CD25-specific antibodies and uses thereof

Abstract

The monoclonal antibody specifically binds to CD25, and the conjugate of anti-CD25 antibody are described.The CD25-specific monoclonal antibody and its conjugate do not block the binding of IL-2 to CD25, and induce little or no antibody-dependent cell-mediated cytotoxicity (ADCC).The anti-CD25 antibody and conjugate can be used, for example, to target photoimmunotherapy to T regulatory (Treg) cells in tumor beds to enhance local host immune response against tumors.

Description

[Technical Field] 【0001】 CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 357,215, filed June 30, 2022, which is incorporated herein by reference in its entirety. 【0002】 Field The present disclosure relates to monoclonal antibodies that specifically bind to CD25 (IL-2Rα) but do not significantly block the binding of interleukin-2 (IL-2) to CD25 and induce little or no antibody-dependent cellular cytotoxicity (ADCC). The present disclosure further relates to the use of CD25-specific antibodies to target CD25-expressing T regulatory (Treg) cells, for example, in tumor beds. 【0003】 Government support approval This invention was made with government support under ZIA BC 010656 awarded by the National Institutes of Health. The government has certain rights in this invention. 【0004】 Incorporation of electronic sequence listings The Electronic Sequence Listing provided herewith as an XML file (245,918 bytes) named 4239-107471-02.xml, created on June 20, 2023, is incorporated herein by reference in its entirety. [Background technology] 【0005】 background Near-infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that uses targeted monoclonal antibody-photoabsorber conjugates. After antibody localization of the antibody-photoabsorber conjugate to tumor cell surface antigens, NIR light is used to induce highly selective cell lysis. NIR-PIT induces rapid necrotic cell death, resulting in innate immune ligands that activate dendritic cells (DCs), consistent with immunogenic cell death (ICD). A description of how NIR-PIT kills tumor cells is described in Sato et al. (ACS Cent. Sci. 4:1559-69, 2018). Briefly, after binding of the antibody-photoabsorber conjugate to its target, activation with NIR light causes a physical change in the shape of the antibody-antigen complex, inducing physical stress within the cell membrane that results in an increase in water flow across the membrane, ultimately resulting in cell burst and necrotic cell death. [Prior art documents] [Non-patent literature] 【0006】 [Non-Patent Document 1] Sato et al. (ACS Cent. Sci. 4:1559-69, 2018) Summary of the Invention [Means for solving the problem] 【0007】 CD25 is a type I transmembrane protein present on activated T cells, activated B cells, some thymocytes, myeloid precursors, and oligodendrocytes. T regulatory (Treg) cells, which also express CD25, play an important role in immunosuppression by allowing tumors to escape immune surveillance. While Treg cell depletion can result in tumor regression, systemic depletion of Treg cells can also induce autoimmune adverse events (Okada et al., Bioconjugate Chem 30(10):2624-2633, 2019). Therefore, there remains a need for a method for targeted depletion of CD25-positive Treg cells, for example, in tumor beds, without negative systemic side effects. overview Disclosed herein are a group of humanized anti-CD25 monoclonal antibodies that specifically bind to CD25 (interleukin-2 receptor alpha chain, also known as IL-2Rα). The disclosed antibodies do not significantly block IL-2 binding to CD25 and induce little or no ADCC. In some examples, the disclosed CD25 antibodies have sub-nM affinity for CD25. The anti-CD25 antibodies can be used, for example, to target photoimmunotherapy to Treg cells in the tumor bed, thereby enhancing the host immune response to the tumor. Thus, in some examples, the disclosed CD25 antibodies can be conjugated to IR700 (CD25 antibody-IR700 conjugate) and used to treat tumors, e.g., immunogenic tumors. In some examples, the treatment is localized, not systemic. 【0008】 Provided herein are monoclonal antibodies that specifically bind to CD25. The disclosed antibodies comprise at least a variable heavy (VH) domain and a variable light (VL) domain. In some embodiments, the complementarity-determining region 1 (CDR), CDR2, and CDR3 sequences of the VH domain comprise SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively, and the CDR1, CDR2, and CDR3 sequences of the VL domain comprise SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively. In some examples, the CDR sequences of the VH and VL domains are those of antibody 14564, 14569, or 14572. In some embodiments, the monoclonal antibody further comprises a constant region, e.g., a heavy chain constant region and / or a light chain constant region. In some embodiments, the antibody does not significantly inhibit IL-2 binding to CD25 and / or induces little or no ADCC. 【0009】 Also provided are conjugates comprising a CD25-specific monoclonal antibody disclosed herein and a photon absorber (referred to herein as a "CD25 antibody-photon absorber conjugate" or "CD25 antibody-IR700 conjugate"), an effector molecule, or a detectable label. In some embodiments, the photon absorber is IR700. 【0010】 Compositions comprising a pharmaceutically acceptable carrier and the disclosed antibodies or conjugates are also provided. 【0011】 Further provided are nucleic acid molecules encoding the CD25-specific monoclonal antibodies disclosed herein, or encoding the VH or VL domains thereof. Also provided are vectors comprising the disclosed nucleic acid molecules, and isolated cells comprising the vectors or nucleic acid molecules disclosed herein. 【0012】 Kits containing the monoclonal antibodies, conjugates, compositions, nucleic acid molecules, vectors, or isolated cells disclosed herein are also provided. In some embodiments, the kits further include buffers, cell culture media, transfection reagent(s), one or more checkpoint inhibitors, one or more immunotherapies, one or more additional anti-cancer reagents (e.g., chemotherapeutic agents or biologics), and / or instructional materials. In one example, the kit further includes a tumor-specific antibody-IR700 conjugate (e.g., cetuximab-IR700, or any other of those provided in Table 1). 【0013】 Also provided are methods of treating cancer in a subject. In some aspects, the methods include administering to the subject a therapeutically effective amount of a CD25 antibody-IR700 conjugate (wherein the CD25 antibody is disclosed herein), followed by administration of a therapeutically effective amount of a CD25 antibody-IR700 conjugate at a wavelength of 660-740 nm and at least 1 J / cm. 2 Or at least 4J / cm 2 In some examples, the method further includes administering a second monoclonal antibody conjugated to IR700, wherein the second monoclonal antibody specifically binds to a tumor antigen (see, e.g., Table 1) expressed by cells of the cancer. 【0014】 The above and other features of the present disclosure will become more apparent from the following detailed description of several embodiments that proceeds with reference to the accompanying drawings. [Brief explanation of the drawings] 【0015】 [Figure 1] Figure 1 is a series of graphs showing the binding of antibodies 14564, 14569, and 14572 to cynomolgus monkey CD25 by surface plasmon resonance. The parental antibody VH2+VL4 was used as a control. Antibodies 14564, 14569, and 14572 bound to CD25 with high affinity. 【0016】 [Figure 2] Figure 2 is a graph showing the binding of antibodies 14564, 14569, and 14572 to CD25-expressing KIT-225 cells (a human T cell line) by FACS analysis. The anti-CD25 antibody basiliximab was used as a positive control. The binding affinities of antibodies 14564, 14569, and 14572 to CD25 were similar to that of basiliximab. 【0017】 [Figure 3] Figure 3 is a graph showing the binding affinity of antibodies 14564, 14569, and 14572 to CD25-expressing KIT-225 cells by FACS analysis. The anti-CD25 antibody basiliximab was used as a positive control. The calculated effective concentration 50 (EC50) of each antibody is also shown. 【0018】 [Figure 4-1] 4A-4B are graphs showing the results of a cytotoxicity assay to determine whether antibodies 14564, 14569, and 14572 induce antibody-dependent cellular cytotoxicity (ADCC) of CD25-expressing cells. Basiliximab was used as a positive control. Only basiliximab induced ADCC of target cells. [Figure 4-2] Same as above. 【0019】 [Figure 5-1]Figures 5A-5C show the results of a study to evaluate the targeting of photoimmunotherapy (PIT) to CD25-expressing cells. (Figure 5A) Binding of anti-hCD25-IR700 to KIT-225 cells. KIT-225 cells (0.5 x 10) were incubated with 1 µg of anti-hCD25 antibody 14564 conjugated to IR700 (anti-hCD25-IR700). For the blocking control ("blocking + anti-hCD25-IR700"), cells were incubated with 10 µg of unconjugated antibody prior to anti-hCD25-IR700 incubation. Cells were stained with a viability dye and analyzed by fluorescence-activated cell sorting (FACS). Unstained cells were used as a negative control. (Figure 5B) Representative flow cytometry plot showing live / dead staining after PIT (0 J = no NIR light; 5 J = irradiation with NIR light at 5 J / cm; 20 J = irradiation with NIR light at 20 J / cm). (Figure 5C) Cell viability after hCD25-targeted PIT. KIT-225 cells (0.5 × 10) were incubated with 1 μg of anti-hCD25-IR700 conjugate and 0, 5, 20, or 50 J / cm of NIR light was applied at 150 mW / cm. Cells were then stained with a viability dye and analyzed by FACS. Results are shown as the mean + SEM of five replicates. [Figure 5-2] Same as above. 