Methods for local and systemic treatment of cancer, tumors and tumor cells - Patents.com

Abstract

Provided herein are conjugates, compositions, methods and uses for treating subjects with cancer, including cancer, such as first tumor, primary tumor, metastatic tumor cells, and / or invasive tumor cells. The methods include administering to a subject a targeting molecule that binds to interleukin-2 receptor alpha chain (CD25) without substantially blocking or interfering with IL-2 signaling, conjugated with a phthalocyanine dye, such as IR700, followed by irradiating the first tumor or primary tumor with a wavelength of light to activate the phthalocyanine dye. The methods and uses described herein provide for the reduction and elimination of tumor and tumor cell growth, including first tumor, primary tumor, metastatic tumor cells, and / or invasive tumor cells. Also provided are methods and uses for enhancing systemic immunity against tumor growth in subjects with cancer, tumor, or lesion. TIFF2024505556000032.tif107170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 144,931, entitled "METHODS FOR LOCAL AND SYSTEMIC TREATMENT OF CANCERS, TUMORS AND TUMOR CELLS," filed February 2, 2021, the contents of which are incorporated by reference in their entirety.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named 751702002240SeqList.txt, created on January 31, 2022, and having a size of 38.2 kilobytes. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to conjugates, compositions, methods and uses for treating a subject with cancer, such as cancer comprising a first tumor, a primary tumor, a metastatic tumor cell, and / or an invasive tumor cell. The method includes administering to the subject a targeting molecule that binds to the interleukin-2 receptor alpha chain (CD25) without substantially blocking or interfering with IL-2 signaling, the targeting molecule being conjugated to a phthalocyanine dye, such as IR700, followed by irradiating the first tumor or primary tumor with a wavelength of light to activate the phthalocyanine dye. The methods and uses described herein provide for the reduction and elimination of tumor and tumor cell growth, including a first tumor, a primary tumor, a metastatic tumor cell, and / or an invasive tumor cell. Also provided are methods and uses for enhancing systemic immunity against tumor growth in a subject with cancer, tumor or lesion. [Background technology]

[0004] background Cancer metastasis is the main cause of cancer-related death.Although there are 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

[0005] overview Compositions, combinations, methods and uses for treating cancer are provided herein. In some of the embodiments, a conjugate is provided herein that includes an antibody or antigen-binding fragment that specifically binds to CD25 without substantially blocking or interfering with IL-2 signaling and a Si-phthalocyanine dye. In some of the embodiments, the conjugate is activated by irradiation at a wavelength of at or about 600 nm to at or about 850 nm to cause cell death. In some of the embodiments, the activated conjugate does not substantially block or interfering with IL-2 signaling. In some of the embodiments, the Si-phthalocyanine dye is IR700. In some of the embodiments, the Si-phthalocyanine dye is represented by the formula (I): It has the structure TIFF2024505556000002.tif71128, or a salt, stereoisomer, or tautomer thereof.

[0006] In some of the optional embodiments, the activated conjugate causes tumor inhibition or killing at a higher level, activity or potency than the unconjugated antibody.

[0007] Provided herein is a conjugate comprising an antibody or antigen-binding fragment that specifically binds to CD25 without substantially blocking or interfering with IL-2 signaling and a Si-phthalocyanine dye, wherein the conjugate is activated by irradiation at a wavelength at or about 600 nm to 850 nm or about 850 nm to cause cell killing.

[0008] In some of the embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable (V H ) region and the light chain variable (V L In some of the optional embodiments, the V H The region is V H The heavy chain complementarity determining region 1 (CDR-H1), the heavy chain complementarity determining region 2 (CDR-H2) and the heavy chain complementarity determining region 3 (CDR-H3) are contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:2. L The CDR-L1 region includes a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2) and a light chain complementarity determining region 3 (CDR-L3) contained within the amino acid sequence of the CDR-L1 region. H The region is V as shown in SEQ ID NO:3. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:4. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:6. L The domains include CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence.

[0009] In some of the optional embodiments, V HThe region is V H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:11. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:12. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:10. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:11. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:10. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:12. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:13. HCDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:16. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:13. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:17. L The domains include CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence.

[0010] In some of the optional embodiments, V H The region is V as shown in SEQ ID NO:13. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:18. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:13. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:19. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:14. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:16. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:14.H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:17. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:14. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:18. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:14. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:19. L The domains include CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence.

[0011] In some of the optional embodiments, V H The region is V as shown in SEQ ID NO:15. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:16. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:15. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:17. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V HThe region is V as shown in SEQ ID NO:15. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:18. L In some of the embodiments, the CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the V H The region is V as shown in SEQ ID NO:15. H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:19. L The domains include CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence.

[0012] In some of the optional embodiments, V H The region includes a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence shown in SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence shown in SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence shown in SEQ ID NO: 22, L The regions include light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO:23, light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO:24, and light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO:25.

[0013] In some of the optional embodiments, V H The region includes CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:26, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:27, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:28, LThe regions include CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:29, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:24, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:30.

[0014] In some of the optional embodiments, V H The region includes CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 31, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 33, L The region comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:34, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:35, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:36. H The region includes CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 37, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 39, L The regions include CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:40, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:41, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:42.

[0015] In some of the optional embodiments, the conjugate comprises a heavy chain variable (V H ) region and the light chain variable (V L In some of the optional embodiments, the antibody or antigen-binding fragment includes an antibody or antigen-binding fragment that includes a V H The region comprises the amino acid sequence shown in SEQ ID NO:1, L The region comprises the amino acid sequence shown in SEQ ID NO:2. H The region comprises the amino acid sequence shown in SEQ ID NO:3, LThe region comprises the amino acid sequence shown in SEQ ID NO:4. H The region comprises the amino acid sequence shown in SEQ ID NO:5, L The region comprises the amino acid sequence shown in SEQ ID NO:6. H The region comprises the amino acid sequence shown in SEQ ID NO:7, L The region comprises the amino acid sequence set forth in SEQ ID NO:8. H The region comprises the amino acid sequence shown in SEQ ID NO:9, L The region comprises the amino acid sequence shown in SEQ ID NO:11. H The region comprises the amino acid sequence shown in SEQ ID NO:9, L The region comprises the amino acid sequence set forth in SEQ ID NO: 12. H The region comprises the amino acid sequence shown in SEQ ID NO:10, L The region comprises the amino acid sequence shown in SEQ ID NO:11. H The region comprises the amino acid sequence shown in SEQ ID NO:10, L The region comprises the amino acid sequence set forth in SEQ ID NO: 12. H The region comprises the amino acid sequence shown in SEQ ID NO:13, L The region comprises the amino acid sequence set forth in SEQ ID NO: 16. H The region comprises the amino acid sequence shown in SEQ ID NO:13, L The region comprises the amino acid sequence set forth in SEQ ID NO: 17. H The region comprises the amino acid sequence shown in SEQ ID NO:13, L The region comprises the amino acid sequence set forth in SEQ ID NO: 18. HThe region comprises the amino acid sequence shown in SEQ ID NO:13, L The region comprises the amino acid sequence set forth in SEQ ID NO:19. H The region comprises the amino acid sequence shown in SEQ ID NO:14, L The region comprises the amino acid sequence set forth in SEQ ID NO: 16. H The region comprises the amino acid sequence shown in SEQ ID NO:14, L The region comprises the amino acid sequence set forth in SEQ ID NO: 17. H The region comprises the amino acid sequence shown in SEQ ID NO:14, L The region comprises the amino acid sequence set forth in SEQ ID NO: 18. H The region comprises the amino acid sequence shown in SEQ ID NO:14, L The region comprises the amino acid sequence set forth in SEQ ID NO:19. H The region comprises the amino acid sequence shown in SEQ ID NO:15, L The region comprises the amino acid sequence set forth in SEQ ID NO: 16. H The region comprises the amino acid sequence shown in SEQ ID NO:15, L The region comprises the amino acid sequence set forth in SEQ ID NO: 17. H The region comprises the amino acid sequence shown in SEQ ID NO:15, L The region comprises the amino acid sequence set forth in SEQ ID NO: 18. H The region comprises the amino acid sequence shown in SEQ ID NO:15, L The region comprises the amino acid sequence shown in SEQ ID NO:19.

[0016] In some of the embodiments, the antibody or antigen-binding fragment comprises MA251, 7G7B6, or an antigen-binding portion thereof. In some of the embodiments, the antibody or antigen-binding fragment is a human antibody or human antigen-binding fragment, a chimeric antibody or chimeric antigen-binding fragment, or a humanized antibody or humanized antigen-binding fragment. In some of the embodiments, the conjugate comprises an IgG1 Fc region or an IgG1 isotype, an IgG2 Fc region or an IgG2 isotype, an IgG3 Fc region or an IgG3 isotype, or an IgG4 Fc region or an IgG4 isotype.

[0017] In some of the embodiments, the antibody or antibody binding fragment comprises an IgG1 Fc region or an IgG1 isotype. In some of the embodiments, the IgG1 Fc region does not exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) effector function.

[0018] In some of the embodiments, the antibody or antibody-binding fragment comprises an IgG2 Fc region or an IgG2 isotype. In some of the embodiments, the IgG2 Fc region comprises a substitution that reduces or abolishes ADCC effector function. In some of the embodiments, the substitution is an asparagine to glutamine substitution in the Fc region at the position corresponding to 297 according to EU numbering (N297Q).

[0019] In some of the embodiments, the conjugate exhibits one or more Fc-mediated effector functions. In some of the embodiments, the conjugate lacks Fc-mediated effector functions, exhibits substantially reduced Fc-mediated effector functions, or exhibits no substantial Fc-mediated effector functions. In some of the embodiments, the activated conjugate is capable of killing cells in the absence of substantial Fc-mediated effector functions. In some of the embodiments, the Fc-mediated effector functions are selected from one or more of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In some of the embodiments, the antibody or antigen-binding fragment comprises a human immunoglobulin Fc region and / or a human antibody framework region.

[0020] Provided herein are methods for treating a tumor or lesion in a subject. In some of the embodiments, the methods provided include administering to the subject any of the conjugates provided herein; administering to a target site within the subject at a wavelength of at or about 600 nm to at or about 850 nm and at a dose of at least 25 J / cm 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 The method includes irradiating the conjugate with a dose of up to, or at or about, 2 J / cm fiber length to up to, or at or about, 500 J / cm fiber length, thereby activating the conjugate.

[0021] administering to a subject a conjugate comprising an antibody or antigen-binding fragment that specifically binds to CD25 without substantially blocking or interfering with IL-2 signaling and a Si-phthalocyanine dye; 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2Provided herein are methods of treating a tumor or lesion in a subject, comprising irradiating the tumor or lesion with a dose of up to, or at or about, 2 J / cm fiber length to up to, or at or about, 500 J / cm fiber length, thereby activating the conjugate.

[0022] In some of the optional embodiments, the Si-phthalocyanine dye is IR700 and the irradiation is performed at a wavelength of 690 nm ± 20 nm.

[0023] In some of the optional embodiments, the Si-phthalocyanine dye is represented by formula (I): TIFF2024505556000003.tif71128, or a salt, stereoisomer, or tautomer thereof, and irradiation is carried out at a wavelength of 660 nm ± 50 nm.

[0024] In some of the optional embodiments, the growth, volume or size of a tumor or lesion is reduced or inhibited.

[0025] In some of the embodiments, the tumor or lesion being treated, or the tumor microenvironment (TME) of the tumor or lesion being treated, contains reduced levels of immune effector cells. In some of the embodiments, the tumor or lesion has a reduced response or is unresponsive to unconjugated antibodies.

[0026] In some of the optional embodiments, the immune effector cells are selected from one or more of macrophages, natural killer (NK) cells, neutrophils, and eosinophils.

[0027] In some of the embodiments, the target site is irradiated within about 24±4 hours after administration of the conjugate. In some of the embodiments, the target site is irradiated with 50 mW / cm 2 Or about 50mW / cm 2 From 200mW / cm 2 Or about 200mW / cm 2In some embodiments, the target site is irradiated for at or about 120 seconds to at or about 600 seconds.

[0028] In some of any of the embodiments, the tumor or lesion is resistant or non-responsive to immune checkpoint inhibitor therapy.

[0029] In some of the optional embodiments, the conjugate comprises an IgG1 Fc region or an IgG1 isotype, an IgG2 Fc region or an IgG2 isotype, an IgG3 Fc region or an IgG3 isotype, or an IgG4 Fc region or an IgG4 isotype.

[0030] In some of the embodiments, the antibody or antibody binding fragment comprises an IgG1 Fc region or an IgG1 isotype. In some of the embodiments, the IgG1 Fc region is not enhanced with respect to ADCC effector function. In some of the embodiments, the antibody or antibody binding fragment comprises an IgG2 Fc region or an IgG2 isotype. In some of the embodiments, the IgG2 Fc region comprises a substitution that reduces or abolishes ADCC effector function. In some of the embodiments, the substitution is an asparagine to glutamine substitution in the Fc region at the position corresponding to 297 according to EU numbering (N297Q).

[0031] In some of the embodiments, the conjugate exhibits one or more Fc-mediated effector functions. In some of the embodiments, the conjugate lacks Fc-mediated effector functions, exhibits substantially reduced Fc-mediated effector functions, or exhibits no substantial Fc-mediated effector functions. In some of the embodiments, the activated conjugate is capable of killing cells in the absence of substantial Fc-mediated effector functions. In some of the embodiments, the Fc-mediated effector functions are selected from one or more of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0032] In some of the embodiments, the methods disclosed herein further include administering immune checkpoint inhibitor therapy after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor therapy is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, or 3 weeks after administration of the conjugate. In some of the embodiments, the immune checkpoint inhibitor therapy is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, or 3 weeks after irradiation. In some of the embodiments, the immune checkpoint inhibitor therapy is administered more than once after administration of the conjugate.

[0033] In some of the embodiments, the immune checkpoint inhibitor therapy includes a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some of the embodiments, the PD-1 inhibitor 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, TSR-042 (ANB011), or an antigen-binding fragment thereof, or any combination thereof.

[0034] In some of any of the embodiments, the population of regulatory T cells (Tregs) in the tumor or lesion or in the tumor microenvironment is reduced as a result of the method.

[0035] In some of any of the embodiments, the reduction or inhibition comprises one or more of an increase in tumor volume or size by less than 20%, or a reduction in tumor volume, size or mass, or a reduction in tumor cell number. In some of any of the embodiments, the growth, volume or size of a tumor or lesion is inhibited or reduced to a greater extent as compared to methods using a conjugate comprising an antibody or antigen-binding fragment that specifically binds to CD25 and substantially blocks or prevents IL-2 signaling.

[0036] In some of any of the embodiments, the method disclosed herein improves the overall survival of the subject.In some of any of the embodiments, the subject has a second tumor or a secondary population of tumor cells, and the growth, volume or size of the second tumor or the secondary population of tumor cells is reduced or inhibited as a result of the method.In some of any of the embodiments, the second tumor or the secondary population of tumor cells is not irradiated or has never been irradiated. [Brief description of the drawings]

