Near-infrared (NIR) photoimmunotherapy (PIT) for the treatment of cancer using anti-CD25 antibody-phthalocyanine dye conjugate and anti-PD1 antibody

Abstract

The present disclosure relates to near-infrared (NIR) photoimmunotherapy (PIT) for treating a subject with cancer, e.g., cancers including primary or primary tumors, metastatic tumor cells, and / or invasive tumor cells. The method includes administering to the subject a targeting molecule that binds to CD25 conjugated to a phthalocyanine dye, such as IR700, and administering an immune checkpoint inhibitor, such as an anti-PD-1 antibody, followed by irradiating the primary or primary tumor with a wavelength of light suitable for activating the phthalocyanine dye.

Description

[Technical field]

[0001] Related Applications This application is a continuation of U.S. Provisional Patent Application No. 62 / 880,514, filed July 30, 2019, entitled “COMPOSITIONS AND METHODS FOR LOCAL AND SYSTEMIC TREATMENT OF CANCERS, TUMORS AND TUMOR CELLS,” U.S. Provisional Patent Application No. 62 / 896,453, filed September 5, 2019, entitled “COMPOSITIONS AND METHODS FOR LOCAL AND SYSTEMIC TREATMENT OF CANCERS, TUMORS AND TUMOR CELLS,” and U.S. Provisional Patent Application No. 62 / 896,453, filed November 6, 2019, entitled “COMPOSITIONS AND METHODS FOR LOCAL AND SYSTEMIC TREATMENT OF CANCERS, TUMORS AND TUMOR CELLS,” This application claims priority to U.S. Provisional Patent Application No. 62 / 931,405, entitled "Microfluidic Device for Imaging and Cells," the contents of which are incorporated by reference in their entireties.

[0002] Field The present disclosure relates to compositions, combinations, methods and uses for treating subjects with cancer, including cancers such as primary or primary tumors, metastatic tumor cells and / or invasive tumor cells. The method comprises administering to the subject a targeting molecule that binds to CD25 conjugated with a phthalocyanine dye, such as IR700, administering an immune checkpoint inhibitor, and then irradiating the primary or primary tumor with a wavelength of light suitable for activating the phthalocyanine dye. The methods and uses described herein provide for the reduction and elimination of tumor and tumor cell growth, including primary or primary tumors, metastatic tumor cells and / or invasive tumor cells. Also provided are compositions, combinations, methods and uses for enhancing systemic immunity against tumor growth in subjects with cancer, including cancers such as primary or primary tumors, metastatic tumor cells and / or invasive tumor cells. [Background technology]

[0003] background Cancer metastasis is the main cause of cancer-related death. Despite some available treatments for certain types of cancer, there is still an urgent need for therapeutic strategies to effectively treat cancer, including cancer with both primary tumor and metastatic tumor. In particular, treating metastatic cancer remains a major clinical challenge. Summary of the Invention

[0004] overview Provided herein are compositions, combinations, methods and uses for treating cancer, including cancers that contain tumors, such as primary tumors, as well as cancers that have secondary populations of tumor cells, such as metastatic tumor cells, invasive tumor cells or invasive cells.

[0005] Provided herein are methods and uses for treating cancer. In some of the embodiments, the methods and uses include administering an immune checkpoint inhibitor to a subject with cancer, including a primary tumor and a secondary population of tumor cells; administering a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody to the subject; and administering the conjugate to the primary tumor at a wavelength of at or about 600 nm to at or about 850 nm and at 25 J / cm. 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of at or about 2 J / cm fiber length to at or about 500 J / cm fiber length, wherein the secondary population is not directly irradiated.

[0006] In some of the embodiments, the primary tumor and / or secondary populations are inhibited to a greater extent compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the immune checkpoint inhibitor alone. In some of the embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof. In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, or an antigen-binding fragment thereof.

[0007] Provided herein are methods and uses for treating cancer. In some of the embodiments, the methods and uses provided include administering an anti-PD-1 antibody to a subject having a cancer, including a primary tumor and a secondary population of tumor cells; administering to the subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody; and administering to the subject, after administration of the conjugate, a phthalocyanine dye at a wavelength at or about 600 nm to 850 nm or about 850 nm and at a concentration of 25 J / cm to the primary tumor. 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or irradiating with a dose of at or about 2 J / cm fiber length to at or about 500 J / cm fiber length, where the secondary population is not directly irradiated; the primary tumor and / or the secondary population are inhibited to a greater extent as compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone.

[0008] In some of the embodiments, the inhibition comprises one or more of an increase in tumor volume, size or mass of less than 20% or less than about 20%, or a reduction in tumor volume, size or mass, or a reduction in the number of tumor cells. In some of the embodiments, the reduction in tumor volume, size or mass, or the number of tumor cells comprises a reduction of 30% or more, or a reduction of about 30% or more.

[0009] In some of the optional aspects, the secondary population comprises metastatic tumor cells. In some of the optional aspects, the secondary population comprises invasive tumor cells. In some of the optional aspects, the secondary population comprises metastatic tumor cells and invasive tumor cells.

[0010] In some of the optional embodiments, the immune checkpoint inhibitor is administered to the subject simultaneously with the conjugate. In some of the optional embodiments, the immune checkpoint inhibitor is administered to the subject within 24-48 hours of administering the conjugate. In some of the optional embodiments, the immune checkpoint inhibitor is administered to the subject within 24 hours ± 4 hours of administering the conjugate.

[0011] In some of the embodiments, the first dose of the immune checkpoint inhibitor is administered prior to administration of the conjugate. In some of the embodiments, the first dose of the immune checkpoint inhibitor is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate.

[0012] In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times. In some of the embodiments, the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by administration of the immune checkpoint inhibitor 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times to the subject after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor is administered simultaneously with administration of the conjugate, followed by administration of the immune checkpoint inhibitor to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate.

[0013] In some of the optional embodiments, the anti-PD-1 antibody is administered to the subject simultaneously with the conjugate. In some of the optional embodiments, the anti-PD-1 antibody is administered to the subject within 24-48 hours of administering the conjugate. In some of the optional embodiments, the anti-PD-1 antibody is administered to the subject within 24 hours ± 4 hours of administering the conjugate.

[0014] In some of the embodiments, the first dose of anti-PD-1 antibody is administered prior to administration of the conjugate. In some of the embodiments, the first dose of anti-PD-1 antibody is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate.

[0015] In some of the embodiments, the anti-PD-1 antibody is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times. In some of the embodiments, the anti-PD-1 antibody is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 administrations of the anti-PD-1 antibody to the subject after administration of the conjugate. In some of the embodiments, the anti-PD-1 antibody is administered simultaneously with administration of the conjugate, followed by administration of the anti-PD-1 antibody to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate. In some of the embodiments, the anti-PD-1 antibody is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate.

[0016] In some of the optional embodiments, the secondary population is comprised in a solid tumor.

[0017] In some of any of the embodiments, the subject exhibits a complete response.

[0018] In some of the embodiments, the anti-CD25 antibody comprises a functional Fc region. In some of the embodiments, the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof. In some of the embodiments, the anti-CD25 antibody is basiliximab.

[0019] In some of the optional embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), pimbalimab (PIM-1), rivaroxaban (RIRV ... Zilizumab (CT-011), genolimzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, and TSR-042 (ANB011).

[0020] In some of the optional embodiments, the phthalocyanine dye is a Si-phthalocyanine dye. In some of the optional embodiments, the Si-phthalocyanine dye is IR700.

[0021] In some optional embodiments, irradiation is performed 30 minutes to 96 hours after administration of the conjugate. In some optional embodiments, irradiation is performed 24 hours ± 4 hours after administration of the conjugate. In some optional embodiments, the primary tumor is irradiated with a wavelength of 690 ± 40 nm. In some optional embodiments, the primary tumor is irradiated with 50 J / cm. 2 Or about 50 J / cm 2 Alternatively, the fiber is irradiated at a dose of 100 J / cm of fiber length or approximately 100 J / cm of fiber length.

[0022] In some of the optional embodiments, the methods and uses also involve the step of (d) administering an additional therapeutic agent or anti-cancer treatment.

[0023] In some optional embodiments, one or more of steps (a), (b), (c) or (d) are repeated.

[0024] In some of the optional aspects, the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell cancer, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerve, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0025] In some of the optional embodiments, the secondary populations are located in one, two, three or more tissues or organs different from the tissue or organ in which the primary tumor is located.

[0026] In some embodiments, the primary tumor and / or secondary populations are inhibited to a greater extent compared to administration of the conjugate followed by radiation alone and compared to administration of the immune checkpoint inhibitor alone, In some embodiments, the primary tumor and / or secondary populations are inhibited to a greater extent compared to administration of the conjugate followed by radiation alone and compared to administration of the anti-PD-1 antibody alone.

[0027] Provided herein is a method and use for generating an enhanced response in a subject with cancer. In some of the optional embodiments, the method and use include the steps of: administering an immune checkpoint inhibitor to a subject with cancer, including a tumor; administering to the subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody, wherein the immune checkpoint inhibitor is administered before or simultaneously with the conjugate; and administering to the tumor, after administration of the conjugate, an immune checkpoint inhibitor at a wavelength of at or about 600 nm to at or about 850 nm and at 25 J / cm. 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of at or about 2 J / cm fiber length to at or about 500 J / cm fiber length.

[0028] In some of any of the embodiments, the enhanced response comprises enhanced systemic immunity in the subject compared to the subject's systemic immunity prior to administration of the conjugate followed by irradiation and the immune checkpoint inhibitor; and / or the enhanced response comprises enhanced inhibition of tumors compared to administration of only the conjugate, only the conjugate followed by irradiation, or only the immune checkpoint inhibitor.

[0029] In some of the embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof. In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, or an antigen-binding fragment thereof.

[0030] Provided herein are methods and uses for generating an enhanced response in a subject with cancer. In some of the optional embodiments, the methods and uses include administering an anti-PD-1 antibody to a subject with cancer, including a tumor; administering to the subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody, where the anti-PD-1 antibody is administered before or simultaneously with the conjugate; and administering to the tumor, after administration of the conjugate, a phthalocyanine dye having a wavelength of at or about 600 nm to at or about 850 nm and a wavelength of at least 25 J / cm. 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or irradiating at a dose of at or about 2 J / cm fiber length to at or about 500 J / cm fiber length; the enhanced response comprises enhanced systemic immunity in the subject compared to the subject's systemic immunity prior to administration of the conjugate followed by irradiation and the anti-PD-1 antibody; and / or the enhanced response comprises enhanced tumor inhibition compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone.

[0031] In some of the optional aspects, the enhanced response is additive or synergistic.

[0032] In some of the embodiments, the inhibition comprises one or more of an increase in tumor volume, size or mass of less than 20% or less than about 20%, or a reduction in tumor volume, size or mass, or a reduction in the number of tumor cells. In some of the embodiments, the reduction in tumor volume, size or mass, or the number of tumor cells comprises a reduction of 30% or more, or a reduction of about 30% or more.

[0033] In some of any of the embodiments, the tumor comprises a primary tumor and a secondary population of tumor cells, where the primary tumor is irradiated and the secondary population is not directly irradiated; the tumor comprises a primary tumor and metastatic tumor cells, where the primary tumor is irradiated and the metastatic tumor cells are not directly irradiated; and / or the tumor comprises a primary tumor and invasive tumor cells, where the primary tumor is irradiated and the invasive tumor cells are not directly irradiated.

[0034] In some of the optional aspects, the enhanced response is a synergistic response, which includes a synergistic reduction in primary tumor growth, tumor volume, tumor size or tumor mass, a synergistic reduction in the number of cells in a secondary population of subjects, a synergistic reduction in metastatic or invasive tumor cell growth, tumor volume, tumor size, tumor mass or number, or any combination thereof.

[0035] In some of the embodiments, the immune checkpoint inhibitor is administered to the subject within 24 hours to 48 hours after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor is administered to the subject within 24 hours ± 4 hours after administration of the conjugate. In some of the embodiments, the first dose of the immune checkpoint inhibitor is administered prior to administration of the conjugate. In some of the embodiments, the first dose of the immune checkpoint inhibitor is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate.

[0036] In some of the optional embodiments, the anti-PD-1 antibody is administered to the subject within 24 hours to 48 hours after administration of the conjugate. In some of the optional embodiments, the anti-PD-1 antibody is administered to the subject within 24 hours ± 4 hours after administration of the conjugate. In some of the optional embodiments, the first dose of the anti-PD-1 antibody is administered prior to administration of the conjugate. In some of the optional embodiments, the first dose of the anti-PD-1 antibody is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate.

[0037] In some of the optional embodiments, systemic immunity can be measured using a cytotoxic T lymphocyte (CTL) activity assay, an intratumoral T cell exhaustion assay, an intratumoral effector T cell proliferation assay, a T cell receptor diversity assay, an activated CD8 + T cell assay, circulating regulatory T cell (Treg) assay, intratumoral Treg assay, or CD8 +In some of the embodiments, systemic immunity is measured by one or more of T cell:Treg assays. In some of the embodiments, systemic immunity is measured by CTL activity assay using splenocytes or peripheral blood cells or bone marrow cells or lymph node cells, optionally collected from the subject 4-28 days after irradiation of the primary tumor in the subject. In some of the embodiments, systemic immunity is measured by intratumoral T cell exhaustion assay using T cells collected from the primary tumor or metastatic tumor cell mass or invasive tumor cell mass, optionally collected from the subject 4-28 days after irradiation of the primary tumor in the subject. In some of the embodiments, systemic immunity is measured by intratumoral effector T cell proliferation assay using T cells collected from the primary tumor or metastatic tumor cell mass or invasive tumor cell mass, optionally collected from the subject 4-28 days after irradiation of the primary tumor in the subject. In some of the optional embodiments, systemic immunity is measured by a T cell receptor diversity assay using T cells collected from the primary tumor or the metastatic tumor cell mass or the invasive tumor cell mass or the peripheral circulation, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0038] In some of the optional embodiments, systemic immunity is measured by determining the presence, number or frequency of regulatory T cells (Treg) in tumor from a primary tumor or a metastatic or invasive tumor cell mass, optionally collected from a subject 4 to 28 days after irradiation of the primary tumor in the subject, and / or the ratio of intratumoral Treg cells to intratumoral CD8+ T cells or intratumoral CD4+ T cells.

[0039] In some of the embodiments, the anti-CD25 antibody comprises a functional Fc region. In some of the embodiments, the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof. In some of the embodiments, the anti-CD25 antibody is basiliximab.

[0040] In some of the optional embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-6372), and the like. 3283), pidilizumab (CT-011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, and TSR-042 (ANB011).

[0041] In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times. In some of the embodiments, the anti-PD-1 antibody is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times.

[0042] In some of the optional embodiments, the phthalocyanine dye is a Si-phthalocyanine dye. In some of the optional embodiments, the Si-phthalocyanine dye is IR700.

[0043] In some of the optional embodiments, irradiation is performed 30 minutes to 96 hours after administration of the conjugate. In some of the optional embodiments, irradiation is performed 24 hours ± 4 hours after administration of the conjugate. In some of the optional embodiments, the tumor is irradiated with a wavelength of 690 ± 40 nm. In some of the optional embodiments, the tumor is irradiated with 50 J / cm. 2 Or about 50 J / cm 2 Alternatively, the fiber is irradiated at a dose of 100 J / cm of fiber length or approximately 100 J / cm of fiber length.

[0044] In some of the optional embodiments, the methods and uses also involve the step of (d) administering an additional therapeutic agent or anti-cancer treatment.

[0045] In some optional embodiments, one or more of steps (a), (b), (c) or (d) are repeated.

[0046] In some of the optional aspects, the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell cancer, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerve, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0047] In some of the embodiments, the enhanced response comprises an additive or synergistic response compared to administration of the conjugate followed by irradiation alone and compared to administration of the immune checkpoint inhibitor alone. In some of the embodiments, the enhanced response comprises an additive or synergistic response compared to administration of the conjugate followed by irradiation alone and compared to administration of the anti-PD-1 antibody alone.

[0048] In some embodiments, a method is provided for treating cancer in a subject using photoimmunotherapy, for example, photoimmunotherapy using a photosensitive phthalocyanine dye-targeting molecule conjugate. In some embodiments, the method comprises administering to a subject having cancer, for example, a cancer including a primary or primary tumor and invasive tumor cells and / or metastatic tumor cells, a conjugate containing a phthalocyanine dye linked to a targeting molecule, such as an antibody or antigen-binding fragment thereof, that binds to CD25 protein on the surface of cells present in the tumor microenvironment. In some embodiments, the conjugate is administered alone or in combination with an immune checkpoint inhibitor (ICI). In some embodiments, after administration of the conjugate, the primary or primary tumor is administered at least 1 J / cm at a wavelength of 500 to 900 nm. 2 or 1 J / cm of fiber length, thereby treating a tumor in a subject. In some embodiments, the wavelength for irradiation is 600 nm to 850 nm, for example, 660 nm to 740 nm.

[0049] In some embodiments, the cancer comprises a primary or multiple primary or multiple primary tumors, invasive tumor cells and / or metastatic tumor cells.

[0050] Methods and uses for treating cancer are provided. In some of the optional embodiments, the methods and uses include: (a) administering to a subject having cancer, including a primary or primary tumor and metastatic tumor cells, a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25; (b) administering to the subject an immune checkpoint inhibitor; and (c) administering to the primary or primary tumor, after administration of the conjugate, an immune checkpoint inhibitor at a wavelength of at or about 600 nm to at or about 850 nm and at 25 J / cm. 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2or at a dose of at or about 2 J / cm fiber length to at or about 500 J / cm fiber length, thereby inhibiting the growth and / or increase in volume of one or both of the primary or primary tumor and metastatic tumor cells in the subject. In some of any of the embodiments, the metastatic tumor cells are not directly irradiated.

[0051] In some of the embodiments, the metastatic tumor cells are comprised in a solid tumor. In some of the embodiments, the growth inhibition of the primary tumor, the metastatic tumor cells, or both, is synergistic compared to administration of only one of the conjugate and the immune checkpoint inhibitor.

[0052] In some of any of the embodiments, the subject exhibits a complete response.

[0053] In some of the embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof or comprises an antigen-binding fragment thereof. In some of the embodiments, the antibody is an anti-CD25 antibody. In some of the embodiments, the anti-CD25 antibody comprises a functional Fc region. In some of the embodiments, the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof. In some of the embodiments, the anti-CD25 antibody is basiliximab.

[0054] In some of the embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0055] In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody or an antigen-binding fragment thereof. In some of the embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab (MK-3475, KEYTRUDA), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), pygismustachomab (PDX-1), serotinib (SEQ ID NO: 1), serotinib (SEQ ID NO: 2), serotinib (SEQ ID NO: 3), serotinib (SEQ ID NO: 4), serotinib (SEQ ID NO: 5), serotinib (SEQ ID NO: 6), serotinib (SEQ ID NO: 7), serotinib (SEQ ID NO: 8), serotinib (SEQ ID NO: 9), serotinib (SEQ ID NO: 10), serotinib (SEQ ID NO: 11), serotinib (SEQ ID NO: 12), serotinib (SEQ ID NO: 13), serotinib (SEQ ID NO: 14), serotinib (SEQ ID NO: 15), serotinib (SEQ ID NO: 16), serotinib (SEQ ID NO: 17), serotinib (SEQ ID NO: 18), serotinib (SEQ ID NO: 19), serotinib (SEQ ID NO: 20), serotinib (SEQ ID NO: 21), serotinib (SEQ ID NO: 22), serotinib (S is selected from the group consisting of tafamidis (CT-011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), GLS-010, CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, MEDI0680, Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, and spartalizumab.

[0056] In some of the optional embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab (MPDL3280A, Tecentriq), avelumab (BAVENCIO), durvalumab (MEDI4736, IMFINZI), LDP, NM-01, STI-3031, KN035, LY3300054, M7824 (MSB0011359C), BMS-936559, M Selected from the group consisting of SB2311, BCD-135, BGB-A333, CBT-502, cosibelimab (CK-301), CS1001, FAZ053, MDX-1105, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, LY3415244, REGN3504, and HLX20.

[0057] In some of the optional embodiments, the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab (YERVOY), tremelimumab, AGEN1181, AGEN1884, ADU-1064, BCD-145, and BCD-217.

[0058] In some of the optional embodiments, the conjugate is administered to the subject prior to, simultaneously with, or following administration of an immune checkpoint inhibitor.

[0059] In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times. In some of the embodiments, the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by administration of the immune checkpoint inhibitor 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times to the subject after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor is administered simultaneously with administration of the conjugate, followed by administration of the immune checkpoint inhibitor to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate.

[0060] In some of the optional embodiments, the phthalocyanine dye is a Si-phthalocyanine dye. In some of the optional embodiments, the Si-phthalocyanine dye is IR700.

[0061] In some of the optional embodiments, the irradiation is performed 30 minutes to 96 hours after administration of the conjugate. In some of the optional embodiments, the irradiation is performed 24 hours ± 4 hours after administration of the conjugate. In some of the optional embodiments, the primary tumor or tumor is irradiated with a wavelength of 690 ± 40 nm. In some of the optional embodiments, the primary tumor or tumor is irradiated with 50 J / cm. 2 Or about 50 J / cm 2 Alternatively, the fiber is irradiated at a dose of 100 J / cm of fiber length or approximately 100 J / cm of fiber length.

[0062] In some optional embodiments, one or more of steps (a), (b) or (c) are repeated.

[0063] In some of any of the embodiments, the methods or uses provided further involve (d) administering an additional therapeutic agent or anti-cancer treatment.

[0064] In some of the embodiments, the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell cancer, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma. In some of the embodiments, the metastatic tumor cells are located in one, two, three, or more than three tissues or organs different from the tissue or organ in which the primary or primary tumor is located.

[0065] (1) administering to a subject having cancer, including a primary or invading tumor cell, a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25; (2) administering to the subject an immune checkpoint inhibitor; and (3) administering to the subject, after administration of the conjugate, an immune checkpoint inhibitor at a wavelength at or about 600 nm to at or about 850 nm and at least 25 J / cm to the primary or invading tumor cell.2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of at or about 2 J / cm fiber length to at or about 500 J / cm fiber length, whereby growth and / or an increase in the volume of one or both of the primary or primary tumor and invasive tumor cells in the subject are inhibited. In some of the optional embodiments, the invasive tumor cells are not directly irradiated.

[0066] Methods and uses for treating cancer are provided. In some of the optional aspects, the methods and uses involve the inclusion of invasive tumor cells in a solid tumor.

[0067] In some of any of the embodiments, the inhibition of growth of the primary tumor, the invasive tumor cells, or both is synergistic compared to administration of only one of the conjugate and the immune checkpoint inhibitor.

[0068] In some of any of the embodiments, the subject exhibits a complete response.

[0069] In some of the embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof or comprises an antigen-binding fragment thereof. In some of the embodiments, the antibody is an anti-CD25 antibody. In some of the embodiments, the anti-CD25 antibody comprises a functional Fc region. In some of the embodiments, the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof. In some of the embodiments, the anti-CD25 antibody is basiliximab.

[0070] In some of the embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0071] In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody or an antigen-binding fragment thereof. Methods and uses for treating cancer are provided. In some of the embodiments, the methods and uses are for treating cancer, the anti-PD-1 antibody being selected from the group consisting of pembrolizumab (MK-3475, KEYTRUDA), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-6372328), and the like. 3), pidilizumab (CT-011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), GLS-010, CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, MEDI0680, Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, and spartalizumab.

[0072] In some of the optional embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab (MPDL3280A, Tecentriq), avelumab (BAVENCIO), durvalumab (MEDI4736, IMFINZI), LDP, NM-01, STI-3031, KN035, LY3300054, M7824 (MSB0011359C), BMS-936559, M Selected from the group consisting of SB2311, BCD-135, BGB-A333, CBT-502, cosibelimab (CK-301), CS1001, FAZ053, MDX-1105, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, LY3415244, REGN3504, and HLX20.

[0073] In some of the optional embodiments, the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab (YERVOY), tremelimumab, AGEN1181, AGEN1884, ADU-1064, BCD-145, and BCD-217.

[0074] Methods and uses for treating cancer are provided. In some of the optional embodiments, the methods and uses involve administering a conjugate to a subject prior to, simultaneously with, or following administration of an immune checkpoint inhibitor.