【0020】 [Figure 6] Anti-CD25 antibodies do not block IL-2 binding to CD25. KIT-225 cells were starved for 5 days in IL-2 before performing proliferation assays. Fifty thousand starved cells in culture medium were preincubated with 0, 5, or 10 μg / ml of 14564, 14569, 14572, or parental anti-CD25 antibodies for 30 minutes at 37°C, and then cultured with or without 50 pM human IL-2 for 1.5 days. Cells cultured without antibody and with or without IL-2 were included as controls. After pulsing with 3H-thymidine (37 kBq), cells were harvested 4 hours later, and incorporated radioactivity was measured using a β-counter. The results show that antibodies 14564, 14569, and 14572 do not inhibit IL-2-induced proliferation of KIT-225. 【0021】 [Figure 7-1] hCD25-targeted PIT kills human Treg cells. Splenocytes obtained from MDA-MD231 tumor-bearing hCD34-NSG humanized mice were incubated with 5 μg of IR700-conjugated anti-CD25 antibody 14564 and then irradiated with 50 J / cm2 of NIR light at 150 mW / cm2. Splenocytes cultured in the absence of antibody-IR700 conjugate and not irradiated were used as a control. The percentage of CD25+ Treg cells was assessed by flow cytometry. (Figure 7A) Representative dot plots showing CD25+ Treg cells for control (top) and CD25 PIT (bottom) splenocytes. (Figure 7B) Graph showing the percentage of CD25+ Treg cells present in control and CD25 PIT splenocyte samples (n=5). (Figure 7C) Gating strategy. [Figure 7-2] Same as above. [Figure 7-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION 【0022】 Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and single-letter codes for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the represented strand. In the accompanying sequence listing: 【0023】 SEQ ID NOs: 1 to 42 are the amino acid sequences of the VH domain and VL domain of anti-CD25 monoclonal antibodies. 【0024】 SEQ ID NOs: 43 to 48 are consensus amino acid sequences of anti-CD25 monoclonal antibody CDRs. 【0025】 SEQ ID NOs: 49-56 are the amino acid sequences of exemplary anti-CD25 monoclonal antibody CDRs. 【0026】 SEQ ID NO:57 is the amino acid sequence of an exemplary leader sequence. 【0027】 SEQ ID NO: 58 is the amino acid sequence of a modified human IgG4 heavy chain constant region. 【0028】 SEQ ID NO: 59 is the amino acid sequence of the human Ig kappa light chain constant region. 【0029】 SEQ ID NOs: 60 to 101 are the amino acid sequences of the heavy and light chains of anti-CD25 antibodies. 【0030】 SEQ ID NOs: 102 to 143 are nucleic acid sequences encoding the VH and VL domains of anti-CD25. 【0031】 SEQ ID NOs: 144-145 are nucleic acid sequences of exemplary leader sequences. 【0032】 SEQ ID NO: 146 is the nucleic acid sequence encoding a modified human IgG4 heavy chain constant region. 【0033】 SEQ ID NO: 147 is the nucleic acid sequence encoding the human Igκ light chain constant region. 【0034】 SEQ ID NOs: 148 to 189 are nucleic acid sequences encoding the heavy and light chains of anti-CD25 monoclonal antibodies. 【0035】 Detailed Description I. Introduction Therapeutic monoclonal antibodies (mAbs) are typically injected systemically to block specific biological functions (e.g., by binding to the ligand-binding site of a specific receptor) or to attract immune cells to targets, resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby killing antibody-bound cells. While there has been some success with these strategies, pharmacological doses of mAbs can also cause unexpected and unwanted side effects. This disclosure describes the development of a humanized anti-CD25 antibody that lacks significant biological or cytotoxic properties and is used as a localized delivery platform (as opposed to a systemic therapy). 【0036】 Two anti-CD25 antibodies developed for systemic therapy have been previously commercialized: daclizimab and basiliximab. Both antibodies block the IL-2α receptor and thus activate the effector CD8 + These two antibodies interfere with T cell maturation. These two antibodies were developed for use as immunosuppressants for autoimmune diseases, such as multiple sclerosis (MS) in the case of daclizumab and organ transplant rejection in the case of basiliximab. However, because daclizumab and basiliximab also block the proliferation of Treg cells, a potential side effect is autoimmune syndrome. In fact, daclizumab was withdrawn from the market in 2018 due to complications related to brain inflammation. Basiliximab continues to be used as induction therapy to reduce the immune response to organ transplants. 【0037】 Photoimmunotherapy (PIT) is a cancer treatment that relies on a completely different principle from surgery, drug therapy (including immunotherapy), or radiation. A specific infrared dye (e.g., IR700) is conjugated to a target-specific antibody, thereby forming an antibody-photoabsorbent conjugate (APC). When injected systemically, the APC binds to target receptors on tumor cell membranes, a process that typically takes about 24 hours in humans. Near-infrared (NIR) light is then applied to the tumor. This causes photochemical changes in the APC, resulting in damage to the portion of the cell membrane bound to the APC. By causing numerous such microperforations in the cell membrane, the cells are irreversibly damaged and die. Within minutes of NIR light exposure, the target cells swell, bleb, and burst, which leads to cell death. This type of treatment completely avoids the side effects of systemic mAb therapy. 【0038】 This disclosure describes humanized CD25-specific mAbs suitable for NIR-PIT because they do not possess any specific biological function without conjugation to an effector molecule (e.g., IR700). CD25 is the IL-2α receptor found on T regulatory (Treg) cells and activated effector T cells, among other immune cell types. Antibodies against CD25 can be blocking or non-blocking with respect to IL-2, a cytokine that induces cell proliferation in immune cell subtypes. Non-IL-2-blocking CD25-specific antibodies bind to CD25 without interfering with IL-2 binding and therefore have no direct pharmacokinetic effect. Therefore, APCs directed against the CD25 protein can selectively deplete Treg cells within the tumor border without systemic effects. 【0039】 The present disclosure provides humanized anti-CD25 antibodies having at least the following features: (i) high-affinity binding to CD25 that is not impaired by IR700 conjugation; (ii) little or no IL-2 blocking (e.g., non-IL-2 blocking); and (iii) little or no ADCC activity (e.g., lack of ADCC activity). Based on these features, the disclosed anti-CD25 antibodies provide excellent delivery platforms for effector molecules, including IR700, for the treatment of, for example, cancer. 【0040】 II. Abbreviations ADCC Antibody-dependent cellular cytotoxicity CDR Complementarity Determining Region DC dendritic cells FACS Fluorescence-activated cell sorting FLT Fluorescence Lifetime J / cm 2 joules per square centimeter IL-2 Interleukin-2 IL-2R Interleukin-2 receptor LED Light Emitting Diode mAb monoclonal antibody NIR near-infrared light PIT photoimmunotherapy RU Response Unit Treg T regulation VH Variable Weight VL Variable Light 【0041】 III. Terminology Overview Unless otherwise specified, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's Genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "an antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." It should be further understood that any and all base or amino acid sizes and all molecular weight or molecular mass values given for nucleic acids or polypeptides are approximate and, unless otherwise indicated, are provided for descriptive purposes. While many methods and materials similar or equivalent to those described herein can be used, certain suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. All sequences associated with GenBank® accession numbers referred to herein are incorporated by reference for sequences available as of June 30, 2022. In order to facilitate review of the various aspects of this disclosure, the following explanations of specific terms are provided: 【0042】 Abscopal effect: Treatment of tumors, e.g., metastases, in parts of the body that are not the direct target of local therapy (e.g., NIR-PIT). For example, irradiation of a specific tumor with NIR light in combination with an appropriate antibody-IR700 conjugate(s) can reduce the size of a different tumor (e.g., metastasis) that is not irradiated with NIR light. In some cases (e.g., in humans), the unirradiated / distant tumor is at least 3 inches away from the tumor treated with NIR light, e.g., at least 4 inches, at least 5 inches, at least 10 inches, at least 12 inches, at least 18 inches, or at least 24 inches away from the tumor treated with NIR light. 【0043】 Administration: Providing or giving an agent, e.g., an antibody, antibody-IR700 conjugate, and / or immunomodulator, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, topical, systemic or local injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, intraosseous, intraprostatic, and intravenous), oral, ocular, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. In one example, administration is intravenous. In one example, administration is intraperitoneal. In one example, administration is not systemic. In one example, administration is local to the tumor or cancer. 