[0037] [Figure 1] Figure 1 shows the average tumor volume over time in mice bearing implanted CT26 tumors. Mice were administered an exemplary IL-2 non-blocking anti-CD25-mIgG1-IR700 conjugate (7D4-IR700; triangles) or an exemplary IL-2 blocking anti-CD25-IgG1-IR700 conjugate (PC61-IR700; squares) alone (solid line) or after the conjugate was irradiated at 690 nm at a dose of 100 J / cm2 (dashed line). Control tumor-bearing mice were administered saline only (open circles). [Diagram 2] 2A-2E show tumor growth in individual mice from FIG. [Diagram 3] FIG. 3 shows survival of the mice from FIG. [Figure 4]Figure 4A shows the average tumor volume over time in mice bearing implanted MCA205 murine fibrosarcoma. Mice were administered anti-PD1 antibody (open circle, dashed line), an exemplary IL-2 non-blocking anti-CD25 antibody (7D4-mIgG2a; closed square, solid line), or an exemplary combination of IL-2 blocking anti-CD25 antibody and anti-PD-1 antibody (7D4-mIgG2a+a-PD1; open square, dashed line). Control tumor-bearing mice were administered saline only (closed circle, solid line). Figure 4B shows the average tumor volume over time in mice bearing implanted MCA205 murine fibrosarcoma. Mice were administered anti-PD1 antibody (open circle, dashed line), an exemplary IL-2 non-blocking anti-CD25-mIgG1-IR700 conjugate followed by irradiation at 690 nm at a dose of 200 J / cm2 (7D4-mIgG2a-IR700+PIT; closed triangle, solid line), or an exemplary IL-2 non-blocking anti-CD25-mIgG1-IR700 conjugate followed by irradiation at 690 nm at a dose of 200 J / cm2 in combination with anti-PD-1 antibody (7D4-mIgG2a-IR700 PIT+a-PD1; open triangle, dashed line). Control tumor-bearing mice were administered saline only (closed circle, solid line). [Diagram 5] Figures 5A-5F show tumor growth in individual mice from Figures 4A-4B. [Figure 6] FIG. 6 shows survival of the mice from FIGS. 4A-4B. [Figure 7] Figure 7A shows the antitumor effects of saline (open triangles, dashed line), an exemplary IL-2 blocking anti-CD25-IR700 conjugate alone (PC61-IR700; open circles, solid line), or conjugation followed by irradiation at 690 nm at a dose of 200 J / cm2 (PC61-IR700 PIT; closed circles, solid line) in irradiated tumors of mice bearing bilaterally implanted MCA205 murine fibrosarcomas. Figure 7B shows the antitumor effects of saline (open triangles, dashed line), an exemplary IL-2 non-blocking anti-CD25-IR700 conjugate alone (7D4-IR700; open squares, solid line), or conjugation followed by irradiation at 690 nm at a dose of 200 J / cm2 (closed squares, solid line) in irradiated tumors of mice bearing bilaterally implanted MCA205 murine fibrosarcomas. [Figure 8] Figure 8A shows the antitumor effect of saline (open triangles, dashed line), an exemplary IL-2 blocking anti-CD25-IR700 conjugate alone (PC61-IR700; open circles, solid line), or conjugation followed by irradiation at 690 nm at a dose of 200 J / cm2 (PC61-IR700 PIT; closed circles, solid line) in the non-irradiated distant tumor of the mouse from Figure 7A. Figure 8B shows the antitumor effect of saline (open triangles, dashed line), an exemplary IL-2 non-blocking anti-CD25-IR700 conjugate alone (7D4-IR700; open squares, solid line), or conjugation followed by irradiation at 690 nm at a dose of 200 J / cm2 (closed squares, solid line) in the non-irradiated distant tumor of the mouse from Figure 7B. [Figure 9] FIG. 9 shows the antibody-dependent cellular cytotoxicity (ADCC) activity of an IL-2 non-blocking anti-CD25 antibody (7D4-mIgG2a) and the corresponding IR700 conjugate (7D4-mIgG2a-IR700). [Figure 10] Figure 10 shows the average tumor volume over time in mice bearing implanted CT26 tumors. Mice were administered an exemplary IL-2 non-blocking antibody with a mouse IgG2a backbone (7D4-mIgG2a; closed squares), a corresponding ADCC / ADCP null IL-2 non-blocking antibody (7D4-mIgG2a-N297Q; open squares), and an exemplary IL-2 non-blocking anti-CD25-mIgG1-IR700 conjugate with a mouse IgG1 backbone alone (7D4-mIgG1-IR700; triangles), or conjugation followed by irradiation at 690 nm at a dose of 100 J / cm2 (7D4-mIgG1-IR700+PIT, dashed line). Control tumor-bearing mice were administered saline only (open circles). [Figure 11] 11A-11E show tumor growth in individual mice from FIG. [Figure 12] FIG. 12 shows the survival of the mice from FIG. [Figure 13]Figure 13 shows the mean tumor volume over time in mice with implanted immunological "cold" tumors. Mice were administered an exemplary IL-2 non-blocking antibody with a mouse IgG2a backbone (7D4-mIgG2a) alone (open squares) or in combination with an anti-PD-1 antibody (closed squares), a corresponding exemplary IL-2 non-blocking anti-CD25-mIgG1-IR700 conjugate followed by irradiation at 690 nm at a dose of 150 J / cm2 alone (7D4-mIgG1-IR700 PIT; open triangles) or in combination with an anti-PD-1 antibody (closed triangles). Control tumor-bearing mice were administered saline alone (open circles) or anti-PD-1 alone (closed circles). [Figure 14] FIG. 14 shows survival of the mice from FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description Provided herein are conjugates, compositions, combinations and methods for treating cancer, including not only first tumor or primary tumor or multiple primary tumors, but also tumors introduced later, such as tumors that contain secondary populations of tumor cells, such as metastatic tumor cells, such as metastatic cancer; and / or not only primary tumor or multiple primary tumors, but also cancers that contain invasive or infiltrating tumor cells, such as invasive or infiltrating cancer.Also provided are conjugates, compositions, combinations and methods for enhancing systemic immunity in subjects, such as subjects with cancer, such as invasive cancer, invasive cancer, or metastatic cancer.Also provided are conjugates, 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, invasive cancer, or metastatic cancer. In some situations, cancers exhibit reduced immune responsiveness (e.g., exhibit low levels or exhausted tumor infiltrating lymphocytes (TILs), insufficient tumor antigen load, and / or an immunosuppressive microenvironment; also referred to as "cold" tumors), and the provided conjugates, compositions, combinations, and / or methods result in treatment of the cancer, enhance the subject's systemic immunity, and / or generate an enhanced response to a treatment or therapy in the subject.

[0039] In any of the embodiments provided, the conjugates described herein can exhibit one or more mechanisms of anti-tumor effect. Thus, the conjugates provided herein present the ability to provide anti-tumor effects in a broader spectrum of tumor types and tumor environments. The conjugates herein target the CD25 antigen on the cell surface and thus can specifically target CD25-expressing T cells for destruction by the methods provided. In some aspects, the conjugates can bind to the CD25 antigen but do not substantially block or interfere with IL-2 signaling. In some aspects, preservation of IL-2 signaling not only allows stimulation of cytotoxic effector T cells and enhanced activation-induced cell death (AICD), but also promotes differentiation of T cells into effector T cells and into memory T cells. In some aspects, the conjugates also include a photoactivated phthalocyanine dye. The conjugated dye allows activation of targeted cell killing in the presence or irradiation of a specific wavelength of light. In some aspects, unlike non-conjugated anti-CD25 antibodies, targeted destruction of CD25-expressing T cells can be localized using light delivery.In addition, the conjugate can provide targeted cell killing in the absence of Fc-mediated effector function.Thus, in some aspects, the conjugate provides cell killing in tumors and tumor microenvironments where antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) are reduced or hindered.

[0040] The conjugates, compositions, combinations, methods and uses provided can be used to treat cancers including first 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 cancers, such as cancers including secondary populations of tumor cells, metastatic cancers and / or invasive cancers, including no need to locate and / or directly irradiate metastatic tumor cells and / or invasive tumor cells. The present disclosure also provides an unexpected feature in enhancing systemic immunity in a subject, for example against cancer recurrence.

[0041] The embodiments provided include or use a targeting molecule, such as an IL-2 non-blocking anti-CD25 targeting antibody or antigen-binding fragment thereof (e.g., also referred to as an IL-2 non-blocking antibody), that specifically binds to CD25 but does not interfere or block, e.g., does not substantially interfere or substantially block, the binding of IL-2 to CD25. The embodiments provided include or use an IL-2 non-blocking anti-CD25 conjugate, such as a conjugate comprising an IL-2 non-blocking anti-CD25 targeting antibody or antigen-binding fragment thereof and a phthalocyanine dye with a silicon-coordinated metal (Si-phthalocyanine dye). In any of the embodiments, the phthalocyanine dye is in its salt or ionic form. In any of the embodiments, the phthalocyanine dye is in its salt, ionic form, stereoisomer, or tautomer.

[0042] CD25, also known as the alpha chain of the interleukin-2 receptor (IL-2Ra or IL-2Rα), is constitutively expressed at high levels on regulatory T cells (Treg) and activated T cells. In some cases, CD25 can also be expressed on cancer cells, for example, on leukemic cells in some acute myeloid leukemias. IL-2 has an essential role in key functions during immune homeostasis, particularly through its direct effects on regulatory T cells as well as optimizing and fine-tuning effector lymphocyte responses (Arenas-Ramirez et al., (2015) Trends Immunol. 36(12):763-777). For example, in the thymus where T cells mature, low levels of IL-2 signaling can promote the differentiation of certain immature T cells into Tregs, while high levels of IL-2 signaling can stimulate cytotoxic effector T cells that can promote antitumor responses. IL-2 can also enhance activation-induced cell death (AICD). IL-2 signaling can also promote differentiation of T cells into effector and memory T cells if early T cells are also stimulated by antigen (Liao et al., (2011) Curr Opin Immunol. 23(5):598-604. IL-2 expression and secretion are tightly regulated and function as part of both transient positive and negative feedback loops in initiating and dampening immune responses. It plays a major role in conferring cell-mediated immunity due to its role in the generation of immunological memory of T cells, which depends on the expansion of numbers and function of antigen-selected T cell clones. Thus, in provided embodiments, the IL-2 non-blocking anti-CD25 antibody or conjugate does not interfere, e.g., does not substantially interfere, with these contributions of IL-2 to the immune response. In some embodiments, the provided methods and uses do not systemically interfere or block, e.g., does not substantially block or block, IL-2 signaling outside of irradiated tumors or lesions.

[0043] In any of the embodiments provided, the conjugates described herein can exhibit one or more Fc-mediated effector functions. Fc-mediated effector functions include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In some embodiments, Fc-mediated effector functions (e.g., ADCC or ADCP) can be induced in CD25-expressing cells when bound to the IL-2 non-blocking anti-CD25 antibody conjugates provided herein. In some embodiments, the IL-2 non-blocking anti-CD25 antibody comprises a functional Fc region to enable the Fc-mediated effector functions (e.g., ADCC or ADCP) of the IL-2 non-blocking anti-CD25 conjugates provided herein.

[0044] In some embodiments, the IL-2 non-blocking anti-CD25 antibodies contain an Fc region that has been modified to enhance one or more Fc-mediated effector functions (e.g., ADCC and / or ADCP) of the provided IL-2 non-blocking anti-CD25 conjugates. Exemplary modifications to the Fc region include one or more amino acid substitutions within the Fc region and / or replacement of the entire Fc region with an Fc region of an antibody of a different isotype. In such embodiments, the methods provided cause targeted elimination of CD25-expressing cells via irradiation and Fc-mediated effector functions (e.g., ADCC or ADCP), such as ADCC-mediated elimination of CD25-expressing cells distal to the irradiation site, while also retaining IL-2 signaling and / or not substantially disrupting IL-2 signaling.

[0045] In some embodiments, the Fc region of the non-IL-2 blocking anti-CD25 antibody is non-functional or has reduced Fc-mediated effector function, e.g., reduced ADCC, ADCP, and / or CDC activity. In some such embodiments, the Fc region of the targeting antibody is modified to reduce or eliminate Fc-mediated effector function (e.g., ADCC and / or ADCP). Such modifications include amino acid substitution, deletion, or truncation of part or all of the Fc region.

[0046] The embodiments provided are based on the observation that in some circumstances, treatment of cancer with a phthalocyanine dye-targeting molecule conjugate, such as an IL-2 non-blocking anti-CD25 antibody-phthalocyanine dye conjugate (e.g., an IL-2 non-blocking anti-CD25-IR700), followed by irradiation of the first or primary tumor, not only results in treatment of the irradiated tumor, e.g., the irradiated first tumor or the irradiated primary tumor, but also results in effective treatment of tumors that are distal to the irradiation site (e.g., metastatic tumors), and tumors introduced after the subject has had a complete remission following treatment of the initial tumor, and exhibits a tumor-specific immune memory response. The provided embodiments are based on the further observation that combination treatment with an IL-2 non-blocking anti-CD25 antibody-phthalocyanine dye conjugate (e.g., IL-2 non-blocking anti-CD25-IR700) and an immune checkpoint inhibitor, such as an anti-PD-1 antibody, results in remarkable synergy in the treatment of both irradiated first or primary tumors and distant or subsequently introduced tumors, such as tumors comprising a secondary population of tumor cells, metastatic tumors and / or invasive tumors.

[0047] The embodiments provided are based on the observation that in some situations, treatment of tumors with IL-2 non-blocking anti-CD25 conjugates (e.g., IL-2 non-blocking anti-CD25-IR700) exhibits at least two modes of action to cause targeted cell killing. In some situations, the tumor is a tumor with reduced immune reactivity (e.g., exhibiting low levels and exhausted tumor infiltrating lymphocytes (TILs), insufficient tumor antigen load, and / or an immunosuppressive microenvironment), such as a "cold" tumor. In some situations, tumors with reduced immune reactivity do not respond or do not substantially respond to immune checkpoint inhibitor therapy, such as one or more of anti-PD-1, anti-PD-L1, or anti-CTLA-4 therapy. Thus, the provided conjugates, compositions, combinations, methods and uses have been demonstrated to provide substantially improved and effective treatment of cancer, including cancers that include not only first tumors or primary tumors or multiple primary tumors, but also metastatic tumor cells, e.g., metastatic cancers; and / or cancers that include not only first tumors or primary tumors or multiple primary tumors, but also invasive tumor cells, e.g., invasive cancers. The provided conjugates, compositions, combinations, methods and uses can result in an enhanced or improved immune response of the subject, for example, the systemic immune response against cancer, including immune memory responses that may occur after treatment and can be effective against tumors.

[0048] The methods and uses provided herein include treating a subject having one or more first tumors, e.g., primary tumors, and optionally a secondary population of cells, 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 first 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 before, simultaneously with, or after administration of the conjugate.

[0049] I. Methods of Treatment with Non-IL-2 Blocking Anti-CD25 Conjugates and Uses Thereof In some embodiments, the methods and uses provided involve administering an anti-CD25 conjugate, such as a conjugate comprising an anti-CD25 antibody (i.e., a non-IL-2 blocking anti-CD25 antibody) and a phthalocyanine dye, which when the antibody is bound does not substantially block access to or binding of IL-2 to CD25 or does not substantially impede or impair IL-2 mediated signaling via CD25, and illuminating the target area with a wavelength of light suitable for use with the phthalocyanine dye, whereby the light excites the dye and results in the killing of cells expressing CD25 on their surface.

[0050] In some aspects, IL-2 non-blocking anti-CD25 conjugates are provided. In such embodiments, IL-2 can bind to CD25, while the anti-CD25 conjugate is also bound to CD25, and IL-2-mediated signaling via binding to CD25 is not impaired in cells within or distal to the irradiated target area. IL-2-mediated activities include cytotoxic T lymphocyte expansion and other immunomodulatory activities, such as those described in Ross and Cantrell (2018) Annu Rev Immunol. 36: 411-433. Such methods result in enhancing, activating, inducing, eliciting, enhancing or supporting immune functions, such as local and / or systemic immunity, reducing or eliminating lesions (e.g., tumors), reducing or inhibiting tumor growth, reducing, inhibiting or eliminating metastasis of tumor cells, or any combination thereof. In some embodiments, the provided methods and uses result in the treatment or treatment of cancer, such as cancer that includes not only a first tumor or multiple tumors (e.g., one or more primary tumors), but also a secondary population of cancer cells, such as metastatic tumor cells (e.g., metastatic cancer), invasive tumor cells (e.g., invasive cancer), or infiltrating tumor cells (e.g., invasive cancer). In some embodiments, the secondary population of cancer cells is related, e.g., directly or indirectly, to the first tumor. In some embodiments, the secondary population of cells is not directly derived from the first tumor.

[0051] The provided methods and uses 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 first tumor) or tumor microenvironment associated with the cancer with a specific wavelength and dose of light. In some aspects, the irradiation (or radiation) causes irradiation-dependent lysis and death of cells expressing CD25 target molecules, resulting in a therapeutic effect or treatment of the 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, in the provided methods and uses for the treatment of cancer, for example, as a combination therapy or combination treatment.

[0052] The uses include the use of the compositions and combinations described herein 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 aspects, such therapeutic methods include combination therapy. In some embodiments, the methods and uses thereby treat cancer in a subject, including a first 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 first tumor. In some embodiments of the methods and uses, more than one tumor is treated. In some aspects, methods and uses of such conjugates, compositions and combinations in enhancing, increasing, boosting, strengthening, increasing, enhancing or supporting immune function, such as systemic immunity, in a subject are also provided.

[0053] The method includes administering to a subject having a first tumor a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25, and irradiating at least the first 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.

[0054] 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 therapy. 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 death, 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.

[0055] 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.

[0056] 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 2J / cm 2 From 400J / cm 2Or about 400 J / cm 2 In some embodiments, the irradiation is at a dose of at least 2 J / cm fiber length to at or about 2 J / cm fiber length to at or about 500 J / cm fiber length. 2 , 5J / cm 2 , 10J / cm 2 , 25J / cm 2 , 50J / cm 2 , 75J / cm 2 , 100J / cm 2 , 150J / cm 2 , 200J / cm 2 , 300J / cm 2 , 400J / cm 2 , or 500 J / cm 2 or at least about 2 J / cm 2 , 5J / cm 2 , 10J / cm 2 , 25J / cm 2 , 50J / cm 2 , 75J / cm 2 , 100J / cm 2 , 150J / cm 2 , 200J / cm 2 , 300J / cm 2 , 400J / cm 2 , or 500 J / cm 2 or the lesion is irradiated with a dose of at least 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.

[0057] In some embodiments, the irradiation is about 25 J / cm 2 to about 400 J / cm 2 or from about 2 J / cm to about 500 J / cm of fiber length. In some embodiments, the irradiation is at a dose of 5 J / cm 2 from 200 J / cm 2 or from 20 J / cm to 500 J / cm of fiber length. In some embodiments, the irradiation is at a dose of about 50 J / cm 2 Or irradiation with a dose of 100 J / cm of fiber length.

[0058] In some embodiments of the methods and uses provided herein, the optical power (or optical fluence) of the light is from at or about 20 J / cm of fiber length to at or about 500 J / cm of fiber length. In some embodiments, the optical power (or optical fluence) of the interstitial light dose is from at or about 100 mW / cm of fiber length to at or about 500 mW / cm of fiber length. In some embodiments, the light is applied for at or about 120 seconds to at or about 600 seconds. In some embodiments, the irradiation is administered for at or about 250 seconds with a dose of at or about 100 J / cm of fiber length and an optical fluence of at or about 400 mW / cm.

[0059] In some embodiments of the methods and uses provided herein, the optical power (or optical fluence) of the light is greater than or equal to 25 J / cm 2 or about 25 J / cm 2 From 400J / cm 2 or about 400 J / cm 2 In some embodiments, the optical power (or optical fluence) of the light dose is up to 50 mW / cm 2 or about 50 mW / cm 2 From 200mW / cm 2 or about 200 mW / cm2 In some embodiments, the light is applied for at or about 120 seconds to at or about 600 seconds. In some embodiments, the irradiation is at or above 50 J / cm. 2 or about 50 J / cm 2 At a dose of 150 mW / cm 2 The optical power is 333 s or approximately 333 s.

[0060] In some embodiments of the methods and uses provided herein, the irradiation is administered after administration of the phthalocyanine dye-targeting molecule conjugate (e.g., a non-IL-2 blocking anti-CD25 antibody-IR700 conjugate). In some embodiments, the irradiation or illumination is administered 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., 30 minutes to 48 hours, 30 minutes to 24 hours, or 12 hours to 48 hours, e.g., 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.

[0061] The methods described herein include irradiating a first tumor in a subject, such as a primary tumor, or the tumor microenvironment (TME) of a first 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.