[0075] In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times. In some of the embodiments, the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by administration of the immune checkpoint inhibitor 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times to the subject after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor is administered simultaneously with administration of the conjugate, followed by administration of the immune checkpoint inhibitor to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate.

[0076] In some of the optional embodiments, the phthalocyanine dye is a Si-phthalocyanine dye. In some of the optional embodiments, the Si-phthalocyanine dye is IR700.

[0077] In some optional embodiments, the irradiation is administered 30 minutes to 96 hours after administration of the conjugate. In some optional embodiments, the irradiation is administered 24 hours ± 4 hours after administration of the conjugate.

[0078] In some of the embodiments, the primary or primary tumor is irradiated with a wavelength of 690±40 nm. In some of the embodiments, the primary or primary tumor is irradiated with a wavelength of 50 J / cm. 2 Or about 50 J / cm 2 Alternatively, the fiber is irradiated at a dose of 100 J / cm of fiber length or approximately 100 J / cm of fiber length.

[0079] In some optional embodiments, one or more of steps (1), (2) or (3) are repeated.

[0080] In some of the optional embodiments, the methods and uses provided also involve (4) administering an additional therapeutic agent or anti-cancer treatment.

[0081] In some of the optional aspects, the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell cancer, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerve, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0082] Also provided are methods and uses for enhancing systemic immunity in a subject with a tumor. In some of the optional embodiments, the methods and uses include: (a) administering to a subject with a tumor a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody; (b) administering to the subject an immune checkpoint inhibitor; and (c) administering to the tumor, after administration of the conjugate, an immune checkpoint inhibitor at a wavelength at or about 600 nm to 850 nm or about 850 nm and at least 25 J / cm. 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of at or about 2 J / cm fiber length to at or about 500 J / cm fiber length, wherein the subject's systemic immunity is enhanced as compared to the subject's systemic immunity prior to administration of the conjugate and the immune checkpoint inhibitor.

[0083] In some of the optional embodiments, systemic immunity is measured by one or more of a cytotoxic T lymphocyte (CTL) activity assay, an intratumor T cell exhaustion assay, an intratumor effector T cell proliferation assay, or a T cell receptor diversity assay.

[0084] In some of any of the embodiments, the tumor comprises a primary or primary tumor and metastatic tumor cells, where the primary or primary tumor is irradiated and the metastatic tumor cells are not directly irradiated; or the tumor comprises a primary or primary tumor and invasive tumor cells, where the primary or primary tumor is irradiated and the invasive tumor cells are not directly irradiated.

[0085] In some of the optional embodiments, systemic immunity is measured by a CTL activity assay using splenocytes or peripheral blood cells or bone marrow cells or lymph node cells, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor or primary tumor in the subject.

[0086] In some of the optional embodiments, systemic immunity is measured by an intratumoral T cell exhaustion assay using T cells collected from a primary or primary tumor or a metastatic or invasive tumor cell mass, optionally collected from a subject 4 to 28 days after irradiation of the primary or primary tumor in the subject.

[0087] In some of the optional embodiments, systemic immunity is measured by an intratumoral effector T cell proliferation assay using T cells collected from a primary or primary tumor or a metastatic or invasive tumor cell mass, optionally collected from a subject 4 to 28 days after irradiation of the primary or primary tumor in the subject.

[0088] In some of the optional embodiments, systemic immunity is measured by a T cell receptor diversity assay using T cells collected from the primary or primary tumor or metastatic or invasive tumor cell mass or peripheral circulation, optionally collected from the subject 4 to 28 days after irradiation of the primary or primary tumor in the subject.

[0089] In some of the optional embodiments, systemic immunity is measured by determining the presence, number or frequency of regulatory T cells (Treg) in tumor from a primary or primary tumor or a metastatic or invasive tumor cell mass, optionally collected from a subject 4 to 28 days after irradiation of the primary or primary tumor in the subject, and / or the ratio of intratumoral Treg cells to intratumoral CD8+ T cells or intratumoral CD4+ T cells.

[0090] In some of the embodiments, the anti-CD25 antibody comprises a functional Fc region. In some of the embodiments, the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof. In some of the embodiments, the anti-CD25 antibody is basiliximab. In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0091] In some of the embodiments, the immune checkpoint inhibitor is or includes an anti-PD-1 antibody or an antigen-binding fragment thereof. In some of the embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), and the like. ), pidilizumab (CT-011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), GLS-010, CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, and TSR-042 (ANB011).

[0092] In some of the optional embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab (MPDL3280A, Tecentriq), avelumab (BAVENCIO), durvalumab (MEDI4736, IMFINZI), LDP, NM-01, STI-3031, KN035, LY3300054, M7824 (MSB0011359C), BMS-936559, M Selected from the group consisting of SB2311, BCD-135, BGB-A333, CBT-502, cosibelimab (CK-301), CS1001, FAZ053, MDX-1105, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, LY3415244, REGN3504, and HLX20.

[0093] In some of the optional embodiments, the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab (YERVOY), tremelimumab, AGEN1181, AGEN1884, ADU-1064, BCD-145, and BCD-217.

[0094] In some of the embodiments, the conjugate is administered to the subject prior to, simultaneously with, or after administration of the immune checkpoint inhibitor. In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times.

[0095] In some of the optional embodiments, the phthalocyanine dye is a Si-phthalocyanine dye. In some of the optional embodiments, the Si-phthalocyanine dye is IR700.

[0096] In some of the optional embodiments, the irradiation is administered 30 minutes to 96 hours after administration of the conjugate and the immune checkpoint inhibitor. In some of the optional embodiments, the irradiation is administered 24 hours ± 4 hours after administration of the conjugate and the immune checkpoint inhibitor.

[0097] In some of the embodiments, the tumor is irradiated with a wavelength of 690±40 nm. In some of the embodiments, the tumor is irradiated with a wavelength of 50 J / cm. 2 Or about 50 J / cm 2 Alternatively, the fiber is irradiated at a dose of 100 J / cm of fiber length or approximately 100 J / cm of fiber length.

[0098] In some optional embodiments, one or more of steps (a), (b), (c) or (d) are repeated.

[0099] In some of the optional embodiments, the tumor is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0100] Also provided are methods and uses for producing a synergistic response in a subject. In some embodiments, the methods and uses include: (1) administering to a subject having cancer, including a primary or primary tumor, a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25; (2) administering to the subject an immune checkpoint inhibitor; and (3) administering to the primary or primary tumor, after administration of the conjugate, an immune checkpoint inhibitor at a wavelength of at or about 600 nm to at or about 850 nm and at 25 J / cm. 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of at or about 2 J / cm fiber length to at or about 500 J / cm fiber length, wherein the growth of the primary tumor and / or the increase in its volume is synergistically reduced compared to administration of the conjugate alone or the immune checkpoint inhibitor alone.

[0101] In some of the embodiments, the cancer further comprises metastatic tumor cells, and the growth and / or increase in volume of the metastatic tumor cells is reduced compared to administration of the conjugate alone or the immune checkpoint inhibitor alone. In some of the embodiments, the reduction in the growth or increase in volume of the metastatic tumor cells is synergistic.

[0102] In some of the embodiments, the cancer further comprises invasive tumor cells, and the growth and / or increase in the volume of the invasive tumor cells is reduced compared to administration of the conjugate alone or the immune checkpoint inhibitor alone. In some of the embodiments, the reduction in the growth or increase in the volume of the invasive tumor cells is synergistic.

[0103] In some of the embodiments, the targeting molecule is or comprises an antibody or an antigen-binding fragment thereof. In some of the embodiments, the antibody is an anti-CD25 antibody. In some of the embodiments, the anti-CD25 antibody comprises a functional Fc region. In some of the embodiments, the anti-CD25 antibody is an antibody fragment. In some of the embodiments, the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof. In some of the embodiments, the anti-CD25 antibody is basiliximab. In some of the embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some of the embodiments, the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0104] In some of the embodiments, the immune checkpoint inhibitor is or includes an anti-PD-1 antibody or an antigen-binding fragment thereof. In some of the embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), and the like. ), pidilizumab (CT-011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), GLS-010, CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, and TSR-042 (ANB011).

[0105] In some of the optional embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab (MPDL3280A, Tecentriq), avelumab (BAVENCIO), durvalumab (MEDI4736, IMFINZI), LDP, NM-01, STI-3031, KN035, LY3300054, M7824 (MSB0011359C), BMS-936559, M Selected from the group consisting of SB2311, BCD-135, BGB-A333, CBT-502, cosibelimab (CK-301), CS1001, FAZ053, MDX-1105, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, LY3415244, REGN3504, and HLX20.

[0106] In some of the optional embodiments, the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab (YERVOY), tremelimumab, AGEN1181, AGEN1884, ADU-1064, BCD-145, and BCD-217.

[0107] In some of the embodiments, the conjugate is administered to the subject prior to, simultaneously with, or after administration of the immune checkpoint inhibitor. In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In some of the embodiments, the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by administration of the immune checkpoint inhibitor to the subject 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor is administered simultaneously with administration of the conjugate, followed by administration of the immune checkpoint inhibitor to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate.

[0108] In some of the optional embodiments, the phthalocyanine dye is a Si-phthalocyanine dye. In some of the optional embodiments, the Si-phthalocyanine dye is IR700.

[0109] In some optional embodiments, the irradiation is administered 30 minutes to 96 hours after administration of the conjugate. In some optional embodiments, the irradiation is administered 24 hours ± 4 hours after administration of the conjugate.

[0110] In some of the embodiments, the primary or primary tumor is irradiated with a wavelength of 690±40 nm. In some of the embodiments, the primary or primary tumor is irradiated with a wavelength of 50 J / cm. 2 Or about 50 J / cm 2 Alternatively, the fiber is irradiated at a dose of 100 J / cm of fiber length or approximately 100 J / cm of fiber length.

[0111] In some optional embodiments, one or more of steps (1), (2) and (3) are repeated.

[0112] In some optional embodiments, the methods and uses also involve (4) administering an additional therapeutic agent or anti-cancer treatment.

[0113] In some of the optional aspects, the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell cancer, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerve, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0114] In some of any of the embodiments, the metastatic tumor cells are located in one, two, three or more tissues or organs different from the tissue or organ in which the primary or initial tumor is located. [The present invention 1001] 1. A method for treating cancer, comprising: (a) administering an anti-PD-1 antibody to a subject having cancer, including a primary tumor and a secondary population of tumor cells; (b) administering to the subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody; (c) After administration of the conjugate, primary tumors were at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm irradiating, wherein the secondary population is not directly irradiated; Including, the primary tumor and / or secondary populations are inhibited to a greater extent as compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone; The method. [The present invention 1002] The method of the present invention 1001, wherein the inhibition comprises one or more of an increase in tumor volume, tumor size or tumor mass of less than 20% or less than about 20%, or a reduction in tumor volume, tumor size or tumor mass, or a reduction in the number of tumor cells. [The present invention 1003] The method of the present invention 1002, wherein the reduction in tumor volume, tumor size or tumor mass, or number of tumor cells comprises a 30% reduction or more, or about a 30% reduction or more. [The present invention 1004] The method of any of claims 1001 to 1003, wherein the secondary population comprises metastatic tumor cells. [The present invention 1005] The method of any of claims 1001 to 1003, wherein the secondary population comprises invasive tumor cells. [The present invention 1006] The method of any of claims 1001 to 1005, wherein the secondary population comprises metastatic tumor cells and invasive tumor cells. [The present invention 1007] The method of any of claims 1001 to 1006, wherein the anti-PD-1 antibody is administered to the subject simultaneously with the conjugate. [The present invention 1008] The method of any of claims 1001 to 1007, wherein the anti-PD-1 antibody is administered to the subject within 24 to 48 hours of administering the conjugate. [The present invention 1009] The method of any of claims 1001 to 1008, wherein the anti-PD-1 antibody is administered to the subject within 24 hours ± 4 hours after administration of the conjugate. [The present invention 1010] The method of any of claims 1001 to 1009, wherein a first dose of the anti-PD-1 antibody is administered prior to administration of the conjugate. [The present invention 1011] The method of the present invention 1010, wherein the first dose of the anti-PD-1 antibody is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate. [The present invention 1012] The method of any of claims 1001 to 1011, wherein the anti-PD-1 antibody is administered to the subject one, two, three, four, five, six, seven, eight, nine, ten or more than ten times. [The present invention 1013] Any of the methods of inventions 1001 to 1012, wherein the anti-PD-1 antibody is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 administrations of the anti-PD-1 antibody to the subject after administration of the conjugate. [The present invention 1014] The method of any of claims 1001 to 1012, wherein the anti-PD-1 antibody is administered simultaneously with administration of the conjugate, followed by administration of the anti-PD-1 antibody to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate. [The present invention 1015] The method of any of claims 1001 to 1012, wherein the anti-PD-1 antibody is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate. [The present invention 1016] The method of any of claims 1001 to 1015, wherein the secondary population is comprised in a solid tumor. [The present invention 1017] The method of any one of 1001 to 1016, wherein the subject exhibits a complete response. [The present invention 1018] The method of any of claims 1001 to 1017, wherein the anti-CD25 antibody comprises a functional Fc region. [The present invention 1019] The method of any of claims 1001 to 1017, wherein the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biological product or biogeneric thereof. [The present invention 1020] The method of claim 1019, wherein the anti-CD25 antibody is basiliximab. [The present invention 1021] Anti-PD-1 antibodies include pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), and pidilizumab ( CT-011), genolimzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG Any of the methods of 1001 to 1020, wherein the antibody is selected from the group consisting of 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, and TSR-042 (ANB011). [The present invention 1022] The method according to any one of claims 1001 to 1021, wherein the phthalocyanine dye is a Si-phthalocyanine dye. [The present invention 1023] The method of claim 1022, wherein the Si-phthalocyanine dye is IR700. [The present invention 1024] The method of any one of claims 1001 to 1023, wherein the irradiation is carried out 30 minutes to 96 hours after administration of the conjugate. [The present invention 1025] The method of claim 1024, wherein the irradiation is performed 24 hours ± 4 hours after administration of the conjugate. [The present invention 1026] The method of any one of claims 1001 to 1025, wherein the primary tumor is irradiated with light of a wavelength of 690±40 nm. [The present invention 1027] Primary tumors were 50 J / cm 2 Or about 50 J / cm 2 Or the method according to any one of claims 1001 to 1026, wherein the radiation is irradiated at a dose of 100 J / cm of fiber length or about 100 J / cm of fiber length. [The present invention 1028] (d) Any of the methods of claims 1001 to 1027, further comprising the step of administering an additional therapeutic agent or anti-cancer treatment. [The present invention 1029] The method of any of claims 1001-1028, wherein one or more of steps (a), (b), (c) or (d) are repeated. [The present invention 1030] Any of the methods of claims 1001 to 1029, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, thyroid cancer, prostate cancer, head and neck cancer, digestive cancer, gastric cancer, small intestine cancer, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, peripheral nerve cancer, brain cancer, skeletal muscle cancer, smooth muscle cancer, bone cancer, adipose tissue cancer, cervical cancer, uterine cancer, reproductive organ cancer, lymphoma, and multiple myeloma. [The present invention 1031] The method of any of claims 1001-1030, wherein the secondary populations are located in one, two, three or more than three tissues or organs different from the tissue or organ in which the primary tumor is located. [The present invention 1032] Any of the methods of claims 1001 to 1031, wherein primary tumors and / or secondary populations are inhibited to a greater extent compared to administration of the conjugate followed by irradiation alone and compared to administration of the anti-PD-1 antibody alone. [The present invention 1033] 1. A method for producing an enhanced response in a subject having cancer, comprising: (a) administering an anti-PD-1 antibody to a subject having cancer, including a tumor; (b) administering to the subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody, wherein an anti-PD-1 antibody is administered prior to or simultaneously with the conjugate; (c) After administration of the conjugate, the tumor at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm Irradiating; Including, the enhanced response comprises an enhancement of the subject's systemic immunity compared to the subject's systemic immunity prior to administration of the conjugate followed by irradiation and the anti-PD-1 antibody; and / or the enhanced response comprises enhanced tumor inhibition compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone. The method. [The present invention 1034] The method of the present invention, wherein the enhanced response is additive or synergistic. [The present invention 1035] The method of any one of claims 1033 to 1034, wherein the inhibition comprises one or more of an increase in tumor volume, tumor size or tumor mass of less than 20% or less than about 20%, or a reduction in tumor volume, tumor size or tumor mass, or a reduction in the number of tumor cells. [The present invention 1036] The method of the present invention 1035, wherein the reduction in tumor volume, tumor size or tumor mass, or the number of tumor cells comprises a 30% reduction or more, or about a 30% reduction or more. [The present invention 1037] the tumor comprises a primary tumor and a secondary population of tumor cells, the primary tumor being irradiated and the secondary population not being directly irradiated; the tumor comprises a primary tumor and metastatic tumor cells, the primary tumor being irradiated and the metastatic tumor cells not being directly irradiated; and / or the tumor comprises a primary tumor and invasive tumor cells, the primary tumor is irradiated and the invasive tumor cells are not directly irradiated; Any of the methods of 1033 to 1036 of the present invention. [The present invention 1038] Any of the methods of claims 1034 to 1037, wherein the enhanced response is a synergistic response, the synergistic response comprising a synergistic reduction in primary tumor growth, tumor volume, tumor size or tumor mass, a synergistic reduction in the number of cells in a secondary population of subjects, a synergistic reduction in metastatic or invasive tumor cell growth, tumor volume, tumor size, tumor mass or number, or any combination thereof. [The present invention 1039] The method of any of claims 1033 to 1038, wherein the anti-PD-1 antibody is administered to the subject within 24 to 48 hours after administration of the conjugate. [The present invention 1040] The method of any of claims 1033 to 1039, wherein the anti-PD-1 antibody is administered to the subject within 24 hours ± 4 hours after administration of the conjugate. [The present invention 1041] The method of any of claims 1033 to 1040, wherein a first dose of the anti-PD-1 antibody is administered prior to administration of the conjugate. [The present invention 1042] The method of the present invention 1041, wherein the first dose of the anti-PD-1 antibody is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate. [The present invention 1043] Systemic immunity was assessed using cytotoxic T lymphocyte (CTL) activity assays, intratumoral T cell exhaustion assays, intratumoral effector T cell proliferation assays, T cell receptor diversity assays, and activated CD8 + T cell assay, circulating regulatory T cell (Treg) assay, intratumoral Treg assay, or CD8 + T cells: Any of the methods of claims 1033 to 1042, as measured by one or more of the Treg assays. [The present invention 1044] Any of the methods of claims 1033 to 1043, wherein systemic immunity is measured by a CTL activity assay using splenocytes or peripheral blood cells or bone marrow cells or lymph node cells, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject. [The present invention 1045] Any of the methods of claims 1033 to 1043, wherein systemic immunity is measured by an intratumoral T cell exhaustion assay using T cells collected from a primary tumor or a metastatic tumor cell mass or an invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject. [The present invention 1046] Any of the methods of claims 1033 to 1043, wherein systemic immunity is measured by an intratumoral effector T cell proliferation assay using T cells collected from a primary tumor or a metastatic tumor cell mass or an invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject. [The present invention 1047] Any of the methods of claims 1033 to 1043, wherein systemic immunity is measured by a T cell receptor diversity assay using T cells collected from a primary tumor or a metastatic tumor cell mass or an invasive tumor cell mass or peripheral circulation, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject. [The present invention 1048] Any of the methods of claims 1033 to 1043, wherein systemic immunity is measured by determining the presence, number or frequency of regulatory T cells (Treg) in tumor from a primary tumor or a metastatic tumor cell mass or an invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject, and / or the ratio of intratumoral Treg cells to intratumoral CD8+ T cells or intratumoral CD4+ T cells. [The present invention 1049] The method of any of claims 1033 to 1048, wherein the anti-CD25 antibody comprises a functional Fc region. [The present invention 1050] The method of any one of claims 1033 to 1049, wherein the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biological product or biogeneric thereof. [The present invention 1051] The method of claim 10, wherein the anti-CD25 antibody is basiliximab. [The present invention 1052] Anti-PD-1 antibodies include pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), Pidilizumab (CT-011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG Any of the methods of the present invention 1033 to 1051, wherein the antibody is selected from the group consisting of 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, and TSR-042 (ANB011). [The present invention 1053] The method of any of claims 1033 to 1052, wherein the anti-PD-1 antibody is administered to the subject one, two, three, four, five, six, seven, eight, nine, ten or more than ten times. [The present invention 1054] The method according to any one of claims 1033 to 1053, wherein the phthalocyanine dye is a Si-phthalocyanine dye. [The present invention 1055] The method of claim 1054, wherein the Si-phthalocyanine dye is IR700. [The present invention 1056] The method of any one of claims 1033 to 1055, wherein the irradiation is performed 30 minutes to 96 hours after administration of the conjugate. [The present invention 1057] The method of claim 1056, wherein the irradiation is performed 24 hours ± 4 hours after administration of the conjugate. [The present invention 1058] The method of any one of claims 1033 to 1057, wherein the tumor is irradiated with light of a wavelength of 690±40 nm. [The present invention 1059] The tumor is 50 J / cm 2 Or about 50 J / cm 2 Or the method of any one of claims 1033 to 1058, wherein the radiation is irradiated at a dose of 100 J / cm of fiber length or about 100 J / cm of fiber length. [The present invention 1060] (d) any of the methods of claims 1033 to 1059, further comprising the step of administering an additional therapeutic agent or anti-cancer treatment. [The present invention 1061] The method of any of claims 1033 to 1060, wherein one or more of steps (a), (b), (c) or (d) are repeated. [The present invention 1062] Any of the methods of claims 1033 to 1061, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, thyroid cancer, prostate cancer, head and neck cancer, digestive cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cancer of the cervix, cancer of the uterus, cancer of the reproductive organs, lymphoma, and multiple myeloma. [The present invention 1063] Any of the methods of claims 1033 to 1062, wherein the enhanced response comprises an additive or synergistic response compared to administration of the conjugate followed by irradiation alone, and compared to administration of the anti-PD-1 antibody alone. [Brief description of the drawings]