【0044】 Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds to (e.g., specifically recognizes and specifically binds to) an antigen, e.g., a tumor-specific protein, or an epitope of a protein differentially expressed on an immune cell, e.g., a T cell (e.g., a T regulatory cell). Mammalian immunoglobulin molecules are composed of heavy (H) and light (L) chains, each of which has a respective variable heavy (V H ) area and variable light (V L ) region. H Area and V LThe domain is responsible for binding to the antigen recognized by the antibody. Typically, naturally occurring mammalian immunoglobulins have heavy and light chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) of mammalian immunoglobulins that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Some mammals, such as camels, alpacas, and llamas, have heavy-chain antibodies that lack light chains. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the predominant antibody produced by birds and reptiles and shares some functionality with mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish such as sharks, while IgX antibodies are found in amphibians. IgNAR antibodies are heavy-chain antibodies. 【0045】 Antibodies, such as those in antibody-IR700 conjugates, include intact immunoglobulins and antibody variants and portions, such as Fab fragments, Fab' fragments, F(ab)' fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which the light chain variable region of an immunoglobulin and the heavy chain variable region of an immunoglobulin are joined by a linker, whereas in dsFv, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997. 【0046】 Antibody variable regions contain "framework" regions and hypervariable regions known as "complementarity-determining regions" or "CDRs." The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody function to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR may be determined by the method described in Kabat et al. (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; "Kabat" numbering scheme), Chothia et al. (Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; "Chothia" numbering scheme), Kunik et al. (Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40:W521-524, 2012; "Paramome CDR boundaries can be readily determined using any of several well-known numbering schemes, including those described by the ImMunoGeneTics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-9, 2001; "IMGT" numbering scheme). The Kabat, Paratome, and IMGT databases are maintained online. Additionally, the AbRSA tool can be used to determine CDR boundaries according to Kabat, IMGT, or Chothia (online at aligncdr.labshare.cn / aligncdr / abrsa.php). 【0047】 A "single domain antibody" refers to an antibody having a single domain (a variable domain) that is capable of specifically binding to an antigen or an epitope of an antigen in the absence of additional antibody domains. Single domain antibodies include, for example, V H Domain antibodies, V NAR Antibody, Camelidae V H H antibodies and V L Domain antibodies are included. NAR Antibodies are produced by cartilaginous fishes that produce heavy chain antibodies (IgNAR), such as nurse sharks, wobbegong sharks, spiny dogfish, and bamboo sharks. H H antibodies are produced by several species, including camels, llamas, alpacas, and guanacos, which produce heavy chain antibodies that naturally lack light chains. 【0048】 A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies are produced by known methods. Monoclonal antibodies include humanized monoclonal antibodies. 【0049】 A "chimeric antibody" has framework residues from one species, eg, human, and CDRs (which generally confer antigen binding) from another species. 【0050】 A "humanized" antibody is an immunoglobulin containing a human framework region and one or more CDRs from a non-human (e.g., mouse, rabbit, rat, shark, camel, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." In one embodiment, all CDRs in a humanized immunoglobulin are from the donor immunoglobulin. Constant regions may be absent, but if present, they are substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or greater. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody providing the CDRs. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. 【0051】 An antibody that "specifically binds" to an antigen refers to the ability of an individual antibody to specifically immunoreact with an antigen, for example, a tumor-specific antigen, compared to binding to an unrelated protein, for example, a non-tumor protein, such as 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 fragments thereof) and other agents that bind substantially only to tumor-specific proteins in the preparation. 【0052】 Binding is the non-random binding reaction between antibody molecules and antigenic determinants such as CD25.Desired binding specificity is typically determined from the reference point of antibody's ability to differentially bind to target antigen and unrelated antigen, and thus distinguish between two different antigens, especially when the two antigens have unique epitopes.An antibody that specifically binds to a specific epitope is called "specific antibody". 【0053】 Antibody-IR700 conjugate: A molecule comprising both an antibody, such as a CD25-specific antibody or a tumor-specific antibody, conjugated to IR700. In some examples, the antibody is a humanized antibody that specifically binds to CD25, such as those disclosed herein. In other examples, the antibody is a monoclonal antibody that specifically binds to a surface protein on a cancer cell, such as a tumor-specific antigen. 【0054】 Antigen (Ag): A compound, composition, or substance capable of stimulating antibody production or T cell responses in an animal, including compositions injected or absorbed into an animal (e.g., those containing tumor-specific proteins or CD25 proteins). An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, e.g., the disclosed antigens. "Epitope" or "antigenic determinant" refers to the region of an antigen against which B and / or T cells respond. In one embodiment, T cells respond to an epitope when presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, and 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. 【0055】 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, antigens include tumor-specific proteins or peptides (for example, those found on the surface of cells, for example, cancer cells) or their immunogenic fragments.In some examples, antigens include immune cell-specific proteins or peptides (for example, those found on the surface of immune cells, for example, T cells, for example, regulatory T cells) or their immunogenic fragments.In one example, antigen is CD25, for example, mammalian CD25, for example, mouse or human CD25. 【0056】 Binding affinity: The affinity of an antibody or other antigen-binding molecule (e.g., an antibody-IR700 conjugate) to an antigen, e.g., CD25 or a tumor-specific antigen. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by antigen / antibody dissociation rate. In another embodiment, binding affinity is measured by competitive radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In some embodiments, binding affinity is measured using the Octet system (ForteBio) based on biolayer interference (BLI) technology. In other embodiments, Kd is measured using a surface plasmon resonance assay using a BIACORES-2000 or BIACORES-3000 (BIAcore, Inc., Piscataway, NJ). In other embodiments, antibody affinity is measured by flow cytometry. An antibody or antibody conjugate that "specifically binds" to an antigen (e.g., CD25) is an antibody or conjugate that binds to the antigen with high affinity and does not significantly bind to other unrelated antigens. In some examples, the antibody (e.g., the antibody in an antibody-IR700 conjugate) has a binding constant at least 10 times higher than the binding constant for other molecules in the sample or subject. 3 M -1 Large, 10 4 M -1Big or 10 5 M -1 Specifically binds to a target (e.g., a cell surface protein, e.g., CD25 or a tumor-specific protein) with a large binding constant. In some examples, the antibody (e.g., mAb) or fragment thereof has an equilibrium constant (Kd) of 1 nM or less. For example, the antibody specifically binds to a target, e.g., CD25 or a tumor-specific protein, with an equilibrium constant (Kd) of at least about 0.1 x 10 -8 M, at least about 0.3 × 10 -8 M, at least about 0.5 × 10 -8 M, at least about 0.75 × 10 -8 M, at least about 1.0 × 10 -8 M, at least about 1.3 × 10 -8 M, at least about 1.5 × 10 -8 M or at least about 2.0 × 10 -8 It binds with a binding affinity of M. 【0057】 Cancer: A malignant tumor characterized by abnormal or uncontrolled cell growth. Other features often associated with cancer include metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretory products, and suppression or exacerbation of inflammatory or immunological responses, infiltration of surrounding or distant tissues or organs, e.g., lymph nodes. "Metastatic disease" refers to cancer cells that leave the original tumor site and migrate to other parts of the body, e.g., via the bloodstream or lymphatic system. In one example, the cells killed by the disclosed compositions and methods are cancer cells. 