[0062] In some aspects, primary tumor can refer to the first tumor or primary 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 tumor or additional tumor can be one solid tumor or multiple solid tumors, can be lymphoma, or can be leukemia.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.

[0063] In some embodiments of the method, the growth of the first or primary tumor is inhibited, the volume of the primary tumor 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 first or primary tumor is inhibited, the volume of the primary tumor 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.

[0064] 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 originating from a tumor, such as a primary tumor, that have invaded surrounding tissue. The method includes administering to a subject having a first 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 first 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 methods include administering an anti-PD-1 antibody prior to, simultaneously with, or following administration of a non-IL-2 blocking anti-CD25 antibody-IR700 conjugate, where administration of the conjugate is followed by irradiation of the first tumor.

[0065] In some aspects, invasive tumor cells refer to cells that originate from primary tumors, and invade the surrounding tissues of the same organ or adjacent organs of the primary tumor in the body of a subject with a first tumor.In some embodiments, the first tumor is a primary tumor, and the invasive tumor cells originate directly or indirectly from the first tumor.In some embodiments, the invasive tumor cells do not originate directly from the first tumor.

[0066] The methods and uses provided herein include irradiation of the first tumor and / or additional tumor, and do not irradiate some or all of the 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 the first tumor is also inhibited, reduced or eliminated, and the volume, size or mass of the first tumor or additional tumor is also reduced, together with the effect on one or more invasive tumor cells.

[0067] 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.

[0068] In some embodiments, the methods and uses provided herein include treating a subject having a first 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 to the subject having a first 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), a conjugate comprising a phthalocyanine dye linked to a targeting molecule, the targeting molecule binding to CD25, and irradiating the first 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 a first tumor and / or a secondary population of tumor cells, such as metastatic tumor cells, is inhibited, reduced or eliminated, resulting in a reduction in one or more volumes, sizes or masses of the first tumor and / or secondary cell populations, or any combination thereof. In some embodiments, inhibition of the first tumor 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.

[0069] In any of the methods and uses herein, the first tumor can be a primary tumor or a secondary tumor. In some embodiments, the first tumor and the secondary population of tumor cells are related. In some embodiments, the secondary cell population is directly or indirectly derived from the first tumor. In some embodiments, the secondary population is not derived from the first tumor. In some embodiments, the first tumor is a primary tumor and the secondary cell population is related to the primary tumor; for example, the secondary cell population is directly or indirectly derived from the primary tumor. In some embodiments, the first tumor is a primary tumor and the secondary population of tumor cells is a secondary primary tumor. In some embodiments, the first 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 originate 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 first tumor. In other aspects, the secondary population of tumor cells is metastatic and is not directly related to the first 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.

[0070] In some embodiments of the methods and uses provided herein, the secondary tumor cells are metastatic tumor cells that are distant from the first tumor, and some or all of the 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 the first tumor is irradiated, and invasive or metastatic tumor cells are not directly irradiated.In some embodiments, more than one tumor is irradiated, including the first tumor, but at least one site of tumor cells, for example, the site containing metastatic tumor cells, is not irradiated.

[0071] 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.

[0072] 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 without blocking, e.g., substantially blocking, IL-2 binding or signaling, and administering an immune checkpoint inhibitor, and irradiating the tumor or cancerous lesion, or tumor microenvironment, after administration of the conjugate. The conditions of irradiation, such as wavelength, dose of irradiation, and timing of irradiation, are, for example, as described herein. The immune checkpoint inhibitor can be administered before, simultaneously, 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 enhanced or synergistic responses to cancer therapy or treatment. 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, as 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 an enhancement of the subject's systemic immunity, as 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, e.g., an additional, additive, or synergistic response, and / or a more complete, more durable, or longer lasting response, to a treatment, as compared to administration of only the conjugate, only the conjugate followed by irradiation, or only the anti-PD-1 antibody.

[0073] 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), intratumor 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, 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.

[0074] 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 an IL-2 non-blocking anti-CD25 PIT (e.g., including light irradiation for activation of the conjugate) and a checkpoint inhibitor (e.g., an 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., an anti-PD-1 antibody) or a single agent, such as an IL-2 blocking or IL-2 non-blocking anti-CD25 conjugate or an IL-2 blocking or IL-2 non-blocking anti-CD25 PIT alone.

[0075] 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, such as certain Tregs. In some aspects, binding of an IL-2 non-blocking anti-CD25 conjugate to the surface of CD25-expressing cells, such as certain Tregs, and irradiation to effect irradiation-dependent lysis and death of the cells expressing CD25 results in a reduction in the number of cells expressing CD25. In some aspects, such a result 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.

[0076] 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 CD4+ Tregs to Tregs is increased after treatment compared to before treatment, systemic immunity to the recurrent tumor is increased or enhanced. In some embodiments, the level, strength, or extent of systemic immunity is determined by measuring the level, strength, or extent of intratumoral CD4+ Tregs to Tregs. + 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.

[0077] 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 first tumor in the subject.

[0078] 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 first 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 first tumor in the subject.

[0079] 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 first 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 first tumor in the subject.

[0080] 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 the first tumor or metastatic tumor cell mass or invasive tumor cell mass or peripheral circulation. In some embodiments, the cells are collected from the subject 4 to 28 days after irradiation of the first tumor in the subject.

[0081] 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 the first tumor or 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 first tumor in the subject.

[0082] In some embodiments, any of the above assays may be used in combination.

[0083] III. Conjugates and Compositions for Use with the Method In some aspects, conjugates, compositions and combinations are provided that include a phthalocyanine dye linked to a targeting molecule, such as a conjugate that includes a phthalocyanine dye linked to a targeting molecule, such as an antibody or an antigen-binding fragment thereof, that binds to CD25 protein but does not block, e.g. does not substantially block, the binding of IL-2 to CD25. In some aspects, the provided compositions or combinations include an immune checkpoint inhibitor, such as an anti-PD-1 antibody. In some aspects, conjugates, compositions and combinations are provided for use in a treatment method 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, the treatment includes a combination treatment. In some aspects, compositions and combinations are provided for use according to the provided methods and uses.

[0084] The methods and uses provided herein use a conjugate comprising a targeting molecule that binds to CD25, e.g., an anti-CD25 antibody or antigen-binding fragment that binds, e.g., specifically binds, to CD25, but does not block, e.g., does not substantially block, IL-2 from also binding to CD25, and / or does not interfere, e.g., does not substantially interfere with IL-2-mediated signal transduction through interaction with CD25. CD25 can be expressed on CD8+ cells, activated T cells, including 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.

[0085] In some aspects, "IL-2 non-blocking antibodies" or "IL-2 non-blocking anti-CD25 antibodies" include anti-CD25 antibodies (e.g., anti-CD25 IL-2 non-blocking antibodies) that can specifically bind to the CD25 subunit of the IL-2 receptor without blocking IL-2 binding to CD25 or IL-2 signaling through CD25. In some aspects, IL-2 non-blocking anti-CD25 antibodies allow IL-2 signaling in response to IL-2 binding to CD25 at least 50% compared to the signaling level in the absence of the anti-CD25 antibody. In some aspects, IL-2 non-blocking anti-CD25 antibodies allow IL-2 signaling in response to IL-2 binding to CD25 at least 75% compared to the signaling level in the absence of the anti-CD25 antibody.

[0086] In some aspects, "IL-2 non-blocking", which may be related to "non-blocking", "not blocking", or grammatical variations thereof, and may be related to non-blocking of IL-2 binding to CD25 in the presence of an anti-CD25 antibody, includes cases where the anti-CD25 antibody or antigen-binding fragment inhibits IL-2 signaling by less than 50% compared to IL-2 signaling in the absence of the antibody. In some aspects, the anti-CD25 antibody or antigen-binding fragment inhibits IL-2 signaling by less than about 40%, 35%, 30%, e.g., less than about 25%, compared to IL-2 signaling in the absence of the anti-CD25 antibody. A non-IL-2 blocking anti-CD25 antibody can bind to CD25 without interfering with IL-2 binding to CD25 or without substantially interfering with IL-2 binding to CD25. In some aspects, a non-IL-2 blocking anti-CD25 antibody is alternatively referred to as an anti-CD25 antibody that "does not inhibit interleukin-2 binding to CD25" or an anti-CD25 antibody that "does not inhibit IL-2 signaling" or an "antibody or antigen-binding fragment that specifically binds to CD25 without substantially blocking or interfering with IL-2 signaling."

[0087] In some aspects, some anti-CD25 antibodies may allow IL-2 to bind to CD25, yet block signaling through the CD25 receptor. In some aspects, such antibodies are not considered IL-2 non-blocking. In some embodiments, an IL-2 non-blocking anti-CD25 antibody allows IL-2 to bind to CD25 and promotes signaling through the CD25 receptor at a level that is at least 50% compared to signaling in the absence of the IL-2 non-blocking anti-CD25 antibody.

[0088] In some aspects, IL-2 signaling through CD25 can be assessed or measured by any known method for assessing or measuring cell signaling, such as by phosphorylation assays, binding assays, reporter assays, T cell activation assays, in vitro effector assays, in vitro antibody-dependent cellular cytotoxicity assays (ADCC assays), in vitro antibody-dependent cellular phagocytosis (ADCP), cytokine secretion assays, target cell killing assays, or model animal experiments. In some aspects, exemplary methods for assessing IL-2 signaling through CD25 include, for example, those described in Rubin et al. (1985) Hybridoma 4(2) 91-102, Van Assche et al., Gut. 2006 Nov; 55(11): 1568-1574; Martin et al., J Immunol July 15, 2010, 185 (2) 1311-1320, WO2019175223, WO2019175220, WO2019175222, WO2019175224, WO2019175216, WO2019175217, WO2019175226, WO2019175215, US10745485, US20210047420, US10738125, US20210009703, US20210040221, US20210009704, US20200407454, and US20210009699. In some aspects, the comparison of IL-2 signaling in the presence and absence of an anti-CD25 antibody agent can be performed under the same or substantially the same conditions.

[0089] In some embodiments, IL-2 signaling can be determined by measuring the level of phosphorylated STAT5 protein in cells using a standard STAT5 phosphorylation assay. For example, a STAT5 phosphorylation assay to measure IL-2 signaling can involve culturing PMBC cells in the presence of a specific concentration of an anti-CD25 antibody and then adding various concentrations of IL-2 (e.g., a serial dilution of IL-2 concentration). The cells can then be permeabilized and the level of STAT5 protein can then be measured using a fluorescently labeled antibody against phosphorylated STAT5 peptide analyzed by flow cytometry. The percentage of blocking IL-2 signaling can be calculated as follows: % blocking = 100 x [(STAT5 without antibody treatment)]. + Cell %-STAT5 with antibody treatment + Cells%) / (STAT5 without antibody treatment + cell%)].

[0090] In some embodiments, the targeting molecule can be any non-IL-2 blocking anti-CD25 antibody. Non-limiting exemplary non-IL-2 blocking anti-CD25 antibodies include, for example, the variable heavy chain (VH), VH, ... H ), variable light chain (V L ), along with corresponding SEQ ID NOs for the amino acid sequences for exemplary corresponding complementarity determining regions (CDRs). In some embodiments, the targeting molecule is an IL-2 non-blocking anti-CD25 antibody or antigen-binding fragment thereof listed in Table 1. In some embodiments, the targeting molecule is an IL-2 non-blocking anti-CD25 antibody or antigen-binding fragment thereof that competes for the same epitope or overlapping epitopes with any one or more of the antibodies listed in Table 1. In some embodiments, the targeting molecule is an IL-2 non-blocking anti-CD25 antibody or antigen-binding fragment thereof that binds to an epitope that does not overlap with one or more of the antibodies listed in Table 1.

[0091] Table 1. Sequence Identifiers (SEQ ID NOs) for Exemplary Anti-CD25 Antibodies TIFF2024505556000004.tif114145

[0092] 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 a non-IL-2 blocking anti-CD25 antibody. In some of the embodiments, the non-IL-2 blocking anti-CD25 antibody comprises a functional Fc region. In some of the embodiments, the anti-CD25 antibody comprises a full-length Fc region. In some of the embodiments, the non-IL-2 blocking anti-CD25 antibody is an antibody fragment. In some of the embodiments provided, the antibody or antibody fragment can be humanized by known methods. In some of the embodiments, the antibody or antibody fragment is a human antibody, a chimeric antibody, or a humanized antibody.

[0093] In some of any of the embodiments, the non-IL-2 blocking anti-CD25 antibody is an antibody listed in Table 1, or a biosimilar, interconvertible, biobetter, copy biologic or biogeneric thereof, or a fragment thereof.

[0094] In some of any of the embodiments, the non-IL-2 blocking anti-CD25 antibody or fragment thereof is selected from the group consisting of those listed in Table 1, e.g., in each row of Table 1. H and V L V including CDR-H1, CDR-H2, and CDR-H3 according to an antibody numbering scheme (e.g., Kabat, Chothia, Contact, IMGT, Aho, or AbM numbering schemes) of an antibody having H , and V comprising CDR-L1, CDR-L2, and CDR-L3 L In some of the embodiments, the non-IL-2 blocking anti-CD25 antibody or fragment thereof comprises a VDR comprising the CDR-H1, CDR-H2, and CDR-H3 of an antibody listed in Table 1. H , and V comprising CDR-L1, CDR-L2, and CDR-L3 LIn some of the embodiments, the non-IL-2 blocking anti-CD25 antibody or fragment thereof comprises a VDR comprising the CDR-H1, CDR-H2 and CDR-H3 of an antibody listed in Table 1. H and V comprising the CDR-L1, CDR-L2 and CDR-L3 of the antibodies listed in Table 1. L In some embodiments, the non-IL-2 blocking anti-CD25 antibody or fragment thereof comprises the V shown in SEQ ID NO: listed in each row of Table 1 below. H and V L Region or V as shown in SEQ ID NO: listed in each row of Table 1 H and V L V having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence H and V L In some of the embodiments, the non-IL-2 blocking anti-CD25 antibody is an antibody that comprises the sequence V of an antibody listed in Table 1, e.g., in each row of Table 1. H and V L V, each of which contains H and V L Includes.

[0095] In some of any of the provided embodiments, the exemplary IL-2 non-blocking anti-CD25 antibody of the conjugate includes any known IL-2 non-blocking anti-CD25 antibody, humanized form thereof, and / or antigen-binding fragment thereof. In some aspects, exemplary IL-2 non-blocking anti-CD25 antibodies of the conjugate include, for example, those described in Rubin et al. (1985) Hybridoma 4(2) 91-102, Van Assche et al., Gut. 2006 Nov; 55(11): 1568-1574; Martin et al., J Immunol July 15, 2010, 185 (2) 1311-1320, WO2019175223, WO2019175220, WO2019175222, WO2019175224, WO2019175216, WO2019175217, WO2019175226, WO2019175215, US10745485, US20210047420, US10738125, US20210009703, US20210040221, US20210009704, US20200407454, and US20210009699, humanized forms thereof, and / or antigen-binding fragments thereof.

[0096] In some of any of the provided embodiments, the IL-2 non-blocking anti-CD25 antibody is 7G7B6 (Rubin et al. (1985) Hybridoma 4(2) 91-102), or a humanized 7G7B6 antibody, a fragment thereof, or is derived from 7G7B6. In some of any of the embodiments, the IL-2 non-blocking anti-CD25 antibody is 7G7B6 comprising an Fc region, or a humanized form thereof (humanized 7G7B6). In some of any of the embodiments, the IL-2 non-blocking anti-CD25 antibody is 7G7B6 or humanized 7G7B6 comprising an Fc region that has been engineered to exhibit antibody-dependent cellular cytotoxicity (ADCC) activity or to exhibit enhanced ADCC activity. In some of any of the embodiments, the IL-2 non-blocking anti-CD25 antibody is 7G7B6 or humanized 7G7B6 comprising a full-length Fc region. In some of any of the embodiments, the non-IL-2 blocking anti-CD25 antibody is 7G7B6 or humanized 7G7B6 that contains an engineered Fc region that exhibits ADCC activity or enhanced ADCC activity. In some of any of the embodiments, the non-IL-2 blocking anti-CD25 antibody is 7G7B6 or humanized 7G7B6 that contains an Fc region that exhibits no, substantially no, or reduced ADCC activity.

[0097] In some of any of the provided embodiments, the non-IL-2 blocking anti-CD25 antibody is MA251 (see, e.g., Van Assche et al., Gut. 2006 Nov; 55(11): 1568-1574; Martin et al., J Immunol July 15, 2010, 185 (2) 1311-1320), or a humanized form thereof (humanized MA251), a fragment thereof, or is derived from MA251. In some of any of the embodiments, the non-IL-2 blocking anti-CD25 antibody is MA251 or humanized MA251 that includes an Fc region. In some of any of the embodiments, the non-IL-2 blocking anti-CD25 antibody is MA251 or humanized MA251 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 any of the embodiments, the non-IL-2 blocking anti-CD25 antibody is MA251 or humanized MA251 that comprises a full-length Fc region. In some of any of the embodiments, the non-IL-2 blocking anti-CD25 antibody is MA251 or humanized MA251 that comprises an Fc region that exhibits ADCC activity or has been engineered to exhibit enhanced ADCC activity. In some of any of the embodiments, the non-IL-2 blocking anti-CD25 antibody is MA251 or humanized MA251 that comprises an Fc region that exhibits no ADCC activity, no substantial ADCC activity, or reduced ADCC activity.

[0098] In some of any of the embodiments provided, the IL-2 non-blocking anti-CD25 antibody is "Clone A" (as set forth in Table 1) or a fragment thereof or is derived from "Clone A". In some of any of the embodiments, the IL-2 non-blocking anti-CD25 antibody is Clone A that includes an Fc region. In some of the embodiments, the IL-2 non-blocking anti-CD25 antibody is "Clone A" 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 IL-2 non-blocking anti-CD25 antibody is "Clone A" that includes a full-length Fc region. In some of the embodiments, the IL-2 non-blocking anti-CD25 antibody is "Clone A" that includes an Fc region that exhibits ADCC activity or that has been engineered to exhibit enhanced ADCC activity. In some of the embodiments, the IL-2 non-blocking anti-CD25 antibody is "Clone A" that includes an Fc region that exhibits no ADCC activity, no substantial ADCC activity, or that exhibits reduced ADCC activity.