[0115] [Figure 1]1A-1B show the in vivo changes in tumor volume (FIG. 1A) and survival curves (FIG. 1B) in the mouse CT26-EphA2 clone c4D10 allograft model after treatment with anti-PD-1 antibody (RMP1-14), PC61-IR700 (anti-CD25 antibody-IR700 conjugate, no irradiation), PC61-IR700 PIT (anti-CD25 antibody-IR700 conjugate and irradiation), PC61-IR700 PIT combined with anti-PD-1 antibody, and saline control. [Diagram 2] Figures 2A-2B show the in vivo changes in tumor volume of irradiated primary tumors (Figure 2A) and non-irradiated distant tumors (Figure 2B) after treatment with anti-PD-1 antibody (RMP1-14), PC61-IR700, PC61-IR700 PIT, and the combination of PC61-IR700 PIT and anti-PD-1 antibody. [Diagram 3] Figures 3A-3B show the effect of exemplary anti-CD25 antibodies having a wild-type rat Fc region (PC61 rat WT), a wild-type mouse Fc region (PC61 mouse WT), or an N297Q mutant mouse Fc region (PC61 mouse N297Q) on circulating regulatory T cells (Tregs) in vivo 1 day (Figure 3A) and 8 days (Figure 3B) after antibody administration. [Figure 4] Figures 4A-4B show the synergistic effect of anti-PD-1 and anti-CD25 (wild-type Fc; mWT PC61)-IR700 PIT (Figure 4A) and anti-PD-1 and N297Q Fc mutant anti-CD25 (N297Q PC61)-IR700 PIT (Figure 4B) on non-irradiated distant tumors. [Diagram 5] FIG. 5 shows the percentage of intratumoral CD3+ CD4+ FoxP3+ regulatory T (Treg) cells among CD45+ cells from tumor-bearing mice treated with anti-CD25-IR700 with irradiation (PIT), anti-CD25-IR700 conjugate without irradiation (conjugate) or saline control. [Figure 6]Figure 6A shows a comparison of intratumoral CD8+ T cells in mice treated with anti-CD25-IR700 PIT, saline control, and conjugate only (no irradiation) control. Figure 6B shows a comparison of intratumoral exhausted CD8+ T cells in mice treated with anti-CD25-IR700 PIT compared to saline control and conjugate only (no irradiation) control. Figure 6C shows a comparison of intratumoral effector CD8+ T cells in mice treated with anti-CD25-IR700 PIT, saline control, and conjugate only (no irradiation) control. [Figure 7] FIG. 7 shows the effect of anti-CD25 PIT on in vivo intratumoral CD8+ T cell activation from tumor-bearing mice treated with anti-CD25-IR700 with irradiation (PIT), anti-CD25-IR700 conjugate without irradiation (conjugate), or saline control. [Figure 8] Figures 8A-8B show the ratios of intratumoral total CD8+ T cells (CD3+ CD8+) to CD3+CD4+FoxP3+ Tregs 2 hours after irradiation (Figure 8A) or 8 days after irradiation (Figure 8B) from tumor-bearing mice treated with anti-CD25-IR700 with irradiation (PIT), anti-CD25-IR700 conjugate without irradiation (conj.) or saline control. [Figure 9] FIG. 9 shows the results for contralateral tumor rechallenge in animals previously treated with anti-CD25 antibody-IR700 and irradiation (PC61-IR700 PIT) or a combination of PC61-IR700 PIT and anti-PD-1 antibody (RMP1-14), both of which resulted in complete responses after initial treatment, as well as the change in tumor volume in naive (control) animals. [Figure 10] FIG. 10 shows the results for tumor rechallenge of a different tumor type (4T1) in mice previously treated with anti-CD25 antibody-IR700 and radiation (PC61-IR700 PIT) in combination with anti-PD-1 antibody (RMP1-14) that showed a complete response after rechallenge with the same tumor type as the initial tumor, as well as the change in tumor volume in naive (control) animals. [Figure 11]FIG. 11 shows cytotoxicity against CT26 or unrelated tumor cells after priming splenocytes from complete response (CR) mice treated with anti-CD25 antibody-IR700 and irradiation (PC61-IR700 PIT) or a combination of PC61-IR700 PIT and anti-PD-1 antibody (RMP1-14) with tumor-specific antigens followed by co-incubation at effector:target ratios of 30:1, 15:1, and 7:1 (or 250:1 for unrelated tumor cells). [Figure 12] Figures 12A-12B show the in vivo changes in tumor volume of irradiated primary tumors (Figure 12A) and non-irradiated distant tumors (Figure 12B) after treatment with anti-PD-1 antibody (RMP1-14), anti-CD25 antibody-IR700 and irradiation (PC61-IR700 PIT), the combination of PC61-IR700 PIT and anti-PD-1 antibody, and the combination of PC61-IR700 PIT and anti-PD-1 antibody under CD8+ T cell depletion conditions. [Figure 13-1] FIG. 13A shows the percentage of total FoxP3+ Tregs among intratumoral CD3+ CD4+ T cells from tumor-bearing mice treated with anti-CD25-IR700 conjugate PIT (CD25 PIT), anti-CD25-IR700 conjugate without irradiation (CD25 Conj.), anti-CD25-IR700 conjugate PIT with anti-PD-1 antibody (CD25 PIT+PD1), anti-CD25-IR700 conjugate without irradiation with anti-PD-1 antibody (CD25 Conj.+PD1) and saline control. FIG. 13B shows the ratio of intratumoral CD4+FoxP3- helper T cells to CD4+FoxP3+ Treg cells from tumor-bearing mice treated with anti-CD25-IR700 conjugate PIT (CD25 PIT), anti-CD25-IR700 conjugate without irradiation (CD25 Conj.), anti-CD25-IR700 conjugate PIT with anti-PD-1 antibody (CD25 PIT+PD1), anti-CD25-IR700 conjugate without irradiation with anti-PD-1 antibody (CD25 Conj.+PD1) and saline control. [Figure 13-2]FIG. 13C shows the ratio of intratumoral CD8+ T cells to FoxP3+ Treg cells from tumor-bearing mice treated with anti-CD25-IR700 conjugate PIT (anti-CD25-IR700 PIT), anti-CD25-IR700 conjugate without irradiation (anti-CD25-IR700), anti-CD25-IR700 conjugate PIT with anti-PD-1 antibody (anti-CD25-IR700 PIT+anti-PD1), anti-CD25-IR700 conjugate without irradiation with anti-PD-1 antibody (anti-CD25-IR700+anti-PD1) and saline control. [Figure 14] FIG. 14 shows an illustration of the proposed mechanism of action for depletion of intratumoral Treg cells by anti-CD25-IR700 PIT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0116] Detailed Description Provided herein are compositions, combinations and methods for treating cancer, such as cancer that includes not only primary tumor or primary tumor or multiple primary tumors but also metastatic tumor cells, such as metastatic cancer; and / or cancer that includes not only primary tumor or multiple primary tumors but also invasive or infiltrating tumor cells, such as invasive cancer or infiltrating cancer.Also provided are compositions, combinations and methods for enhancing systemic immunity in subjects, such as subjects with cancer, such as invasive cancer, infiltrating cancer or metastatic cancer.Also provided are compositions, combinations and methods for generating enhanced responses, such as enhanced responses to treatment or therapy in subjects, such as subjects with cancer or tumor, such as invasive cancer, infiltrating cancer or metastatic cancer.

[0117] The provided compositions, combinations, methods and uses can be used to treat cancer, including primary or primary tumors, metastatic tumor cells and / or invasive tumor cells.In some embodiments, the phthalocyanine dye conjugated with a targeting molecule that binds to CD25 is used alone or in combination with an immune checkpoint inhibitor.The methods and uses described herein provide various advantages in treating cancer, such as metastatic cancer and / or invasive cancer, including not needing to locate and / or directly irradiate metastatic tumor cells and / or invasive tumor cells.The present disclosure also provides unexpected features in enhancing systemic immunity in a subject, for example against cancer recurrence.

[0118] The embodiments provided are based on the observation that in some circumstances, treatment of cancer with a phthalocyanine dye-targeting molecule conjugate, e.g., an anti-CD25 antibody-IR700 conjugate, followed by irradiation of the primary or primary tumor, not only results in treatment of the irradiated tumor, e.g., the irradiated primary or primary tumor, but also results in effective treatment of tumors distal to the irradiation site (e.g., metastatic tumors), and tumors derived after the subject has had a complete response following treatment of the initial tumor, suggesting a tumor-specific immune memory response. The embodiments provided are based on the further observation that combination treatment of an anti-CD25 antibody-IR700 conjugate with an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, results in significant synergistic effects in the treatment of both the irradiated primary or primary tumor and distant or later derived tumors, e.g., tumors containing a secondary population of tumor cells, metastatic tumors, and / or invasive tumors. Therefore, it is demonstrated that the provided compositions, combinations, methods and uses provide substantially improved and effective treatment of cancer, including cancer that includes not only primary tumor or primary tumor or multiple primary tumors but also metastatic tumor cells, such as metastatic cancer; and / or cancer that includes not only primary tumor or primary tumor or multiple primary tumors but also invasive tumor cells, such as invasive cancer.The provided compositions, combinations, methods and uses can result in the subject's immune response, for example, the enhancement or improvement of the systemic immune response against cancer, including immune memory response, which can be effective against tumors that may develop after treatment.

[0119] The methods and uses provided herein include treating a subject having one or more primary tumors, e.g., primary tumors, and optionally secondary cell populations, e.g., metastatic tumor cells and / or invasive tumor cells, with a conjugate comprising a phthalocyanine dye linked to a targeting molecule, such as a targeting molecule that binds to CD25, and irradiating the one or more primary tumors or primary tumors with a wavelength of light suitable for use with the phthalocyanine dye after administration of the conjugate. Some embodiments of the method include administering an immune checkpoint inhibitor prior to, simultaneously with, or subsequent to administration of the conjugate.

[0120] I. Methods and Uses for Treating Cancer, E.g., Invasive or Metastatic Cancer In some embodiments, methods and uses are provided for the use of a composition containing a phthalocyanine dye-targeting molecule conjugate, where the targeting molecule binds to CD25 (e.g., an anti-CD25 antibody-IR700 conjugate), for the treatment or treatment of cancer, for example, cancer that includes not only a primary tumor or multiple tumors (e.g., one or more primary tumors), but also a secondary population of cancer cells, for example, metastatic tumor cells (e.g., metastatic cancers), invasive tumor cells (e.g., invasive cancers), or invasive tumor cells (e.g., invasive cancers). In some embodiments, the secondary population of cancer cells is associated, for example, directly or indirectly, with the primary tumor. In some embodiments, the secondary cell population is not directly derived from the primary tumor. The methods and uses provided include therapeutic methods and uses, for example, therapeutic methods and uses involving administration of the conjugate to a subject with cancer, followed by irradiation (or radiation) of a tumor (such as a primary tumor) or tumor microenvironment associated with the cancer, using a specific wavelength and dose of light. In some aspects, irradiation (or radiation) causes irradiation-dependent lysis and death of cells expressing target molecules (e.g., CD25), resulting in a therapeutic effect or treatment of cancer (sometimes referred to as photoimmunotherapy (PIT)). In some aspects, the method also involves administering an immunomodulator, such as an immune checkpoint inhibitor (e.g., anti-PD-1 antibody), in combination with the phthalocyanine dye-targeting molecule conjugate. In some aspects, a combination of a phthalocyanine dye-targeting molecule conjugate and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody) is used in the provided methods and uses, for example, the provided methods and uses for the treatment of cancer.

[0121] The uses include the use of the compositions and combinations in such methods and treatments, and the use of such compositions and combinations in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the methods and uses thereby treat cancer in a subject, including a primary tumor (either primary or non-primary tumor) and one or more secondary populations of tumor cells (e.g., metastatic tumor cells and / or invasive tumor cells), such as tumors and cancers that include metastatic and / or invasive cancers. In some embodiments, the secondary tumor cells are associated with the primary tumor. In some embodiments of the methods and uses, more than one tumor is treated. In some aspects, methods and uses of such compositions and combinations in enhancing, increasing, boosting, strengthening, increasing, boosting or supporting immune function, such as systemic immunity, in a subject are also provided.

[0122] The method includes administering to a subject having a primary tumor a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25, and irradiating at least the primary tumor with a wavelength of light appropriate for the selected phthalocyanine dye after administration of the conjugate. In some embodiments, the method includes administering an immune checkpoint inhibitor, such as an anti-PD-1 antibody, prior to, simultaneously with, or subsequent to administration of the conjugate. In some embodiments, the method further comprises administering an additional therapeutic agent or anti-cancer treatment.

[0123] In some embodiments, the method involves irradiating a tumor or tumor microenvironment (tumor microenvironment; TME) associated with cancer, or cells present in the TME, with light. In some aspects, the tumor or TME is irradiated with a wavelength of light suitable for treatment or treatment. In some embodiments, the wavelength of light suitable for use with the phthalocyanine dye includes light having a wavelength that achieves activation of the dye-conjugate by irradiating with absorbed light, and excites the photosensitizer to cause cell killing, thereby reducing or eliminating the lesion (e.g., tumor), reducing or inhibiting tumor growth, reducing, inhibiting or eliminating secondary populations of tumor cells such as tumor cell metastasis, reducing, inhibiting or eliminating invasive and / or metastatic tumor cells, or any combination thereof.

[0124] In some embodiments, the radiation is at a wavelength of about 500 nm to 900 nm, about 600 nm to 850 nm, about 650 nm to 800 nm, or about 660 nm to 740 nm. In some embodiments, the radiation is at a wavelength of 690+50 nm or at or about 690+20 nm.

[0125] The irradiation should be at least 1 J / cm 2 or 1 J / cm of fiber length. In some embodiments, the lesion is irradiated at a dose of 2 J / cm 2 Or about 2 J / cm 2 to about 400 J / cm 2 or at a dose of about 2 J / cm to about 500 J / cm of fiber length. In some embodiments, the irradiation is at least 2 J / cm 2 , 5J / cm 2 , 10J / cm 2 , 25J / cm 2 , 50J / cm 2 , 75J / cm 2 , 100J / cm 2 , 150J / cm 2 , 200J / cm 2 , 300J / cm 2 , 400J / cm 2 Or 500 J / cm2 , or at least about 2 J / cm 2 , 5J / cm 2 , 10J / cm 2 , 25J / cm 2 , 50J / cm 2 , 75J / cm 2 , 100J / cm 2 , 150J / cm 2 , 200J / cm 2 , 300J / cm 2 , 400J / cm 2 Or 500 J / cm 2 or the lesion is irradiated with a dose of at least or at least about 2 J / fiber length cm, 5 J / fiber length cm, 10 J / fiber length cm, 25 J / fiber length cm, 50 J / fiber length cm, 75 J / fiber length cm, 100 J / fiber length cm, 150 J / fiber length cm, 200 J / fiber length cm, 250 J / fiber length cm, 300 J / fiber length cm, 400 J / fiber length cm, or 500 J / fiber length cm.

[0126] In some embodiments, the irradiation is 25 J / cm 2 to 400 J / cm 2 or irradiation at a dose of about 2 J / cm fiber length to about 500 J / cm fiber length. In some embodiments, the irradiation is at a dose of 5 J / cm 2 from 200 J / cm 2 or irradiation at a dose of 20 J / cm to 500 J / cm of fiber length. In some embodiments, the irradiation is at about 50 J / cm 2 Or irradiation with a dose of 100 J / cm of fiber length.

[0127] In some embodiments of the methods and uses provided herein, the irradiation is accomplished after administration of the phthalocyanine dye-targeting molecule conjugate. In some embodiments, the irradiation or exposure is performed or accomplished 30 minutes to 96 hours or about 30 minutes to 96 hours after administration of the phthalocyanine dye-targeting molecule conjugate (e.g., an IR700-anti-CD25 antibody conjugate), for example, 30 minutes to 48 hours, 30 minutes to 24 hours, or 12 hours to 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 more after administration of the conjugate. In some embodiments, irradiation is performed within about 24 hours after administration of the conjugate, or within 24 hours ±4 hours after administration of the conjugate, or within about 20, 21, 22, 23, 24, 24, 26, 27, or 28 hours after administration of the conjugate.

[0128] The methods described herein include irradiating a primary tumor in a subject, such as a primary tumor, or the tumor microenvironment (TME) of a primary tumor. In some embodiments, the methods and uses provided herein include treating a subject with one or more tumors. A subject may have one, two, three or more than three tumors. Such tumors may be in one or more tissues or organs, for example, in one tissue or organ, in two different tissues or organs, in three different tissues or organs, or in more than three different tissues or organs.

[0129] In some aspects, primary tumor can refer to the first or original tumor in a subject, but also to one or more tumors selected for irradiation by the methods and uses provided herein.In some embodiments, the first or additional tumor can be one solid tumor or multiple solid tumors, can be lymphoma, or can be leukemia.The tumor can be lung, stomach, liver, pancreas, breast, esophagus, head and neck, brain, peripheral nerve, skin, small intestine, colon, rectum, anus, ovary, uterus, bladder, prostate, adipose tissue, skeletal muscle, smooth muscle, blood vessel, bone, bone marrow, eye, tongue, lymph node, spleen, kidney, cervix, male reproductive organs, female reproductive organs, testis, or tumor of unknown primary origin.

[0130] In some embodiments of the method, the growth of the primary or primary tumor is inhibited, the volume of the primary or primary tumors is reduced, or both the growth and volume of the tumor are reduced. In some embodiments of the method, the growth of the primary or primary tumor is inhibited, the volume of the primary or primary tumors is reduced, or both the growth and volume of the tumor are reduced compared to a monotherapy, such as administration of only the conjugate, only the conjugate followed by radiation, or only the anti-PD-1 antibody.

[0131] In some embodiments, the methods and uses provided herein include treating a subject having one or more tumors and also a secondary population of tumor cells, such as invasive tumor cells. In some such embodiments, the secondary population includes cells arising from a tumor, such as a primary tumor, that have invaded surrounding tissue. The method includes administering to a subject having a primary tumor and invasive tumor cells a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25, and irradiating the primary tumor with a wavelength appropriate for the selected phthalocyanine dye after administration of the conjugate. In some such embodiments, the secondary population of tumor cells is not directly irradiated. In some embodiments, the method includes administering an immune checkpoint inhibitor before, simultaneously with, or following administration of the conjugate. In some embodiments, the method includes administering an anti-PD-1 antibody before, simultaneously with, or following administration of an anti-CD25 antibody-IR700 conjugate, and administration of the conjugate is followed by irradiation of the primary tumor.

[0132] In some aspects, invasive tumor cells refer to cells that originate from a primary tumor and invade the surrounding tissues of the same organ or adjacent organ of the primary tumor in the body of a subject with the primary tumor.In some embodiments, the primary tumor is a primary tumor, and the invasive tumor cells are derived directly or indirectly from the primary tumor.In some embodiments, the invasive tumor cells are not derived directly from the primary tumor.

[0133] The methods and uses provided herein include irradiation of primary tumor and / or additional tumor, but not irradiating some or all of invasive tumor cells.In some embodiments, the growth of invasive tumor cells is inhibited, reduced or eliminated, the volume, size or mass of one or more invasive tumors is reduced, or any combination thereof is brought about.In some embodiments, the growth of primary tumor is inhibited, reduced or eliminated, and the volume, size or mass of primary tumor or additional tumor is reduced, together with the effect on one or more invasive tumor cells.

[0134] In some embodiments, the invasive tumor cells are contained in solid tumors. In some embodiments, the invasive tumor cells are contained in body fluids, including but not limited to peritoneal fluid, pleural fluid and cerebrospinal fluid. In some embodiments, the invasive tumor cells are contained in the effusion of one or more body cavities, including but not limited to peritoneal effusion (ascites), pleural effusion and pericardial effusion.

[0135] In some embodiments, the methods and uses provided herein include treating a subject having a primary tumor and also a secondary population of tumor cells, such as invasive and / or metastatic tumor cells (e.g., a secondary population of associated tumor cells). The method includes administering a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25, to the subject having a primary tumor and also a secondary population of tumor cells, such as invasive and / or metastatic tumor cells (e.g., a secondary population of associated tumor cells), and irradiating the primary tumor with a wavelength appropriate for the selected phthalocyanine dye after administration of the conjugate. In some such embodiments, the secondary population of tumor cells is not directly irradiated. In some embodiments, the method includes administering an immune checkpoint inhibitor, such as an anti-PD-1 antibody, before, simultaneously with, or subsequent to administration of the conjugate. In such methods, the growth (volume, size or mass) of primary tumors and / or secondary populations of tumor cells, such as metastatic tumor cells, is inhibited, reduced or eliminated, resulting in a reduction in the volume, size or mass of one or more of the primary tumor and / or secondary cell populations, or any combination thereof. In some embodiments, inhibition of primary tumors and / or secondary populations is achieved to a greater extent than inhibition achieved by administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone. In some embodiments, inhibition is achieved when the tumor exhibits less than a 20% increase in tumor volume, tumor size or tumor mass; when there is no change in tumor volume, size or mass (i.e., halted tumor growth or progression); or when the tumor is reduced in terms of volume, size or mass; or when there is a reduction in the number of tumor cells. In some aspects, the reduction in tumor volume, tumor size or tumor mass, or the number of tumor cells, comprises a 30% reduction or more, or about a 30% reduction or more.

[0136] In any of the methods and uses herein, the primary tumor can be a primary tumor or a secondary tumor. In some embodiments, the primary tumor and the secondary population are related. In some embodiments, the secondary cell population is derived directly or indirectly from the primary tumor. In some embodiments, the secondary population is not derived from the primary tumor. In some embodiments, the primary tumor is a primary tumor and the secondary cell population is related to the primary tumor; for example, the secondary cell population is derived directly or indirectly from the primary tumor. In some embodiments, the primary tumor is a primary tumor and the secondary population of tumor cells is a secondary primary tumor. In some embodiments, the primary tumor is a secondary tumor and the secondary cell population is related to the secondary tumor. In some aspects, the secondary population of tumors includes cells that arise from the primary tumor and invade nearby or distant healthy tissues (i.e., invasive tumor cells), or cells that spread to one or more distant tissues or organs in the body of a subject with a primary tumor, such as tissues or organs located remotely or far away from the primary tumor (i.e., metastatic tumor cells). In some aspects, the secondary population of tumor cells is both invasive and metastatic. In some aspects, the secondary population of tumor cells is invasive. In some aspects, the secondary population of tumor cells is metastatic and is directly or indirectly related to, e.g., derived from, the primary tumor. In other aspects, the secondary population of tumor cells is metastatic and is not directly related to the primary tumor. Metastatic tumor cells can be located in one or more locations of lung, stomach, liver, pancreas, breast, esophagus, head and neck, brain, peripheral nerve, skin, small intestine, colon, rectum, anus, ovary, uterus, bladder, prostate, adipose tissue, skeletal muscle, smooth muscle, blood vessel, bone, bone marrow, eye, tongue, lymph node, spleen, kidney, cervix, male reproductive organs, female reproductive organs, testis, blood, bone marrow, cerebrospinal fluid, or any other tissue or organ.In some embodiments, metastatic tumor cells are contained in solid tumors.In some embodiments, metastatic tumor cells are circulating tumor cells, liquid tumors, or are not associated with tumor mass.

[0137] In some embodiments of the methods and uses provided herein, secondary tumor cells are metastatic tumor cells that are distant from primary tumors, and some or all of metastatic tumor cells are not irradiated, for example, are not directly irradiated.In some embodiments of the methods and uses, after administration of the conjugate, only primary tumor is irradiated, and invasive or metastatic tumor cells are not directly irradiated.In some embodiments, more than one tumor is irradiated, including primary tumor, but at least one site of tumor cells, for example, the site containing metastatic tumor cells, is not irradiated.

[0138] II. Methods for enhancing systemic immunity and / or responses Also provided herein are methods and uses of compositions and combinations in enhancing, increasing, boosting or supporting immune function, such as systemic immunity, in a subject, for example, a subject with cancer or tumor. In some embodiments, the methods and uses herein include enhancing systemic immunity in a subject with cancer, tumor or cancerous lesion. In some aspects, "systemic immunity" refers to the ability of a subject's immune system to respond systemically to immunological attacks, including those associated with cancer or tumor. In some aspects, systemic immunity can include a systemic response of the subject's adaptive immune system and / or innate immune system. In some aspects, systemic immunity can include immune responses across various tissues, including bloodstream, lymph nodes, bone marrow, spleen and / or tumor microenvironment, and in some cases, includes tissues and organs and coordinated responses between various cells and factors of tissues and organs. Also provided herein are methods and uses of compositions and combinations in enhancing, increasing or boosting response to treatment or therapy in a subject, for example, a subject with cancer or tumor.

[0139] In some aspects, the methods and uses provided herein include administering to a subject a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25, administering an immune checkpoint inhibitor, and irradiating the tumor or cancerous lesion, or tumor microenvironment, after administration of the conjugate. The conditions of irradiation, with respect to wavelength, dose of irradiation, and timing of irradiation, are, for example, as described herein. The immune checkpoint inhibitor can be administered prior to, simultaneously with, or subsequent to administration of the conjugate, such as described herein. In some aspects, the methods and uses provided herein result in enhanced systemic immunity in the subject, which in turn can result in an enhanced or synergistic response to a cancer treatment or therapy. In some embodiments, the methods and uses provided herein result in an enhanced response, e.g., a synergistic response, to a cancer or tumor treatment or therapy, compared to administration of only the conjugate, only the conjugate followed by irradiation, or only the anti-PD-1 antibody. In some aspects, the enhanced response includes enhanced systemic immunity in the subject compared to the subject's systemic immunity prior to administration of the conjugate followed by irradiation and the anti-PD-1 antibody. In some aspects, the enhanced response includes an enhanced response to treatment, e.g., an additional, additive or synergistic response, and / or a more complete, more durable or longer lasting response, compared to administration of only the conjugate, only the conjugate followed by irradiation, or only the anti-PD-1 antibody.