【0058】 CD25 (IL-2 receptor alpha chain): A type I transmembrane protein present on activated T cells, activated B cells, some thymocytes, myeloid precursors, and oligodendrocytes (e.g., OMIM 147730). CD25 has been used as a marker to identify CD4+FoxP3+ regulatory T cells in mice. CD25 is found on the surface of some cancer cells, including B-cell neoplasms, some acute nonlymphocytic leukemias, neuroblastomas, mastocytosis, and tumor-infiltrating lymphocytes. It functions as a receptor for HTLV-1 and, as a result, is expressed on neoplastic cells in adult T-cell lymphoma / leukemia. Exemplary CD25 sequences can be found in the GenBank® database (e.g., accession numbers CAA44297.1, NP_000408.1, and NP_001295171.1). 【0059】 Complementarity-Determining Region (CDR): A region of hypervariable amino acid sequence that determines the binding affinity and specificity of an antibody. The light and heavy chains of mammalian immunoglobulins each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. 【0060】 Conjugate: In the context of this disclosure, a "conjugate" is an antibody or antibody fragment (e.g., an antigen-binding fragment) covalently linked to an effector molecule or a second protein (e.g., a second antibody). The effector molecule can be, for example, a photon absorber (e.g., IR700), a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. 【0061】 Conservative variant: In the context of the present disclosure, a "conservative" amino acid substitution is one that does not substantially affect or reduce the affinity of an antibody for a protein, e.g., CD25. As an example, a monoclonal antibody that specifically binds to CD25 may contain at most about one, at most about two, at most about five, at most about 10, at most about 15, at most about 20, or at most about 25 conservative substitutions and specifically binds to a CD25 polypeptide (e.g., with sub-nM affinity) but does not block IL-2 binding to CD25 or induce ADCC. The term "conservative variant" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the variant retains activity. Non-conservative substitutions are those that reduce the activity (e.g., affinity) of a protein. 【0062】 Conservative amino acid substitution tables providing functionally similar amino acids are well known. The following six groups are examples of amino acids that are considered conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W). 【0063】 In some aspects herein, provided are amino acid sequences that contain no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution compared to any amino acid sequence disclosed herein. 【0064】 Contacting: To bring into direct physical association, including both in solid and liquid form. Contacting can occur in vitro, for example, with isolated cells, e.g., tumor cells, or in vivo by administration to a subject (e.g., a subject with a tumor, e.g., cancer). 【0065】 Effector molecule: The portion of an antibody conjugate intended to have a desired effect on cells to which the conjugate is targeted. Effector molecules can be, for example, detectable labels, photon absorbers (e.g., IR700), or toxins. Effector molecules are also known as effector moieties, therapeutic agents, diagnostic agents, or similar terms. Therapeutic agents (or drugs) include compounds such as small molecules, nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, nanoparticles, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single- or double-stranded DNA, and triplex-forming oligonucleotides. Alternatively, effector molecules can be contained within an encapsulation system, such as a nanoparticle, liposome, or micelle, conjugated to the antibody. The encapsulation shields the effector molecule from direct exposure to the circulatory system. Means for preparing antibody-bound liposomes are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,957,735; and Connor et al., Pharm Ther 28:341-365, 1985). Diagnostic agents or moieties include radioisotopes and other detectable labels (e.g., fluorophores, chemiluminescent agents, and enzymes). Radioisotopes include: 35 S, 11 C. 13 N, 15 O. 18 F, 19 F, 99m Tc, 131 I, 3 H, 14 C. 15 N, 90 Y, 99 Tc, 111 In and125 Contains I. 【0066】 Framework region: Amino acid sequences inserted between the CDRs. Framework regions include variable light and variable heavy framework regions. Framework regions function to hold the CDRs in the proper orientation for antigen binding. 【0067】 Heterologous: Derived from separate genetic sources or species. 【0068】 Host cell: A cell in which a vector can be propagated and its DNA expressed. The cell can be prokaryotic or eukaryotic. In some examples, a prokaryotic cell is an E. coli cell. In some examples, a eukaryotic cell is a human cell. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell since there may be mutations that occur during replication. However, when the term "host cell" is used, such progeny is included. 【0069】 Immune response: A response of a cell of the immune system, e.g., a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, e.g., a CD4 + Response or CD8 + In another embodiment, the response is a B cell response, resulting in the production of specific antibodies. 【0070】 Immune Modulator: An immune modulator is a substance that alters (e.g., increases or decreases) one or more functions of the immune system. In some examples, an immune modulator activates the immune system. In other examples, an immune modulator inhibits the activity of immune suppressor cells (or kills immune suppressor cells). In some examples, an immune modulator is a mAb that can be conjugated to IR700 to form mAb-IR700, which can be used in the disclosed methods to target cells that express a protein recognized by the mAb. Exemplary immune modulators include the immune activators IL-15 and IFN-γ. 【0071】 IR700 (IRDye® 700DX): A dye having the formula: [ka] 【0072】 Amino-reactive IR700 is a relatively hydrophilic dye that can be covalently conjugated to antibodies using the NHS ester of IR700. IR700 also reacts with conventional photosensitizers, such as the hematoporphyrin derivative Photofrin® (1.2×10 at 630 nm). 3 M -1 cm -1 ), meta-tetrahydroxyphenyl chlorin; Foscan® (2.2 × 10 at 652 nm) 4 M -1 cm -1 ) and mono-L-aspartyl chlorin e6; NPe6 / Laserphyrin® (4.0 × 10 at 654 nm) 4 M -1 cm -1 ) extinction coefficient (2.1 × 10 at absorption maximum at 689 nm) 5 M -1 cm -1 ) 【0073】 Label (or detectable label): A detectable compound or composition that is directly or indirectly conjugated to another molecule, such as an antibody or protein, to facilitate detection of that molecule. Specific non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioisotopes. In one example, a "labeled antibody" refers to the incorporation of another molecule into an antibody. For example, the label is the incorporation of a detectable marker, such as a radiolabeled amino acid, or the attachment of a biotinyl moiety to a polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionucleotides (e.g., 35 S, 11 C. 13 N, 15 O. 18 F, 19 F, 99m Tc, 131 I, 3 H, 14 C. 15 N, 90 Y, 99 Tc, 111 In and 125 I), fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide fluorophores), enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, e.g., gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. 【0074】 Pharmaceutical composition: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when appropriately administered to a subject. A pharmaceutical composition may contain a therapeutic agent, for example, one or more antibodies, antibody-IR700 conjugates, and / or one or more immune activators. A therapeutic or pharmaceutical agent is an agent that, alone or together with additional compound(s), induces a desired response (e.g., induces a therapeutic or prophylactic effect when administered to a subject). In a particular example, a pharmaceutical composition contains a therapeutically effective amount of at least one antibody-IR700 conjugate. 【0075】 Pharmaceutically acceptable vehicles: Pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, such as one or more of the antibodies or antibody-IR700 conjugates disclosed herein. 【0076】 Generally, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually contain an injectable fluid as a vehicle, which includes pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate. 【0077】 Photoimmunotherapy (PIT): A molecularly targeted therapy that utilizes a target-specific photosensitizer based on the near-infrared (NIR) phthalocyanine dye IRDye700DX (IR700) conjugated to a monoclonal antibody (mAb) that targets a cellular protein (e.g., one on the cell surface, e.g., CD25 on the surface of Treg cells, or a tumor-specific protein). In one example, the cell surface protein is one that is specifically found on cancer cells or immune cells, and thus PIT can be used to kill such cells. Cell death occurs when the antibody-IR700 conjugate binds to the cell and the cell is irradiated with NIR, but cells that do not express the cell surface protein recognized by the antibody-IR700 conjugate are not killed in significant numbers. 