[0099] In some of any of the embodiments provided, the IL-2 non-blocking anti-CD25 antibody is "clone B" (as described in Table 1) or a fragment thereof or is derived from "clone B". In some of any of the embodiments, the IL-2 non-blocking anti-CD25 antibody is "clone B" that includes an Fc region. In some of any of the embodiments, the IL-2 non-blocking anti-CD25 antibody is "clone B" that includes an Fc region that exhibits antibody-dependent cellular cytotoxicity (ADCC) activity or has been engineered to exhibit enhanced ADCC activity. In some of any of the embodiments, the IL-2 non-blocking anti-CD25 antibody is "clone B" that includes a full-length Fc region. In some of any of the embodiments, the IL-2 non-blocking anti-CD25 antibody is "clone B" that includes an Fc region that exhibits ADCC activity or has been engineered to exhibit enhanced ADCC activity. In some of any of the embodiments, the IL-2 non-blocking anti-CD25 antibody is "clone B" that includes an Fc region that exhibits no ADCC activity, no substantial ADCC activity, or has reduced ADCC activity.

[0100] In some embodiments, the targeting molecule can be an antibody or antibody fragment that includes the "complementarity determining region" or "CDR" of an IL-2 non-blocking anti-CD25 antibody, such as any of the antibodies or antigen-binding fragments thereof described in Table 1. CDRs are typically responsible for binding to an epitope of an antigen. The exact amino acid sequence boundaries of a given CDR can be easily determined using any of several well-known schemes. Exemplary numbering schemes include Kabat, Chothia, Contact, IMGT, Aho, and AbM numbering schemes. The boundaries of a given CDR can vary depending on the scheme used to identify it. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the most common antibody region sequence length, and insertions are accommodated by an insertion letter, e.g., "30a", and deletions occur in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in differential numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on those used by Oxford Molecular's AbM antibody modeling software. Any antibody numbering scheme can be used to identify the CDR regions of the antibodies and conjugates described herein and can be used in the methods and uses provided.

[0101] The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, are numbered consecutively starting from the N-terminus, and are also commonly identified by the chain in which the particular CDR is located. Thus, the heavy chain variable region (V H ) CDR3 (also called CDR-H3) is located in the variable domain of the heavy chain of the antibody in which it is found, whereas the light chain variable region (V L) CDR1 (also called CDR-L1) is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities, such as different binding sites for different antigens, have different CDRs. Although it is the CDRs that vary between antibodies, 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 CDRs from 7G7B6, MA251, or "clone A" or "clone B", such as those listed in Table 1, according to the Chothia numbering scheme. In some embodiments, the targeting molecule comprises CDRs from 7G7B6, MA251, or "clone A" or "clone B", according to a different numbering scheme. In some embodiments, the antibody of the conjugate is a biosimilar, interconvertible or biobetter of any of the anti-CD25 antibodies described herein, e.g., 7G7B6, MA251, "Clone A, or "Clone B," or an antigen-binding fragment thereof. Such antibodies also include copy biologics and biogenerics of any of the anti-CD25 antibodies described herein, or antigen-binding fragments thereof.

[0102] In some embodiments of the methods and uses provided herein, the IL-2 non-blocking anti-CD25 antibody comprises a functional Fc region. In some embodiments of the methods and uses provided herein, the IL-2 non-blocking anti-CD25 antibody comprises a full-length Fc region. In some embodiments, the IL-2 non-blocking anti-CD25 antibody comprises an IgG1 Fc region. In some embodiments, the IL-2 non-blocking anti-CD25 antibody comprises an IgG2 Fc region. In some embodiments, the IgG2 Fc region is an IgG2a Fc region. In some embodiments, the IgG2 Fc region is an IgG2a / b Fc region. In some embodiments, the IgG2 Fc region is an IgG2a Fc region. In some embodiments of the methods and uses provided herein, the IL-2 non-blocking anti-CD25 antibody comprises an IgG3 Fc region. In some embodiments of the methods and uses provided herein, the IL-2 non-blocking anti-CD25 antibody comprises an IgG4 Fc region. In some embodiments, the Fc region is modified to modulate the effector function of the antibody portion of the conjugate. Such modifications, such as any of those described in Wang et al., (2018) Protein Cell. 9(1): 63-73, are contemplated for the antibodies, antibody fragments, and conjugates described herein.

[0103] In some embodiments of the methods and uses provided herein, the non-IL-2 blocking anti-CD25 antibody does not contain a functional Fc region. In some such examples, the non-IL-2 blocking antibody does not contain an Fc region or contains an Fc region that is modified so that it does not bind to an Fc receptor and / or does not induce substantial Fc effector functions (e.g., ADCC, ADCP, and / or CDC). In some such embodiments, the non-IL-2 blocking antibody contains an Fc receptor that contains an amino acid substitution to eliminate a glycosylation site at a position corresponding to position 297 of the heavy chain based on the EU numbering described by Edelman et al., (1969) Proc Natl Acad Sci US A. 63(1):78-85. For example, a non-IL-2 blocking antibody can include an Fc receptor that contains an asparagine to glutamine substitution at or corresponding to position 297 of the antibody sequence in EU numbering (N297Q), an asparagine to alanine substitution at or corresponding to position 297 of the antibody sequence in EU numbering (N297A), or an asparagine to glycine substitution at or corresponding to position 297 of the antibody sequence in EU numbering (N297G).

[0104] In some embodiments of the methods and uses provided herein, the IL-2 non-blocking anti-CD25 antibody comprises an Fc region that exhibits enhanced Fc-mediated effector function, such as ADCC, ADCP, and / or CDC activity, and / or exhibits preferential binding to Fc gamma receptors. In some embodiments, the IL-2 non-blocking anti-CD25 antibody exhibits enhanced function due to increased Fc receptor engagement. In some embodiments, the Fc region contains one or more of the following mutations: a serine to aspartic acid substitution at position 239 (S239D), an alanine to leucine substitution at position 330 (A330L), an isoleucine to glutamic acid substitution at position 332 (I332E), a glutamic acid to alanine substitution at position 333 (E333A), a lysine to alanine substitution at position 334 (K334A), an arginine to alanine substitution at position 255 (S255A), a threonine to alanine substitution at position 256 (T256A), a cytosine to alanine substitution at position 267 (K334 ... A serine to alanine substitution (S267A), a serine to alanine substitution at position 298 (S298A), an asparagine to serine substitution at position 325 (N325S), a leucine to phenylalanine substitution at position 328 (L328F), an alanine to leucine substitution at position 330 (A330L), an isoleucine to glutamic acid substitution at position 333 (E333A), a glutamic acid to alanine substitution at position 333 (E333A), a lysine to alanine substitution at position 334 (K334), and / or an alanine to glutamine substitution at position 378 (A378Q). In some embodiments, the Fc region contains a serine to alanine substitution at position 298 and a lysine to alanine substitution at position 334 (S298A / K334) with respect to the EU numbering of the antibody heavy chain. In some embodiments, the Fc region contains a glutamic acid to alanine substitution at position 333 and a lysine to alanine substitution at position 334 (E33A / K334) relative to EU numbering of the antibody heavy chain. In some embodiments, the Fc region contains an arginine to alanine substitution at position 255 and a serine to alanine substitution at position 267 (R255A / S267A) relative to EU numbering of the antibody heavy chain.In some embodiments, the Fc region contains a threonine to alanine substitution at position 256 (T256A) relative to the EU numbering of the antibody heavy chain. In some embodiments, the Fc region contains a lysine to alanine substitution at position 334 and an alanine to glutamine substitution at position 378 (K334 / A378) relative to the EU numbering of the antibody heavy chain. In some embodiments, the Fc region contains a serine to aspartic acid substitution at position 239, an alanine to leucine substitution at position 330, and an isoleucine to glutamic acid substitution at position 332 (S239D / A330L / I332E) relative to the EU numbering of the antibody heavy chain. In some embodiments, the Fc region contains a glycine to alanine substitution at position 236, a serine to aspartic acid substitution at position 239, and an isoleucine to glutamic acid substitution at position 332 (S239D / A330L / I332E) relative to EU numbering of the antibody heavy chain. In some embodiments, the Fc region contains an arginine to serine substitution at position 325 and a leucine to phenylalanine substitution at position 328 (N325S / L328F) relative to EU numbering of the antibody heavy chain.

[0105] The conjugate used in the methods and uses provided herein comprises a phthalocyanine dye. In some embodiments of the methods and uses provided herein, the phthalocyanine dye is a phthalocyanine dye with a silicon-coordinated metal (Si-phthalocyanine dye). In any of the embodiments, the phthalocyanine dye is a salt, stereoisomer, or tautomer of any of the dyes described herein. In any of the embodiments, the phthalocyanine dye is an ionic form of any of the dyes described herein.

[0106] In some embodiments, the phthalocyanine dye has the formula: TIFF2024505556000005.tif85128, wherein L is a linker; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7, and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from 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 is alkylene.

[0107] In some embodiments, the phthalocyanine dye has the formula: TIFF2024505556000006.tif75137, wherein X 1 and X 4 each independently represents a C1-C alkyl group optionally interrupted by a heteroatom; 10 is alkylene; R 2 , R3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of contains a water-soluble group; and R 16 , R 17 , R 18 and R 19 are each independently selected from among hydrogen, halogen, optionally substituted alkylthio, optionally substituted alkylamino, and optionally substituted alkoxy.

[0108] In some embodiments, the phthalocyanine dye is a Si-phthalocyanine dye that is IRDye 700DX (IR700). In some embodiments, the phthalocyanine dye containing a reactive group is IR700 NHS ester, e.g., IRDye 700DX NHS ester (LiCor 929-70010, 929-70011). In some embodiments, the dye has the formula: TIFF2024505556000007.tif105159, or a salt, ionic form, stereoisomer, or tautomer thereof.

[0109] 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.

[0110] In some embodiments, the phthalocyanine dye is any of the dyes described in WO 2021 / 207691. In some embodiments, the dye is a silicon phthalocyanine dye described in WO 2021 / 207691.

[0111] In some embodiments, the phthalocyanine dye has the formula (X): TIFF2024505556000008.tif49128 or a salt, ionic form, stereoisomer, or tautomer thereof, wherein X is TIFF2024505556000009.tif22128; Y is TIFF2024505556000010.tif21128; R 1 and R 2 are each independently optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, or optionally substituted heteroaralkyl; R 3 , R 4 or R 5 is selected from the substituent (a) or the substituent (b), (a)R 3 is hydrogen, -L 3 -H, -L 3 -A, or -L 3 -Z; R 4 -L 4 -H, -(NH) m -L 4 -A, -(NH) m-L 4 -Z, -(O) m -L 4 -A or -(O) m -L 4 -Z; R 5 -L 5 -H or -L 5 -A; and (b)R 3 -L 3 -H, or -L 3 -A; R 4 -L 4 -H, -(NH) m -L 4 -A, or -(O) m -L 4 -A; where R 3 and R 4 are connected by a bond, -L 4 -A forms a substituted heterocyclyl; and R 5 -L 5 -H or -L 5 -A; However, R 3 , R 4 and R 5 at least one of is a group containing A; A is a reactive group, or a protected form thereof, or a reacted form thereof, capable of forming a covalent bond with a thiol, hydroxyl, carboxyl, or amino group of a second moiety; R 6 and R 7 are each independently optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, or optionally substituted heteroaralkyl; R 8 , R 9 or R 10 is selected from the substituent (a) or the substituent (b), (a)R8 is hydrogen, -L 8 -H or -L 8 -Z; R 9 -L 9 -H, -(NH) n -L 9 -Z or -(O) n -L 9 -Z; R 10 -L 10 -Z; and (b)R 8 and R 9 are connected by a bond, -L 9 -Z forms a substituted heterocyclyl, R 10 -L 10 -H or -L 10 -Z; However, R 8 , R 9 and R 10 at least one of is a group containing Z; Z is a water-soluble group which may be substituted with A or L'-A; L 1 and L 2 are each independently an optionally substituted alkylene, an optionally substituted heteroalkylene, an optionally substituted alkenylene, an optionally substituted heteroalkenylene, an optionally substituted cycloalkyl, or an optionally substituted heterocyclyl; L 3 , L 4 , L 5 , L 8 , L 9 and L 10are each independently an optionally substituted alkylene, an optionally substituted heteroalkylene, an optionally substituted alkenylene, an optionally substituted heteroalkenylene, an optionally substituted cycloalkylene, an optionally substituted heterocyclene, an optionally substituted arylene, an optionally substituted aralkylene, an optionally substituted heteroaralkylene, or an optionally substituted heteroarylene, wherein a carbon atom of the alkylene, heteroalkylene, alkenylene, heteroalkenylene, cycloalkylene, heterocyclene, arylene, aralkylene, heteroaralkylene, or optionally substituted heteroarylene is optionally further substituted with Z, and each nitrogen atom of the heteroalkylene or heteroalkenylene is optionally substituted with one or two L'-Z; L', at each occurrence, is independently an optionally substituted alkylene, an optionally substituted heteroalkylene, an optionally substituted alkenylene, an optionally substituted heteroalkenylene, an optionally substituted cycloalkylene, an optionally substituted heterocyclene, an optionally substituted arylene, an optionally substituted aralkylene, an optionally substituted heteroaralkylene, or an optionally substituted heteroarylene; a is 0 or 1; b is 0 or 1; c is 0 or 1; d is 0 or 1; m is 0 or 1; n is 0 or 1; provided that when b is 1, a is 0; If d is 1, then c is 0; When m is 1, b is 1; When n is 1, c is 1.

[0112] In one particular embodiment, the silicon phthalocyanine dye has the formula: TIFF2024505556000011.tif152150TIFF2024505556000012.tif246134TIFF2024505556000013.tif206136, or a salt, ionic form, stereoisomer, or tautomer thereof.

[0113] In one particular embodiment, the silicon phthalocyanine dye has the formula: TIFF2024505556000014.tif111128 or a salt, ionic form, stereoisomer, or tautomer thereof.

[0114] In some embodiments, the dye is a silicon phthalocyanine dye selected from the compounds provided in Table A, or a salt, ionic form, stereoisomer, or tautomer thereof.

[0115] (Table A) TIFF2024505556000015.tif202164TIFF2024505556000016.tif213164TIFF20245055560 00017.tif222164TIFF2024505556000018.tif198164TIFF2024505556000019.tif104164

[0116] In certain embodiments, the phthalocyanine dye containing a reactive group has formula (I): It has a structure of TIFF2024505556000020.tif72128.

[0117] In certain embodiments, the phthalocyanine dye containing a reactive group has the formula (II): It has a structure of TIFF2024505556000021.tif70128.

[0118] In some embodiments, the compositions for use with the methods and uses provided herein comprise a conjugate comprising a Si-phthalocyanine dye linked to a targeting molecule, wherein the targeting molecule binds to CD25. In some embodiments, the targeting molecule is an IL-2 non-blocking anti-CD25 antibody or an antigen-binding fragment thereof. In some embodiments, the compositions comprise an IL-2 non-blocking anti-CD25-Si-phthalocyanine dye conjugate. In some embodiments, the compositions comprise an IL-2 non-blocking anti-CD25-IR700 conjugate. In some embodiments, the compositions comprise an IL-2 non-blocking anti-CD25-IR700 conjugate, wherein the anti-CD25 moiety is 7G7B6, MA251, "clone A" or "clone B". In some embodiments, the compositions comprise an IL-2 non-blocking anti-CD25-IR700 conjugate, wherein the IL-2 non-blocking anti-CD25 moiety contains a functional Fc region. In some embodiments, the composition is an IL-2 non-blocking anti-CD25-IR700 conjugate, where the IL-2 non-blocking anti-CD25 moiety contains an Fc region, such as a full-length Fc region. In some embodiments, the composition is an IL-2 non-blocking anti-CD25-IR700 conjugate, where the IL-2 non-blocking anti-CD25 moiety contains an Fc region, such as an Fc region that exhibits antibody-dependent cellular cytotoxicity (ADCC) activity or that has been engineered to exhibit enhanced ADCC activity.

[0119] IV. Checkpoint inhibitor combination therapy The methods and uses provided herein can include administration of an immune checkpoint inhibitor before, simultaneously, or after 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, such as a Si-phthalocyanine dye, linked to a targeting molecule, where the targeting molecule binds to CD25 without blocking IL-2, and irradiating the first 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, where the targeting molecule binds to CD25 without blocking IL-2, and irradiating the first 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 administration of an immune checkpoint inhibitor simultaneously with administration of the conjugate. In some aspects, the provided combinations can include a phthalocyanine dye-targeting molecule conjugate (e.g., a non-IL-2 blocking anti-CD25 antibody-IR700 conjugate) and an immune checkpoint inhibitor. In some aspects, such combinations can be used in the provided methods or uses, such as those involving combination therapy or treatment.

[0120] 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.

[0121] 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), or an antigen-binding fragment thereof, or any combination thereof.

[0122] 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-936559, MSB2 311, 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, or an antigen-binding fragment thereof, or any combination thereof.

[0123] 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, or antigen-binding fragments thereof, or any combination thereof.

[0124] 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 IL-2 non-blocking 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 IL-2 non-blocking anti-CD25-IR700 conjugate, where the IL-2 non-blocking anti-CD25 portion of the conjugate is or is derived from 7G7B6. 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 IL-2 non-blocking anti-CD25-IR700 conjugate, where the anti-CD25 portion of the conjugate is or is derived from or competes with 7G7B6, and the antibody portion of the conjugate comprises a functional Fc region. In some embodiments of the method, the conjugate is an IL-2 non-blocking anti-CD25-IR700 conjugate, where the IL-2 non-blocking anti-CD25 portion of the conjugate is, is derived from, or competes with 7G7B6 that includes a functional Fc region. In some embodiments of the method, the conjugate is an IL-2 non-blocking anti-CD25-IR700 conjugate, where the IL-2 non-blocking anti-CD25 portion of the conjugate is derived from, or competes with 7G7B6 that includes a functional Fc region, and the anti-PD-1 antibody is pembrolizumab (MK-3475, KEYTRUDA), nivolumab (OPDIVO), or cemiplimab (LIBTAYO), or an antigen-binding fragment thereof.