[0140] In some embodiments of the methods and uses provided herein, systemic immunity to recurrent tumors is increased or enhanced. In some aspects, the level, strength or extent of systemic immunity is increased or enhanced by intratumoral CD8 + T lymphocyte count, CD8 + T lymphocytes and regulatory T cells (T reg ), intratumoral T lymphocyte exhaustion (e.g., CD3+ expressing PD-1 and / or CTLA4 markers), + CD8 +cells), intratumoral activated CD8 + The number or percentage of T lymphocytes (e.g., CD45 + Ki67 as a percentage of cells + or CD69 + CD8 cells), expansion of cytotoxic intratumoral T lymphocytes based on splenic cell cytotoxicity against tumor cells (e.g., CD3 + CD8 + In some aspects, the intratumor CD8 + T lymphocytes are CD3 + CD8 + Intratumoral exhausted T lymphocytes, including PD-1 + CTLA-4 + CD3 + CD8 + Activated intratumoral CD8+ T lymphocytes contain CD3 + CD8 + Ki67 + and / or CD3 + CD8 + CD69 + The expansion of cytotoxic T lymphocytes, including PD-1 - CTLA-4 - CD3 + CD8 + In some aspects, intratumoral CD8 + T lymphocytes, exhausted intratumoral T lymphocytes, activated CD8 + T lymphocytes, or expanded cytotoxic intratumoral T lymphocytes, are expressed by leukocytes (CD45 + cells) and / or total CD8 + T cells (e.g., CD3 + CD8 + CD45 +The number or percentage is measured as a percentage of the total number of cells (cells). Determining such a number or percentage can be accomplished using several well-known methods, including those described herein. For example, such a number or percentage can be determined by generating a single cell suspension, such as by mechanical dissociation of a tumor and / or tissue biopsy or a collection of blood samples containing circulating immune cells, followed by staining and flow cytometric analysis or mass cytometry. Other methods can include multiplexed immunofluorescence imaging of tissue and / or tumor biopsies.

[0141] In some such embodiments, the strength or extent of immunity is compared to the strength or extent of immunity in the same subject before treatment. In some such embodiments, the strength or extent of immunity is compared to a population of subjects. In some such embodiments, the strength or extent of immunity is compared to a threshold value. In some embodiments, the strength or extent of immunity after combination therapy, such as the combination of administration of anti-CD25 PIT and checkpoint inhibitor (e.g., anti-PD-1 antibody), is compared to the strength or extent of immunity after treatment with a monotherapy, such as administration of an immune checkpoint inhibitor (e.g., anti-PD-1 antibody) or a single agent, such as anti-CD25 conjugate or anti-CD25 PIT alone.

[0142] In some embodiments, the strength or extent of immunity is determined by CD3 + CD8 + Intratumoral CD8 such as T lymphocytes + Measured by the number of T lymphocytes, CD45 + Intratumoral CD8 cells as a percentage of total cells + T lymphocytes (e.g., CD3 + CD8 + If the proportion of intratumoral CD8 T lymphocytes is increased after treatment compared to before treatment, systemic immunity to the recurrent tumor is increased or augmented. In some such instances, intratumoral CD8 + T lymphocytes (e.g., CD3 + CD8 + T lymphocytes) are CD45 +At least 30% or at least about 30% of the total number of cells, e.g., CD45 + Systemic immunity against recurrent tumors is increased or enhanced if at least or at least about 30%, 35%, 36%, 37%, 38% 39%, 40%, 41%, 42% 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% or more of the total number of cells. In some embodiments, intratumor CD8 + The proportion of T lymphocytes expressing intratumoral CD45 + At least 40% of the cell population. In some embodiments, intratumoral CD45 + Intratumoral CD3 cell population + CD8 + If the proportion of T cells is increased after treatment compared to before treatment, systemic immunity to recurrent tumors is increased or augmented. In some such embodiments, intratumoral CD45 + Intratumoral CD3 cell population + CD8 + The percentage of T cells is increased by at least or at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% or more after treatment compared to before treatment. + Intratumoral CD3 cell population + CD8 + The percentage of T cells is increased by at least 10% after treatment compared to before treatment.

[0143] In some embodiments, the strength or extent of immunity is determined by the expression of exhausted intratumoral CD8 +Number of T lymphocytes, e.g., intratumoral CD8 + PD-1 as a percentage of T lymphocytes (e.g., CD3+CD8+ T lymphocytes) + CTLA-4 + CD3 + CD8 + Intratumoral CD3 + CD8 + PD-1 on T cells + CTLA-4 + CD3 + CD8 + If the proportion of cells decreases after treatment compared to before treatment, systemic immunity to the recurrent tumor is increased or enhanced. In some such examples, intratumoral CD3 + CD8 + PD-1 on T cells + CTLA-4 + CD3 + CD8 + If the percentage of cells is less than or about 20%, e.g., less than 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, systemic immunity to the recurrent tumor is increased. + CD8 + PD-1 on T cells + CTLA-4 + CD3 + CD8 + If the proportion of cells is reduced after treatment compared to before treatment, systemic immunity to the recurrent tumor is increased or enhanced. In some such embodiments, intratumoral CD3 + CD8 + PD-1 on T cells + CTLA-4 + CD3 + CD8 +The percentage of intratumoral CD3 cells is reduced by at least or at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% or more compared to before treatment. + CD8 + Exhausted intratumoral CD8 T cell population + T cells (PD-1 + CTLA-4 + CD3 + CD8 + The percentage of inflammatory cells (IFN-glucose, ...

[0144] In some embodiments, the strength or extent of immunity is measured by the expression of activated intratumoral CD8+ T lymphocytes, e.g., CD3 + CD8 + Ki67 + and / or CD3 + CD8 + CD69 + Measured by the number of T lymphocytes and intratumoral CD45 + Activated intratumoral CD8+ T lymphocytes as a percentage of white blood cells, e.g., CD3 + CD8 + Ki67 + and / or CD3 + CD8 + CD69 + If the number of T lymphocytes is increased after treatment compared to before treatment, systemic immunity to the recurrent tumor is increased or augmented. In some such embodiments, intratumoral CD3 + CD8 + Ki67 + The number of cells was increased after treatment by CD45 + At least 0.15% or at least about 0.15% of the total number of cells, e.g., intratumoral CD45 +If the total number of cells is at least 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% or more, or at least about 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% or more, systemic immunity to recurrent tumors is increased or enhanced. In other such embodiments, intratumoral CD3 + CD8 + CD69 + The number of cells was increased after treatment by CD45 + At least 0.5% or at least about 0.5% of the total number of cells, e.g., intratumoral CD45 + at least 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0% or more of the total number of cells, or at least about 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0% or more Systemic immunity to recurrent tumors is increased or enhanced if intratumoral CD45+ cells are 0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0% or more, e.g., at least about 1.0% of the total number of intratumoral CD45+ cells. + Intratumoral CD3 cells + CD8 + Ki67 + and / or CD3 + CD8 + CD69 + If the proportion of T lymphocytes is increased after treatment compared to before treatment, systemic immunity to the recurrent tumor is increased or augmented. In some such embodiments, intratumoral CD45 + CD3 on cells + CD8 + Ki67 + and / or CD3 + CD8 + CD69 +The percentage of T lymphocyte cells was determined by the pretreatment intratumoral CD45 + CD3 on cells + CD8 + Ki67 + and / or CD3 + CD8 + CD69 + at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 or more times higher than the percentage of T lymphocyte cells In some embodiments, intratumor CD45 is increased by at least about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold or more. + Intratumoral CD3 cells + CD8 + Ki67 + The percentage of T lymphocyte cells was determined by the pretreatment intratumoral CD45 + CD3 on cells + CD8 + Ki67 + In some embodiments, the percentage of intratumoral CD45 + Intratumoral CD3 cells + CD8 + CD69 + The percentage of T lymphocyte cells was determined by the pretreatment intratumoral CD45 + CD3 on cells + CD8 + CD69 + The proportion of T lymphocytes is increased by at least 5-fold compared to that of T lymphocytes.

[0145] In some embodiments, the strength or extent of immunity is measured by the expression of intratumoral cytotoxic T lymphocytes, e.g., PD-1 - CTLA-4 - CD3 + CD8+ Measured by cell expansion, CD8 + T cells (e.g., CD3 + CD8 + T cells) in the tumor. - CTLA-4 - CD3 + CD8 + If the proportion of intratumoral cytotoxic T lymphocytes (e.g., PD-1 cells) increases after treatment compared to before treatment, systemic immunity to the recurrent tumor is increased or augmented. In some such examples, intratumoral cytotoxic T lymphocytes (e.g., PD-1 - CTLA-4 - CD3 + CD8 + The number of CD3 + CD8 + At least 20% or at least about 20% of the total number of T cells, e.g., CD45 + at least 25%, 30%, 35%, 40%, 41%, 42% 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65% or more of the total number of cells, or at least about 25%, 30%, 35%, 40%, 41%, 42% If intratumoral PD-1 is 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65% or more, systemic immunity to the recurrent tumor is increased or augmented. - CTLA-4 - CD3 + CD8 + The percentage of cells in the tumor was + CD8 + At least about or at least 40%, 45%, 50% or 55% of the T cell population. In some embodiments, intratumoral CD3 + CD8 + Intratumoral PD-1 in T cell populations - CTLA-4 -CD3 + CD8 + If the proportion of cells is increased after treatment compared to before treatment, systemic immunity to the recurrent tumor is increased or augmented. In some such embodiments, intratumoral CD3 + CD8 + Intratumoral PD-1 in T cell populations - CTLA-4 - CD3 + CD8 + The percentage of cells in the tumor before treatment was + CD8 + Intratumoral PD-1 in T cell populations - CTLA-4 - CD3 + CD8 + In some embodiments, the percentage of intratumoral CD3 + CD8 + Intratumoral PD-1 in T cell populations - CTLA-4 - CD3 + CD8 + The percentage of cells in the tumor before treatment was + CD8 + Intratumoral PD-1 in T cell populations - CTLA-4 - CD3 + CD8 + The percentage of cells that is increased by at least 30% after treatment.

[0146] In some embodiments, treatment according to the methods and uses provided herein leads to cell death or a reduction in the number of regulatory T cells (Tregs), e.g., intratumoral CD4+FoxP3+ Tregs. Thus, in some embodiments, the level, strength, or extent of systemic immunity is increased by the increase in intratumoral or circulating regulatory T cells (T reg) can be measured based on the number or percentage of CD25-expressing cells. In some aspects, binding of anti-CD25 conjugates to the surface of CD25-expressing cells, such as certain Tregs, and irradiation to effect radiation-dependent lysis and death of the cells expressing CD25 results in a reduction in the number of cells expressing CD25. In some aspects, such results can lead to a reduction in the number of immunosuppressive cells, such as Tregs, in the tumor, thus alleviating or reversing immunosuppression in the tumor. In some aspects, such a reduction in immunosuppressive cells can lead to activation and proliferation of intratumoral T cells, such as intratumoral CD8+ cytotoxic T cells or CD4+ helper T cells, which can eliminate tumor cells, leading to a reduction in tumor volume and / or elimination of the tumor. In some aspects, treatment according to the provided embodiments can result in a reduction in intratumoral Tregs and / or an increase in the ratio of intratumoral CD8+ to Tregs or the ratio of intratumoral CD4+ to Tregs.

[0147] In some aspects, treatment according to the methods and uses provided herein can result in a sustained or permanent decrease in intratumoral Tregs. In some aspects, treatment according to the methods and uses provided herein can result in a sustained or permanent increase in the ratio of intratumoral CD8+ to Tregs or the ratio of intratumoral CD4+ to Tregs. In some embodiments, the level, strength or extent of systemic immunity is determined by the increase in intratumoral CD8+ to Tregs or the increase in intratumoral CD4+ to Tregs. + The intratumoral CD8 + If the ratio of intratumoral CD8 + The ratio of CD8+ / Tregs in the tumor before treatment +and / or Tregs by at least 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold or more, or by at least about ... Systemic immunity to the recurrent tumor is increased or enhanced if the level, strength or extent of systemic immunity is increased by 1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4.0x or more. In some embodiments, the level, strength or extent of systemic immunity is increased or enhanced if the level, strength or extent of systemic immunity is increased by 1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4.0x or more. + The intratumoral CD4 + If the ratio of intratumoral Tregs to CD45 Tregs is increased after treatment compared to before treatment, systemic immunity to recurrent tumors is increased or enhanced. In some embodiments, the level, strength, or extent of systemic immunity is determined by the ratio of intratumoral Tregs to CD45 Tregs. + The tumor phenotype can be measured by determining the ratio of intratumoral Tregs to CD45 + If the ratio of is decreased after treatment compared to before treatment, systemic immunity against recurrent tumors is increased or enhanced. In some aspects, such increase or decrease can last for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or 3, 4, 5, 6, 7, or 8 weeks or longer, or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or 3, 4, 5, 6, 7, or 8 weeks or longer.

[0148] In some aspects, the level, strength or extent of systemic immunity can be measured by CTL activity assay using splenocytes or peripheral blood cells or bone marrow cells or lymph node cells. In some embodiments, the cells are collected from the subject 4-28 days after irradiation of the primary tumor in the subject.

[0149] In some aspects, the level, strength or extent of systemic immunity can be measured by an intratumoral T cell exhaustion assay using T cells collected from a primary tumor or a metastatic tumor cell mass or an invasive tumor cell mass. In some embodiments, the cells are collected from a subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0150] In some aspects, the level, strength or extent of systemic immunity can be measured by an intratumoral effector T cell proliferation assay using T cells collected from a primary tumor or a metastatic or invasive tumor cell mass. In some embodiments, the cells are collected from a subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0151] In some aspects, the level, strength or extent of systemic immunity can be measured by a T cell receptor diversity assay using T cells collected from a primary tumor or a metastatic tumor cell mass or an invasive tumor cell mass or peripheral circulation. In some embodiments, the cells are collected from a subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0152] In some aspects, the level, strength or extent of systemic immunity can be measured by determining the presence, number or frequency of regulatory T cells (Treg) in the tumor from a primary tumor or a metastatic or invasive tumor cell mass and / or the ratio of intratumoral Treg cells to intratumoral CD8+ T cells or intratumoral CD4+ T cells. In some embodiments, the cells are collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0153] In some embodiments, any of the above assays may be used in combination.

[0154] III. Compositions for use according to the methods herein In some aspects, compositions and combinations are provided, comprising a conjugate comprising a phthalocyanine dye linked to a targeting molecule, such as an antibody or antigen-binding fragment thereof that binds to CD25 protein; and an immune checkpoint inhibitor, such as an anti-PD-1 antibody. In some aspects, compositions and combinations are provided for use in a method of treatment or treatment regimen according to the provided methods and uses, or in the manufacture of a medicament for the treatment of cancer or tumor. In some aspects, compositions and combinations are provided for use according to the provided methods and uses.

[0155] The methods and uses provided herein use a conjugate that includes a targeting molecule that binds to CD25, such as an anti-CD25 antibody or antigen-binding fragment that binds (e.g., specifically binds) to CD25. CD25 can be expressed on activated T cells, including CD8+ cells, CD4+FoxP3+ regulatory T cells, activated B cells, some thymocytes, myeloid progenitor cells, and oligodendrocytes. CD25 is also known as interleukin 2 receptor alpha chain (IL2RA), IDDM10, IL2R, TCGFR, p55, or IMD41.

[0156] In some embodiments, the targeting molecule can be an anti-CD25 antibody, such as basiliximab (Simulect®), daclizumab, PC61, or an antigen-binding fragment of an anti-CD25 antibody, such as basiliximab (Simulect®), daclizumab, or PC61. In some of the optional embodiments, the anti-CD25 antibody is daclizumab. In some embodiments, the anti-CD25 antibody is basiliximab that includes an Fc region.

[0157] In some of the embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof or includes an antigen-binding fragment thereof. In some of the embodiments, the antibody is an anti-CD25 antibody. In some of the embodiments, the anti-CD25 antibody includes a functional Fc region. In some of the embodiments, the anti-CD25 antibody includes a full-length Fc region. In some of the embodiments, the anti-CD25 antibody is an antibody fragment. In some of the embodiments, the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof. In some of the embodiments, the anti-CD25 antibody is basiliximab. In some of the embodiments, the anti-CD25 antibody is basiliximab that includes an Fc region. In some of the embodiments, the anti-CD25 antibody is basiliximab that includes an Fc region that exhibits antibody-dependent cellular cytotoxicity (ADCC) activity or that has been engineered to exhibit enhanced ADCC activity. In some of the embodiments, the anti-CD25 antibody is basiliximab with a full-length Fc region. In some of the embodiments, the anti-CD25 antibody is basiliximab with an Fc region that is engineered to exhibit ADCC activity or enhanced ADCC activity. In some of the embodiments, the anti-CD25 antibody is basiliximab with an Fc region that does not exhibit ADCC activity or exhibits reduced ADCC activity. In some of the embodiments, the anti-CD25 antibody is daclizumab. In some of the embodiments, the anti-CD25 antibody is daclizumab with an Fc region. In some of the embodiments, the anti-CD25 antibody is daclizumab with a functional Fc region. In some of the embodiments, the anti-CD25 antibody is daclizumab with a full-length Fc region. In some embodiments, the targeting molecule can be an antibody or antibody fragment that contains the "complementarity determining regions" or "CDRs" of an anti-CD25 antibody, such as any of the described antibodies or antigen-binding fragments thereof. The CDRs are typically involved in binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus, and are also generally identified by the chain in which the particular CDR is located. Thus, the heavy chain variable region (VH ) CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, but not in the light chain variable region (V L ) CDR1 is the CDR1 derived from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities, e.g., different binding sites for different antigens, have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). In some embodiments, the targeting molecule comprises a CDR derived from basiliximab (Simulect®), daclizumab, or PC61. In some embodiments, the targeting molecule is basiliximab (Simulect®). In some embodiments, the antibody of the conjugate is a biosimilar, interchangeable, or biobetter of any of the anti-CD25 antibodies described herein, e.g., basiliximab (Simulect®) or an antigen-binding fragment thereof. Such antibodies also include copy biologics and biogenerics of any of the anti-CD25 antibodies described herein, such as basiliximab (Simulect®) or an antigen-binding fragment thereof.

[0158] In some embodiments of the methods and uses provided herein, the anti-CD25 antibody comprises a functional Fc region. In some embodiments of the methods and uses provided herein, the anti-CD25 antibody comprises a full-length Fc region.

[0159] The conjugates used in the methods and uses provided herein include a phthalocyanine dye. In some embodiments of the methods and uses provided herein, the phthalocyanine dye is a phthalocyanine dye having a silicon-coordinated metal (Si-phthalocyanine dye). In some embodiments, the phthalocyanine dye has the formula: TIFF0007627259000001.tif92128[In the formula, L is a linker; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7 , and R 8 are each independently selected from among 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 among hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from among hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy; and X 2 and X 3 each independently represents a C1-C alkyl group optionally interrupted by a heteroatom; 10 It includes alkylene.

[0160] In some embodiments, the phthalocyanine dye has the formula: TIFF0007627259000002.tif89150[In the formula, X 1 and X 4each independently represents a C1-C alkyl group optionally interrupted by a heteroatom; 10 is alkylene; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from among hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 are each independently selected from among hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy.

[0161] In some embodiments of the methods and uses provided herein, the Si-phthalocyanine dye is IRDye 700DX (IR700). In some embodiments, the phthalocyanine dye containing a reactive group is IR700 NHS ester, such as IRDye 700DX NHS ester (LiCor 929-70010, 929-70011). In some embodiments, the dye is a compound having the formula: TIFF0007627259000003.tif106160

[0162] For purposes herein, the term "IR700," "IRDye 700," or "IRDye 700DX" includes the above formula when the dye is conjugated to an antibody or the like, for example via a reactive group.

[0163] In some embodiments, the composition for use with the methods and uses provided herein comprises a conjugate comprising a Si-phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25. In some embodiments, the composition is an anti-CD25-Si-phthalocyanine dye conjugate. In some embodiments, the composition is an anti-CD25-IR700 conjugate. In some embodiments, the composition is an anti-CD25-IR700 conjugate, where the anti-CD25 moiety is basiliximab. In some embodiments, the composition is an anti-CD25-IR700 conjugate, where the anti-CD25 moiety is basiliximab containing a functional Fc region. In some embodiments, the composition is an anti-CD25-IR700 conjugate, where the anti-CD25 moiety is basiliximab containing an Fc region, e.g., a full-length Fc region. In some embodiments, the composition is an anti-CD25-IR700 conjugate, where the anti-CD25 moiety is basiliximab, which contains an Fc region, e.g., an Fc region that exhibits antibody-dependent cellular cytotoxicity (ADCC) activity or that has been engineered to exhibit enhanced ADCC activity. In some embodiments, the composition is an anti-CD25-IR700 conjugate, where the anti-CD25 moiety is daclizumab.

[0164] IV. Checkpoint inhibitor combination therapy The methods and uses provided herein can include administering an immune checkpoint inhibitor before, simultaneously, or subsequently to administration of the conjugate. For example, the method can include administering one or more doses of an immune checkpoint inhibitor, administering a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25, and irradiating the primary tumor and optionally one or more additional tumors after administration of the conjugate. The method can include first administering a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25, and irradiating the primary tumor and optionally one or more additional tumors after administration of the conjugate, and then administering an immune checkpoint inhibitor following either administration of the conjugate or irradiation (e.g., radiation). The method can also include administering an immune checkpoint inhibitor simultaneously with administration of the conjugate.

[0165] In some embodiments, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a combination thereof. In some embodiments, the immune checkpoint inhibitor is selected from an antibody or antigen-binding fragment that binds to PD-1, an antibody or antigen-binding fragment that binds to PD-L1, or an antibody or antigen-binding fragment that binds to CTLA-4, or a combination thereof.

[0166] In some embodiments of the methods and uses provided herein, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody for use with the methods is selected from the group consisting of pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ -63723283), pidilizumab (CT-011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, TSR-042 (ANB011), and any combination thereof.

[0167] In some embodiments of the methods and uses provided herein, the immune checkpoint inhibitor is an anti-PD-L1 antibody. Anti-PD-L1 antibodies that can be used in the methods and uses provided herein include atezolizumab (MPDL3280A, Tecentriq, RG7446), avelumab (BAVENCIO, MSB0010718C; M7824), durvalumab (MEDI4736, IMFINZI), LDP, NM-01, STI-3031 (IMC-001; STI-A1015), KN035, LY3300054, M7824 (MSB0011359C), BMS-93655 9, MSB2311, BCD-135, BGB-A333, CBT-502 (TQB-2450), cosibelimab (CK-301), CS1001 (WPB3155), FAZ053, MDX-1105, SHR-1316 (HTI-1088), TG-1501, ZKAB001 (STI-A1014), INBRX-105, MCLA-145, KN046, LY3415244, REGN3504, HLX20, and any combination thereof.

[0168] In some embodiments of the methods and uses provided herein, the immune checkpoint inhibitor is an anti-CTLA-4 antibody.Exemplary anti-CTLA-4 antibodies are ipilimumab (YERVOY), tremelimumab (ticilimumab, CP-675,206), AGEN1181, AGEN1884, ADU-1064, BCD-145, BCD-217, ADG116, AK104, ATOR-1015, BMS-986218, KN046, MGD019, MK-1308, REGN4659, XmAb20717, XmAb22841, and any combination thereof.

[0169] In some embodiments of the methods and uses provided herein, the immune checkpoint inhibitor is selected from an antibody or antigen-binding fragment that binds to PD-1, PD-L1, or CTLA-4, and the conjugate is an anti-CD25-IR700 conjugate. In some embodiments of the methods and uses provided herein, the immune checkpoint inhibitor is an antibody or antigen-binding fragment that binds to PD-1, and the conjugate is an anti-CD25-IR700 conjugate, where the anti-CD25 portion of the conjugate is or is derived from basiliximab. In some embodiments of the methods and uses provided herein, the immune checkpoint inhibitor is an antibody or antigen-binding fragment that binds to PD-1, and the conjugate is an anti-CD25-IR700 conjugate, where the anti-CD25 portion of the conjugate is or is derived from basiliximab, and the antibody portion of the conjugate comprises a functional Fc region. In some embodiments of the method, the conjugate is an anti-CD25-IR700 conjugate, where the anti-CD25 portion of the conjugate is or is derived from basiliximab that includes a functional Fc region. In some embodiments of the method, the conjugate is an anti-CD25-IR700 conjugate, where the anti-CD25 portion of the conjugate is or is derived from basiliximab that includes a functional Fc region, and the anti-PD-1 antibody is pembrolizumab (MK-3475, KEYTRUDA), nivolumab (OPDIVO), or cemiplimab (LIBTAYO).