【0078】 Reducing agent: An element or compound that loses electrons in a redox chemical reaction (or "donates" electrons to an electron recipient (oxidizing agent)). Thus, a reducing agent becomes oxidized when it loses electrons in a redox reaction. A reducing agent "reduces" an oxidizing agent (or is "oxidized" by an oxidizing agent). Thus, one or more reducing agents can be used in vitro and in vivo in combination with NIR-PIT to reduce unwanted nonspecific tissue damage caused by reactive oxygen species generated from IR700 after exposure to NIR light. For example, a reducing agent can inactivate reactive oxygen species that are unbound or not in the tumor area, reducing unwanted acute inflammation in other areas of the body. Exemplary reducing agents that can be used in the methods provided herein include L-cysteine, sodium L-ascorbate (L-NaAA), ascorbic acid (e.g., L- or R-ascorbic acid), and glutathione. In some cases, sodium azide is not used in vivo due to its toxicity. Thus, in some examples, the reducing agent used in the disclosed methods is not sodium azide. In one example, the reducing agent used in the disclosed methods is L-NaAA. 【0079】 Sequence identity: The similarity between amino acid sequences or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise called sequence identity. Sequence identity is often measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of polypeptides or nucleic acid molecules have a relatively high degree of sequence identity when aligned using standard methods. 【0080】 Methods for aligning sequences for comparison are known. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. 【0081】 The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to determine sequence identity using this program is available on the internet at the NCBI website. 【0082】 Homologs and variants of antibodies that specifically bind to CD25 are typically characterized by possessing at least about 75%, e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the antibody's amino acid sequence, as measured over the entire length of the alignment, using NCBI Blast 2.0, gapped blastp, set to default parameters. For comparison of amino acid sequences longer than about 30 amino acids, the Blast 2 alignment function is used with the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, and per-residue gap cost of 1). When aligning short peptides (shorter than approximately 30 amino acids), alignments should be performed using the Blast 2 alignment function with the PAM30 matrix set to default parameters (open gap penalty of 9, extension gap penalty of 1). Proteins with even greater similarity to the reference sequence, when assessed by this method, exhibit increasing percentage identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is compared for sequence identity, homologs and variants typically share at least 80% sequence identity over a short window of 10-20 amino acids, and may share at least 85%, or at least 90%, or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available online at the NCBI website. These sequence identity ranges are provided for guidance only; it is entirely possible that strong and significant homologs may be obtained that fall outside the provided ranges. 【0083】 Subject or patient: This term includes humans and non-human mammals. In one example, the subject is a human or veterinary subject, such as a mouse, rat, dog, cat or non-human primate. In some examples, the subject is a mammal (e.g., a human) that has cancer or is being treated for cancer. 【0084】 Therapeutically effective amount: An amount of a composition (e.g., a CD25-specific antibody-IR700 conjugate), alone or in combination with additional therapeutic agent(s) (e.g., an anti-tumor antigen-specific antibody-IR700 conjugate, an anti-CTLA4-IR700 molecule, an anti-PD-L1-IR700 molecule, an immunomodulator, a checkpoint inhibitor, and / or a reducing agent), sufficient to achieve a desired effect in a subject or cell being treated with the agent. The effective amount of an agent (e.g., an antibody-IR700 conjugate, alone or in combination with other agents) can depend on several factors, including, but not limited to, the subject or cell being treated, the particular therapeutic agent, and the mode of administration of the therapeutic composition. In one example, a therapeutically effective amount or concentration of an antibody-IR700 conjugate is an amount or concentration sufficient to prevent progression (e.g., metastasis), delay progression, or cause regression of the disease, or that is capable of reducing symptoms caused by a disease such as cancer. In one example, a therapeutically effective amount or concentration of an antibody-IR700 conjugate is an amount or concentration sufficient to increase survival time of a tumor-bearing patient. In one example, a therapeutically effective amount or concentration of a reducing agent is an amount or concentration sufficient to reduce edema, an acute inflammatory response, or both, in a subject treated with NIR-PIT. 【0085】 In one example, the desired response is to reduce or inhibit one or more symptoms associated with cancer. For a composition to be effective, one or more symptoms do not need to be completely eliminated. For example, treatment with the modalities provided herein, in some cases, reduces the size of a tumor (e.g., the volume or weight of a tumor or tumor metastasis) by, for example, at least 20%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, compared to the tumor size in the absence of treatment (or in the presence of only a subset of the treatments provided herein). In a specific example, treatment with the modalities provided herein kills a population of cells (e.g., cancer cells), for example, by killing at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% of the cells, compared to cell killing in the absence of treatment (or in the presence of only a subset of the treatments provided herein). In a particular example, treatment with a modality provided herein increases survival time of a patient bearing a tumor (or a patient from whom a tumor has recently been removed) compared to survival time in the absence of treatment (or in the presence of only a subset of the treatments provided herein), e.g., increases survival by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at least 200%, or at least 500%. In some examples, treatment with a modality provided herein increases the amount of memory T cells in a subject (e.g., an increase of at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at least 200%, or at least 500%) compared to the amount of memory T cells in the absence of treatment (or in the presence of only a subset of the treatments provided herein).In some examples, treatment with the modalities provided herein reduces the number of Treg cells (e.g., FOXP3) in the targeted tumor / tumor bed. + CD25 + CD4 + The amount of CD8+ / Treg cells in the targeted tumor / tumor bed is reduced (e.g., by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100%) compared to the amount of Treg cells in the targeted tumor / tumor bed in the absence of treatment (or in the presence of only a subset of the therapies provided herein). In some examples, treatment with a modality provided herein increases the CD8+ / Treg ratio in the treated subject compared to the CD8+ / Treg ratio in the absence of treatment (or in the presence of only a subset of the therapies provided herein) (e.g., by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at least 200%, or at least 500%). In some examples, a combination of these effects is achieved by the disclosed methods. 【0086】 The effective amount of a therapeutic agent administered to a human or veterinary subject can vary depending on several factors related to the subject, such as the subject's overall health. The effective amount of an agent can be determined by varying the dosage of a composition(s) and measuring the resulting therapeutic response, such as tumor regression. The effective amount can also be determined through various in vitro, in vivo, or in situ immunoassays. The disclosed agents can be administered in a single dose or in several doses as needed to obtain the desired response. However, the effective amount can depend on the treatment being applied, the subject being treated, the severity and type of the condition being treated, and the mode of administration. 