[0125] 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 an IL-2 non-blocking anti-CD25-Si phthalocyanine dye conjugate (e.g., an IL-2 non-blocking anti-CD25-IR700 conjugate), simultaneously with administration of an IL-2 non-blocking anti-CD25-Si phthalocyanine dye conjugate, after administration of an IL-2 non-blocking anti-CD25-Si phthalocyanine dye conjugate, or any combination thereof.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] In some embodiments of the methods and uses provided herein, the IL-2 non-blocking 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 first 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 the first tumor or one or more additional tumors, residual disease such as residual cells or masses from the primary tumor, invasive cancer cells, or metastatic tumor cells remain in the subject. In some embodiments, administration of the conjugate is repeated if residual lesions persist 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.

[0130] In some embodiments of the methods and uses provided herein, an IL-2 non-blocking anti-CD25 conjugate, such as an IL-2 non-blocking 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, such as 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 IL-2 non-blocking anti-CD25-IR700 conjugate and administering a checkpoint inhibitor, such as 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 first 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 first 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.

[0131] In some embodiments, the method produces an enhanced effect, e.g., an additive effect, or a synergistic effect, on the first tumor, 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., the first tumor).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 a first 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).

[0132] 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), intratumor 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.

[0133] In some embodiments, the method comprises administering an IL-2 non-blocking anti-CD25-IR700 conjugate and an immune checkpoint inhibitor to achieve a synergistic effect. In some embodiments, the method comprises administering an IL-2 non-blocking 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 or metastatic tumor cells). In some embodiments, administration of a non-IL-2 blocking anti-CD25-IR700 conjugate 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 an anti-PD-1 antibody 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.

[0134] 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.

[0135] 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.

[0136] 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, poly(I:C), 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.

[0137] 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.

[0138] In some embodiments, the immunomodulatory agent is a Toll-like receptor (TLR) agonist. In some embodiments, such agonists can include TLR4 agonists, TLR8 agonists, or TLR9 agonists. Such agonists can be selected from peptidoglycan, poly(I:C), CpG, 3M003, flagellin, and Leishmania homolog of eukaryotic ribosome elongation and initiation factor 4a (LeIF).

[0139] 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 anthracyclines (e.g., 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).

[0140] 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.

[0141] 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.

[0142] In some embodiments, the anticancer drug is 5-fluorouracil / leucovorin, oxaliplatin, irinotecan, regorafenib, ziv-aflibercept, capecitabine, cisplatin, paclitaxel, topotecan, carboplatin, gemcitabine, docetaxel, 5-FU, ifosfamide, mitomycin, pemetrexed, vinorelbine, carmustine wafers, wager), temozolomide, methotrexate, capecitabine, lapatinib, etoposide, dabrafenib, vemurafenib, liposomal cytarabine, cytarabine, interferon alpha, erlotinib, vincristine, cyclophosphamide, lomustine, procarbazine, sunitinib, somatostatin, 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 aurorapyrimidines.

[0143] In some embodiments, anticancer drug is an alkylating agent.Alkylating agent is a compound that directly damages DNA by forming covalent bond with nucleic acid and inhibiting DNA synthesis.Exemplary alkylating agents include but are not limited to mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan and thiotepa, and nitrosourea alkylating agents such as carmustine and lomustine.

[0144] In some embodiments, the anti-cancer agent is an antibody or an antigen-binding antibody fragment.

[0145] 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.

[0146] 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®).

[0147] 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.

[0148] 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.

[0149] 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®).

[0150] 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®).

[0151] In some embodiments, the anti-cancer agent is another type of chemotherapeutic agent, such as a proteasome inhibitor, a kinase inhibitor, or a histone deacetylase inhibitor, hi other embodiments, the anti-cancer agent is a biologic, such as an antibody, used in cancer treatment.

[0152] VI. Irradiation and Devices for Use with the Present Methods and Compositions In some aspects, a device that can be used with the provided embodiments includes a light diffusing device that provides illumination (sometimes also referred to as radiation) at a wavelength (or wavelengths) of light suitable for use with dye conjugate compositions, such as phthalocyanine dye conjugates (e.g., IL-2 non-blocking anti-CD25-IR700 conjugates such as those described herein). The illumination device can include a light source (e.g., a laser) and a means for transmitting light to an area of ​​interest (e.g., one or more fibers for irradiating an isolated area or isolated lesion or tumor of a subject). Exemplary illumination devices are described in U.S. Pat. Nos. 10,295,719; 10,527,771; and 10,416,366, which are incorporated herein by reference. Such devices deliver light to a target area of ​​a subject using a light diffusing device that contains a non-circular core optical fiber operatively connected to a laser. In some embodiments, the core optical fiber is circular and is coiled or bent before interfacing with the light diffusing device. In certain aspects, the device delivers a "top hat" core irradiance distribution to deliver uniform light to the illuminated area. The light diffusing device can be for use as a cylindrical diffuser, for example, for intratumoral or interstitial radiation. In some embodiments, the light diffusing device is a frontal diffuser with a lens, where the illumination is projected through the lens of the frontal diffuser at the end of the optical fiber. The projected light can be a parallel or divergent light beam.

[0153] In some embodiments, the target area, e.g., tumor, near tumor, lymph node, near lymph node, is from 400 nm or about 400 nm to 900 nm or about 900 nm, e.g., from 500 nm or about 500 nm to 900 nm or about 900 nm, e.g., from 600 nm or about 600 nm to 850 nm or about 850 nm, e.g., from 600 nm or about 600 nm to 740 nm or about 740 nm, e.g., from 660 nm or about 660 nm to 740 nm or about 740 nm, from 660 nm or about 660 nm to 710 nm or about 710 nm, The optical fiber is irradiated with light having a wavelength within the range of from at or about 660 nm to at or about 700 nm, from at or about 660 to at or about 685, from at or about 685, from at or about 665 to at or about 680, from at or about 670 to at or about 685, from at or about 670 to at or about 685, from at or about 670 to at or about 690 nm, from at or about 670 to at or about 680, from at or about 680 to at or about 740 nm, or from at or about 690 nm to at or about 710 nm. In some embodiments, a target area, e.g., a tumor, near a tumor, lymph node, near a lymph node, or the tumor microenvironment, is irradiated with light having a wavelength of from at or about 600 nm to at or about 850 nm, e.g., from at or about 660 nm to at or about 740 nm. In some embodiments, a target area, e.g., a tumor, near a tumor, lymph node, near a lymph node, or tumor microenvironment, is irradiated with light of a wavelength of at least, or at least about, 600 nm, 620 nm, 640 nm, 660 nm, 680 nm, 700 nm, 720 nm, or 740 nm, e.g., 690±50 nm or about 690±50 nm, or 690±40 nm or about 690±40 nm, e.g., 690 nm or about 690 nm, or 680 nm or about 680 nm.In some embodiments, the target area, e.g., a tumor, near a tumor, lymph node, near a lymph node, or tumor microenvironment, is irradiated with light having a wavelength of at or about 670±50 nm, or at or about 670±40 nm, e.g., 670 nm or about 670 nm. In some embodiments, the target area, e.g., a tumor, near a tumor, lymph node, near a lymph node, or tumor microenvironment, is irradiated with light having a wavelength of less than 685 nm or 680 nm, or less than about 685 nm or 680 nm.

[0154] In some of the optional embodiments, the wavelength of light used for irradiation depends on the phthalocyanine dye. For example, if the Si-phthalocyanine dye is IR700, the irradiation can be performed at a wavelength of 690 nm ± 20 nm. In some cases, the Si-phthalocyanine dye is represented by formula (I): When comprising TIFF2024505556000022.tif69128, or a salt, stereoisomer, or tautomer thereof, irradiation can be carried out at a wavelength of 660 nm ± 50 nm.

[0155] In some embodiments of the methods and uses provided herein, the interstitial irradiation is performed using cylindrical diffusing fibers with a diffuser length of 0.5 cm or about 0.5 cm to 10 cm or about 10 cm and spaced 1.8±0.2 cm or about 1.8±0.2 cm apart. In some embodiments, the light (irradiation) dose is from 2 J / cm or about 2 J / cm to 500 J / cm or about 500 J / cm. In some embodiments, the interstitial light (irradiation) dose is from 20 J / cm or about 20 J / cm to 500 J / cm or about 500 J / cm. In some embodiments, the optical power (or light fluence) of the interstitial light dose is from 100 mW / cm to 500 mW / cm or about 500 mW / cm. In some embodiments, the light is applied for at or about 120 seconds to at or about 600 seconds. In some embodiments, the light is applied for at least or for at least about 100 seconds, 120 seconds, 150 seconds, 180 seconds, 200 seconds, 220 seconds, 250 seconds, 270 seconds, 300 seconds, 310 seconds, 330 seconds, 340 seconds, 350 seconds, 370 seconds, 380 seconds, 400 seconds, 420 seconds, 440 seconds, 460 seconds, 480 seconds, or 500 seconds. In some embodiments, the tumor is greater than or about 10 mm deep or is a subcutaneous tumor.

[0156] In some embodiments, the methods provided include interstitial irradiation of a target area, such as a tumor, in a subject, with a cylindrical diffusing fiber comprising a diffuser length of 0.5 cm or about 0.5 cm to 10 cm or about 10 cm and placed 1.8±0.2 cm or about 1.8±0.2 cm apart, at a light dose of 100 J / cm or about 100 J / cm of fiber length, at a light fluence of 400 mW / cm or about 400 mW / cm for 250 seconds or about 250 seconds. In some embodiments, the target area is a tumor that is greater than 10 mm or about 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 or about 1.8±0.2 cm apart in the tumor. In some embodiments, the catheter is optically transparent.

[0157] In some embodiments, a target area, e.g., a tumor, near a tumor, lymph node, near a lymph node, or the tumor microenvironment, receives at least 1 J / cm 2 or at least about 1 J / cm 2 , e.g. at least 10 J / cm 2 Or at least about 10 J / cm 2 , at least 30 J / cm 2 Or at least about 30 J / cm 2 , at least 50 J / cm 2 Or at least about 50 J / cm 2 , at least 100 J / cm 2 Or at least about 100 J / cm 2 , or at least 500 J / cm 2 Or at least about 500 J / cm 2 In some embodiments, the radiation dose is from 1 or about 1 to 2 J / cm 2 Or about J / cm 2 up to 1 or about 1 to 500 J / cm 2 Or about 500 J / cm 2 Up to 5 or about 5 to 200 J / cm 2 Or about 200 J / cm 2Up to 10 or about 10 to 100 J / cm 2 Or about 100 J / cm 2 Up to 10 or about 10 to 50 J / cm 2 Or about 50 J / cm 2 Up to, or from 25 or about 25 to 400 J / cm 2 Or about 400 J / cm 2 In some embodiments, the target area is at least 2 J / cm 2 , 5J / cm 2 , 10J / cm 2 , 25J / cm 2 , 30J / cm 2 , 50J / cm 2 , 75J / cm 2 , 100J / cm 2 , 150J / cm 2 , 200J / cm 2 , 300J / cm 2 , 400J / cm 2 , or 500 J / cm 2 , or at least about 2 J / cm 2 , 5J / cm 2 , 10J / cm 2 , 25J / cm 2 , 30J / 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 In some embodiments, the light (irradiation) dose is 25 J / cm 2 or about 25 J / cm 2 From 400J / cm 2 or about 400 J / cm 2 In some embodiments, the optical power (or optical fluence) of the light dose is up to 50 mW / cm 2 or about 50 mW / cm 2 From 200mW / cm 2 or about 200 mW / cm 2up to, for example, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mW / cm 2 , or about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mW / cm 2 In some embodiments, the light is applied for a period of at or about 120 seconds to at or about 600 seconds. In some embodiments, the light is applied for at least or for at least about 100 seconds, 120 seconds, 150 seconds, 180 seconds, 200 seconds, 220 seconds, 250 seconds, 270 seconds, 300 seconds, 310 seconds, 330 seconds, 340 seconds, 350 seconds, 370 seconds, 380 seconds, 400 seconds, 420 seconds, 440 seconds, 460 seconds, 480 seconds, or 500 seconds.

[0158] In some embodiments, the target area 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 or superficial irradiation. In some embodiments, the light irradiation dose is 5 J / cm 2 or about 5 J / cm 2 From 200J / cm 2 or about 200 J / cm 2 In some embodiments, the light exposure dose is up to 25 J / cm 2 or about 25 J / cm 2 From 400J / cm 2 or about 400 J / cm 2 In some embodiments, the optical power of the light dose is up to 50 mW / cm 2 or about 50 mW / cm 2 From 200mW / cm 2 or about 200 mW / cm 2In some embodiments, the light exposure dose is up to 50 J / cm 2 or about 50 J / cm 2 , 150mW / cm 2 or about 150 mW / cm 2 of optical power for 333 seconds or about 333 seconds.

[0159] In some embodiments, a target area, e.g., a tumor, near a tumor, lymph node, near a lymph node, or the tumor microenvironment, is irradiated with a dose of at least or at least about 1 J / cm fiber length, e.g., at least or at least about 10 J / cm fiber length, at least or at least about 50 J / cm fiber length, at least or at least about 100 J / cm fiber length, at least or at least about 250 J / cm fiber length, or at least or at least about 500 J / cm fiber length. In some embodiments, the radiation dose is from 1 or about 1 to 1000 J / cm of fiber length, from 1 or about 1 to 500 J / cm of fiber length, from 2 or about 2 to 500 J / cm of fiber length, from 50 or about 50 to 300 J / cm of fiber length, from 10 or about 10 to 100 J / cm of fiber length, or from 10 or about 10 to 50 J / cm of fiber length. In some embodiments, a target area, e.g., a tumor, near a tumor, lymph node, near a lymph node, or tumor microenvironment, is at least 2 J / cm fiber length, 5 J / cm fiber length, 10 J / cm fiber length, 25 J / cm fiber length, 50 J / cm fiber length, 75 J / cm fiber length, 100 J / cm fiber length, 150 J / cm fiber length, 200 J / cm fiber length, 250 J / cm fiber length, 300 J / cm fiber length, 400 J / cm fiber length, 500 J / cm fiber length, 600 J / cm fiber length, 700 J / cm fiber length, 800 J / cm fiber length, 900 J / cm fiber length, 1000 J / cm fiber length, 1500 J / cm fiber length, 2000 J / cm fiber length, 2500 J / cm fiber length, 3000 J / cm fiber length, 4000 J / cm fiber length, 5000 J / cm fiber length, 6000 J / cm fiber length, 7000 J / cm fiber length, 8000 J / cm fiber length, 9000 J / cm fiber length, 1000 J / cm fiber length, 1500 J / cm fiber length, 2000 J / cm fiber length, 2500 J / cm fiber length, 3000 J / cm fiber length, 4000 J / cm fiber length, 5000 J / cm fiber length, 6000 J / cm fiber length, 8000 J / cm fiber length, 9000 J / cm fiber length, 10000 J / cm fiber length, 10000 J / cm fiber length, 15000 J / cm fiber length, 20000 J / cm fiber length, 25000 J / cm fiber length, 30000 J / cm fiber length, 40000 J m or 500 J / cm of fiber length, or at least about 2 J / cm, 5 J / cm, 10 J / cm, 25 J / cm, 50 J / cm, 75 J / cm, 100 J / cm, 150 J / cm, 200 J / cm, 250 J / cm, 300 J / cm, 400 J / cm, or 500 J / cm of fiber length.In some embodiments, the irradiation is administered at a dose of, or about, 100 J / cm, at a fluence rate of, or about, 400 mW / cm, for a duration of, or about, 250 seconds.

[0160] In some embodiments, methods provided include using a microlens-tipped fiber for surface illumination, with a maximum output of 5 J / cm 2 or about 5 J / cm 2 From 200J / cm 2 or about 200 J / cm 2 In some embodiments, the light irradiation dose is up to 25 J / cm. 2 or about 25 J / cm 2 From 400J / cm 2 or about 400 J / cm 2 In some embodiments, the light exposure dose is up to 50 J / cm 2 or about 50 J / cm 2 In some embodiments, irradiation of superficial tumors is at 50 J / cm 2 or about 50 J / cm 2 At a dose of 150 mW / cm 2 or about 150 mW / cm 2 The dose is administered at a fluence rate of 333 seconds or approximately 333 seconds.

[0161] It has been found that in some cases the radiation dose required to achieve PIT in human subjects can be lower than the radiation dose required for PIT in mice. For example, in some cases, 50 J / cm in an in vivo tumor mouse model. 2 or about 50 J / cm 2 (50J / cm 2 ) dosimetry is not effective for PIT, which is in contrast to what can be observed in clinical practice in human patients.