[0170] In some embodiments of the methods and uses provided herein, the immune checkpoint inhibitor can be administered to a subject having cancer prior to administration of the anti-CD25-IR700 conjugate, simultaneously with administration of the anti-CD25-IR700 conjugate, after administration of the anti-CD25-IR700 conjugate, or any combination thereof.

[0171] In some embodiments of the methods and uses provided herein, the immune checkpoint inhibitor is administered to a subject one, two, three, four, five, six, seven, eight, nine, ten or more than ten times.

[0172] In some embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, or more than 5 times prior to administration of the conjugate. In some embodiments, the immune checkpoint inhibitor is administered to the subject 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 4 weeks prior to administration of the conjugate, or about 12 hours, 24 hours, 48 ​​hours, 96 hours, 1 week, 2 weeks, 3 weeks, or 4 weeks prior to administration of the conjugate. In some embodiments, the immune checkpoint inhibitor is administered to the subject less than 1-4 weeks, or less than 1-3 weeks, or less than 1-2 weeks prior to administration of the conjugate.

[0173] In some embodiments, the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5 or more than 5 times after administration of the conjugate. In some embodiments, the immune checkpoint inhibitor is administered to the subject before and after administration of the conjugate.

[0174] In some embodiments of the methods and uses provided herein, the anti-CD25 conjugate is administered to the subject once or more than once. In some embodiments, the conjugate is administered to the subject once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more than ten times. In some embodiments, the primary tumor(s) is irradiated after each administration of the conjugate, for example, within 24 hours ± 4 hours after each administration of the conjugate. In some embodiments, the conjugate is administered more than once if residual disease, such as the primary tumor or one or more additional tumors, residual cells or masses from the primary tumor, invasive cancer cells, or metastatic tumor cells, remains in the subject. In some embodiments, administration of the conjugate is repeated if residual disease remains for more than 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more than 1 year after the previous administration of the conjugate.

[0175] In some embodiments of the methods and uses provided herein, an anti-CD25 conjugate, for example, an anti-CD25-IR700 conjugate, is administered, and the method also includes administration of an immune checkpoint inhibitor, and the dual administration results in an enhanced effect, for example, an additive, additive, or synergistic effect. An additive, additive, or synergistic effect refers to an effect that exceeds the effect of either monotherapy alone (i.e., administering only the conjugate to a subject, administering only the conjugate followed by irradiation, or administering only the checkpoint inhibitor). For example, administering an anti-CD25-IR700 conjugate and administering a checkpoint inhibitor, for example, an anti-PD-1 antibody, produces a greater effect than when only the anti-CD25-IR700 conjugate or only the checkpoint inhibitor is administered, or when only the anti-CD25-IR700 conjugate followed by irradiation is administered. In some aspects, the provided methods and uses result in an additive, additive or synergistic anti-tumor response; for example, the growth of one or both of the primary tumor and the secondary population in the subject and / or the increase in the volume, size or mass thereof and the number of cells in the secondary population is inhibited to a greater extent or extent compared to administration of the conjugate alone, the conjugate followed by irradiation alone, and / or the anti-PD-1 antibody alone. In some aspects of the provided methods or uses, the growth of one or both of the primary tumor and / or the increase in the volume, size or mass thereof and the increase in the volume, size or number of cells in the secondary population in the subject is inhibited to a greater extent compared to administration of the conjugate followed by irradiation alone and compared to administration of the anti-PD-1 antibody alone. In some embodiments, the inhibition includes one or more of: an increase in tumor volume, tumor size or tumor mass of less than 20%; no change in tumor volume, size or mass (i.e., halted tumor growth or progression); or a reduction in tumor volume, tumor size or tumor mass, or a reduction in the number of tumor cells. In some embodiments, the inhibition comprises one or more of less than a 20% increase in the number of tumor cells. In some embodiments, the method produces an enhanced response, e.g., a more complete response, a more durable response, or a longer lasting response.

[0176] In some embodiments, the method produces an enhanced effect, e.g., an additive effect, or a synergistic effect, on primary tumors, including one or more of the following: inhibition, reduction, or elimination of tumor growth, reduction of tumor volume, size, or mass, or an increase in the number of subjects with complete response, and any combination thereof.In some embodiments, the method produces an enhanced effect, e.g., an additive effect, or a synergistic effect, on invasive tumor cells, including one or more of the following: inhibition, reduction, or elimination of tumor cell growth, reduction of the number or volume, size, or mass of invasive tumor cells, and any combination thereof (including in combination with the effect on one or more irradiated tumors, e.g., primary tumors).In some embodiments, the method produces an enhanced effect, e.g., an additive effect, or a synergistic effect, on metastatic tumor cells, including one or more of the following: inhibition, reduction, or elimination of metastatic tumor cell growth, reduction of the number or volume, size, or mass of metastatic tumor cells, and any combination thereof (including in combination with the effect on one or more irradiated tumors). In some embodiments, a synergistic effect is achieved for tumors directly exposed to irradiation (i.e., irradiation or radiation with a selected wavelength of light). In some embodiments, an enhanced effect is achieved for tumor cells that were not irradiated (e.g., where the primary tumor is irradiated) and a synergistic effect is achieved for non-irradiated metastatic or invasive tumor cells (e.g., non-irradiated metastatic or invasive tumor cells located distal to the irradiated tumor).

[0177] In some embodiments, the method produces a synergistic effect on increasing or enhancing systemic immunity. In some embodiments, the method uses a measure of systemic immunity as described herein or known, such as any of those described in Section II herein, for example, intratumoral CD8 + T lymphocyte count, CD8 + T lymphocytes and regulatory T cells (T reg ), intratumoral T lymphocyte exhaustion (e.g., CD3+ expressing PD-1 and / or CTLA4 markers), +CD8 + cells), intratumoral activated CD8 + The number or percentage of T lymphocytes (e.g., CD45 + Ki67 on cells + or CD69 + CD8 cells), expansion of cytotoxic intratumoral T lymphocytes based on splenic cell cytotoxicity against tumor cells (e.g., CD3 + CD8 + In some aspects, the intratumoral CD8 + T lymphocytes are CD3 + CD8 + Intratumoral exhausted T lymphocytes, including PD-1 + CTLA-4 + CD3 + CD8 + Activated intratumoral CD8+ T lymphocytes contain CD3 + CD8 + Ki67 + and / or CD3 + CD8 + CD69 + The expansion of cytotoxic T lymphocytes, including PD-1 - CTLA-4 - CD3 + CD8 + Contains cells.

[0178] In some embodiments, the method comprises administering an anti-CD25-IR700 conjugate and an immune checkpoint inhibitor to achieve a synergistic effect. In some embodiments, the method comprises administering an anti-CD25-IR700 conjugate and an anti-PD-1 antibody to achieve an enhanced effect, e.g., a synergistic or additive effect. In some embodiments, the anti-CD25-IR700 conjugate comprises basiliximab with a functional Fc region, and the immune checkpoint inhibitor is an anti-PD-1 antibody, such as nivolumab, and the enhanced effect, e.g., an additive, additive or synergistic effect, is found as a reduction in tumor growth, a reduction in tumor volume, a reduction in tumor size, a reduction in tumor mass or a complete response in the irradiated tumor, and / or a reduction in the growth, number, volume, size or mass of non-irradiated tumor cells in a secondary population of tumor cells (such as invasive tumor cells or metastatic tumor cells). In some embodiments, administration of anti-CD25-IR700 conjugates and irradiation results in a reduction in the number of intratumoral Tregs and / or an increase in the ratio of intratumoral CD8+ to Tregs or CD4+ to Tregs. In some embodiments, combination treatment with anti-PD-1 antibodies can result in a synergistic effect with respect to a reduction in the number of intratumoral Tregs and / or an increase in the ratio of intratumoral CD8+ to Tregs or CD4+ to Tregs.

[0179] V. Additional Therapeutic Agents In some aspects, the methods and uses provided involve administration of an additional therapeutic agent or anti-cancer treatment. In some aspects, the additional therapeutic agent is an immunomodulatory agent. In some aspects, the additional therapeutic agent is an anti-cancer treatment.

[0180] In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the immunomodulatory agent is a cytokine or an agent that induces increased expression of cytokines in the tumor microenvironment. In some aspects, "cytokine" refers to a collective term for proteins released by a cell population that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among these are growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; Müllerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor; transforming growth factors such as TGF-alpha and TGF-beta. Cytokines include transforming growth factor (TGF); insulin-like growth factor-I and -II; erythropoietin (EPO); bone morphogenetic factor; interferons such as interferon-alpha, beta and gamma; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors, such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines.For example, the immunomodulatory agent is a cytokine, the cytokine being IL-4, TNF-a, GM-CSF or IL-2.

[0181] In some embodiments, the immunomodulatory agent is selected from among GM-CSF, CpG-ODN (CpG oligodeoxynucleotide), lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), alum, recombinant Leishmania polyprotein, imiquimod, MF59, poly I:C, poly A:U, type 1 IFN, Pam3Cys, Pam2Cys, complete Freund's adjuvant (CFA), alpha-galactosylceramide, RC-529, MDF2P, loxoribine, anti-CD40 agonist, SIRPa antagonist, AS04, AS03, flagellin, resiquimod, DAP (diaminopimelic acid), MDP (muramyl dipeptide), and CAF01 (cationic adjuvant formulation-01). In some embodiments, the immunomodulatory agent is a Toll-like receptor (TLR) agonist, adjuvant or cytokine. In some embodiments, the immunomodulatory agent is a TLR agonist, and the TLR agonist is a TLR4 agonist, a TLR7 agonist, a TLR8 agonist or a TLR9 agonist. In some embodiments, the TLR agonist is selected from among triacylated lipoproteins, diacylated lipopeptides, lipoteichoic acid, peptidoglycan, zymosan, Pam3CSK4, dsRNA, polyLC, polyG10, polyG3, CpG, 3M003, flagellin, lipopolysaccharide (LPS), Leishmania homolog of eukaryotic ribosome elongation and initiation factor 4a (LeIF), MED 19197, SD-101, and imidazoquinoline TLR agonists.

[0182] In some embodiments, the immunomodulatory agent can contain one or more interleukins or other cytokines. For example, the interleukins can include Leukocyte Interleukin Injection (Multikine), which is a combination of natural cytokines.

[0183] In some embodiments, the immunomodulatory agent is a Toll-like receptor (TLR) agonist. In some embodiments, such agonist can include TLR4 agonist, TLR8 agonist, or TLR9 agonist. Such agonist can be selected from peptidoglycan, polyLC, CpG, 3M003, flagellin, and Leishmania homolog of eukaryotic ribosome elongation and initiation factor 4a (LeIF).

[0184] In some embodiments, the immunomodulatory agent can be one that enhances the immunogenicity of tumor cells, such as patupilone (epothilone B), epidermal growth factor receptor (EGFR) targeting monoclonal antibody 7A7.27, histone deacetylase inhibitors (e.g., vorinostat, romidepsin, panobinostat, belinostat, and entinostat), n3-polyunsaturated fatty acid docosahexaenoic acid, proteasome inhibitors (e.g., bortezomib), shikonin (the main component of the root of Lithospermum erythrorhizon) and oncolytic viruses, such as TVec (talimogene laherparepvec). In some embodiments, immunomodulators such as anthracyclins (doxorubicin, mitoxantrone), BK channel agonists, bortezomib, bortezomib + mitomycin C + hTert-Ad, cardiac glycosides + non-ICD inducers, cyclophosphamide, GADD34 / PP1 inhibitors + mitomycin, LV-tSMAC, and oxaliplatin activate immunogenic cell death of cancer or tumor. In some embodiments, the immunomodulator can be an epigenetic therapy, for example, a DNA methyltransferase inhibitor (e.g., decitabine, 5-aza-2'-deoxycytidine).

[0185] For example, in some embodiments, the immunomodulatory agent can be a DNA methyltransferase inhibitor, which can regulate the expression of tumor-associated antigens (TAA). TAA is an antigenic substance produced in tumor cells that triggers an immune response. TAA is often downregulated in tumors by DNA methylation to evade the immune system. Reversal of DNA methylation restores TAA expression and increases the immunogenicity of tumor cells. For example, demethylating agents such as decitabine (5-aza-2'-deoxycytidine) can upregulate the expression of TAA in tumor cells and increase immune recognition of cancerous cells. Photoimmunotherapy will further expose TAA to the immune system by destroying cells.

[0186] In some embodiments, the additional therapeutic agent is an agent or compound used in anti-cancer treatment, such as an anti-cancer drug. These include any agent that can alleviate, reduce, improve, suppress, or place or maintain in a state of efficacy clinical symptoms or diagnostic markers associated with tumors and cancer, and can be used in the combinations and compositions provided herein. In some embodiments, the anti-cancer drug is one whose therapeutic effect is generally related to the penetration or delivery of the anti-cancer drug into the tumor microenvironment or tumor space. In some embodiments, the anti-cancer drug is an alkylating agent, a platinum agent, an antimetabolite, an antitumor antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a corticosteroid, a proteasome inhibitor, a kinase inhibitor, a histone deacetylase inhibitor, or an antibody or an antigen-binding antibody fragment thereof. In some embodiments, the anti-cancer drug is a peptide, a protein, or a small molecule drug.

[0187] In some embodiments, the anticancer drug is 5-fluorouracil / leucovorin, oxaliplatin, irinotecan, regorafenib, ziv-afibercept, capecitabine, cisplatin, paclitaxel, toptecan, carboplatin, gemcitabine, docetaxel, 5-FU, ifosfamide, mitomycin, pemetrexed, vinorelbine, carmustine wafers. wager), temozolomide, methotrexate, capacitabine, lapatinib, etoposide, dabrafenib, vemurafenib, liposomal cytarabine, cytarabine, interferon alpha, erlotinib, vincristine, cyclophosphamide, lomusin, procarbazine, sunitinib, somastostatin, doxorubicin, pegylated liposomal doxorubicin, epirubicin, eribulin, albumin-bound paclitaxel, ixabepilone, cotrimoxazole, taxanes, vinblastine, temsirolimus, temozolomide, bendamustine , oral etoposide, everolimus, octreotide, lanreotide, dacarbazine, mesna, pazopanib, eribulin, imatinib, regorafenib, sorafenib, nilotinib, dazantinib, celecoxib, tamoxifen, toremifene, dactinomycin, sirolimus, crizotinib, certinib, enzalutamide, abiraterone acetate, mitoxantrone, cabazitaxel, fluoropyrimidines, oxaliplatin, leucovorin, afatinib, ceritinib, gefitinib, cabozantinib, oxaliplatin or auroropyrimidines.

[0188] In some embodiments, the anticancer drug is an alkylating agent.Alkylating agents are compounds that directly damage DNA by forming covalent bonds with nucleic acid and inhibiting DNA synthesis.Exemplary alkylating agents include, but are not limited to, mechlorethamine, cyclophosphamide, ifosamide, melphalan, chlorambucil, busulfan and thiotepa, and nitrosurea alkylating agents such as carmustine and lomustine.

[0189] In some embodiments, the anti-cancer agent is an antibody or an antigen-binding antibody fragment.

[0190] In some embodiments, anticancer drug is a platinum agent.Platinum agent binds to DNA and causes DNA cross-linking, which ultimately triggers apoptosis.Exemplary platinum agents include but are not limited to cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin and lipoplatin.

[0191] In some embodiments, the anticancer drug is an antimetabolite. Antimetabolites disrupt the growth of DNA and RNA by replacing the normal components of RNA and DNA. These agents damage cells during the S phase, when the chromosomes of the cell are being copied. In some cases, antimetabolites can be used to treat leukemia, breast, ovarian and intestinal cancer, and other types of cancer. Exemplary antimetabolites include, but are not limited to, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, and pemetrexed (Alimta®).

[0192] In some embodiments, the anti-cancer drug is an anti-tumor antibiotic. Anti-tumor antibiotics act by altering the DNA in cancer cells, preventing them from growing and multiplying. Anthracyclines are anti-tumor antibiotics that interfere with the enzymes involved in DNA replication. These drugs generally act in all stages of the cell cycle. They can be widely used against a wide variety of cancers. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin, epirubicin, and idarubicin. Other anti-tumor antibiotics include actinomycin-D, bleomycin, mitomycin-C, and mitoxantrone.

[0193] In some embodiments, the anticancer drug is a topoisomerase inhibitor. These drugs interfere with the enzyme called topoisomerase, which helps separate DNA strands so that they can copy during S-phase. Topoisomerase inhibitors can be used to treat certain leukemias, as well as lung cancer, ovarian cancer, gastrointestinal cancer and other cancers. Exemplary topoisomerase inhibitors include, but are not limited to, doxorubicin, topotecan, irinotecan (CPT-11), etoposide (VP-16), teniposide and mitoxantrone.

[0194] In some embodiments, the anti-cancer drug is a mitotic inhibitor. Mitotic inhibitors are often plant alkaloids and other compounds derived from natural plant products. They act by stopping mitosis in the M phase of the cell cycle, but in some cases can damage cells at all stages by preventing enzymes from making proteins necessary for cell reproduction. Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol®), docetaxel (Taxotere®), ixabepilone (Ixempra®), vinblastine (Velban®), vincristine (Oncovin®), vinorelbine (Navelbine®), and estramustine (Emcyt®).

[0195] In some embodiments, the anti-cancer drug is a corticosteroid. Corticosteroids, often simply called steroids, are natural hormones and hormone-like drugs that are useful in the treatment of many types of cancer. Corticosteroids can also be used before chemotherapy to help prevent allergic reactions, and during and after chemotherapy to help prevent nausea and vomiting. Exemplary corticosteroids include, but are not limited to, prednisone, methylprednisolone (Solumedrol®), and dexamethasone (Decadron®).

[0196] In some embodiments, the anti-cancer agent is another type of chemotherapy agent, such as a proteosome inhibitor, a kinase inhibitor, or a histone deacetylase inhibitor. In other embodiments, the anti-cancer agent is a biologic, such as an antibody, used in cancer treatment.

[0197] VI. DEVICES FOR USE WITH THE METHODS AND COMPOSITIONS In some aspects, devices that can be used with the methods and compositions herein include a light diffusing device that provides illumination at one wavelength (or multiple wavelengths) of light suitable for use with a dye conjugate composition, e.g., a phthalocyanine dye conjugate (e.g., an anti-CD25-IR700 conjugate such as those described herein). The illumination device can include a light source (e.g., a laser) and a means for delivering light to an area of ​​interest (e.g., one or more fibers for irradiating an isolated area or an isolated lesion or tumor of a subject).

[0198] In some embodiments of the methods and uses provided herein, the irradiation is performed using a cylindrical diffusing fiber with a diffuser length of 0.5 cm to 10 cm and placed 1.8±0.2 cm apart. In some embodiments, the light irradiation dose is 20 J / cm fiber length or about 20 J / cm fiber length to about 500 J / cm fiber length. In some embodiments, the lesion is a tumor that is a stromal tumor, and the irradiation is performed using a cylindrical diffusing fiber. In some embodiments, the tumor is more than 10 mm deep or is a subcutaneous tumor.

[0199] In some embodiments, the methods provided include irradiating a stromal tumor in a subject with a cylindrical diffusing fiber comprising a diffuser length of 0.5 cm to 10 cm and placed 1.8±0.2 cm apart with a light dose of at or about 100 J / cm fiber length or a fluence rate of at or about 400 mW / cm. In some embodiments, the tumor is greater than 10 mm deep or is a subcutaneous tumor. In some embodiments, the cylindrical diffusing fiber is placed in a catheter positioned 1.8±0.2 cm apart in the tumor. In some embodiments, the catheter is optically transparent.

[0200] In some embodiments, the lesion is a tumor that is a superficial tumor. In some embodiments, the tumor is less than 10 mm thick. In some embodiments, the irradiation is performed using a microlens-tipped fiber for surface irradiation. In some embodiments, the light irradiation dose is 5 J / cm. 2 or about 5 J / cm 2 to about 200 J / cm 2 It is.

[0201] In some embodiments, the methods provided involve administering 5 J / cm 2 of a microlens-tipped fiber for surface illumination to a superficial tumor in a subject. 2 or about 5 J / cm 2 to about 200 J / cm 2 In some embodiments, the light exposure dose is 50 J / cm. 2 or about 50 J / cm 2 It is.

[0202] In some embodiments, the illumination (also referred to herein as irradiating) uses a device having a "top hat" irradiance distribution profile, such as those described in WO2018 / 080952 and US20180239074.

[0203] VII. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, but the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is commonly understood in the art.

[0204] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." Aspects and variations described herein are understood to include "consisting of" and / or "consisting essentially of" aspects and variations.

[0205] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, when a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included within the claimed subject matter. This applies regardless of the breadth of the range.

[0206] The term "about" as used herein refers to the normal error range for each value that is evident to a person skilled in the art. Reference herein to a value or parameter with "about" includes (and describes) the embodiment directed to that value or parameter itself. For example, a description that refers to "about X" includes a description of "X".

[0207] As used herein, "conjugate" refers to a targeting molecule that is directly or indirectly linked to a photoactivatable dye, such as those produced by chemical conjugates and those produced by any other method.For example, conjugate can refer to a phthalocyanine dye (such as an IR700 molecule) that is directly or indirectly linked to one or more targeting molecules (such as a polypeptide that binds to or targets cell surface proteins).Targeting molecules can be a polypeptide, more than one polypeptide, an antibody, or a chemical moiety.

[0208] As used herein, an "anti-CD25 conjugate" refers to a conjugate having a targeting molecule that binds to CD25. An anti-CD25 conjugate can have a targeting molecule that is an antibody, antigen-binding fragment, or other moiety that binds to CD25.

[0209] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0210] "Specifically bind" refers to the ability of an individual antibody to specifically immunoreact with an antigen, such as a tumor-specific antigen, compared to binding to unrelated proteins, such as non-tumor proteins (e.g., β-actin). For example, a CD25-specific binding agent binds substantially only to CD25 protein in vitro or in vivo. As used herein, the term "tumor-specific binding agent" includes tumor-specific antibodies and other agents that bind substantially only to tumor-specific proteins in the preparation.

[0211] An "antibody-IR700 molecule" or "antibody-IR700 conjugate" refers to a molecule that includes both an antibody, such as a tumor-specific antibody, conjugated to IR700. In some examples, the antibody is a humanized antibody (such as a humanized monoclonal antibody) that specifically binds to a surface protein on a cancer cell.

[0212] "Antigen" refers to a compound, composition or substance capable of stimulating the production of antibodies or a T-cell response in an animal, including compositions (such as those containing tumor-specific proteins) that are injected or absorbed into an animal. Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens such as the disclosed antigens. "Epitope" or "antigenic determinant" refers to a region of an antigen to which B cells and / or T cells respond. In one embodiment, T cells respond to an epitope when the epitope is presented with an MHC molecule. Epitopes can be formed from both contiguous contiguous or non-contiguous amino acids by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes typically contain at least 3, more usually at least 5, about 9 or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and nuclear magnetic resonance.

[0213] Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids that contain antigenic determinants, such as those recognized by immune cells. In some examples, the antigen comprises a tumor-specific peptide (such as one found on the surface of a cancer cell) or an immunogenic fragment thereof.

[0214] "Immune checkpoint inhibitors" refers to a class of drugs that block certain proteins made by some immune system cells, such as T cells, and some cancer cells. These proteins help suppress the immune response and can prevent T cells from killing cancer cells. When these proteins are blocked, the "brakes" on the immune system are released and T cells are better able to kill cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2. Several immune checkpoint inhibitors are used to treat cancer.

[0215] As used herein, combination refers to any association between two or more items.Combination can be two or more separate items, such as two compositions or two collections, or can be a mixture of two or more items, such as a single mixture of two or more items, or a variant thereof.The components of combination are generally functionally related or associated.

[0216] As used herein, "combination therapy" refers to a treatment in which two or more therapeutic agents, such as at least two or at least three therapeutic agents, are given to a subject to treat a single disease.In some embodiments, each therapy can produce an independent pharmaceutical effect, and together can produce an additive or synergistic pharmaceutical effect.

[0217] As used herein, "treating" a subject with a disease or condition means that the subject's symptoms are partially or totally alleviated or remain stable after treatment. Thus, treating includes prevention, therapy and / or cure. Prevention refers to suppressing potential disease and / or suppressing the worsening of disease symptoms or progression.

[0218] As used herein, "treatment" means any manner in which the symptoms of a condition, disorder or disease or other indication are ameliorated or otherwise beneficially altered.