【0087】 In particular examples, a therapeutically effective dose of the antibody-IR700 conjugate, when administered, for example, intravenously, is at least 0.5 milligrams per 60 kilograms (mg / kg), at least 5 mg / 60 kg, at least 10 mg / 60 kg, at least 20 mg / 60 kg, at least 30 mg / 60 kg, at least 50 mg / 60 kg, at least 75 mg / 60 kg, at least 100 mg / 60 kg, at least 125 mg / 60 kg, at least 150 mg / 60 kg, at least 175 mg / 60 kg, at least 200 mg / 60 kg, or at least The dose is at least 225 mg / 60 kg, at least 250 mg / 60 kg, at least 300 mg / 60 kg, at least 350 mg / 60 kg, or at least 400 mg / 60 kg, e.g., 0.5 to 250 mg / 60 kg, e.g., 1 mg / 60 kg, 2 mg / 60 kg, 5 mg / 60 kg, 20 mg / 60 kg, 50 mg / 60 kg, 75 mg / 60 kg, 100 mg / 60 kg, 125 mg / 60 kg, 150 mg / 60 kg, 175 mg / 60 kg, 200 mg / 60 kg, 300 mg / 60 kg, or 400 mg / 60 kg. In one non-limiting example, a therapeutically effective dose of an antibody-IR700 conjugate, e.g., an antibody-IR700 conjugate containing a disclosed CD25 antibody, is 100 to 200 mg / 60 kg. In another example, a therapeutically effective dose of the antibody-IR700 conjugate, e.g., when administered intratumorally or i.p., is at least 10 μg / kg, e.g., at least 100 μg / kg, at least 500 μg / kg or at least 500 μg / kg, e.g., a dose of 10 μg / kg to 1000 μg / kg, e.g., 100 μg / kg, 250 μg / kg, about 500 μg / kg, 750 μg / kg or 1000 μg / kg. In one example, the therapeutically effective dose of the antibody-IR700 conjugate is administered in a topical solution and is at least 1 μg / ml, e.g., at least 500 μg / ml, e.g., between 20 μg / ml and 100 μg / ml, e.g., 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml or 100 μg / ml.However, those skilled in the art will recognize that higher or lower dosages may also be used, depending, for example, on the particular antibody-IR700 conjugate. In certain examples, such daily dosages are administered in one or more divided doses (e.g., two, three, or four doses) or in a single formulation. The disclosed antibody-IR700 conjugates may be administered alone, in the presence of a pharmaceutically acceptable carrier, or in the presence of other therapeutic agents (e.g., other anti-neoplastic agents and / or reducing agents). 【0088】 Generally, a suitable dose of irradiation following administration of one or more antibody-IR700 conjugates (and in some cases also a reducing agent) is at least 1 J / cm at a wavelength of 660-740 nm. 2 , e.g., at least 4 J / cm at wavelengths of 660-740 nm 2 , at least 10 J / cm at wavelengths of 660-740 nm 2 , at least 20 J / cm at wavelengths of 660-740 nm 2 , at least 25 J / cm at wavelengths of 660-740 nm 2 , at least 50 J / cm at wavelengths of 660-740 nm 2 , or at least 100 J / cm at wavelengths between 660 and 740 nm 2 For example, 1 to 500 J / cm at wavelengths of 660 to 740 nm 2 , 10-100J / cm at wavelengths of 660-740nm 2 Or 20-60 J / cm at wavelengths of 660-740 nm 2 In some examples, the wavelength is 660-710 nm, e.g., 680 nm or 690 nm. In a specific example, a suitable dose of irradiation after administration of an antibody-IR700 conjugate is at least 1 J / cm at a wavelength of 680 or 690 nm. 2 , e.g., at least 4 J / cm at a wavelength of 680 or 690 nm 2 , at least 10 J / cm at wavelengths of 680 or 690 nm 2 , at least 20 J / cm at wavelengths of 680 or 690 nm 2 , at least 25 J / cm at wavelengths of 680 or 690 nm 2, at least 50 J / cm at wavelengths of 680 or 690 nm 2 , or at least 100 J / cm at a wavelength of 680 or 690 nm 2 , for example, 4 to 100 J / cm at a wavelength of 680 or 690 nm 2 , 4~50J / cm 2 Or 10-25J / cm 2 In one non-limiting example, a suitable dose of irradiation after administration of an antibody-IR700 conjugate is 50 J / cm at a wavelength of 690 nm. 2 In particular examples, multiple irradiations (e.g., at least two, at least three, or at least four irradiations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate administrations) are administered after administration of one or more antibody-IR700 conjugates (and also, in some examples, after administration of a second (tumor antigen-specific) monoclonal antibody-IR700 conjugate, an anti-CTLA4-IR700 conjugate, an anti-PD-L1-IR700 conjugate, an immune activator, a checkpoint inhibitor, and / or a reducing agent). 【0089】 Treat: A term used to refer to the treatment of cells or tissues with a therapeutic agent, including contacting or incubating one or more agents (e.g., one or more CD25 antibody-IR700 conjugates and, in some cases, a second (tumor antigen-specific) monoclonal antibody-IR700 conjugate, an anti-CTLA4-IR700 conjugate, an anti-PD-L1-IR700 conjugate, an immune activator, a checkpoint inhibitor, and / or a reducing agent) with the cells or tissue, and / or administering one or more agents to a subject, e.g., a subject with cancer. Treated cells are cells that have been contacted with a desired composition in an amount and under conditions sufficient to produce a desired response. In one example, treated cells are cells that have been exposed to an antibody-IR700 conjugate under conditions sufficient for the antibody to bind to a surface protein on the cell, optionally contacted with a reducing agent, and irradiated with NIR light until sufficient cell death is achieved. In other examples, a treated subject is one to which one or more antibody-IR700 conjugates have been administered under conditions sufficient for the antibodies to bind to surface proteins on cells, and optionally one or more reducing agents, and irradiated with NIR light until sufficient cell kill is achieved. 【0090】 Tumor, neoplasia, malignancy, or cancer: A neoplasm is an abnormal growth of tissue or cells resulting from excessive cell division. Neoplastic growth can give rise to a tumor. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors. Tumors that do not metastasize are called "benign." Tumors that can invade surrounding tissues and / or metastasize are called "malignant." "Non-cancerous tissue" is tissue from the same organ in which a malignant neoplasm forms, but does not have the characteristic pathology of a neoplasm. Generally, non-cancerous tissue appears histologically normal. "Normal tissue" is tissue from an organ that is not afflicted with cancer or another disease or disorder of that organ. A "cancer-free" subject has not been diagnosed with cancer in that organ and does not have detectable cancer. 【0091】 Tumors include original (primary) tumors, recurrent tumors, and metastatic (secondary) tumors. Tumor recurrence is the return of a tumor at the same site as the original (primary) tumor, for example, after the tumor has been surgically removed, by drug or other treatment, or otherwise eliminated. Metastasis is the spread of a tumor from one part of the body to another. Tumors formed from the spread cells are called secondary tumors and contain cells similar to those in the original (primary) tumor. Recurrence of either a primary tumor or metastasis can occur. 【0092】 Exemplary tumors, e.g., cancers, that can be treated with the disclosed methods include solid tumors, e.g., breast cancer (e.g., lobular carcinoma and duct carcinoma), sarcoma, lung carcinoma (e.g., non-small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma), pulmonary mesothelioma, colorectal adenocarcinoma, head and neck cancer (e.g., adenocarcinoma, squamous cell carcinoma, and adenocarcinoma), and tumors of the breast (e.g., ovarian cancer ... carcinoma), metastatic squamous cell, e.g., cancers caused by HPV or Epstein-Barr virus, e.g., HPV16; which may include cancers of the mouth, tongue, cheek, nasopharynx, throat, hypopharynx, oropharynx, larynx, and trachea), gastric cancer, prostate adenocarcinoma, ovarian cancer (e.g., serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumor, testicular cancer and germ cell tumor, pancreatic adenocarcinoma, biliary tract adenocarcinoma, hepatocellular carcinoma, bladder cancer (including, e.g., transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma), renal cell adenocarcinoma, endometrial cancer (including, e.g., adenocarcinoma and mixed Müllerian tumor (carcinosarcoma)), endocervical, ectocervix, and vaginal carcinoma (e.g., their respective adenocarcinoma and squamous cell carcinoma), tumors of the skin (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin appendage tumor The tumor may include, for example, esophageal cancer, nasopharyngeal and oropharyngeal carcinoma (including squamous cell carcinoma and adenocarcinoma thereof), Kaposi's sarcoma, cutaneous lymphoma, skin adnexal tumor, and various types of sarcoma and Merkel cell carcinoma), esophageal cancer, nasopharyngeal and oropharyngeal carcinoma (including squamous cell carcinoma and adenocarcinoma thereof), salivary gland cancer, brain and central nervous system tumors (including, for example, tumors of glial, neuronal, and meningeal origin), peripheral nerve tumors, soft tissue sarcomas, and sarcomas of bone and cartilage, and lymphatic tumors (including B-cell and T-cell malignant lymphoma). In one example, the tumor is an adenocarcinoma. In one example, the tumor is oropharyngeal cancer. In one example, the tumor is squamous cell carcinoma, for example, of the head and neck or skin. 【0093】 These methods can also be used to treat liquid tumors (e.g., hematological malignancies), such as lymphatic, leukemia, or other types of leukemia. In specific examples, the tumors treated are hematological tumors, such as leukemias (e.g., acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia), lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), and myelomas. 【0094】 Under conditions sufficient for: A phrase used to describe any environment that allows for a desired activity. In one example, "under conditions sufficient for" includes administration of an antibody-IR700 conjugate to a subject sufficient to allow binding of the antibody-IR700 conjugate to its targeted cell surface protein (e.g., a tumor-specific antigen or an immune cell-specific antigen, e.g., CD25). In certain embodiments, the desired activity is killing of cells to which the antibody-IR700 conjugate binds following therapeutic irradiation of the cells. 