[0162] In some embodiments, the radiation dose following administration of the composition comprising the phthalocyanine dye-targeting molecule conjugate is at least 1 J / cm at a wavelength of 660-740 nm or about 660-740 nm.2 Or at least about 1 J / cm 2 or at least 1 J / cm of fiber length or at least about 1 J / cm of fiber length, e.g., at least 10 J / cm at wavelengths of 660-740 nm or about 660-740 nm 2 Or at least about 10 J / cm 2 or at least 10 J / cm of fiber length or at least about 10 J / cm of fiber length, at wavelengths of 660-740 nm or at least 50 J / cm 2 Or at least about 50 J / cm 2 or at least 50 J / cm of fiber length or at least about 50 J / cm of fiber length, or at least 100 J / cm at wavelengths of 660-740 nm or at about 660-740 nm 2 Or at least about 100 J / cm 2 or at least 100 J / cm of fiber length or at least about 100 J / cm of fiber length. In some embodiments, the wavelength is 660-710 nm. In some embodiments, the radiation dose following administration of the composition comprising the phthalocyanine dye-targeting molecule conjugate is at least 1.0 J / cm at a wavelength of 690 nm or about 690 nm. 2 Or at least about 1.0 J / cm 2 or at least 1 J / cm of fiber length or at least about 1 J / cm of fiber length, e.g., at least 10 J / cm at a wavelength of 690 nm or about 690 nm 2 Or at least about 10 J / cm 2 or at least 10 J / cm fiber length or at least about 10 J / cm fiber length, at a wavelength of 690 nm or about 690 nm, at least 50 J / cm 2 Or at least about 50 J / cm 2 or at least 50 J / cm of fiber length or at least about 50 J / cm of fiber length, or at least 100 J / cm at a wavelength of 690 nm or about 690 nm 2 Or at least about 100 J / cm 2or at least about 100 J / cm of fiber length, e.g., 1.0 to 500 J / cm at a wavelength of 690 nm or about 690 nm 2 or 1.0-500 J / cm of fiber length. Exemplary irradiation following administration of a conjugate or composition provided herein is at least 1 J / cm at wavelengths at or about 660 nm to at or about 740 nm. 2 Or at least about 1 J / cm 2 or irradiating the target area with a dose of at least or at least about 1 J / cm of fiber length.

[0163] In some embodiments, the radiation dose following administration of the composition comprising the phthalocyanine dye-targeting molecule conjugate is at least 1 J / cm at a wavelength of 600-800 nm or about 600-800 nm. 2 Or at least about 1 J / cm 2 or at least 1 J / cm of fiber length or at least about 1 J / cm of fiber length, e.g., at or about 620-720 nm wavelengths, at least 1 J / cm 2 Or at least about 1 J / cm 2 or at least 1 J / cm of fiber length or at least about 1 J / cm of fiber length, at wavelengths of 620-720 nm or at least 10 J / cm 2 Or at least about 10 J / cm 2 or at least 10 J / cm of fiber length or at least about 10 J / cm of fiber length, at wavelengths of 620-720 nm or at least 50 J / cm 2 Or at least about 50 J / cm 2 or at least 50 J / cm of fiber length or at least about 50 J / cm of fiber length, or at least 100 J / cm at wavelengths of 620-720 nm or at about 620-720 nm 2 Or at least about 100 J / cm 2or at least 100 J / cm of fiber length or at least about 100 J / cm of fiber length. In some embodiments, the wavelength is 640-700 nm. In some embodiments, the radiation dose after administration of the composition comprising the phthalocyanine dye-targeting molecule conjugate is at least 1.0 J / cm at a wavelength of 670 nm or about 670 nm. 2 Or at least about 1.0 J / cm 2 or at least 1 J / cm of fiber length or at least about 1 J / cm of fiber length, e.g., at least 10 J / cm at a wavelength of 670 nm or about 670 nm 2 Or at least about 10 J / cm 2 or at least 10 J / cm fiber length or at least about 10 J / cm fiber length, at a wavelength of 670 nm or about 670 nm, at least 50 J / cm 2 Or at least about 50 J / cm 2 or at least 50 J / cm of fiber length or at least about 50 J / cm of fiber length, or at least 100 J / cm at a wavelength of 670 nm or about 670 nm 2 Or at least about 100 J / cm 2 or at least about 100 J / cm of fiber length, e.g., 1.0 to 500 J / cm at a wavelength of 670 nm or about 670 nm. 2 or 1.0-500 J / cm of fiber length. Exemplary irradiation following administration of a conjugate or composition provided herein is at least 1 J / cm at wavelengths at or about 620 nm to 720 nm or about 720 nm. 2 Or at least about 1 J / cm 2 or irradiating the target area with a dose of at least or at least about 1 J / cm of fiber length.

[0164] In some embodiments, the irradiating step comprises irradiating at a wavelength of at or about 600 nm to at or about 850 nm with 25 J / cm 2 Or about 25J / cm 2 From 400J / cm 2Or about 400 J / cm 2 In some embodiments, the target area is irradiated with a wavelength of 690±40 nm. In some embodiments, the target area is irradiated with a dose 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.

[0165] In some embodiments, light or laser can be applied to the dye molecule, e.g., to the cell containing the conjugate, for 5 seconds or about 5 seconds to 5 minutes or about 5 minutes. For example, in some embodiments, light or laser is applied for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 seconds, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 seconds, or within a range between any two of such values, to activate the dye molecule. In some embodiments, light or laser is applied for 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 minutes, or about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 minutes, or longer, or within a range between any two of such values. In some embodiments, the length of time that light or laser is applied can vary depending on the energy, e.g., the wattage, of the light or laser. For example, a light or laser with a lower wattage can be applied for a longer period of time to activate the dye molecules.

[0166] In some embodiments, the light or laser may be applied at or about 30 minutes to or about 96 hours after administration of the conjugate. For example, in some embodiments, the light or laser is applied at or about 30, 35, 40, 45, 50, or 55 minutes after administration of the conjugate, or within a range between any two of such values. In some embodiments, the light or laser is applied at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after administration of the conjugate, or within a range between about any two of such values, e.g., between at or about 20 hours to at or about 28 hours, or about 24 hours ± 4 hours. In some embodiments, the light or laser is applied between or about 1-24 hours, e.g., between 1 or about 1-12 hours, between 12 or about 12-24 hours, between 6 or about 6-12 hours, or greater than 24 hours, or greater than about 24 hours, after administration of the conjugate. In some embodiments, the light or laser is applied at or about 36, 48, 72, or 96 hours after administration of the conjugate. In some embodiments, the light or laser is applied at or about 24 hours ±4 hours after administration of the conjugate.

[0167] In some embodiments, a target area, such as a tumor, a tumor vicinity, a lymph node, a lymph node vicinity, or a tumor microenvironment, or a subject, can be irradiated once or multiple times. Thus, irradiation can be completed in one day, or repeated on multiple days with the same or different doses, such as at least 2 different times, 3 different times, 4 different times, 5 different times, or 10 different times, or at least about 2 different times, 3 different times, 4 different times, 5 different times, or 10 different times. In some embodiments, repeated irradiation can be performed on the same day, on consecutive days, or every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or at longer intervals. In some embodiments, multiple irradiations, such as at least 2 times, at least 3 times, or at least 4 times, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate administrations, are performed.

[0168] In some embodiments, the dose or method of radiation varies depending on the type or morphology of the target area, eg, tumor, near-tumor, lymph node, near-lymph node.

[0169] In some embodiments, the irradiation is with a device having a "top hat" irradiance distribution profile, such as those described in WO2018 / 080952 and US20180239074.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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".

[0174] 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.

[0175] As used herein, a "non-IL-2 blocking anti-CD25 conjugate" refers to a conjugate having a targeting molecule that binds to CD25 but does not substantially or significantly block IL-2 binding to CD25 and / or does not substantially or significantly block or interfere with IL-2 signaling. A non-IL-2 blocking anti-CD25 conjugate can have a targeting molecule that is an antibody, antigen-binding fragment or other moiety that binds to CD25 but does not substantially or significantly block IL-2 binding to CD25.

[0176] 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.

[0177] "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.

[0178] 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.

[0179] "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.

[0180] 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.

[0181] "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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] As used herein, the term "subject" refers to animals, including mammals such as humans.

[0190] 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.

[0191] 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.

[0192] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0193] VIII. Illustrative Embodiments Among the aspects provided herein are the following: 1. A method for the treatment of a pulmonary artery disease comprising administering to a patient a therapeutically effective amount of a therapeutic agent comprising administering to said patient an antibody or antigen-binding fragment that specifically binds to CD25 without substantially blocking or interfering with IL-2 signaling, and a Si-phthalocyanine dye; Activated by irradiation with wavelengths at or about 600 nm to at or about 850 nm, causing cell death; Conjugate. 2. The conjugate of embodiment 1, wherein the activated conjugate does not substantially block or interfere with IL-2 signaling. 3. The conjugate of embodiment 1 or 2, wherein the Si-phthalocyanine dye is IR700. 4. The Si-phthalocyanine dye has the formula (I): 3. The conjugate of embodiment 1 or 2, having the structure of TIFF2024505556000023.tif71128, or a salt, stereoisomer, or tautomer thereof. 5. The conjugate of any of embodiments 1-4, wherein the activated conjugate causes tumor inhibition or killing with a higher level, activity or potency than the unconjugated antibody. 6. The antibody or antigen-binding fragment has a heavy chain variable (V H ) region and the light chain variable (V L ) region, where: Applicable V H The region is V H The heavy chain complementarity determining region 1 (CDR-H1), the heavy chain complementarity determining region 2 (CDR-H2) and the heavy chain complementarity determining region 3 (CDR-H3) are contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:2 L including light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2) and light chain complementarity determining region 3 (CDR-L3) contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:3 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:4 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:5 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:6 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:7.H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:8 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:9 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:11 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:9 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:12. L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:10 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:11 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:10 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:12. L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:13 HCDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:16 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:13 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:17. L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:13 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:18 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:13 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:19. L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:14 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:16 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:14 HCDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:17. L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:14 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:18 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:14 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:19. L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:15 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:16 L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:15 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:17. L including CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; Applicable V H The region is V as shown in SEQ ID NO:15 HCDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:18 L comprising CDR-L1, CDR-L2 and CDR-L3 contained within the region amino acid sequence; or Applicable V H The region is V as shown in SEQ ID NO:15 H CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the V L The region is V as shown in SEQ ID NO:19. L CDR-L1, CDR-L2 and CDR-L3 are contained within the amino acid sequence of the region, The conjugate according to any one of embodiments 1 to 5. 7. The antibody or antigen-binding fragment has a heavy chain variable (V H ) region and the light chain variable (V L ) region, where: Applicable V H The V region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 20; a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO: 21; and a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO: 22; L the region comprises light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO:23; light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO:24; and light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO:25; Applicable V H The region comprises CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 26; CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 27; and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 28, Lthe region comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:29; CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:24; and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:30; Applicable V H The region comprises CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 31; CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 32; and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 33, L the region comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:34; a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:35; and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:36; or Applicable V H The region comprises CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 37; CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 38; and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 39, L The region comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 40; CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 41; and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 42; The conjugate of any one of embodiments 1 to 6. 8. The antibody or antigen-binding fragment has a heavy chain variable (V H ) region and the light chain variable (V L ) region, where: Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO:1, L The region comprises the amino acid sequence shown in SEQ ID NO:2; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO:3, L The region comprises the amino acid sequence shown in SEQ ID NO:4; Applicable V HThe region comprises the amino acid sequence shown in SEQ ID NO:5, L The region comprises the amino acid sequence shown in SEQ ID NO:6; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO:7, L The region comprises the amino acid sequence shown in SEQ ID NO:8; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO:9, L The region comprises the amino acid sequence shown in SEQ ID NO:11; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO:9, L The region comprises the amino acid sequence shown in SEQ ID NO:12; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 10, L The region comprises the amino acid sequence shown in SEQ ID NO:11; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 10, L The region comprises the amino acid sequence shown in SEQ ID NO:12; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 13, L The region comprises the amino acid sequence shown in SEQ ID NO:16; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 13, L The region comprises the amino acid sequence shown in SEQ ID NO:17; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 13, L The region comprises the amino acid sequence shown in SEQ ID NO:18; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 13, LThe region comprises the amino acid sequence shown in SEQ ID NO:19; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 14, L The region comprises the amino acid sequence shown in SEQ ID NO:16; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 14, L The region comprises the amino acid sequence shown in SEQ ID NO:17; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 14, L The region comprises the amino acid sequence shown in SEQ ID NO:18; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 14, L The region comprises the amino acid sequence shown in SEQ ID NO:19; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 15, L The region comprises the amino acid sequence shown in SEQ ID NO:16; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 15, L The region comprises the amino acid sequence shown in SEQ ID NO:17; Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 15, L the region comprises the amino acid sequence set forth in SEQ ID NO:18; or Applicable V H The region comprises the amino acid sequence shown in SEQ ID NO: 15, L The region comprises the amino acid sequence shown in SEQ ID NO:19, The conjugate of any one of embodiments 1 to 7. 9. The conjugate of any of embodiments 1-8, wherein the antibody or antigen-binding fragment comprises MA251, 7G7B6, or an antigen-binding portion thereof. 10. The conjugate of any of embodiments 1-9, which exhibits one or more Fc-mediated effector functions. 11. Lacking Fc-mediated effector functions; exhibit substantially reduced Fc-mediated effector function, or does not exhibit substantial Fc-mediated effector functions; The conjugate of any one of embodiments 1 to 9. 12. The conjugate of embodiment 11, wherein the activated conjugate is capable of killing a cell in the absence of substantial Fc-mediated effector function. 13. The conjugate of any of embodiments 10-12, wherein the Fc-mediated effector function is selected from one or more of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC). 14. The conjugate of any of embodiments 1-13, which has at least two modes of action for causing cell death when activated. 15. The conjugate of embodiment 14, wherein one of said at least two modes of action is ADCC-independent. 16. The conjugate of any of embodiments 1-15, wherein the activated conjugate exhibits at least one mode of cell killing or tumor inhibition that is not present in the unconjugated antibody. 17. The conjugate of any of embodiments 1-16, comprising an IgG1 Fc region or an IgG1 isotype, an IgG2 Fc region or an IgG2 isotype, an IgG3 Fc region or an IgG3 isotype, or an IgG4 Fc region or an IgG4 isotype. 18. The conjugate of any of embodiments 1-17, wherein the antibody or antibody-binding fragment comprises an IgG1 Fc region or an IgG1 isotype. 19. The conjugate of embodiment 18, wherein the IgG1 Fc region does not exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) effector function. 20. The conjugate of any of embodiments 1-17, wherein the antibody or antibody-binding fragment comprises an IgG2 Fc region or an IgG2 isotype. 21. The conjugate of embodiment 20, wherein the IgG2 Fc region comprises a substitution that reduces or abolishes ADCC effector function. 22. The conjugate of embodiment 21, wherein the substitution is an asparagine to glutamine substitution in the Fc region at the position corresponding to 297 according to EU numbering (N297Q). 23. The conjugate of any of aspects 1 to 22, wherein the antibody or antigen-binding fragment is a human antibody or human antigen-binding fragment, a chimeric antibody or chimeric antigen-binding fragment, or a humanized antibody or humanized antigen-binding fragment. 24. The conjugate of any of embodiments 1-23, wherein the antibody or antigen-binding fragment comprises a human immunoglobulin Fc region and / or a human antibody framework region. 25. (a) administering to a subject a conjugate according to any one of embodiments 1 to 24; and (b) at a wavelength of at or about 600 nm to at or about 850 nm and at 25 J / cm to a target site within a subject; 2 Or about 25J / cm 2 From 400J / cm 2 Or about 400 J / cm 2 or at or about 2 J / cm fiber length to at or about 500 J / cm fiber length, thereby activating the conjugate. 1. A method of treating a tumor or lesion in a subject, comprising: thereby reducing or inhibiting the growth, volume or size of said tumor or lesion; The method. 26. (a) administering to a subject a conjugate comprising an antibody or antigen-binding fragment that specifically binds to CD25 without substantially blocking or interfering with IL-2 signaling and a Si-phthalocyanine dye; (b) at a wavelength of at or about 600 nm to at or about 850 nm and at 25 J / cm to a target site within a subject; 2 Or about 25J / cm 2 From 400J / cm 2Or about 400 J / cm 2 or at or about 2 J / cm fiber length to at or about 500 J / cm fiber length, thereby activating the conjugate. 1. A method of treating a tumor or lesion in a subject, comprising: thereby reducing or inhibiting the growth, volume or size of said tumor or lesion; The method. 27. The method of embodiment 25 or 26, wherein the tumor or lesion being treated, or the tumor microenvironment (TME) of the tumor or lesion being treated, contains reduced levels of immune effector cells. 28. The method of embodiment 26 or 27, wherein the Si-phthalocyanine dye is IR700 and the irradiation is performed at a wavelength of 690 nm ± 20 nm. 29. The Si-phthalocyanine dye is represented by the formula (I) TIFF2024505556000024.tif71128, or a salt, stereoisomer, or tautomer thereof, and wherein irradiation is carried out at a wavelength of 660 nm ± 50 nm. 30. The method of any of aspects 27-29, wherein the immune effector cells are selected from one or more of macrophages, natural killer (NK) cells, neutrophils, and eosinophils. 31. The method of any of aspects 25-30, wherein the target site is irradiated within about 24±4 hours after administering the conjugate. 32. The target area is 50mW / cm 2 or about 50 mW / cm 2 From 200mW / cm 2 or about 200 mW / cm 2 32. The method of any of embodiments 25 to 31, wherein the optical power is up to 100 .mu.m. 33. The method of any of embodiments 25-31, wherein the target site is irradiated with an optical power of from at or about 100 mW / cm fiber length to at or about 500 mW / cm fiber length. 34. The method of any of embodiments 25-33, wherein the target site is irradiated for a period of from at or about 120 seconds to at or about 600 seconds. 35. The method of any of aspects 25-34, wherein the tumor or lesion is resistant or non-responsive to immune checkpoint inhibitor therapy. 36. The method of any of aspects 25-34, wherein the tumor or lesion has a reduced response or is unresponsive to the unconjugated antibody. 37. The method of any of embodiments 26-36, wherein the conjugate exhibits one or more Fc-mediated effector functions. 38. The method of any of embodiments 26-36, wherein the conjugate lacks Fc-mediated effector function, exhibits substantially reduced Fc-mediated effector function, or exhibits no substantial Fc-mediated effector function. 39. The method of embodiment 38, wherein the activated conjugate is capable of killing a cell in the absence of substantial Fc-mediated effector function. 40. The method of any of aspects 37-39, wherein the Fc-mediated effector function is selected from one or more of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). 41. The method of any of embodiments 26-40, wherein the conjugate comprises an IgG1 Fc region or an IgG1 isotype, an IgG2 Fc region or an IgG2 isotype, an IgG3 Fc region or an IgG3 isotype, or an IgG4 Fc region or an IgG4 isotype. 42. The method of any of embodiments 26-41, wherein the antibody or antibody-binding fragment comprises an IgG1 Fc region or an IgG1 isotype. 43. The method of embodiment 42, wherein the IgG1 Fc region is not enhanced with respect to ADCC effector function. 44. The method of any of embodiments 26-43, wherein the antibody or antibody-binding fragment comprises an IgG2 Fc region or an IgG2 isotype. 45. The method of embodiment 44, wherein the IgG2 Fc region comprises a substitution that reduces or abolishes ADCC effector function. 46. ​​The method of embodiment 45, wherein the substitution is an asparagine to glutamine substitution in the Fc region at the position corresponding to 297 according to EU numbering (N297Q). 47. The method of any of aspects 25-46, further comprising administering immune checkpoint inhibitor therapy after administration of the conjugate. 48. The method of embodiment 47, wherein the immune checkpoint inhibitor therapy is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, or 3 weeks after administration of the conjugate. 49. The method of embodiment 47 or 48, wherein the immune checkpoint inhibitor therapy is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, or 3 weeks after radiation. 50. The method of any of aspects 47-49, wherein the immune checkpoint inhibitor therapy is administered more than once following administration of the conjugate. 51. The method of any of aspects 47-50, wherein the immune checkpoint inhibitor therapy comprises a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. 52. PD-1 inhibitors 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, TSR-042 (ANB011), or an antigen-binding fragment thereof, or any combination thereof. 53. The method of any of aspects 25-52, wherein the regulatory T cell (Treg) population in the tumor or lesion, or in the tumor microenvironment, is reduced as a result of the method. 54. The method of any of aspects 25-53, wherein the reduction or inhibition comprises one or more of: an increase in tumor volume or tumor size of less than 20%, or a reduction in tumor volume, tumor size or tumor mass, or a reduction in tumor cell number. 55. The method of any of aspects 25-54, wherein the growth, volume or size of a tumor or lesion is inhibited or reduced to a greater extent as compared to a method using a conjugate comprising an antibody or antigen-binding fragment that specifically binds to CD25 and substantially blocks or prevents IL-2 signaling. 56. A method according to any one of aspects 25 to 55, which improves survival of a subject. 57. The method of any of embodiments 25-56, wherein the subject comprises a second tumor or a secondary population of tumor cells, and the growth, volume, or size of the second tumor or secondary population of tumor cells is reduced or inhibited as a result of the method. 58. The method of embodiment 57, wherein the second tumor or secondary population of tumor cells has not been irradiated. EXAMPLES