[0219] As used herein, "therapeutic effect" means an effect resulting from treatment of a subject that alters, typically improves or ameliorates, the symptoms of a disease or condition or cures the disease or condition.

[0220] As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" refers to an amount of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect, and thus is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder.

[0221] As used herein, amelioration of symptoms of a particular disease or disorder by treatment, e.g., by administration of a pharmaceutical composition or other therapeutic agent, refers to any alleviation, whether permanent or temporary, lasting or transient, of symptoms that may result from or be associated with the administration of the composition or therapeutic agent.

[0222] As used herein, the term "subject" refers to animals, including mammals such as humans.

[0223] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when said event or circumstance occurs and instances when it does not occur. For example, an optionally substituted group means that the group is unsubstituted or substituted.

[0224] All publications referenced in this application, including patent documents, scientific papers and databases, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication had been individually incorporated by reference. To the extent that the definitions set forth herein are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications and other publications incorporated herein by reference, the definitions set forth herein shall take precedence over the definitions incorporated herein by reference.

[0225] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0226] VIII. Illustrative Embodiments Among the aspects provided herein are the following:

[0227] 1. A method for treating cancer, comprising: (a) administering to a subject having cancer, including primary tumor and metastatic tumor cells, a conjugate comprising a phthalocyanine dye linked to a targeting molecule that binds to CD25; (b) administering an immune checkpoint inhibitor to a subject; (c) After administration of the conjugate, the primary tumor at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm Irradiating; Including, The growth and / or increase in the volume of primary and / or metastatic tumor cells in a subject is inhibited; The method.

[0228] 2. The method of embodiment 1, wherein the metastatic tumor cells are not directly irradiated.

[0229] 3. The method of embodiment 1 or 2, wherein the metastatic tumor cells are contained in a solid tumor.

[0230] 4. The method of any of aspects 1-3, wherein inhibition of growth of the primary tumor, the metastatic tumor cells, or both is synergistic compared to administration of only one of the conjugate and the immune checkpoint inhibitor.

[0231] 5. The method of any of aspects 1-4, wherein the subject exhibits a complete response.

[0232] 6. The method of any of aspects 1-5, wherein the targeting molecule is an antibody or an antigen-binding fragment thereof, or comprises an antigen-binding fragment thereof.

[0233] 7. The method of embodiment 6, wherein the antibody is an anti-CD25 antibody.

[0234] 8. The method of embodiment 7, wherein the anti-CD25 antibody comprises a functional Fc region.

[0235] 9. The method of embodiment 7 or 8, wherein the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic or biogeneric thereof.

[0236] 10. The method of any of aspects 7-9, wherein the anti-CD25 antibody is basiliximab.

[0237] 11. The method of any of aspects 1-10, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0238] 12. The method of any of aspects 1-11, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0239] 13. The method of embodiment 12, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0240] 14. Anti-PD-1 antibodies include pembrolizumab (MK-3475, KEYTRUDA), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), and pidilizumab (CT 14. The method of embodiment 12 or 13, wherein the therapeutic agent is selected from the group consisting of: -011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), GLS-010, CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, MEDI0680, Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, and spartalizumab.

[0241] 15. Anti-PD-L1 antibodies include atezolizumab (MPDL3280A, Tecentriq), avelumab (Bavencio), durvalumab (MEDI4736, Imfinzi), LDP, NM-01, STI-3031, KN035, LY3300054, M7824 (MSB0011359C), BMS-936559, MSB2311, and BCD. 13. The method of embodiment 12, wherein the IL-135, BGB-A333, CBT-502, cosibelimab (CK-301), CS1001, FAZ053, MDX-1105, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, LY3415244, REGN3504, and HLX20.

[0242] 16. The method of embodiment 12, wherein the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab (Yervoy), tremelimumab, AGEN1181, AGEN1884, ADU-1064, BCD-145, and BCD-217.

[0243] 17. The method of any of embodiments 1-16, wherein the conjugate is administered to the subject prior to, simultaneously with, or following administration of the immune checkpoint inhibitor.

[0244] 18. The method of any of aspects 1-17, wherein the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times.

[0245] 19. The method of any of embodiments 1-18, wherein the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 administrations of the immune checkpoint inhibitor to the subject after administration of the conjugate.

[0246] 20. The method of any of embodiments 1-18, wherein the immune checkpoint inhibitor is administered simultaneously with administration of the conjugate, followed by administration of the immune checkpoint inhibitor to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate.

[0247] 21. The method of any of embodiments 1-18, wherein the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate.

[0248] 22. The method of any one of aspects 1 to 21, wherein the phthalocyanine dye is a Si-phthalocyanine dye.

[0249] 23. The method of embodiment 22, wherein the Si-phthalocyanine dye is IR700.

[0250] 24. The method of any of aspects 1-23, wherein the irradiation is performed 30 minutes to 96 hours after administration of the conjugate.

[0251] 25. The method of any of embodiments 1-24, wherein the irradiation is performed 24 hours ± 4 hours after administration of the conjugate.

[0252] 26. The method of any of aspects 1-25, wherein the primary tumor is irradiated with light of a wavelength of 690±40 nm.

[0253] 27. Primary tumor is 50 J / cm 2 Or about 50 J / cm 2 or the method of any of embodiments 1-26, wherein the fiber is irradiated with a dose of at or about 100 J / cm of fiber length.

[0254] 28. The method of any of embodiments 1-27, wherein one or more of steps (a), (b), or (c) are repeated.

[0255] 29. The method of any of embodiments 1-28, further comprising the step of (d) administering an additional therapeutic agent or anti-cancer treatment.

[0256] 30. The method of any of embodiments 1-29, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0257] 31. The method of any of embodiments 1-30, wherein the metastatic tumor cells are located in one, two, three or more tissues or organs different from the tissue or organ in which the primary tumor is located.

[0258] 32. A method for treating cancer, comprising: (1) administering to a subject having cancer, including a primary tumor and invasive tumor cells, a conjugate comprising a phthalocyanine dye linked to a targeting molecule that binds to CD25; (2) administering an immune checkpoint inhibitor to a subject; (3) After administration of the conjugate, the primary tumor is at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm Irradiating; Including, The growth and / or increase in the volume of primary tumor and / or invasive tumor cells in the subject is inhibited; The method.

[0259] 33. The method of embodiment 32, wherein the invasive tumor cells are not directly irradiated.

[0260] 34. The method of embodiment 32 or 33, wherein the invasive tumor cells are contained in a solid tumor.

[0261] 35. The method of any of aspects 32-34, wherein inhibition of growth of the primary tumor, the invasive tumor cells, or both, is synergistic compared to administration of only one of the conjugate and the immune checkpoint inhibitor.

[0262] 36. The method of any of aspects 32-35, wherein the subject exhibits a complete response.

[0263] 37. The method of any of aspects 32-36, wherein the targeting molecule is, or comprises, an antibody or antigen-binding fragment thereof.

[0264] 38. The method of embodiment 37, wherein the antibody is an anti-CD25 antibody.

[0265] 39. The method of embodiment 38, wherein the anti-CD25 antibody comprises a functional Fc region.

[0266] 40. The method of embodiment 38 or 39, wherein the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof.

[0267] 41. The method of any of aspects 38-40, wherein the anti-CD25 antibody is basiliximab.

[0268] 42. The method of any of aspects 32-41, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0269] 43. The method of any of aspects 32-42, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0270] 44. The method of embodiment 43, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0271] 45. Anti-PD-1 antibodies include pembrolizumab (MK-3475, KEYTRUDA), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), and pidilizumab (CT 45. The method of embodiment 43 or 44, wherein the therapeutic agent is selected from the group consisting of: -011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), GLS-010, CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, MEDI0680, Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, and spartalizumab.

[0272] 46. ​​Anti-PD-L1 antibodies include atezolizumab (MPDL3280A, Tecentriq), avelumab (Bavencio), durvalumab (MEDI4736, Imfinzi), LDP, NM-01, STI-3031, KN035, LY3300054, M7824 (MSB0011359C), BMS-936559, MSB2311, and BCD. 44. The method of embodiment 43, wherein the IL-135, BGB-A333, CBT-502, cosibelimab (CK-301), CS1001, FAZ053, MDX-1105, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, LY3415244, REGN3504, and HLX20.

[0273] 47. The method of embodiment 43, wherein the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab (Yervoy), tremelimumab, AGEN1181, AGEN1884, ADU-1064, BCD-145, and BCD-217.

[0274] 48. The method of any of aspects 32-47, wherein the conjugate is administered to the subject prior to, simultaneously with, or following administration of the immune checkpoint inhibitor.

[0275] 49. The method of any of aspects 32-48, wherein the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times.

[0276] 50. The method of any of embodiments 32-49, wherein the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 administrations of the immune checkpoint inhibitor to the subject after administration of the conjugate.

[0277] 51. The method of any of aspects 32-49, wherein the immune checkpoint inhibitor is administered simultaneously with administration of the conjugate, followed by administration of the immune checkpoint inhibitor to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate.

[0278] 52. The method of any of aspects 32-49, wherein the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate.

[0279] 53. The method of any one of aspects 32 to 52, wherein the phthalocyanine dye is a Si-phthalocyanine dye.

[0280] 54. The method of embodiment 53, wherein the Si-phthalocyanine dye is IR700.

[0281] 55. The method of any of aspects 32-54, wherein irradiation is performed 30 minutes to 96 hours after administration of the conjugate.

[0282] 56. The method of any of aspects 32-55, wherein irradiation is performed 24 hours ± 4 hours after administration of the conjugate.

[0283] 57. The method of any of aspects 32-56, wherein the primary tumor is irradiated with light of a wavelength of 690±40 nm.

[0284] 58. The primary tumor is 50 J / cm 2 Or about 50 J / cm 2 or the method of any of embodiments 32 to 57, wherein the fiber is irradiated at a dose of 100 J / cm of fiber length or about 100 J / cm of fiber length.

[0285] 59. The method of any of embodiments 32-58, wherein one or more of steps (1), (2), or (3) are repeated.

[0286] 60. (4) The method of any of aspects 32 to 59, further comprising the step of administering an additional therapeutic agent or anti-cancer treatment.

[0287] 61. The method of any of aspects 32-60, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0288] 62. A method for enhancing systemic immunity in a tumor-bearing subject, comprising: (a) administering to a tumor-bearing subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody; (b) administering an immune checkpoint inhibitor to a subject; (c) After administration of the conjugate, the tumor at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm Irradiating; Including, the subject's systemic immunity is enhanced as compared to the subject's systemic immunity prior to administration of the conjugate and the immune checkpoint inhibitor; The method.

[0289] 63. The method of embodiment 62, wherein systemic immunity is measured by one or more of a cytotoxic T lymphocyte (CTL) activity assay, an intratumor T cell exhaustion assay, an intratumor effector T cell proliferation assay, or a T cell receptor diversity assay.

[0290] 64. The tumor comprises a primary tumor and metastatic tumor cells, the primary tumor is irradiated and the metastatic tumor cells are not directly irradiated; or the tumor comprises a primary tumor and invasive tumor cells, the primary tumor is irradiated and the invasive tumor cells are not directly irradiated; The method of embodiment 62 or 63.

[0291] 65. The method of any of embodiments 62-64, wherein systemic immunity is measured by a CTL activity assay using splenocytes or peripheral blood cells or bone marrow cells or lymph node cells, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0292] 66. The method of any of aspects 62-64, wherein systemic immunity is measured by an intratumoral T cell exhaustion assay using T cells collected from a primary tumor or a metastatic or invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0293] 67. The method of any of aspects 62-64, wherein systemic immunity is measured by an intratumoral effector T cell proliferation assay using T cells collected from a primary tumor or a metastatic or invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0294] 68. The method of any of aspects 62-64, wherein systemic immunity is measured by a T cell receptor diversity assay using T cells collected from the primary tumor or metastatic tumor cell mass or invasive tumor cell mass or peripheral circulation, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0295] 69. The method of any of embodiments 62-64, wherein systemic immunity is measured by determining the presence, number or frequency of regulatory T cells (Treg) in tumor from a primary tumor or a metastatic tumor cell mass or an invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject, and / or the ratio of intratumoral Treg cells to intratumoral CD8+ T cells or intratumoral CD4+ T cells.

[0296] 70. The method of any of embodiments 62-69, wherein the anti-CD25 antibody comprises a functional Fc region.

[0297] 71. The method of any of aspects 62-70, wherein the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof.

[0298] 72. The method of any of aspects 62-71, wherein the anti-CD25 antibody is basiliximab.

[0299] 73. The method of any of aspects 62-72, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0300] 74. The method of embodiment 73, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0301] 75. Anti-PD-1 antibodies include pembrolizumab (MK-3475, KEYTRUDA), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), and pidilizumab (CT 75. The method of embodiment 73 or 74, wherein the therapeutic agent is selected from the group consisting of: -011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), GLS-010, CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, MEDI0680, Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, and spartalizumab.

[0302] 76. Anti-PD-L1 antibodies include atezolizumab (MPDL3280A, Tecentriq), avelumab (Bavencio), durvalumab (MEDI4736, Imfinzi), LDP, NM-01, STI-3031, KN035, LY3300054, M7824 (MSB0011359C), BMS-936559, MSB2311, and BCD. 74. The method of embodiment 73, wherein the IL-135, BGB-A333, CBT-502, cosibelimab (CK-301), CS1001, FAZ053, MDX-1105, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, LY3415244, REGN3504, and HLX20.

[0303] 77. The method of embodiment 73, wherein the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab (Yervoy), tremelimumab, AGEN1181, AGEN1884, ADU-1064, BCD-145, and BCD-217.

[0304] 78. The method of any of embodiments 62-77, wherein the conjugate is administered to the subject prior to, simultaneously with, or following administration of the immune checkpoint inhibitor.

[0305] 79. The method of any of embodiments 62-78, wherein the immune checkpoint inhibitor is administered to the subject one, two, three, four, five, six, seven, eight, nine, ten or more than ten times.

[0306] 80. The method of any one of aspects 62-79, wherein the phthalocyanine dye is a Si-phthalocyanine dye.

[0307] 81. The method of embodiment 80, wherein the Si-phthalocyanine dye is IR700.

[0308] 82. The method of any of aspects 62 to 81, wherein irradiation is performed 30 minutes to 96 hours after administration of the conjugate and the immune checkpoint inhibitor.

[0309] 83. The method of any of aspects 62 to 82, wherein irradiation is performed 24 hours ± 4 hours after administration of the conjugate and the immune checkpoint inhibitor.

[0310] 84. The method of any of aspects 62-83, wherein the tumor is irradiated with light of a wavelength of 690±40 nm.

[0311] 85. The tumor is 50 J / cm 2 Or about 50 J / cm 2 or the method of any of embodiments 62 to 84, wherein the fiber is irradiated at a dose of 100 J / cm of fiber length or about 100 J / cm of fiber length.

[0312] 86. The method of any of embodiments 62-85, wherein one or more of steps (a), (b), (c), or (d) are repeated.

[0313] 87. The method of any of embodiments 62-86, wherein the tumor is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0314] 88. A method for producing a synergistic response in a subject, comprising: (1) administering to a subject having cancer, including a primary tumor, a conjugate comprising a phthalocyanine dye linked to a targeting molecule that binds to CD25; (2) administering an immune checkpoint inhibitor to a subject; (3) After administration of the conjugate, the primary tumor is at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm Irradiating; Including, The growth of the primary tumor and / or its increase in volume is synergistically reduced compared to administration of the conjugate alone or the immune checkpoint inhibitor alone; The method.

[0315] 89. The method of embodiment 88, wherein the cancer further comprises metastatic tumor cells, and the growth of the metastatic tumor cells and / or the increase in their volume is reduced compared to administration of only the conjugate or only the immune checkpoint inhibitor.

[0316] 90. The method of embodiment 89, wherein the reduction in the growth of metastatic tumor cells or the increase in their volume is synergistic.

[0317] 91. The method of embodiment 88, wherein the cancer further comprises invasive tumor cells, and the growth of the invasive tumor cells and / or the increase in their volume is reduced compared to administration of only the conjugate or only the immune checkpoint inhibitor.

[0318] 92. The method of embodiment 91, wherein the reduction in the growth of invasive tumor cells or the increase in their volume is synergistic.

[0319] 93. The method of any of embodiments 88-84, wherein the targeting molecule is, or comprises, an antibody or antigen-binding fragment thereof.

[0320] 94. The method of any of aspects 88-93, wherein the antibody is an anti-CD25 antibody.

[0321] 95. The method of embodiment 94, wherein the anti-CD25 antibody comprises a functional Fc region.

[0322] 96. The method of any of aspects 88-95, wherein the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof.

[0323] 97. The method of any of aspects 88-96, wherein the anti-CD25 antibody is basiliximab.

[0324] 98. The method of any of aspects 88-97, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0325] 99. The method of any of aspects 88-98, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0326] 100. The method of embodiment 99, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0327] 101. Anti-PD-1 antibodies include pembrolizumab (MK-3475, KEYTRUDA), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), and pidilizumab (CT -011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), GLS-010, CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, MEDI0680, Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, and spartalizumab.

[0328] 102. Anti-PD-L1 antibodies include atezolizumab (MPDL3280A, Tecentriq), avelumab (Bavencio), durvalumab (MEDI4736, Imfinzi), LDP, NM-01, STI-3031, KN035, LY3300054, M7824 (MSB0011359C), BMS-936559, MSB2311, and BCD. 99. The method of embodiment 99, wherein the IL-135, BGB-A333, CBT-502, cosibelimab (CK-301), CS1001, FAZ053, MDX-1105, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, LY3415244, REGN3504, and HLX20.

[0329] 103. The method of embodiment 99, wherein the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab (Yervoy), tremelimumab, AGEN1181, AGEN1884, ADU-1064, BCD-145, and BCD-217.

[0330] 104. The method of embodiment 90, wherein the conjugate is administered to the subject prior to, simultaneously with, or following administration of an immune checkpoint inhibitor.

[0331] 105. The method of any of embodiments 88-104, wherein the immune checkpoint inhibitor is administered to the subject one, two, three, four, five, six, seven, eight, nine, ten or more than ten times.

[0332] 106. The method of any of embodiments 88-105, wherein the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 administrations of the immune checkpoint inhibitor to the subject after administration of the conjugate.

[0333] 107. The method of any of embodiments 88-105, wherein the immune checkpoint inhibitor is administered simultaneously with administration of the conjugate, followed by administration of the immune checkpoint inhibitor to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate.

[0334] 108. The method of any of embodiments 88-105, wherein the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate.

[0335] 109. The method of any one of embodiments 88 to 108, wherein the phthalocyanine dye is a Si-phthalocyanine dye.

[0336] 110. The method of embodiment 109, wherein the Si-phthalocyanine dye is IR700.

[0337] 111. The method of any of embodiments 88 to 110, wherein irradiation is performed 30 minutes to 96 hours after administration of the conjugate.

[0338] 112. The method of any of embodiments 88-111, wherein irradiation is performed 24 hours ± 4 hours after administration of the conjugate.

[0339] 113. The method of any of aspects 88-112, wherein the primary tumor is irradiated with light of a wavelength of 690±40 nm.

[0340] 114. Primary tumor is 50 J / cm 2 Or about 50 J / cm 2 or the method of any of embodiments 88 to 113, wherein the fiber is irradiated with a dose of at or about 100 J / cm of fiber length.

[0341] 115. The method of any of embodiments 88-114, wherein one or more of steps (1), (2), and (3) are repeated.

[0342] 116. (4) The method of any of embodiments 88 to 115, further comprising the step of administering an additional therapeutic agent or anti-cancer treatment.

[0343] 117. The method of any of embodiments 88-116, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0344] 118. The method of any of embodiments 89-117, wherein the metastatic tumor cells are located in one, two, three, or more than three tissues or organs different from the tissue or organ in which the primary tumor is located.

[0345] 119. A method for treating cancer, comprising: (a) administering an immune checkpoint inhibitor to a subject having cancer, the cancer comprising a primary tumor and a secondary population of tumor cells; (b) administering to the subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody; (c) After administration of the conjugate, primary tumors were at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm irradiating, wherein the secondary population is not directly irradiated; The method comprising:

[0346] 120. The method of embodiment 119, wherein primary tumors and / or secondary populations are inhibited to a greater extent compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the immune checkpoint inhibitor alone.

[0347] 121. The method of embodiment 119 or 120, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0348] 122. The method of any of embodiments 119-121, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0349] 123. The method of embodiment 122, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0350] 124. A method for treating cancer, comprising: (a) administering an anti-PD-1 antibody to a subject having cancer, including a primary tumor and a secondary population of tumor cells; (b) administering to the subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody; (c) After administration of the conjugate, primary tumors were at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm irradiating, wherein the secondary population is not directly irradiated; Including, the primary tumor and / or secondary populations are inhibited to a greater extent as compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone; The method.

[0351] 125. The method of any of embodiments 119-124, wherein the inhibition comprises one or more of: an increase in tumor volume, tumor size, or tumor mass of less than 20% or less than about 20%, or a reduction in tumor volume, tumor size, or tumor mass, or a reduction in the number of tumor cells.

[0352] 126. The method of embodiment 125, wherein the reduction in tumor volume, tumor size or tumor mass, or number of tumor cells comprises a 30% reduction or more, or about a 30% reduction or more.

[0353] 127. The method of any of embodiments 119-126, wherein the secondary population comprises metastatic tumor cells.

[0354] 128. The method of any of embodiments 119-126, wherein the secondary population comprises invasive tumor cells.

[0355] 129. The method of any of embodiments 119-128, wherein the secondary population comprises metastatic tumor cells and invasive tumor cells.

[0356] 130. The method of any of embodiments 119-129, wherein an immune checkpoint inhibitor is administered to the subject simultaneously with the conjugate.

[0357] 131. The method of any of embodiments 119-130, wherein an immune checkpoint inhibitor is administered to the subject within 24 to 48 hours of administering the conjugate.

[0358] 132. The method of any of embodiments 119-131, wherein the immune checkpoint inhibitor is administered to the subject within 24 hours ± 4 hours of administering the conjugate.

[0359] 133. The method of any of embodiments 119-132, wherein a first dose of the immune checkpoint inhibitor is administered prior to administration of the conjugate.

[0360] 134. The method of embodiment 133, wherein the first dose of the immune checkpoint inhibitor is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate.

[0361] 135. The method of any of embodiments 119-134, wherein the immune checkpoint inhibitor is administered to the subject one, two, three, four, five, six, seven, eight, nine, ten or more than ten times.

[0362] 136. The method of any of embodiments 119-135, wherein the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 administrations of the immune checkpoint inhibitor to the subject after administration of the conjugate.

[0363] 137. The method of any of embodiments 119-135, wherein the immune checkpoint inhibitor is administered simultaneously with administration of the conjugate, followed by administration of the immune checkpoint inhibitor to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate.

[0364] 138. The method of any of embodiments 119-135, wherein the immune checkpoint inhibitor is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate.

[0365] 139. The method of any of embodiments 122-129, wherein the anti-PD-1 antibody is administered to the subject simultaneously with the conjugate.

[0366] 140. The method of any of embodiments 122-130, wherein an anti-PD-1 antibody is administered to the subject within 24 to 48 hours of administering the conjugate.

[0367] 141. The method of any of embodiments 122-131, wherein the anti-PD-1 antibody is administered to the subject within 24 hours ± 4 hours of administering the conjugate.

[0368] 142. The method of any of embodiments 122-132, wherein a first dose of the anti-PD-1 antibody is administered prior to administration of the conjugate.

[0369] 143. The method of embodiment 142, wherein the first dose of the anti-PD-1 antibody is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate.

[0370] 144. The method of any of embodiments 122-143, wherein the anti-PD-1 antibody is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times.

[0371] 145. The method of any of embodiments 122-144, wherein the anti-PD-1 antibody is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to administration of the conjugate, followed by zero, one, two, three, four, five, six, seven, eight, nine, ten, or more than ten administrations of the anti-PD-1 antibody to the subject after administration of the conjugate.

[0372] 146. The method of any of embodiments 122-144, wherein the anti-PD-1 antibody is administered simultaneously with administration of the conjugate, followed by administration of the anti-PD-1 antibody to the subject 0, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times after administration of the conjugate.

