanine dye comprises IRDye 700DX (IR700).

13. The container according to any one of claims 1 to 12, wherein the targeting molecule is an antibody or an antibody fragment.

14. The cell surface target molecules include cell membrane phospholipids, prokaryotic peptidoglycans, bacterial cell envelope proteins, viral capsid proteins, ACTHR, endothelial cell Anxa-1, aminopeptidase N, IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B cell receptors BB1, BB2, BB4, calcitonin receptor, and cancer antigen 125 (CA). 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell surface annexin-1, cell surface plectin-1, crypto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, endothialin, EP2, EP 4, EpCAM, EphA2, ET receptor, fibronectin, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GLP-1 receptor, Family A G protein-coupled receptor (rhodopsin-like), Family B G protein-coupled receptor (secretin receptor-like), Family C G protein-coupled receptor (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV16 / 18 and E6 / E7 antigens, hTERT, IL11-R, IL-13R, ITGAM, kallikrein-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE1, MAGE2, MAGE3, MAGE4, ​​MART1, MC1R, mesothelin, MUC1, MUC16, Neu (cell surface nucleolin), neprilysin, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-E SO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32 (p32 / gC1q-R / HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptor, PDT, protease-cleaved collagen IV, proteinase 3, inhibitor, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family, P2X1-5, mutant Ras, RAMP1, RAMP2, RAMP3 Selected from patched, RET receptor, plexin, smoothed, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP, TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3, TSH receptor, VEGF receptor, VEGFR1 or Flt-1, VEGFR2 or FLK-1 / KDR, VEGF-3 or FLT-4, voltage-gated ion channel, VPAC1, VPAC2, Wilms tumor 1, Y1, Y2, Y4, and Y5. A container according to any one of claims 1 to 13.

15. The container according to any one of claims 1 to 14, wherein the cell surface target molecule is selected from PD-L1, HER1 / EGFR, HER2, CD20, CD25, CD33, CD52, and prostate-specific membrane antigen (PSMA).

16. The container according to any one of claims 1 to 15, wherein the cell surface target molecule is HER1 / EGFR.

17. The container according to any one of claims 1 to 16, wherein the targeting molecule is cetuximab or an antigen-binding fragment thereof.

18. The container according to any one of claims 1 to 17, wherein the concentration of the conjugate in the composition is 0.01 mg / mL to 200 mg / mL.

19. The container according to any one of claims 1 to 18, wherein the concentration of the conjugate in the composition is 0.5 mg / mL to 10 mg / mL.

20. A packaging system for protecting phthalocyanine dye-targeted molecule conjugates from light, A first container comprising the container according to any one of claims 1 to 19; and The second container, which includes the first container. Includes, The second container is protected from light transmission such that its light transmittance is less than 50%. The packaging system.

21. The packaging system according to claim 20, wherein the second container is protected from light transmission such that the light transmittance is less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.

22. The packaging system according to claim 20 or 21, wherein the second container is green, blue, or amber in color.

23. The packaging system according to any one of claims 20 to 22, wherein the second container is covered with a material that is translucent, opaque, or has a light transmittance of less than 50%.

24. The packaging system according to any one of claims 20 to 23, wherein the second container is protected from the transmission of light having wavelengths of 250 nm to 800 nm.

25. The packaging system according to any one of claims 20 to 24, wherein the second container is protected from the transmission of light having wavelengths of 250 nm to 450 nm, 400 nm to 800 nm, 450 nm to 650 nm, or 600 nm to 720 nm.

26. A container according to any one of claims 1 to 19 or a packaging system according to any one of claims 20 to 25; and A photoprotective cover capable of coating a device that can administer a composition containing a phthalocyanine dye-targeted molecule conjugate. A kit that includes this.

27. The kit according to claim 26, including an instruction manual.

28. A container according to any one of claims 1 to 19, a packaging system according to any one of claims 20 to 25, or a kit according to claim 26 or 27, The pharmaceutical composition is a pharmaceutical composition for use in a method for treating hyperplasia or tumors in human subjects, The measure is, (a) a step of administering a therapeutically effective amount of the composition to a subject, wherein the conjugate is not exposed to ambient light of an intensity exceeding 500 lux prior to the administration step; and (b) The hyperplasia or tumor is exposed to at least 1 J cm at a wavelength of 600–800 nm. -2 Alternatively, the process involves irradiating the target with a dose of 1 J / cm of fiber length, thereby treating the hyperplasia or tumor in the target. including, The container, the packaging system, or the kit.

29. The container, packaging system, or kit according to claim 28, wherein the tumor is a cancer located in the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung.

30. The container, packaging system, or kit according to claim 28 or 29, wherein the hyperplasia or tumor is irradiated with a wavelength of 690 ± 50 nm or 690 ± 20 nm.

31. The hyperplasia or tumor is 2 J cm -2 ~400J cm -2 A container, packaging system, or kit according to claim 28 or 29, which is irradiated with a dose of , or a dose of 2 J / cm fiber length to 500 J / cm fiber length.

32. The pharmaceutical composition contains 25 mg / m 2 ~2000mg / m² 2 A container, packaging system, or kit according to any one of claims 28 to 31, administered in a dose between [number].

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