[0194] IX. Working Example The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0195] Example 1 : Generation of IRDye 700-conjugated anti-CD25 antibodies This example describes methods for preparing conjugates containing the exemplary phthalocyanine dye IRDye 700DX (IR700) linked to the anti-CD25 antibodies PC61 (an exemplary IL-2 blocking anti-CD25 antibody) and 7D4 (an exemplary non-IL-2 blocking anti-CD25 antibody) of mouse IgG1 and IgG2a isotypes, making PC61-mIgG1-IR700, PC61-mIgG2a-IR700, 7D4-mIgG1-IR700, and 7D4-mIgG2a-IR700. SEQ ID NOs corresponding to the amino acid sequences of the variable heavy chain (VH), constant heavy chain (CH1-CH3), variable light chain (VL), and constant light chain (CL) of the antibodies are shown in Table 2.

[0196] (Table 2) TIFF2024505556000025.tif33167

[0197] PC61 or 7D4, rat monoclonal antibodies (mAbs) against mouse CD25, of isotype mIgG1 or mIgG2a, were incubated with IRDye 700DX NHS ester (IR700; LI-COR Bioscience, Lincoln, NE) (52.1–71.9 μg, [27.2–36.8 nmol], 5 mmol / L in DMSO for each 1 mg [6.8 nmol] of antibody) in 0.1 mol / L Na2HPO4 (pH 8.5) for 30–120 min at room temperature. Conjugates were prepared in batches ranging from 50–182 mg of antibody. 1 mol / L C2H5NO2 (pH 9.0) was then added to this mixture to a target of 20 mmol / L for 2–16 h at room temperature. The mixture was buffer exchanged using Amicon centrifugal spin filters (30 kD; Millipore Sigma). Protein concentration and dye-to-antibody ratio (DAR) were determined by analytical size exclusion (SEC-HPLC) by measuring absorbance at 280 nm and 690 nm. Protein concentration was measured using absorbance at 280 nm corrected for the effect of dye absorption at 690 nm. The average DAR was calculated as the ratio between the peak areas at the measured wavelengths. The average number of IR700 per mAb (PC61 or 7D4) was approximately 3.

[0198] The purity of the PC61-IR700 and 7D4-IR700 conjugates 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 using a Shodex KW-803 column (New York, NY) eluted with phosphate-buffered saline (PBS) at 1.0 mL / min for 20 min. The conjugate preparations showed strong association and contained no detectable mAb aggregates as determined by SE-HPLC.

[0199] To determine the in vitro binding properties of the IR700 conjugates, use the Indo-Gen procedure. 125 I labeling was performed. Minimal loss of mAb was observed upon IR700 conjugation. Immunoreactivity assays were performed. 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) or 125 I-7D4-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 of unconjugated unlabeled mAb).

[0200] Example 2 : Photoimmunotherapy (PIT) using a non-IL-2 blocking anti-CD25 conjugate is more effective at inhibiting tumor growth than IL-2 blocking PIT This example compares the activity of an exemplary IL-2 non-blocking anti-CD25-IR700 conjugate and an exemplary IL-2 blocking anti-CD25-IR700 conjugate, with and without light irradiation, against primary colon cancer tumors.

[0201] 1 × 10 6 CT26 mouse colon cancer cells were inoculated subcutaneously. The allograft tumors were approximately 150 mm 3When tumors had grown to a size of 100 μL (approximately 6 days after tumor implantation), mice were administered saline (100 μL; n=12; control), the IL-2 non-blocking anti-CD25 antibody 7D4-mIgG1-IR700 conjugate (7D4-IR700; 100 μg; n=24) produced as described in Example 1 above, or the IL-2 blocking anti-CD25 antibody PC61-IgG1-IR700 conjugate (PC61-IR700; 100 μg; n=24) by retro-orbital (RO) injection. Twenty-four hours after administration of the conjugate, tumors in half of the mice that had received the conjugate were dosed with 100 J / cm at 690 nm. 2 (photoimmunotherapy (PIT) group). Tumor growth for all mice was measured every 2-3 days, and tumor volume was calculated using the formula: tumor volume = (minor diameter x minor diameter) x major diameter / 2. Survival of mice was also recorded over time.

[0202] The mean tumor growth and individual tumor growth for each treatment group are shown in Figure 1 and Figure 2A-2E, respectively. In mice administered an exemplary IL-2 non-blocking anti-CD25-IR700 conjugate in combination with irradiation (7D4-IR700 PIT; filled triangles, dashed line), the mean tumor growth was substantially inhibited compared to the mean tumor growth inhibition in control mice receiving saline (open circles) or 7D4-IR700 conjugate alone without irradiation (filled triangles, solid line). Mice administered 7D4-IR700 conjugate alone without irradiation also showed reduced tumor growth compared to saline control mice. In contrast, the mean tumor growth in mice administered IL-2 blocking anti-CD25-IR700 conjugate alone (PC61-IR700 conjugate) without irradiation was indistinguishable from saline control mice (filled squares, open circles vs. solid line). Tumors in mice receiving IL-2 blocking anti-CD25-IR700 conjugate and irradiation (PC61-IR700 PIT; black squares, dashed line) showed a greater reduction in tumor growth than the IL-2 non-blocking anti-CD25 conjugate alone, but less than the reduction in tumor growth observed with IL-2 non-blocking anti-CD25 PIT. As shown in Figures 2A-2E, which plot the growth of individual tumors, 7 of 12 mice receiving IL-2 non-blocking anti-CD25 PIT (7D4-IR700 PIT) achieved complete remission (CR) (Figure 2D), while 4 of 12 mice receiving IL-2 non-blocking anti-CD25 conjugate alone or IL-2 blocking anti-CD25 PIT achieved CR (Figures 2B and 2E, respectively), 1 of 12 mice receiving IL-2 blocking anti-CD25 conjugate alone achieved CR (Figure 2C), and 0 of 12 control mice receiving saline achieved CR (Figure 2A).Consistent with these results, as shown in FIG. 3, mice receiving IL-2 non-blocking anti-CD25 PIT (7D4-IR700 PIT) showed the highest survival (closed triangles, dashed line), followed by mice receiving IL-2 blocking anti-CD25 PIT (PC61-IR700 PIT; closed squares, dashed line), IL-2 non-blocking anti-CD25 conjugate alone (without irradiation) (7D4-IR700 conjugate; closed triangles, solid line), IL-2 blocking anti-CD25 conjugate alone (PC61-IR700 conjugate; closed squares, solid line), and saline control (saline; open circles, solid line). Taken together, these results indicate that IL-2 non-blocking anti-CD25 PIT is more effective than IL-2 blocking anti-CD25 PIT and more effective than the IL-2 non-blocking anti-CD25 conjugate alone in inhibiting tumor growth and promoting survival in tumor-bearing mice.

[0203] Example 3 : Non-IL-2 blocking anti-CD25 photoimmunotherapy (PIT) and anti-PD-1 antibody synergistically inhibit tumor growth in vivo This example describes the synergistic inhibitory effect of an exemplary IL-2 non-blocking anti-CD25-IR700 photoimmunotherapy (PIT) in combination with an anti-PD-1 antibody compared to treatment with a naked (unconjugated) anti-CD25 antibody on tumor growth in an anti-PD-1 resistant mouse tumor model.

[0204] 5 × 10 5 MCA205 mouse fibrosarcoma cells were inoculated subcutaneously (day 0). The allograft tumors were approximately 150 mm 3When tumors had grown to a size of 100 nm (day 7), mice were administered saline (group 1), anti-PD-1 antibody (group 2), naked IL-2 non-blocking 7D4-mIgG2a antibody (group 3), 7D4-mIgG2a-IR700 conjugate (group 4), naked 7D4-mIgG2a antibody and anti-PD-1 antibody (group 5), or 7D4-mIgG2a-IR700 conjugate and anti-PD-1 antibody (group 6) as detailed in Table 3. Conjugates were generated as described in Example 1. Saline, antibody, and conjugate were administered via retro-orbital (RO) injection. As shown in Table 3, 24 hours after administration of the conjugate (day 8), tumors in half of the mice that received the 7D4-mIgG2a-IR700 conjugate received 200 J / cm at 690 nm. 2 On day 7, mice in groups 2, 5, and 6 were administered anti-PD-1 antibody and received repeat doses three times a week throughout the study. Tumor growth for all mice was measured every 2-3 days, and tumor volume was calculated using the formula: tumor volume = (minor diameter x minor diameter) x major diameter / 2. Survival was also recorded.

[0205] Table 3: Treatment groups TIFF2024505556000026.tif56167

[0206] The mean tumor growth and individual tumor growth for each treatment group are shown in Figures 4A-4B and 5A-5E, respectively. Figure 6 shows survival. As shown in Figure 4A and Figures 5A, 5D, 5B, and 5E, administration of saline (Figure 4A; filled circles, solid line and Figure 5A), anti-PD-1 antibody alone (Figure 4A; open circles, dashed line and Figure 5D), naked 7D4-mIgG2a antibody (Figure 4A; filled squares, solid line and Figure 5B), or naked 7D4-mIgG2a antibody and anti-PD-1 antibody (Figure 4A; open squares, dashed line and Figure 5E) was ineffective in reducing tumor growth, and no mice in any of these treatment groups achieved CR. These results were also consistent with the survival results, where only 7D4-mIgG2a antibody treatment improved survival (Figure 6; filled squares, solid line). These results indicate that MCA205 cells are resistant to anti-PD-1 and non-IL-2 blocking anti-CD25 antibody immunotherapy, either as monotherapy or in combination. In contrast, 7D4-mIgG2a-IR700 PIT substantially reduced tumor growth compared to saline control (Figure 4B; filled triangles, solid line and Figure 5C), with 3 out of 10 mice achieving CR and substantially improving survival (Figure 6; filled triangles, dashed line). The combination of 7D4-mIgG2a-IR700 PIT and anti-PD-1 inhibited tumor growth even further (Figure 4B; open triangles, dashed line and Figure 5E), with 7 out of 10 mice achieving CR and resulting in 100% survival (Figure 6; open triangles, dashed line). Taken together, these results indicate that IL-2 non-blocking anti-CD25 PIT is more effective at inhibiting tumor growth and promoting survival than naked IL-2 non-blocking anti-CD25 antibody alone. Furthermore, IL-2 non-blocking anti-CD25 PIT acted synergistically with anti-PD-1 treatment to cause greater inhibition of tumor growth compared to either monotherapy alone. IL-2 non-blocking anti-CD25 PIT in combination with anti-PD-1 treatment was more effective at inhibiting tumor growth and promoting survival than naked IL-2 non-blocking anti-CD25 antibody treatment with or without anti-PD-1 treatment. In this anti-PD-1 resistant tumor model, treatment with naked IL-2 non-blocking anti-CD25 antibody was ineffective at reducing tumor growth.Administration of anti-PD-1 antibody in addition to naked IL-2 non-blocking anti-CD25 antibody treatment had no substantial effect on inhibiting tumor growth or promoting survival in this anti-PD-1 resistant model.

[0207] Example 4 : Photoimmunotherapy (PIT) using a non-IL-2 blocking anti-CD25 conjugate is more effective in inducing systemic immune responses than IL-2 blocking PIT In this example, photoimmunotherapy (PIT) using a conjugate that uses an IL-2 blocking anti-CD25 antibody as the targeting molecule is compared to PIT using a conjugate that uses a non-IL-2 blocking anti-CD25 antibody as the targeting molecule in terms of efficacy on directly treated tumors and on non-irradiated distant tumors (abscopal effect).

[0208] Immunocompetent BALB / c mice were injected with 1 × 10 6 MCA-205 murine fibrosarcoma cells were inoculated subcutaneously. Bilateral allograft tumors were approximately 150 mm in volume. 3 When tumors had grown to 100 mm Hb (5 days after tumor cell inoculation), mice were administered saline (100 μL), an exemplary IL-2 blocking anti-CD25 antibody conjugated to IR700 (PC61-mIgG1-IR700; 100 μg) or an exemplary IL-2 non-blocking anti-CD25 antibody conjugated to IR700 (7D4-mIgG1-IR700; 100 μg) produced as described in Example 1 above, intravenously. Twenty-four hours after administration of the conjugates, tumors in the right flank of half of the animals that received PC61-IR700 or 7D4-IR700 were dosed with 200 J / cm at 690 nm. 2 The tumor was irradiated at a dose of 0.01 mg / kg / day, while the tumor on the left flank was shielded from irradiation. The growth of the irradiated tumor (target tumor) and the non-irradiated tumor (distal tumor) was monitored over time, and tumor volume was calculated using the formula: tumor volume = (minor diameter x major diameter) x height / 2.

[0209] As shown in Figures 7A and 7B, tumors in saline-treated animals showed rapid growth. Tumor growth in mice treated with IL-2-blocking PC61-IR700 conjugate alone (without irradiation) was indistinguishable from that observed for saline control animals (Figure 7A; open circles), while tumor growth in animals receiving PC61-IR700 PIT showed substantial inhibition of tumor growth of irradiated tumors (Figure 7A; closed circles). Tumor growth in mice treated with IL-2-nonblocking 7D4-IR700 conjugate alone (without irradiation) showed slightly reduced tumor growth compared to saline controls (Figure 7B; open squares). Tumors treated with 7D4-IR700 PIT showed nearly complete inhibition of tumor growth of irradiated tumors after treatment (Figure 7B; closed squares).

[0210] Non-irradiated tumors distal to the irradiated site (distal tumors) in mice treated with saline (Figures 8A and 8B; open triangles) and mice treated with IL-2 blocking PC61-IR700 conjugate alone (Figure 8A; open circles) showed continued tumor growth. Non-irradiated distal tumors in mice treated with PC61 PIT (Figure 8A; closed circles) or IL-2 non-blocking 7D4 conjugate alone (no irradiation) (Figure 8B; open squares) showed a small reduction in tumor growth compared to saline controls. Non-irradiated distal tumors in mice treated with IL-2 non-blocking 7D4-IR700 conjugate PIT showed approximately 50% reduction in tumor growth compared to saline controls (Figure 8B; closed squares). The results indicate that treatment with anti-CD25 PIT, particularly IL-2 non-blocking anti-CD25 PIT, is effective in reducing tumor growth of irradiated and non-irradiated distal tumor lesions. Based on the abscopal effect (i.e., tumor growth inhibition of distant non-irradiated lesions), these effects indicate that IL-2 non-blocking anti-CD25 PIT is more effective than IL-2 blocking anti-CD25 PIT in inducing a systemic immune response.

[0211] Furthermore, the results show that IL-2 non-blocking anti-CD25 PIT treatment is more effective at inhibiting tumor growth in irradiated target lesions and non-irradiated distant lesions than conjugate treatment alone without irradiation.

[0212] Example 5 : Antibody-dependent cellular cytotoxicity (ADCC) of non-IL-2 blocking anti-CD25 antibodies and corresponding non-IL-2 blocking anti-CD25 conjugates In this example, the antibody-dependent cellular cytotoxicity (ADCC) activity of an exemplary naked (unconjugated) IL-2 non-blocking anti-CD25 antibody and a conjugate comprising an IL-2 non-blocking anti-CD25 antibody and the phthalocyanine dye IR700 was evaluated.