[0373] 147. The method of any of embodiments 122-144, wherein the anti-PD-1 antibody is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times after administration of the conjugate.

[0374] 148. The method of any of embodiments 119-147, wherein the secondary population is contained in a solid tumor.

[0375] 149. The method of any of embodiments 119-148, wherein the subject exhibits a complete response.

[0376] 150. The method of any of embodiments 119-149, wherein the anti-CD25 antibody comprises a functional Fc region.

[0377] 151. The method of any of embodiments 119-150, wherein the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof.

[0378] 152. The method of embodiment 151, wherein the anti-CD25 antibody is basiliximab.

[0379] 153. Anti-PD-1 antibodies include pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), Pidilizumab (CT-011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, and TSR-042 (ANB011).

[0380] 154. The method of any one of embodiments 119 to 153, wherein the phthalocyanine dye is a Si-phthalocyanine dye.

[0381] 155. The method of embodiment 154, wherein the Si-phthalocyanine dye is IR700.

[0382] 156. The method of any of embodiments 119 to 155, wherein irradiation is performed 30 minutes to 96 hours after administration of the conjugate.

[0383] 157. The method of embodiment 156, wherein irradiation is performed 24 hours ± 4 hours after administration of the conjugate.

[0384] 158. The method of any of aspects 119-157, wherein the primary tumor is irradiated with light of 690±40 nm.

[0385] 159. Primary tumors were irradiated at 50 J / cm 2 Or about 50 J / cm 2 or the method of any of embodiments 119 to 158, wherein the fiber is irradiated at a dose of 100 J / cm of fiber length or about 100 J / cm of fiber length.

[0386] 160. The method of any of embodiments 119 to 159, further comprising the step of (d) administering an additional therapeutic agent or anti-cancer treatment.

[0387] 161. The method of any of embodiments 119-160, wherein one or more of steps (a), (b), (c), or (d) are repeated.

[0388] 162. The method of any of embodiments 119-161, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0389] 163. The method of any of embodiments 119-162, wherein the secondary population is located in one, two, three, or more than three tissues or organs different from the tissue or organ in which the primary tumor is located.

[0390] 164. The method of any of embodiments 119-163, wherein primary tumors and / or secondary populations are inhibited to a greater extent compared to administration of the conjugate followed by irradiation alone and compared to administration of an immune checkpoint inhibitor alone.

[0391] 165. The method of any of embodiments 122-164, wherein primary tumors and / or secondary populations are inhibited to a greater extent compared to administration of the conjugate followed by radiation alone, and compared to administration of the anti-PD-1 antibody alone.

[0392] 166. A method for producing an enhanced response in a subject having cancer, comprising: (a) administering an immune checkpoint inhibitor to a subject having cancer, including a tumor; (b) administering to the subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody, wherein an immune checkpoint inhibitor is administered prior to or simultaneously with the conjugate; (c) After administration of the conjugate, the tumor at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm Irradiating; The method comprising:

[0393] 167. The enhanced response comprises an enhancement of the subject's systemic immunity compared to the subject's systemic immunity prior to administration of the conjugate followed by irradiation and an immune checkpoint inhibitor; and / or The enhanced response includes enhanced tumor inhibition compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the immune checkpoint inhibitor alone. The method of embodiment 166.

[0394] 168. The method of embodiment 166 or 167, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0395] 169. The method of any of embodiments 166-168, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.

[0396] 170. The method of embodiment 169, wherein the immune checkpoint inhibitor is or comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0397] 171. A method for producing an enhanced response in a subject having cancer, comprising: (a) administering an anti-PD-1 antibody to a subject having cancer, including a tumor; (b) administering to the subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody, wherein an anti-PD-1 antibody is administered prior to or simultaneously with the conjugate; (c) After administration of the conjugate, the tumor at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm Irradiating; Including, the enhanced response comprises an enhancement of the subject's systemic immunity compared to the subject's systemic immunity prior to administration of the conjugate followed by irradiation and the anti-PD-1 antibody; and / or the enhanced response comprises enhanced tumor inhibition compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone. The method.

[0398] 172. The method of any of embodiments 166-171, wherein the enhanced response is additive or synergistic.

[0399] 173. The method of any of embodiments 166-172, wherein the inhibition comprises one or more of an increase in tumor volume, tumor size, or tumor mass of less than 20% or less than about 20%, or a reduction in tumor volume, tumor size, or tumor mass, or a reduction in the number of tumor cells.

[0400] 174. The method of embodiment 173, wherein the reduction in tumor volume, tumor size or tumor mass, or number of tumor cells comprises a 30% reduction or more, or about a 30% reduction or more.

[0401] 175. The tumor comprises a primary tumor and a secondary population of tumor cells, the primary tumor being irradiated and the secondary population not being directly irradiated; the tumor comprises a primary tumor and metastatic tumor cells, the primary tumor being irradiated and the metastatic tumor cells not being directly irradiated; and / or the tumor comprises a primary tumor and invasive tumor cells, the primary tumor is irradiated and the invasive tumor cells are not directly irradiated; The method of any one of embodiments 166 to 174.

[0402] 176. The method of any of embodiments 167-175, wherein the enhanced response is a synergistic response, the synergistic response comprising a synergistic reduction in primary tumor growth, tumor volume, tumor size or tumor mass, a synergistic reduction in the number of cells in a secondary population of subjects, a synergistic reduction in metastatic or invasive tumor cell growth, tumor volume, tumor size, tumor mass or number, or any combination thereof.

[0403] 177. The method of any of embodiments 166-176, wherein an immune checkpoint inhibitor is administered to the subject within 24 to 48 hours of administering the conjugate.

[0404] 178. The method of any of embodiments 166-177, wherein the immune checkpoint inhibitor is administered to the subject within 24 hours ± 4 hours of administering the conjugate.

[0405] 179. The method of any of embodiments 166-178, wherein a first dose of the immune checkpoint inhibitor is administered prior to administration of the conjugate.

[0406] 180. The method of embodiment 179, wherein the first dose of the immune checkpoint inhibitor is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate.

[0407] 181. The method of any of embodiments 169-180, wherein the anti-PD-1 antibody is administered to the subject within 24 hours to 48 hours of administering the conjugate.

[0408] 182. The method of any of embodiments 169-181, wherein the anti-PD-1 antibody is administered to the subject within 24 hours ± 4 hours of administering the conjugate.

[0409] 183. The method of any of embodiments 169-182, wherein a first dose of the anti-PD-1 antibody is administered prior to administration of the conjugate.

[0410] 184. The method of embodiment 183, wherein the first dose of the anti-PD-1 antibody is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate.

[0411] 185. Systemic immunity is measured by cytotoxic T lymphocyte (CTL) activity assay, intratumoral T cell exhaustion assay, intratumoral effector T cell proliferation assay, T cell receptor diversity assay, activated CD8 + T cell assay, circulating regulatory T cell (Treg) assay, intratumoral Treg assay, or CD8 + The method of any of embodiments 166-184, wherein T cells: are measured by one or more of the Treg assays.

[0412] 186. The method of any of embodiments 166-185, wherein systemic immunity is measured by a CTL activity assay using splenocytes or peripheral blood cells or bone marrow cells or lymph node cells, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0413] 187. The method of any of embodiments 166-185, wherein systemic immunity is measured by an intratumoral T cell exhaustion assay using T cells collected from a primary tumor or a metastatic or invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0414] 188. The method of any of embodiments 166-185, wherein systemic immunity is measured by an intratumoral effector T cell proliferation assay using T cells collected from a primary tumor or a metastatic or invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0415] 189. The method of any of embodiments 166-185, wherein systemic immunity is measured by a T cell receptor diversity assay using T cells collected from the primary tumor or a metastatic tumor cell mass or an invasive tumor cell mass or peripheral circulation, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject.

[0416] 190. The method of any of embodiments 166-185, wherein systemic immunity is measured by determining the presence, number or frequency of regulatory T cells (Treg) in tumor from a primary tumor or a metastatic tumor cell mass or an invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject, and / or the ratio of intratumoral Treg cells to intratumoral CD8+ T cells or intratumoral CD4+ T cells.

[0417] 191. The method of any of embodiments 166-190, wherein the anti-CD25 antibody comprises a functional Fc region.

[0418] 192. The method of any of embodiments 166-191, wherein the anti-CD25 antibody is basiliximab or daclizumab, or a biosimilar, interchangeable, biobetter, copy biologic, or biogeneric thereof.

[0419] 193. The method of embodiment 192, wherein the anti-CD25 antibody is basiliximab.

[0420] 194. Anti-PD-1 antibodies include pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), Pidilizumab (CT-011), genolimuzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, and TSR-042 (ANB011).

[0421] 195. The method of any of embodiments 166-194, wherein the immune checkpoint inhibitor is administered to the subject one, two, three, four, five, six, seven, eight, nine, ten or more than ten times.

[0422] 196. The method of any of embodiments 169-195, wherein the anti-PD-1 antibody is administered to the subject one, two, three, four, five, six, seven, eight, nine, ten or more than ten times.

[0423] 197. The method of any one of embodiments 166-196, wherein the phthalocyanine dye is a Si-phthalocyanine dye.

[0424] 198. The method of embodiment 197, wherein the Si-phthalocyanine dye is IR700.

[0425] 199. The method of any of embodiments 166 to 198, wherein irradiation is performed 30 minutes to 96 hours after administration of the conjugate.

[0426] 200. The method of embodiment 199, wherein irradiation is performed 24 hours ± 4 hours after administration of the conjugate.

[0427] 201. The method of any of aspects 166-200, wherein the tumor is irradiated with light of a wavelength of 690±40 nm.

[0428] 202. The tumor is 50 J / cm 2 Or about 50 J / cm 2 Or the method of any of embodiments 166 to 201, wherein the fiber is irradiated at a dose of 100 J / cm of fiber length or about 100 J / cm of fiber length.

[0429] 203. The method of any of embodiments 166-202, further comprising the step of (d) administering an additional therapeutic agent or anti-cancer treatment.

[0430] 204. The method of any of embodiments 166-203, wherein one or more of steps (a), (b), (c), or (d) are repeated.

[0431] 205. The method of any of embodiments 166-204, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell carcinoma, thyroid cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cervical cancer, uterine cancer, cancer of the reproductive organs, lymphoma, and multiple myeloma.

[0432] 206. The method of any of embodiments 166-205, wherein the enhanced response comprises an additive or synergistic response compared to administration of the conjugate followed by irradiation alone, and compared to administration of the immune checkpoint inhibitor alone.

[0433] 207. The method of any of embodiments 166-206, wherein the enhanced response comprises an additive or synergistic response compared to administration of the conjugate followed by irradiation alone, and compared to administration of the anti-PD-1 antibody alone. EXAMPLES

[0434] IX. Working Example The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0435] Example 1: Generation of anti-CD25 antibody-IRDye 700 conjugates This example describes a method for preparing a conjugate containing IRDye 700DX (IR700) linked to the exemplary anti-CD25 antibody PC61, thus producing PC61-IRDye 700DX (PC61-IR700 conjugate).

[0436] The rat monoclonal antibody (mAb) PC61 (1 mg, 6.8 nmol) directed against mouse CD25 was incubated with IRDye 700DX NHS ester (IR700; LI-COR Bioscience, Lincoln, NE) (66.8 μg, 34.2 nmol, 5 mmol / L in DMSO) in 0.1 mol / L Na2HPO4 (pH 8.5) for 30–120 min at room temperature. The mixture was purified using a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, NJ). Protein concentration was determined by measuring absorbance at 595 nm with a UV-Vis system (8453 Value System; Agilent Technologies, Palo Alto, CA) using a Coomassie Plus protein assay kit (Pierce Biotechnology, Rockford, IL). The concentration of IR700 was measured by absorption with a UV-Vis system to confirm the number of fluorophore molecules conjugated to each PC61 molecule. The number of IR700 per PC61 was approximately 3.

[0437] The purity of the PC61-IR700 conjugate was confirmed by analytical size-exclusion HPLC (SE-HPLC). SE-HPLC was performed using an Agilent 1100 HPLC system (Santa Clara, CA) equipped with a PDA detector controlled by Chemstation software. SE chromatography was performed on a Shodex KW-803 column (New York, NY) eluted with phosphate-buffered saline (PBS) at 1.0 mL / min for 20 min. The PC61-IR700 preparation showed strong association and contained no detectable mAb aggregates as determined by SE-HPLC.

[0438] To determine the in vitro binding properties of the IR700 conjugates, the Indo-Gen procedure was used to characterize the conjugates. 125 I-labeling was performed. Minimal loss of mAb-IR700 conjugation was observed. Immunoreactivity assays were performed as previously described. Briefly, after trypsinization, 2 × 10 6 Tumor cells were resuspended in PBS containing 1% bovine serum albumin (BSA). 125 I-PC61-IR700 (1 mCi, 0.2 μg) was added and incubated on ice for 1 h. Cells were washed, pelleted, the supernatant was decanted, and cells were counted in a 2470 Wizard gamma counter (Perkin Elmer, Shelton, CT). Nonspecific binding to cells was examined under the condition of excess unlabeled antibody (200 μg unlabeled PC61).

[0439] Example 2: Anti-CD25-IR700 PIT and anti-PD-1 antibodies synergistically inhibit in vivo tumor growth and survival This example illustrates the synergistic inhibitory effect of an exemplary anti-CD25 antibody-IR700 PIT in combination with an exemplary anti-PD-1 antibody on tumor growth in vivo.

[0440] Immunocompetent BALB / c mice aged 6–8 weeks were treated with 3 × 10 6 CT26-EphA2 clone c4D10 murine colon cancer cells / mouse were inoculated subcutaneously into the right hind flank. Allograft tumors grew to approximately 150 mm 3 Once tumors had grown to 100 μL, mice were administered saline (100 μg), PC61-IR700 conjugate produced substantially as described in Example 1 above (100 μg), anti-PD-1 antibody clone RMP1-14 (100 μg), or a combination of PC61-IR700 conjugate (100 μg) and RMP1-14 (100 μg). PC61-IR700 conjugate was administered on day 4, and RMP1-14 on days 4, 6, 8, and 11. Twenty-four hours after administration of the PC61-IR700 conjugate, tumors in the PIT group were dosed with 690 nm and 100 J / cm. 2 The tumors were irradiated at a dose of 100 ng / mL. Tumor growth and survival were monitored for 22 days. Tumor volume was calculated using the formula: tumor volume = (width x length) x height / 2.

[0441] In mice receiving PC61-IR700-PIT or anti-PD-1 (RMP1-14) monotherapy, tumor growth was substantially inhibited compared to tumor growth in control mice receiving saline or PC61-IR700 conjugate alone and no PIT (Figure 1A; compare filled and open triangles with open and closed circles, respectively). Notably, tumor growth was further inhibited synergistically in mice treated with the combination of PC61-IR700-PIT and anti-PD-1 antibody (Figure 1A, filled squares). In addition to examining overall tumor growth, complete response (CR) rates were also compared between treatment groups. After 25 days of treatment, 7 / 20 (35%) of mice treated with PC61-IR700 PIT and 1 / 12 (8.3%) of mice treated with anti-PD-1 (RMP1-14) monotherapy achieved CR, whereas 1 / 16 (6.25%) or 0 / 12 (0%) of animals achieved CR in the control groups with saline only or PC61-IR700 conjugate alone and no PIT. Strikingly, the combination of PC61-IR700 conjugate and anti-PD-1 treatment resulted in CR in 73.9% (17 / 23) of mice (Figure 1A). The combination therapy had a substantially greater CR rate than either PC61-IR700-PIT or anti-PD-1 monotherapy, and the effects of these two treatments were synergistic. In support of these results, mice receiving PC61-IR700-PIT or anti-PD-1 (RMP1-14) monotherapy (closed triangles and closed circles, respectively) showed extended survival compared to control mice receiving saline or PC61-IR700 conjugates alone and no PIT (Figure 1B, compare closed and open triangles with open circles and closed circles, respectively), and mice treated with the combination of PC61-IR700 PIT and PD-1 antibody showed even better (100%) survival (Figure 1B, closed squares). These results support a synergistic effect between anti-CD25 PIT and anti-PD-1 therapy.

[0442] Example 3: Anti-CD25-IR700 PIT and anti-PD-1 synergistically inhibit the in vivo growth of non-irradiated distant tumors This example illustrates the synergistic inhibitory effect of an exemplary anti-CD25 antibody-IR700 PIT in combination with an exemplary anti-PD-1 antibody (RMP1-14) on the growth of non-irradiated distant tumors.

[0443] BALB / c mice aged 6–8 weeks were treated with 3 × 10 6 CT26-EphA2 clone c4D10 cells / mouse were inoculated subcutaneously into both the right and left hind flanks. Mice had bilateral allograft tumors of approximately 150 mm 3 Once tumors had grown to a size of 100 μL, mice were administered saline (100 μL), anti-CD25 antibody PC61-IR700 conjugate (100 μg), RMP1-14 (100 μg), or a combination of PC61-IR700 conjugate (100 μg) and RMP1-14 (100 μg). PC61-IR700 conjugate was administered on day 4, and RMP1-14 on days 4, 6, 8, and 11. Twenty-four hours after administration of PC61-IR700 conjugate, tumors in the right flank of mice in the PC61-IR700 PIT group were injected with 690 nm and 100 J / cm. 2 The right flank tumor was irradiated at 100x the primary tumor site, whereas the left flank tumor was not irradiated. Thus, the right flank tumor served as the primary tumor and the left flank tumor served as the distant or metastatic tumor.

[0444] As shown in Figures 2A-2B, in mice treated with PC61-IR700 conjugates with irradiation (PC61-IR700 PIT; closed triangles) or RMP1-14 monotherapy (open triangles), the growth of irradiated primary tumors (Figure 2A) and distant non-irradiated tumors (Figure 2B) was substantially inhibited compared to mice treated with PC61-IR700 conjugates without irradiation (closed circles). In mice treated with PC61-IR700 conjugates with irradiation (PC61-IR700 PIT) and anti-PD-1 (RMP1-14) in combination, the growth of irradiated primary tumors (Figure 2A) and distant non-irradiated tumors (Figure 2B) was further inhibited, and this inhibitory effect was synergistic (closed squares).

[0445] Example 4: Effect of anti-CD25 antibodies on circulating regulatory T cell levels This example describes the effect of an exemplary anti-CD25 antibody on circulating regulatory T cells (Tregs).

[0446] BALB / c mice were administered 100 μg of anti-CD25 antibody (PC61 antibody) (PC61 WT; n=8), or PC61 antibody in which the rat Fc region is replaced by the wild-type mouse Fc region (PC61 mouse WT; n=8), or PC61 antibody in which the rat Fc region is replaced by the N297Q mutant mouse Fc region lacking effector function / ADCC activity (PC61 mouse N297Q; n=8). Control mice were administered 100 μl of saline for comparison (n=6). Blood samples were collected on days 1 and 8 after antibody administration and isolated lymphocytes were stained for cell markers including CD25, FoxP3, CD3 and CD4. Isotype controls were also used for staining. Stained cells were analyzed using flow cytometry to determine CD3 + CD4 + CD25 on T cells + FoxP3 + The ratio was determined for each condition.

[0447] CD25 in mice administered PC61 carrying wild-type rat or mouse Fc domains + FoxP3 + The percentage of CD25 T cells was reduced compared to saline controls the day after treatment (Figure 3A), and the reduction persisted on day 8 (Figure 3B). + FoxP3 + The proportion of T cells was reduced to a lesser extent the day after administration of PC61 with mutant Fc than with an antibody with a wild-type Fc domain (PC61 mouse N297Q; FIG. 3A). Unlike mice treated with an antibody with a wild-type Fc domain, the reduction after administration of PC61 mouse N297Q was greater than that of recovered CD25 + FoxP3 + was transient, as the proportion of T cells was similar to control levels on day 8 ( Fig. 3B ).

[0448] Example 5: Synergistic Effects of Anti-CD25-IR700 PIT and Anti-PD-1 on Non-irradiated Distant Tumors This example demonstrates the efficacy of circulating regulatory T cells (TCR) in synergistic anti-cancer effects of an exemplary anti-CD25 antibody-IR700 PIT in combination with an exemplary anti-PD-1 antibody (RMP1-14) on the growth of non-irradiated distant tumors. reg ) Explain the role of depletion.

[0449] BALB / c mice aged 6–8 weeks were treated with 3 × 10 6 CT26-EphA2 clone c4D10 cells / mouse were inoculated subcutaneously into both the right and left hind flanks. Mice had bilateral homograft tumors of approximately 130 mm 3 ~140mm 3 Once the mice had grown to a size of 100 μL (day 6 post-implantation), they were administered saline (100 μL), anti-CD25 antibody PC61-IR700 conjugates containing a murine wild-type (mWT) Fc region (mWT PC61-IR700) or anti-CD25 antibody PC61-IR700 conjugates containing a N297Q mutant Fc region lacking effector function (N297Q) (N297Q PC61-IR700) (100 μg), anti-PD-1 (RMP1-14; 100 μg), or a combination of PC61-IR700 conjugates containing WT or N297Q Fc regions (100 μg) and anti-PD-1 (100 μg). mWT PC61-IR700 and N297Q conjugates were administered on day 6, and RMP1-14 on days 6, 8, 10, and 12. Twenty-four hours after administration of the PC61-IR700 conjugate, the right flank tumors of the mice in the PC61-IR700 PIT group were treated with 690 nm and 100 J / cm 2 The right flank tumor was irradiated at 100 nm, while the tumor on the left flank was shielded with black foil to prevent irradiation. Thus, the right flank tumor served as the primary tumor and the left flank tumor served as the non-irradiated distant tumor. Tumor volumes were measured every 2-3 days until 18 days post-implantation. Results for mice receiving treatment with a conjugate bearing the mWT Fc domain are shown in Figure 4A. Results for mice receiving treatment with a conjugate bearing the mutant N297Q Fc domain lacking ADCC capabilities are shown in Figure 4B.

[0450] Mice administered saline alone (open circles; Figures 4A and 4B) showed progressive tumor growth throughout the course of the study. Mice administered conjugate alone (mWT or N297Q) (open triangles, Figures 4A and 4B, respectively) also showed progressive tumor growth. Combined administration of conjugate (mWT or N297Q) and anti-PD-1 without irradiation (open squares, Figures 4A and 4B, respectively) resulted in somewhat reduced tumor growth compared to saline controls. PIT with mWT conjugate (closed triangles, Figure 4A) resulted in reduced tumor growth compared to saline controls, while PIT with N297Q conjugate without ADCC capability (closed triangles, Figure 4B) had no effect on tumor growth. Combining PIT with mWT conjugate and anti-PD-1 treatment (closed squares, Figure 4A) virtually halted tumor growth. The efficacy of the PIT-anti-PD-1 combination treatment was substantially reduced when the N297Q conjugate was administered (filled squares, FIG. 4B). These results, taken together with those of Example 4, suggest that the anti-cancer activity of anti-CD25-IR700 PIT as monotherapy or in combination with anti-PD-1 treatment is consistent with the concomitant sustained circulating T reg It is shown that the expression level of β-lactamase was significantly increased with depletion of β-lactamase.

[0451] Example 6: Effect of anti-CD25-IR700 PIT on intratumoral regulatory T cells This example describes the expression of regulatory T cells (T reg ) in vivo depletion.

[0452] BALB / c mice, 1 × 10 6 4T1-EpCAM tumor cells were inoculated subcutaneously into the right hind flank. Tumors grew to an average of approximately 180 mm 3 Once the volume was reached, the mice were treated with saline, anti-CD25 antibody PC61-IR700 conjugate alone, or PC61-IR700 conjugate with irradiation (PC61-IR700 PIT). 24 hours after administration of the conjugate, tumors in mice in the irradiation (PIT) group were irradiated with 100 J / cm2. 2The tumors were exposed to 1000 ng of 690 nm light. Two hours after irradiation, tumors from all groups were excised and processed into single cell suspensions. The suspended cells were then reg Staining was performed for the markers CD3, CD4, CD45, and FoxP3. Stained cells were analyzed using flow cytometry, and CD3 + CD4 + The percentage of FoxP3 cells among lymphocytes was determined.