[0213] The ADCC activity for naked IL-2 non-blocking anti-CD25 antibody (7D4-mIgG2a) and IL-2 non-blocking anti-CD25-IR700 conjugate (7D4-mIgG2a-IR700) produced as described in Example 1 above was measured using the ADCC Reporter Bioassay Kit (Promega) essentially as described in the vendor's protocol. Briefly, target HT-2 cells were plated in 96-well white-walled plates at a density of 25,000 cells / well. The next day, the medium was replaced with 25 μL of ADCC Assay Buffer, followed by 25 μL of 9 serially diluted concentrations of 7D4-mIgG2a antibody or 7D4-mIgG2a-IR700 conjugate at 3-fold higher concentrations than the final concentration. Serial dilutions were prepared starting at 6 μg / mL (1×=2 μg / mL). Induction of the ADCC response was initiated by adding 25 μL (75,000) effector cells to achieve an effector to target cell ratio of 3:1. The mixture was incubated at 37° C. for 6 hours. To quantify the ADCC response, the plates were brought to room temperature and 75 μL of Bio-Glo Luciferase Assay Reagent was added to the wells containing the cells and to three surrounding cell-free wells for background determination. Luminescence was read on a Tecan Spark multiplate reader (Tecan Life Sciences) after 20 minutes of incubation at ambient temperature. To analyze the data, background was subtracted from the cell-free wells and the data was fitted with a four-parameter nonlinear curve fit algorithm (GraphPad Prism software).

[0214] The results are shown in Figure 9. Both the naked antibody and the conjugate showed a dose-dependent increase in ADCC activity. Naked 7D4-mIgG2a had an EC 50 (R 2 EC of 23.85 compared to the 7D4-mIgG2a-IR700 conjugate, which had an EC of 0.9949. 50 (R 2 : 0.9896), with a higher saturation point exhibited by the naked 7D4-mIgG2a antibody compared to the 7D4-mIgG2a-IR700 conjugate. These results indicate that the IR700 conjugate exhibits weaker ADCC activity compared to the naked antibody.

[0215] Example 6 : IL-2 non-blocking anti-CD25 photoimmunotherapy (PIT) does not require antibody-dependent cell-mediated cytotoxicity (ADCC) / antibody-dependent cellular phagocytosis (ADCP) for antitumor activity This example evaluates the effect of antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) on the anti-tumor activity of treatment with an exemplary non-IL-2 blocking anti-CD25 antibody and non-IL-2 blocking anti-CD25 photoimmunotherapy (PIT).

[0216] The antitumor activity of mIgG2a backbone ADCC / ADCP-competent and ADCC / ADCP-null IL-2 non-blocking anti-CD25 antibodies and mIgG1 backbone IL-2 non-blocking anti-CD25 conjugates with and without irradiation was compared using mouse primary colon cancer tumors. The mIgG2a isotype has a higher binding affinity to mouse Fc gamma receptors than the mIgG1 isotype and exhibits a higher level of ADCC / ADCP activity than the mIgG1 isotype (Stewart et al., (2014) J. Immunotherapy Cancer 2, 29). An asparagine to glutamine substitution in the Fc region at position corresponding to 297 according to EU numbering (N297Q) abrogates the interaction of the Fc region with the Fc gamma receptor and nullifies the ADCC / ADCP activity of the IgG2a isotype.

[0217] 1 × 10 6CT26 mouse colon cancer cells were inoculated subcutaneously. The allograft tumors were approximately 150 mm 3 When tumors had grown to a size of 10 μL (approximately 6 days after tumor implantation), mice were administered saline (100 μL; n=12; control) or the IL-2 non-blocking anti-CD25 antibody 7D4-mIgG1-IR700 conjugate (7D4-IR700; 100 μg; n=24) produced as described in Example 1 above by retro-orbital (RO) injection; or naked (unconjugated) IL-2 non-blocking anti-CD25 antibody 7D4-mIgG2a (10 mg / kg; n=11) or 7D4-mIgG2a-ADCC / ADCP null (7D4-mIgG2a-N297Q; 10 mg / kg; n=12) by intraperitoneal (IP) administration. Twenty-four hours after administration of the antibody or conjugate, tumors in half of the mice that received the conjugate were dosed with 100 J / cm at 690 nm. 2 (photoimmunotherapy (PIT) group). Tumor growth for all mice was measured every 2-3 days, and tumor volume was calculated using the formula: tumor volume = (minor diameter x minor diameter) x major diameter / 2. Survival of mice was also recorded over time.

[0218] The mean and individual tumor growth for each treatment group is shown in Figure 10 and Figures 11A-11E, respectively. Mice administered naked 7D4-mIgG2a antibody showed substantial tumor growth inhibition compared to saline control mice (Figure 10; open circles versus closed squares), with 10 of 11 mice achieving complete remission (Figure 11D). This effect was almost completely abrogated in mice administered 7D4-mIgG2a-ADCC / ADCP null (same naked antibody in the context of an ADCC null mutant mIgG2a backbone; Figure 10; open squares and Figure 11B), indicating that the tumor growth inhibition observed with 7D4-mIgG2a was dependent on ADCC activity.

[0219] Mice administered 7D4-mIgG1-IR700 conjugate without irradiation showed similar mean tumor growth as unconjugated 7D4-mIgG2a antibody, which lacks ADCC / ADCP activity (Figure 10; filled triangles, solid line (conjugate) vs. open squares, solid line (ADCC / ADCP null antibody)). However, 4 of 12 mice administered 7D4-mIgG1-IR700 conjugate achieved complete remission (Figure 11C), compared to 1 of 12 mice that received 7D4-mIgG2a-N297Q (ADCC null) (Figure 11B). This result further supports the idea that tumor growth inhibition is dependent on ADCC / ADCP activity, since mIgG1 antibodies still exhibit low levels of ADCC / ADCP activity. However, mice administered 7D4-mIgG1-IR700 followed by irradiation (7D4-mIgG1-IR700+PIT) showed substantially greater tumor growth inhibition than the conjugate without irradiation (Figure 10 filled triangles, dashed line), with 7 of 12 mice achieving CR (Figure 11E).

[0220] As shown in Figure 12, survival curves confirmed the observed tumor growth inhibition. Consistent with the tumor growth inhibition results, mice receiving IL-2 non-blocking anti-CD25 mIgG2a antibody with increased ADCC activity showed the greatest survival (7D4-mIgG2a; closed squares), followed by IL-2 non-blocking anti-CD25 PIT (7D4-mIgG1-IR700 PIT; closed triangles, dashed line), IL-2 non-blocking anti-CD25 conjugate alone (without irradiation) (7D4-mIgG1-IR700; closed triangles, solid line), IL-2 blocking anti-CD25-IgG2a-ADCC / ADCP null antibody (7D4-mIgG2a-N297Q; open squares), and saline control (saline; open circles).

[0221] These results indicate that IL-2 non-blocking anti-CD25 PIT can effectively suppress tumor growth even in the presence of limited ADCC / ADCP activity, and thus, unlike unconjugated IL-2 non-blocking anti-CD25 antibodies, ADCC / ADCP activity is not required for the efficacy of IL-2 non-blocking anti-CD25 PIT.

[0222] Example 7 : Effect of IL-2 non-blocking anti-CD25 antibody and IL-2 non-blocking anti-CD25 photoimmunotherapy (PIT) on cold tumors with and without anti-PD-1 antibody treatment This example describes the effect of naked (unconjugated) IL-2 non-blocking anti-CD25 antibodies and IL-2 non-blocking anti-CD25 PIT on immune "cold" tumors, i.e., tumors characterized by low immune reactivity (low and exhausted tumor infiltrating lymphocytes (TILs), poor tumor antigen load, and an immunosuppressive microenvironment). Cold tumors are characteristically poorly responsive to immune checkpoint inhibitor therapy.

[0223] BALB / c mice (16–18 g) were injected with 1 × 10 5 4T1 cells / mouse were inoculated subcutaneously. Allograft tumors grew to approximately 140 mm 3 When tumors had grown to a volume of 100 μL (6 days after tumor cell inoculation), mice were administered saline (100 μL via intraperitoneal (IP) injection), 7D4-mIgG2a (200 μg via IP injection), or 7D4-mIgG2a-IR700 (100 μg via retroorbital injection). Half of each treatment group was also treated with the anti-PD-1 antibody RMP1-14 (10 mg / kg) three times per week (days 6, 9, 13, etc.). Twenty-four hours after administration of the conjugate, tumors in the group receiving 7D4-mIgG2a-IR700 were dosed with 150 J / cm at 690 nm. 2 Tumor growth (Figure 13) and survival (Figure 14) were measured over time.

[0224] 7D4-mIgG2a PIT, alone (open triangles) or in combination with anti-PD-1 (closed triangles), substantially inhibited the growth of "cold" tumors compared to saline (open circles) or anti-PD-1 monotherapy (closed circles) (Figure 13). 7D4-mIgG2a antibody, alone (open squares) or in combination with anti-PD-1 (closed squares), had virtually no inhibitory effect on the growth of "cold" tumors (Figure 13). Survival curves were consistent with the anti-tumor effect of each treatment (Figure 14). These data support the finding that IL-2 non-blocking anti-CD25 PIT effectively inhibited the growth of cold tumors, whereas IL-2 non-blocking antibody alone had minimal effect on the growth of cold tumors, with or without PD-1 treatment.

[0225] 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 description 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.

[0226] array TIFF2024505556000027.tif237170TIFF2024505556000028.tif244170TIFF2024505556000029.tif178170

Claims

A medicament comprising a conjugate comprising a Si-phthalocyanine dye for use in a method of treating a tumor or lesion in a subject, wherein the method comprises (a) administering the conjugate to the subject, and (b) irradiating a target site within the subject with a wavelength from 600 nm or about 600 nm to 850 nm or about 850 nm, and at a dose from 25 J / cm 2 or about 25 J / cm 2 to 400 J / cm 2 or about 400 J / cm 2 up to, or at a dose from 2 J / fiber length cm or about 2 J / fiber length cm to 500 J / fiber length cm or about 500 J / fiber length cm, thereby activating the conjugate comprises whereby the growth, volume or dimensions of the tumor or lesion are reduced or inhibited, said medicament. A medicament comprising an antibody or antigen-binding fragment that specifically binds to CD25 without substantially blocking or interfering with IL-2 signaling and a conjugate comprising a Si-phthalocyanine dye for use in a method of treating a tumor or lesion in a subject, wherein the method comprises a) administering the conjugate to the subject, (b) irradiating a target site within the subject with a wavelength from 600 nm or about 600 nm to 850 nm or about 850 nm, and at a dose from 25 J / cm 2 or about 25 J / cm 2 to 400 J / cm 2 or about 400 J / cm 2 up to, or at a dose from 2 J / fiber length cm or about 2 J / fiber length cm to 500 J / fiber length cm or about 500 J / fiber length cm, thereby activating the conjugate comprising a method of treating a tumor or lesion in a subject, whereby the growth, volume or dimensions of the tumor or lesion are reduced or inhibited, said medicament. The medicament according to claim 1, wherein the conjugate, when activated, does not substantially block or interfere with IL-2 signaling.

4. The Si-phthalocyanine dye comprises a structure of formula (X): or a salt, ionic form, stereoisomer, or tautomer thereof, wherein X is ; Y is ; R1 and R2 are each independently optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, or optionally substituted heteroaralkyl; R3, R4 or R5 is selected from substituent (a) or substituent (b), wherein a) R3 is hydrogen, -L3-H, -L3-A, or -L3-Z; R4 is -L4-H, -(NH)m-L4-A, -(NH)m-L4-Z, -(O)m-L4-A or -(O)m-L4-Z; R5 is -L5-H or -L5-A; and b) R3 is -L3-H, or -L3-A; R 4 is -L 4 -H, -(NH) m -L 4 -A, or -(O) m -L 4 -A; wherein R 3 and R 4 are connected by a bond to form a heterocyclyl substituted with -L 4 -A; and R 5 is -L 5 -H or -L 5 -A; provided that at least one of R 3, R 4 and R 5 is a group containing A; A is a reactive group capable of forming a covalent bond with a thiol, hydroxyl, carboxyl or amino group of the second moiety, or a protected form or a reactive form thereof; R 6 and R 7 are each independently optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl or optionally substituted heteroaralkyl; R 8, R 9 or R 10 is selected from substituent (a) or substituent (b), wherein (a) R 8 is hydrogen, -L 8 -H or -L 8 -Z; R 9 is -L 9 -H, -(NH) n -L 9 -Z or -(O) n -L 9 -Z; R 10 is -L 10 -Z; and (b) R 8 and R 9 are connected by a bond to form a heterocyclyl substituted with -L 9 -Z, and R 10 is -L 10 -H or -L 10 -Z; provided that at least one of R 8, R 9 and R 10 is a group containing Z; Z is a water-soluble group optionally substituted with A or L'-A; L 1 and L 2 are each independently optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted cycloalkyl, or optionally substituted heterocyclyl; L 3, L 4, L 5, L 8, L 9 and L 10 are each independently optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted cycloalkylene, optionally substituted heterocycle, optionally substituted arylene, optionally substituted aralkylene, optionally substituted heteroaralkylene, or optionally substituted heteroarylene, wherein the carbon atoms of the alkylene, heteroalkylene, alkenylene, heteroalkenylene, cycloalkylene, heterocycle, arylene, aralkylene, heteroaralkylene, or optionally substituted heteroarylene may be further substituted with Z, and each nitrogen atom of the heteroalkylene or heteroalkenylene may be substituted with 1 or 2 L'-Z; L' is each independently optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted cycloalkylene, optionally substituted heterocycle, optionally substituted arylene, optionally substituted aralkylene, optionally substituted heteroaralkylene, or optionally substituted heteroarylene; a is 0 or 1; b is 0 or 1; c is 0 or 1; d is 0 or 1; m is 0 or 1; n is 0 or 1; provided that when b is 1, a is 0; when d is 1, c is 0; when m is 1, b is 1; and when n is 1, c is 1, The medicament according to any one of claims 1 to 3, comprising.

5. The Si-phthalocyanine dye comprises the formula (I) , or a salt, stereoisomer, or tautomer thereof, and the irradiation is carried out at a wavelength of 660 nm ± 50 nm. The medicament according to any one of claims 1 to 4.

6. The medicament according to any one of 1 to 5, wherein the irradiation is carried out at a wavelength of 690 nm ± 20 nm.

7. The medicament according to any one of 1 to 6, wherein the Si-phthalocyanine dye is IR700.

8. The tumor or lesion is resistant or non-responsive to immune checkpoint inhibitor therapy. The medicament according to any one of claims 1 to 7.

9. The pharmaceutical according to any one of claims 1 to 8, wherein the conjugate exhibits one or more Fc-mediated effector functions.

10. The pharmaceutical according to any one of claims 1 to 9, wherein the conjugate lacks an Fc-mediated effector function, exhibits a substantially reduced Fc-mediated effector function, or does not exhibit a substantial Fc-mediated effector function.

11. The pharmaceutical according to any one of claims 1 to 10, wherein the activated conjugate is capable of killing cells in the absence of a substantial Fc-mediated effector function.

12. The pharmaceutical according to any one of claims 9 to 11, wherein the Fc-mediated effector function is selected from one or more of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

13. The pharmaceutical according to any one of claims 1 to 12, wherein the conjugate, when activated, has at least two modes of action for causing cell death.

14. The pharmaceutical according to claim 13, wherein one of the at least two modes of action is ADCC-independent.

15. The pharmaceutical according to any one of claims 1 to 14, wherein the conjugate, when activated, exhibits at least one mode of cell death or tumor inhibition that does not exist in the unconjugated antibody.

16. The pharmaceutical according to any one of claims 1 to 15, wherein the conjugate comprises an IgG1 Fc region or IgG1 isotype, an IgG2 Fc region or IgG2 isotype, an IgG3 Fc region or IgG3 isotype, or an IgG4 Fc region or IgG4 isotype.

17. The pharmaceutical according to any one of claims 1 to 16, wherein the antibody or antibody-binding fragment comprises an IgG1 Fc region or IgG1 isotype.

18. The pharmaceutical according to claim 17, wherein the IgG1 Fc region is not enhanced with respect to the ADCC effector function.

19. The pharmaceutical according to any one of claims 1 to 18, wherein the antibody or antibody-binding fragment comprises an IgG2 Fc region or IgG2 isotype.

20. The pharmaceutical according to claim 19, wherein the IgG2 Fc region comprises substitutions that reduce or suppress the ADCC effector function.

21. The medicament according to claim 20, wherein the substitution is a substitution from asparagine to glutamine (N297Q) in the Fc region at a position corresponding to 297 by EU numbering.

22. The medicament according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment is a human antibody or human antigen-binding fragment, a chimeric antibody or chimeric antigen-binding fragment, or a humanized antibody or humanized antigen-binding fragment.

23. The medicament according to any one of claims 1 to 22, wherein the antibody or antigen-binding fragment comprises the Fc region of a human immunoglobulin and / or a human antibody framework region.

24. The medicament according to any one of claims 1 to 23, further comprising the step of administering an immune checkpoint inhibitor therapy after administration of the conjugate.

25. The medicament according to any one of claims 1 to 24, wherein the tumor or lesion to be treated, or the tumor microenvironment (TME) of the tumor or lesion to be treated, contains a reduced level of immune effector cells.

26. The medicament according to claim 25, wherein the immune effector cells are selected from one or more of macrophages, natural killer (NK) cells, neutrophils, and eosinophils.

27. The medicament according to claim 25 or 26, wherein the reduction or inhibition comprises one or more of an increase in tumor volume or tumor dimension of less than 20%, a reduction in tumor volume, tumor dimension, or tumor mass, or a reduction in the number of tumor cells.

28. The medicament according to any one of claims 1 to 27, wherein the subject comprises a second tumor or a secondary population of tumor cells, the growth, volume, or dimension of the second tumor or secondary population of tumor cells is reduced or inhibited as a result of the method, and the second tumor or secondary population of tumor cells has not been irradiated.

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