[0453] As shown in Figure 5, in tumor-bearing mice treated with PC61-IR700 PIT (circles), FoxP3 + CD3 + CD4 + The number of T cells was significantly reduced compared to mice treated with saline (squares) or PC61-IR700 conjugate alone (diamonds) (P<0.01 and P<0.0001, respectively), indicating an immediate reduction in Tregs in the tumor after anti-CD25 PIT (Figure 5). + No reduction in T cells was observed (data not shown). The results showed that anti-CD25 PIT suppressed intratumoral regulatory T cells (T reg ) leading to acute depletion.

[0454] Example 7: Anti-CD25-IR700 PIT inhibits intratumoral CD8 + Alleviating T cell exhaustion This example demonstrates in vivo intratumoral CD8 + 1 illustrates the mitigating effect of an exemplary anti-CD25 antibody-IR700 PIT on T cell exhaustion.

[0455] BALB / c mice were inoculated with CT26-EphA2 clone c4D10 tumor cells. Tumors grew to an average of 150 mm 3 Once the volume was reached, the mice were treated with saline, anti-CD25 antibody PC61-IR700 conjugate alone, or PC61-IR700 conjugate with irradiation (PC61-IR700 PIT). 24 hours after administration of the conjugate, tumors in mice in the irradiation (PIT) group were dosed with 690 nm and 100 J / cm. 2Two days after irradiation, tumors from all groups were excised and processed into single cell suspensions. The suspended cells were then stained for cell markers including CD3, CD45, CD8a, PD-1, and CTLA4. Isotype controls were also used for staining. Stained cells were analyzed using flow cytometry.

[0456] As shown in Figures 6A-6B, mice treated with PC61-IR700 PIT had significantly increased intratumoral CD8 + T cells (CD3 + CD8 + The percentage of exhausted CD8 + Cell (PD1 + CTLA-4 + ) was significantly decreased after PC61-IR700 PIT compared with saline or conjugate alone controls (P<0.0001) (Figure 6B).

[0457] Example 8: Anti-CD25-IR700 PIT inhibits non-exhausted intratumoral effector CD8 + Increases T lymphocytes This example illustrates the stimulatory effect of an exemplary anti-CD25 antibody-IR700 PIT on the expansion of intratumoral effector CD8+ T lymphocytes in vivo.

[0458] BALB / c mice were inoculated with CT26-EphA2 clone c4D10 tumor cells. Tumors averaged approximately 150 mm 3 Once the volume was reached, the mice were treated with saline, PC61-IR700 conjugate alone or PC61-IR700 conjugate with irradiation (PC61-IR700 PIT). 24 hours after administration of the conjugate, tumors in mice in the irradiation (PIT) group were irradiated with 100 J / cm 2The tumors were exposed to 1000 x 690 nm light for 8 days. Eight days after irradiation, tumors from all groups were excised and processed into single cell suspensions. The suspended cells were then stained for cell markers including CD3, CD45, CD8a, PD-1, and CTLA4. Isotype controls were also used for staining. The stained cells were analyzed using flow cytometry.

[0459] As shown in Figure 6C, tumor-bearing mice treated with PC61-IR700 PIT showed no exhausted PD-1 in the tumors. - CTLA-4 - CD3 + CD8 + The number of T cells was significantly increased (P<0.001) compared with those treated with saline or PC61-IR700 conjugate alone (Conj.; without irradiation), indicating that PC61-IR700 PIT promotes the expression of effector CD8 T cells in the tumor microenvironment. + These results indicate that the IL-1 induced T cell expansion (Figure 6C).

[0460] Example 9: Anti-CD25-IR700 PIT increases activated CD8 + Bringing T cells This example describes the effect of an exemplary anti-CD25 antibody-IR700 PIT on intratumoral CD8+ T cell activation in vivo.

[0461] BALB / c mice were inoculated with 4T1-EpCAM tumor cells. Tumors reached an average size of approximately 150 mm. 3 Once the volume was reached, the mice were treated with saline, anti-CD25 antibody PC61-IR700 conjugate alone, or PC61-IR700 conjugate with irradiation (PC61-IR700 PIT). 24 hours after administration of the conjugate, tumors in mice in the irradiation (PIT) group were dosed with 690 nm and 100 J / cm. 2 Seven days after irradiation, tumors from all groups were excised and processed into single cell suspensions. The suspended cells were then stained for cell markers including CD3, CD69, CD45, CD8a, and Ki67. Isotype controls were also used for staining. Stained cells were analyzed using flow cytometry.

[0462] As shown in Figure 7, mice treated with PC61-IR700 PIT (circles) showed increased expression of activated CD8 + T cells (CD3 + CD8 + Ki67 + , left panel; and CD3 + CD8 + CD69 + , right panel) was significantly increased (P<0.001) compared to mice receiving saline (squares) or PC61-IR700 conjugate alone (Conj.; no irradiation) (diamonds). These results indicate that anti-CD25 PIT results in increased activated CD8 T cells in irradiated tumors.

[0463] Example 10: Anti-CD25-IR700 PIT induces persistent intratumoral increased CD8 + / T reg Bring about the ratio This example demonstrates that in vivo intratumoral CD8 is a predictive marker of clinical response to treatment. + T cells and regulatory T cells (T reg ) ratio.

[0464] BALB / c mice, 1 × 10 6 CT26-EphA2 clone c4D10 tumor cells were inoculated subcutaneously into the right hind flank. Tumors averaged approximately 150 mm 3 Once the volume was reached, mice were treated with saline, PC61-IR700 conjugate (100 μg) alone (Conj.), or PC61-IR700 conjugate (100 μg) with irradiation (PC61-IR700 PIT). 24 hours after administration of the conjugate, tumors in mice in the irradiated (PIT) group were injected with 690 nm and 100 J / cm. 2At 2 hours or 8 days after irradiation, tumors from all groups were excised and processed into single cell suspensions. The suspended cells were then stained for cell markers including CD3, CD45, CD8a, CD4 and FoxP3. Isotype controls were also used for staining. Flow cytometry was used to analyze the stained cells and to identify intratumoral CD8 + T cells and T reg The ratio was determined (Figures 8A to 8B).

[0465] As shown in Figure 8A, at 2 hours post-irradiation, tumors from mice treated with PC61-IR700 PIT (triangles) showed increased intratumoral CD8+ cells compared with mice receiving saline (circles; P ≤ 0.05) or PC61-IR700 conjugate alone (no irradiation) (squares; P ≤ 0.05). + / T reg These results indicate that anti-CD25 PIT results in intratumoral CD8 T cell activation. Increased CD8 + / T reg The ratio persisted up to 8 days after irradiation (FIG. 8B; P≦0.0001). These results suggest that treatment with anti-CD25 PIT alone significantly increased the CD8 + / T reg These results show that this leads to a persistent increase in the ratio.

[0466] Example 11: Rejection of rechallenged tumor growth in mice with complete response after anti-CD25-PIT700 PIT and anti-PD-1 treatment This example describes the rejection of growth of freshly inoculated tumors in mice that achieved a complete response following initial treatment with an exemplary anti-CD25 antibody-IR700 PIT alone or in combination with an exemplary anti-PD-1 antibody (RMP1-14) treatment.

[0467] BALB / c mice aged 6–8 weeks were treated with 3 × 10 6 CT26-EphA2 clone c4D10 cells / mouse were inoculated subcutaneously into the right hind flank. Allograft tumors grew approximately 150 mm 3Once tumors had grown to a size of 10 μL, mice were administered saline (100 μL), anti-CD25 antibody PC61-IR700 conjugate (100 μg), anti-PD-1 antibody RMP1-14 (100 μg), or a combination of PC61-IR700 conjugate (100 μg) and RMP1-14 (100 μg). PC61-IR700 conjugate was administered on day 4, and RMP1-14 on days 4, 6, 8, and 11. Twenty-four hours after administration of PC61-IR700, tumors were dosed with 690 nm and 100 J / cm. 2 42 days after initial tumor inoculation (38 days after treatment), mice that achieved a complete response (with disappearance of tumor) (CR mice) and naïve mice were rechallenged with CT26-EphA2 cells in the left hind flank. For animals that did not show tumor growth after rechallenge with CT26-EphA2 cells, 78 days after initial tumor inoculation (74 days after treatment), 4T1 tumors (a syngeneic mouse tumor line of different origin from CT26) were inoculated into the right axilla.

[0468] In CR mice reloaded with the same type of tumor cells, CT26-EphA2, the tumor cells were completely rejected, and no tumor growth was observed in the initial treatment groups with PC61-IR700 PIT alone (5 / 5, 100%) and PC61-IR700 PIT in combination with RMP1-14 (17 / 17, 100%), whereas tumor volume increased in naive mice not previously exposed to treatment (Figure 9). After inoculation of 4T1 tumor cells (a different type of tumor cells than those initially inoculated) into the right axilla, 4T1 tumor growth increased similarly between treated mice and naive mice not previously exposed to treatment (Figure 10). These data indicated that the rejection of reloaded tumor cell growth was tumor-specific.

[0469] Example 12: Anti-CD25-IR700 PIT and anti-PD-1 enhance systemic immunity This example illustrates the stimulatory effect of an exemplary anti-CD25 antibody-IR700 PIT in combination with an exemplary anti-PD-1 antibody on systemic immunity in vivo.

[0470] A. Cytotoxic T Lymphocyte (CTL) Assay A CTL assay was designed to assess the tumor-specific cytotoxic activity of splenocytes from mice inoculated with CT26-EphA2 clone c4D10 tumors. Cytotoxicity was assessed using the CytoTox™ 96 Non-Radioactive Cytotoxicity Assay (Promega; Cat. # G1780). This kit measured the levels of lactate dehydrogenase (LDH) in the wells, which is released from cells upon cell death. Spleens were harvested from tumor-bearing mice that had achieved a complete response after treatment with anti-CD25 antibody PC61-IR700 PIT monotherapy or PC61-IR700 PIT in combination with anti-PD-1 (RMP1-14) (CR mice) or from tumor-bearing mice that were treatment naive. Single cell suspensions were prepared by mechanical dissociation of spleens over a 70 μm pore size cell strainer. The resulting flow-through fraction was collected and red blood cells were lysed. Suspended splenocytes were primed with CT26 antigen AH1 peptide in vitro for 4 days. Cytotoxicity assays were then performed by co-incubating splenocytes (effector cells) with CT26-ephA2 clone c4D10 target cells at various effector:target cell ratios (E:T ratios) for 4 hours. Splenocytes were then harvested and the LDH levels released by the target cells were measured. Human pancreatic cancer cell line BxPC3 cells were used as irrelevant control target cells.

[0471] B. Results In CR mice treated with PC61-IR700 PIT alone or in combination with RMP1-14, splenocytes demonstrated ex vivo tumor-specific immune responses against target tumor cells (Figure 11). For splenocytes derived from CR mice treated with PC61-IR700 PIT alone, the results showed a clear E:T ratio-dependent cytotoxic effect against target tumor cells, capable of killing 100% of target cells at an E:T ratio of 31:1 and 92% at an E:T ratio of 7.8:1 (Figure 11). For splenocytes derived from CR mice previously treated with PC61-IR700 PIT together with RMP1-14, the results showed a clear E:T ratio-dependent cytotoxic effect against target tumor cells, capable of killing 91% of target cells at an E:T ratio of 31:1 and 75% at an E:T ratio of 7.8:1. For splenocytes from untreated naive mice, the results showed minimal cytotoxicity against target tumor cells, killing less than 11% of the target cells at any E:T ratio (Figure 11). Furthermore, against BxPC3 cells (an unrelated tumor cell type serving as a control for target tumor cells), E:T ratios as high as 250:1 only produced less than 15% cytotoxicity. These results clearly demonstrated that anti-CD25-IR700 PIT treatment alone or in combination with anti-PD-1 resulted in an increase in tumor-specific cytotoxic T cells in the spleen.

[0472] Example 13: CD8 of anti-CD25-conjugate and anti-PD-1 treatment + T Cell Activity and Efficacy This example demonstrates the effect of an exemplary anti-CD25 antibody-IR700 PIT in combination with an exemplary anti-PD-1 antibody on in vivo tumor growth by inhibiting functional CD8 + This demonstrates that the expression of β-lactamase inhibitors was dependent on the T cell population.

[0473] BALB / c mice, 3 × 10 6 CT26-EphA2 clone c4D10 cells were inoculated subcutaneously into both the right and left hind flanks per mouse. CD8a was induced by intraperitoneal injection of anti-CD8a antibody (BioXCell, clone 2.43, catalog# BP0061) (100 μg / mouse) on days 4 and 7 after tumor cell inoculation.+ T cell-depleted mice were generated. The mice had bilateral allograft tumors of approximately 150 mm 3 Once the tumors had grown to a size of 100 μg, the mice were administered saline, anti-CD25 antibody PC61-IR700 conjugate (100 μg), anti-PD-1 antibody RMP1-14 (100 μg), or a combination of PC61-IR700 conjugate (100 μg) and RMP1-14 (100 μg). PC61-IR700 conjugate was administered on day 4, and RMP1-14 was administered on days 4, 6, 8, and 11. Twenty-four hours after administration of PC61-IR700 conjugate, tumors in the right flank of mice in the PIT group were implanted with 690 nm and 100 J / cm. 2 The right flank tumor was irradiated at a dose of 100 mg / kg / day, whereas the left flank tumor was not irradiated. Thus, the right flank tumor served as the primary tumor and the left flank tumor served as the distant tumor.

[0474] As shown in Figures 12A-12B, combined treatment of PC61-IR700 conjugate with irradiation (PC61-IR700 PIT) and anti-PD-1 (RMP1-14) substantially and synergistically inhibited tumor growth of both irradiated tumors (right flank; Figure 12A) and non-irradiated distal tumors (left flank; Figure 12B) in immunocompetent BALB / c mice (black squares) compared to individual PC61-IR700 PIT (black triangles) or anti-PD-1 (white triangles) monotherapy. Growth of both irradiated tumors (right flank) and non-irradiated distal tumors (left flank) was substantially inhibited compared to control saline or PC61-IR700 conjugate only (non-irradiated) groups (data not shown). Surprisingly, CD8 + In T cell-depleted mice, the inhibitory effect of the combination of PC61-IR700 PIT and anti-PD-1 on tumor growth was completely abolished (Figures 12A and 12B; closed diamonds), indicating that the effect of the combination treatment was limited to CD8 + This indicates that it was mediated by T cells.

[0475] Example 14: Reduction of immunosuppressive intratumoral regulatory T cells (Tregs) by CD25 PIT and anti-PD-1 treatment This example describes the effect of an exemplary anti-CD25 conjugate PIT treatment in combination with an anti-PD-1 antibody on the ratio of intratumoral CD8 T cells to Tregs.

[0476] BALB / c mice, 1 × 10 6 CT26-EphA2 clone c4D10 cells were inoculated subcutaneously into the right hind flank. Allograft tumors grew approximately 150 mm 3 Once tumors had grown to a size of 100 μg, mice were administered saline control, anti-CD25 antibody PC61-IR700 conjugate (100 μg), or a combination of PC61-IR700 conjugate (100 μg) and anti-PD-1 antibody (clone RMP1-14, 100 μg). PC61-IR700 conjugate was administered on day 7, and anti-PD-1 antibody was administered on days 7, 9, 11, and 13. Twenty-four hours after administration of PC61-IR700 conjugate, tumors in the right flank of mice in the PIT group were implanted with 690 nm and 100 J / cm. 2 The irradiation was performed at a dose of .

[0477] Tumors were excised from the first and second subsets of mice from each of the treatment groups, respectively, 8 days after irradiation. The excised tumors were processed into single cell suspensions. The suspended cells were then stained for cell markers, including CD3, CD45, CD4, and FoxP3. Isotype controls were also used for staining. The stained cells were analyzed using flow cytometry.

[0478] Flow cytometry analysis showed that the combination of anti-CD25 conjugate PIT treatment and anti-PD-1 antibody (CD25 PIT+PD1) significantly increased the expression of FoxP3 + We demonstrated that combination treatment provided a durable reduction in FoxP3 Tregs (Figure 13A). + The reduction in Tregs was significant with combined conjugate and anti-PD-1 antibody treatment compared with no irradiation (CD25 Conj.+PD1) (P≦0.01) and with anti-CD25 conjugate PIT treatment alone without anti-PD-1 antibody (CD25 PIT) (P≦0.05).

[0479] Combination treatment (CD25 PIT+PD1) also increased CD4 + Helper T cells (CD4 + FoxP3 - ) and Treg cells (CD4 + FoxP3 + ) ratio (Figure 13B). This increase was significant when combined with the conjugate and anti-PD-1 antibody, compared with no irradiation (CD25 Conj.+PD1) (P≦0.05), and when combined with anti-CD25 conjugate PIT treatment alone without anti-PD-1 antibody (CD25 PIT) (P≦0.05). The effect of using anti-CD25 conjugate PIT with anti-PD-1 antibody was not only to reduce intratumoral CD4 + and Treg ratio.

[0480] Anti-CD25 conjugate PIT treatment alone (CD25 PIT) or in combination with anti-PD-1 antibody (CD25 PIT+PD1) resulted in a substantial increase in the ratio of intratumoral CD8 T cells to FoxP3 Tregs compared with the control group (Figure 13C).

[0481] These results indicate that combined treatment with anti-CD25 conjugated PIT and anti-PD-1 antibody treatment induces a durable reduction in intratumoral Tregs. The proposed mechanism of action is depicted in FIG.

[0482] The present invention should not be limited in scope by the embodiments disclosed herein, which are intended as one illustration of each aspect of the present invention, and any functional equivalents are within the scope of the present invention.In addition to those described herein, various modifications to the compositions and methods of the present invention will be apparent to those skilled in the art from the foregoing descriptions and teachings, and are intended to be included within the scope of the present invention as well.Such modifications and other embodiments can be practiced without departing from the true scope and spirit of the present invention.

Claims

1. A medicament for treating cancer comprising a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody, (a) the conjugate, in combination with an anti-PD-1 antibody, is administered to a subject having cancer, including a primary tumor and a secondary population of tumor cells; and (b) after administration of the conjugate, the primary tumor is at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm is directly irradiated and the secondary population is not directly irradiated, the primary tumor and / or secondary populations are inhibited to a greater extent as compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone; The above medicine.

2. A medicament for treating cancer, comprising an anti-PD-1 antibody, (a) the anti-PD-1 antibody is administered in combination with a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody to a subject having cancer, including a primary tumor and a secondary population of tumor cells; and (b) after administration of the conjugate, the primary tumor is at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm is directly irradiated and the secondary population is not directly irradiated, the primary tumor and / or secondary populations are inhibited to a greater extent as compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone; The above medicine.

3. A medicament for treating cancer, comprising a combination of a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody and an anti-PD-1 antibody, (a) the conjugate, in combination with an anti-PD-1 antibody, is administered to a subject having cancer, including a primary tumor and a secondary population of tumor cells; and (b) after administration of the conjugate, the primary tumor is at or about 600 nm to 850 nm or about 850 nm; and 25J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at a dose of 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm is directly irradiated and the secondary population is not directly irradiated, the primary tumor and / or secondary populations are inhibited to a greater extent as compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone; The above medicine.

4. The method of any one of claims 1 to 3, wherein the secondary population comprises (i) metastatic tumour cells, (ii) invasive tumour cells, or (iii) metastatic and invasive tumour cells.

5. The method of any one of claims 1 to 4, wherein a first dose of the anti-PD-1 antibody is administered prior to administration of the conjugate.

6. 6. The pharmaceutical composition of claim 5, wherein the first dose of the anti-PD-1 antibody is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks prior to administration of the conjugate.

7. Anti-PD-1 antibodies, (i) is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times (a) simultaneously with administration of the conjugate; or (b) prior to administration of the conjugate; and (ii) The pharmaceutical agent of any one of claims 1 to 6, wherein following administration of the conjugate, the subject is subsequently administered 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 administrations of the anti-PD-1 antibody.

8. The method of any one of claims 1 to 4, wherein the first dose of the anti-PD-1 antibody is administered subsequent to administration of the conjugate.

9. The method of any one of claims 1 to 8, wherein the secondary population is comprised in a solid tumor.

10. The pharmaceutical of any one of claims 1 to 9, wherein the anti-CD25 antibody comprises a functional Fc region.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the anti-CD25 antibody is basiliximab or daclizumab.

12. The pharmaceutical composition of claim 11, wherein the anti-CD25 antibody is basiliximab.

13. Anti-PD-1 antibodies include pembrolizumab (MK-3475, KEYTRUDA; lambrolizumab), nivolumab (OPDIVO), cemiplimab (LIBTAYO), toripalimab (JS001), HX008, SG001, GLS-010, dostallimab (TSR-042), tislelizumab (BGB-A317), cetrelimab (JNJ-63723283), and pidilizumab ( CT-011), genolimzumab (APL-501, GB226), BCD-100, cemiplimab (REGN2810), F520, sintilimab (IBI308), CS1003, LZM009, camrelizumab (SHR-1210), SCT-I10A, MGA012, AK105, PF-06801591, AMP-224, AB122, AMG 404, BI 754091, HLX10, JTX-4014, AMP-514 (MEDI0680), Sym021, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, spartalizumab, BCD-217, HX009, IBI308, PDR001, REGN2810, and TSR-042 (ANB011).

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the phthalocyanine dye is a Si-phthalocyanine dye.

15. The pharmaceutical composition of claim 14, wherein the Si-phthalocyanine dye is IR700.

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

17. The pharmaceutical agent according to any one of claims 1 to 16, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, skin cancer, lung cancer, non-small cell lung cancer, renal cell cancer, thyroid cancer, prostate cancer, head and neck cancer, digestive cancer, gastric cancer, cancer of the small intestine, spindle cell neoplasm, hepatocellular carcinoma, liver cancer, cancer of the peripheral nerves, brain cancer, cancer of skeletal muscle, cancer of smooth muscle, bone cancer, cancer of adipose tissue, cancer of the cervix, cancer of the uterus, cancer of the reproductive organs, lymphoma, and multiple myeloma.

18. administering to a subject a conjugate comprising a phthalocyanine dye linked to an anti-CD25 antibody and an anti-PD-1 antibody results in an enhanced response; the enhanced response comprises an enhancement of the subject's systemic immunity compared to the subject's systemic immunity prior to administration of the conjugate followed by irradiation and an anti-PD-1 antibody; and / or the enhanced response comprises enhanced tumor inhibition compared to administration of the conjugate alone, the conjugate followed by irradiation alone, or the anti-PD-1 antibody alone. The pharmaceutical composition according to any one of claims 1 to 17.

19. The pharmaceutical agent of claim 18, wherein the enhanced response is a synergistic response, the synergistic response comprising a synergistic reduction in primary tumor growth, tumor volume, tumor size or tumor mass, a synergistic reduction in the number of cells in a secondary population of subjects, a synergistic reduction in metastatic or invasive tumor cell growth, tumor volume, tumor size, tumor mass or number, or any combination thereof.

20. Systemic immunity, (a) Cytotoxic T lymphocyte (CTL) activity assay, intratumoral T cell exhaustion assay, intratumoral effector T cell proliferation assay, T cell receptor diversity assay, activated CD8 + T cell assay, circulating regulatory T cell (Treg) assay, intratumoral Treg assay, or CD8 + T cells: one or more of the Treg assays; (b) CTL activity assay using splenocytes or peripheral blood cells or bone marrow cells or lymph node cells, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject; (c) an intratumoral T cell exhaustion assay using T cells collected from a primary tumor or a metastatic or invasive tumor cell mass, optionally collected from a subject between 4 and 28 days after irradiation of the primary tumor in the subject; (d) an intratumoral effector T cell proliferation assay using T cells collected from a primary tumor or a metastatic or invasive tumor cell mass, optionally collected from a subject between 4 and 28 days after irradiation of the primary tumor in the subject; (e) a T cell receptor diversity assay using T cells collected from the primary tumor or metastatic tumor cell mass or invasive tumor cell mass or peripheral circulation, optionally collected from the subject between 4 days and 28 days after irradiation of the primary tumor in the subject; or (f) determining the presence, number or frequency of regulatory T cells (Treg) in tumor from the primary tumor or metastatic or invasive tumor cell mass, optionally collected from the subject 4 to 28 days after irradiation of the primary tumor in the subject, and / or the ratio of intratumoral Treg cells to intratumoral CD8+ T cells or intratumoral CD4+ T cells; The pharmaceutical composition according to claim 18 or 19, wherein the measurement is performed by

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