【0095】 Untreated cells: Cells that have never been in contact with a therapeutic agent, for example, an antibody-IR700 conjugate and / or irradiation. In one example, an untreated cell is a cell that receives a vehicle in which a therapeutic agent(s) is delivered. Similarly, an untreated subject is a subject that has not been administered a therapeutic agent, for example, an antibody-IR700 conjugate and / or irradiation. In one example, an untreated subject is a subject that receives a vehicle in which a therapeutic agent(s) is delivered. 【0096】 Vector: A nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain a nucleic acid sequence that allows it to replicate in a host cell, such as a replication origin. A vector may also contain one or more selectable marker genes and other genetic elements. In some examples, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector. 【0097】 The disclosure of a particular specific example is not meant to exclude other embodiments. Furthermore, any treatment described herein may be combined with other bioactive agents or treatment modalities, although this does not necessarily exclude other treatments. 【0098】 IV. Monoclonal antibodies specific for CD25 Described herein are monoclonal antibodies that bind with high affinity to human and cynomolgus monkey CD25 (interleukin-2 receptor alpha; IL-2Rα). The disclosed antibodies specifically bind to CD25 but do not inhibit CD25 binding to IL-2, and the CD25-specific antibodies do not induce ADCC. In some examples, the CD25 antibodies provided herein, including variants thereof, have sub-nM affinity for CD25, e.g., human, cynomolgus monkey, or mouse CD25. The amino acid sequences of the VH and VL domains of 21 exemplary CD25-specific monoclonal antibodies are set forth herein as SEQ ID NOS: 1-42. The position of each CDR, as determined by IMGT, is underlined in the following sequences. In each VH domain sequence, the IMGT CDR1, CDR2, and CDR3 sequences are located at positions 31-35, 50-66, and 99-112, respectively; in each VL domain sequence, the IMGT CDR1, CDR2, and CDR3 sequences are located at positions 24-33, 49-55, and 88-96, respectively. Other numbering schemes, such as Chothia or Kabat, can also be used to determine the boundaries of each CDR. Consensus and exemplary CDR sequences are also provided below. 14554-VH (SEQ ID NO: 1) [ka] 14554-VL (SEQ ID NO: 2) [ka] 14555-VH (SEQ ID NO: 3) [ka] 14555-VL (SEQ ID NO: 4) [ka] 14556-VH (SEQ ID NO: 5) [ka] 14556-VL (SEQ ID NO: 6) [ka] 14557-VH (SEQ ID NO: 7) [ka] 14557-VL (SEQ ID NO: 8) [ka] 14558-VH (SEQ ID NO: 9) [ka] 14558-VL (SEQ ID NO: 10) [ka] 14559-VH (SEQ ID NO: 11) [ka] 14559-VL (SEQ ID NO: 12) [ka] 14560-VH (SEQ ID NO: 13) [ka] 14560-VL (SEQ ID NO: 14) [ka] 14561-VH (SEQ ID NO: 15) [ka] 14561-VL (SEQ ID NO: 16) [ka] 14562-VH (SEQ ID NO: 17) [ka] 14562-VL (SEQ ID NO: 18) [ka] 14563-VH (SEQ ID NO: 19) [ka] 14563-VL (SEQ ID NO: 20) [ka] 14564-VH (SEQ ID NO: 21) [ka] 14564-VL (SEQ ID NO: 22) [ka] 14565-VH (SEQ ID NO: 23) [ka] 14565-VL (SEQ ID NO: 24) [ka] 14566-VH (SEQ ID NO: 25) [ka] 14566-VL (SEQ ID NO: 26) [ka] 14567-VH (SEQ ID NO: 27) [ka] 14567-VL (SEQ ID NO: 28) [ka] 14568-VH (SEQ ID NO: 29) [ka] 14568-VL (SEQ ID NO: 30) [ka] 14569-VH (SEQ ID NO: 31) [ka] 14569-VL (SEQ ID NO: 32) [ka] 14570-VH (SEQ ID NO: 33) [ka] 14570-VL (SEQ ID NO: 34) [ka] 14571-VH (SEQ ID NO: 35) [ka] 14571-VL (SEQ ID NO: 36) [ka] 14572-VH (SEQ ID NO: 37) [ka] 14572-VL (SEQ ID NO: 38) [ka] 14573-VH (SEQ ID NO: 39) [ka] 14573-VL (SEQ ID NO: 40) [ka] U579-WT-VH (SEQ ID NO: 41) [ka] U579-WT-VL (SEQ ID NO: 42) [ka] 【0099】 Below are the consensus amino acid sequences of each VH domain CDR (HCDR) and each VL domain CDR (LCDR) based on an alignment of 21 antibody sequences. Consensus HCDR1:SXGMS, where X is W or Y (SEQ ID NO:43) Consensus HCDR2: TINGYGDTXYYPDSVKG, where X is W or T (SEQ ID NO: 44) Consensus HCDR3: DRDYX1NSYYYAX2DY, where X1 is S or G; X2 is L or H (SEQ ID NO: 45) Consensus LCDR1: RASSXVSFMH, where X is S, W, or Y (SEQ ID NO: 46) Consensus LCDR2: ATXNLAS, where X is S, A, Q, or M (SEQ ID NO: 47) Consensus LCDR3: QQWSSNPPA (SEQ ID NO: 48). 【0100】 Below are specific amino acid sequences of exemplary VH domain CDRs (HCDRs) and exemplary VL domain CDRs (LCDRs). Exemplary HCDR1: SWGMS (SEQ ID NO: 49) or SYGMS (SEQ ID NO: 50) Exemplary HCDR2: TINGYGDTTYYPDSVKG (SEQ ID NO: 51) Exemplary HCDR3: DRDYSNSYYYAHDY (SEQ ID NO: 52) or DRDYTNSYYYAHDY (SEQ ID NO: 53) Exemplary LCDR1: RASSYVSFMH (SEQ ID NO: 54) Exemplary LCDR2: ATANLAS (SEQ ID NO: 55) or ATMNLAS (SEQ ID NO: 56) Exemplary LCDR3: QQWSSNPPA (SEQ ID NO: 48). 【0101】 Provided herein is a monoclonal antibody that specifically binds to human CD25. The monoclonal antibody comprises a variable heavy (VH) domain and a variable light (VL) domain. In some embodiments, the VH domain comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3, with the amino acid sequences of CDR1, CDR2, and CDR3 comprising SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively; and / or the VL domain comprises CDR1, CDR2, and CDR3, with the amino acid sequences of CDR1, CDR2, and CDR3 comprising SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively. 【0102】 In some examples, the amino acid sequences of CDR1, CDR2, and CDR3 of the VH domain comprise SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:52, respectively; and / or the amino acid sequences of CDR1, CDR2, and CDR3 of the VL domain comprise SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:48, respectively. In particular examples, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:21, and the CDR1, CDR2, and CDR3 of the VH domain comprise SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:52, respectively; and / or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO:22, and the CDR1, CDR2, and CDR3 of the VL domain comprise SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:48, respectively. 【0103】 In other examples, the amino acid sequences of CDR1, CDR2, and CDR3 of the VH domain comprise SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:52, respectively; and / or the amino acid sequences of CDR1, CDR2, and CDR3 of the VL domain comprise SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:48, respectively. In particular examples, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:31, and the CDR1, CDR2, and / or CDR3 of the VH domain comprise SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:52, respectively; and / or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO:32, and the CDR1, CDR2, and CDR3 of the VL domain comprise SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:48, respectively. 【0104】 In other examples, the amino acid sequences of CDR1, CDR2, and CDR3 of the VH domain comprise SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:53, respectively; and / or the amino acid sequences of CDR1, CDR2, and CDR3 of the VL domain comprise SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:48, respectively. In particular examples, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:37, and the CDR1, CDR2, and CDR3 of the VH domain comprise SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:53, respectively; and / or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO:38, and the CDR1, CDR2, and CDR3 of the VL domain comprise SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:48, respectively. 【0105】 In some embodiments, the monoclonal antibody comprises at least a portion of the amino acid sequence set forth as any one of SEQ ID NOs: 1-42, e.g., one or more (e.g., all three) CDR sequences from any one of SEQ ID NOs: 1-42. In some examples, the CDR positions are determined by IMGT, Kabat, Paratome, Chothia, or a combination of one or more thereof. 【0106】 In some examples, the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 1, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 2; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 3, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 4; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 5, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 6; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 7, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 8; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 9, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 10. the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 10; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 11 and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 12; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 13 and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 14; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 15 and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 16; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 17 and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 18; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 19, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 20; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 21, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 22; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 23, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 24; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 25, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 26; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 27, The VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 28; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 29, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 30; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 31, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 32; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 33, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 34; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 35, and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 36;The VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 37 and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 38; the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 39 and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 40; or the VH domain of the antibody comprises the CDR sequence of SEQ ID NO: 41 and the VL domain of the antibody comprises the CDR sequence of SEQ ID NO: 42. In particular examples, the CDR1, CDR2, and CDR3 of the VH domain are located at positions 31-35, 50-66, and 99-112, respectively, of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and / or the CDR1, CDR2, and CDR3 of the VL domain are located at positions 24-33, 49-55, and 88-96, respectively, of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; 【0107】 In specific examples, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 1, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 2; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 3, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 4; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 5, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 6; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 7, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 8; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 9, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 10; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 11, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 12; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 13, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 14; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 15, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 16; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 17, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 18; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 19, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 20; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 21, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 22; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 23, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 24; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 25, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 26; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 27, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 28;the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:29 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:30; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:31 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:32; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:33 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:34; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:35 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:36; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:37 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:38; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:39 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:40; or the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:41 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:42; 【0108】 In some embodiments, a CD25-specific monoclonal antibody does not significantly block the binding of IL-2 to CD25. In the context of the present disclosure, an antibody that does not significantly block the binding of IL-2 to CD25 is an antibody that reduces the binding of IL-2 to CD25 by less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0% (no detectable reduction in binding) compared to the binding of IL-2 to CD25 in the absence of the antibody. In some examples, a CD25-specific monoclonal antibody does not prevent the maturation or proliferation of T cells, e.g., CD8+ T cells. 【0109】 In some embodiments, a CD25-specific monoclonal antibody induces little or no ADCC. In the context of the present disclosure, an antibody that induces little or no ADCC is an antibody that induces cell death of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0% (no detectable cell death) of target cells in a cytotoxicity assay. In some examples, a CD25-specific monoclonal antibody induces cell death of less than 2% of target cells, for example, less than 1.5%, 1%, 0.5%, or 0.25% of target cells. Cytotoxicity assays that can be used to evaluate antibodies for ADCC activity are well known, and exemplary assays are described in Example 3. Suitable target cells are cells expressing human CD25, such as KIT-225 cells. 【0110】 In some embodiments of the disclosed monoclonal antibodies, the monoclonal antibody is an antigen-binding fragment selected from a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain variable fragment (scFv), and a disulfide-stabilized variable fragment (dsFv). 【0111】 In some embodiments, the monoclonal antibody is an IgG, e.g., an IgG4. In other embodiments, the monoclonal antibody is an IgA, IgD, IgE, or IgM. 【0112】 In some aspects, the monoclonal antibody is a humanized, chimeric, or synthetic monoclonal antibody. 【0113】 Also provided herein are heavy and light chain sequences comprising the VH and VL domains, respectively, of the CD25-specific monoclonal antibodies disclosed herein. In some embodiments, the heavy and / or light chain amino acid sequence comprises a leader sequence, e.g., MGWSCIILFLVATATGVHS (SEQ ID NO: 57). In some embodiments, the heavy chain comprises a modified human IgG4 constant region set forth as SEQ ID NO: 58. In some embodiments, the light chain comprises a human Ig kappa constant region set forth as SEQ ID NO: 49. In other embodiments, the heavy chain constant region is a constant region from a different isotype, e.g., IgG1, but modified to prevent induction of ADCC. 【0114】 Human (modified) IgG4 constant region (SEQ ID NO: 58) [ka] Human Ig kappa constant region (SEQ ID NO: 59) [ka] 【0115】 The heavy and light chain sequences for each CD25-specific antibody are provided below, including the leader sequence (SEQ ID NO: 57), VH or VL domain (underlined), and modified human IgG4 constant region (SEQ ID NO: 58; for heavy chain sequences) or human Ig kappa constant region (SEQ ID NO: 59; for light chain sequences). 【0116】 14554-VH (SEQ ID NO: 60) [ka] 【0117】 14554-VL (SEQ ID NO: 61) [ka] 【0118】 14555-VH (SEQ ID NO: 62) [ka] 【0119】 14555-VL (SEQ ID NO: 63) [ka] 【0120】 14556-VH (SEQ ID NO: 64) [ka] 【0121】 14556-VL (SEQ ID NO: 65) [ka] 【0122】 14557-VH (SEQ ID NO: 66) [ka] 【0123】 14557-VL (SEQ ID NO: 67) [ka] 【0124】 14558-VH (SEQ ID NO: 68) [ka] 【0125】 14558-VL (SEQ ID NO: 69) [ka] 【0126】 14559-VH (SEQ ID NO: 70) [ka] 【0127】 14559-VL (SEQ ID NO: 71) [ka] 【0128】 14560-VH (SEQ ID NO: 72) [ka] 【0129】 14560-VL (SEQ ID NO: 73) [ka] 【0130】 14561-VH (SEQ ID NO: 74) [ka] 【0131】 14561-VL (SEQ ID NO: 75) [ka] 【0132】 14562-VH (SEQ ID NO: 76) [ka] [ka] 【0133】 14562-VL (SEQ ID NO: 77) [ka] 【0134】 14563-VH (SEQ ID NO: 78) [ka] 【0135】 14563-VL (SEQ ID NO: 79) [ka] 【0136】 14564-VH (SEQ ID NO: 80) [ka] 【0137】 14564-VL (SEQ ID NO: 81) [ka] 【0138】 14565-VH (SEQ ID NO: 82) [ka] 【0139】 14565-VL (SEQ ID NO: 83) [ka] 【0140】 14566-VH (SEQ ID NO: 84) [ka] 【0141】 14566-VL (SEQ ID NO: 85) [ka] 【0142】 14567-VH (SEQ ID NO: 86) [ka] 【0143】 14567-VL (SEQ ID NO: 87) [ka] 【0144】 14568-VH (SEQ ID NO: 88) [ka] 【0145】 14568-VL (SEQ ID NO: 89) [ka] 【0146】 14569-VH (SEQ ID NO: 90) [ka] 【0147】 14569-VL (SEQ ID NO: 91) [ka] 【0148】 14570-VH (SEQ ID NO: 92) [ka] 【0149】 14570-VL (SEQ ID NO: 93) [ka] 【0150】 14571-VH (SEQ ID NO: 94) [ka] 【0151】 14571-VL (SEQ ID NO: 95) [ka] 【0152】 14572-VH (SEQ ID NO: 96) [ka] 【0153】 14572-VL (SEQ ID NO: 97) [ka] 【0154】 14573-VH (SEQ ID NO: 98) [ka] 【0155】 14573-VL (SEQ ID NO: 99) [ka] 【0156】 U579-WT-VH (SEQ ID NO: 100) [ka] 【0157】 U579-WT-VL (SEQ ID NO: 101) [ka] 【0158】 In some embodiments disclosed herein, the monoclonal antibody further comprises a constant region, e.g., a constant region comprising at least one modification that increases the half-life, stability, and / or function of the monoclonal antibody. In some examples, the constant region comprises a heavy chain constant region and / or a light chain constant region. In a specific example, the heavy chain constant region is a human IgG4 constant region, e.g., a human IgG4 constant region having the amino acid sequence of SEQ ID NO: 58, and / or the light chain constant region is a human Igκ light chain, e.g., a human Igκ light chain having the amino acid sequence of SEQ ID NO: 59. 【0159】 In a specific, non-limiting example, the monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain and light chain comprise or consist of the amino acid sequences set forth as follows, respectively: SEQ ID NO:60 and SEQ ID NO:61; SEQ ID NO:62 and SEQ ID NO:63; SEQ ID NO:64 and SEQ ID NO:65; SEQ ID NO:66 and SEQ ID NO:67; SEQ ID NO:68 and SEQ ID NO:69; SEQ ID NO:70 and SEQ ID NO:71; SEQ ID NO:72 and SEQ ID NO:73; SEQ ID NO:74 and SEQ ID NO:75; SEQ ID NO:76 and SEQ ID NO:77; SEQ ID NO:78 and SEQ ID NO:79; SEQ ID NO:80 and SEQ ID NO:81; SEQ ID NO:82 and SEQ ID NO:83; SEQ ID NO:84 and SEQ ID NO:85; SEQ ID NO:86 and SEQ ID NO:87; SEQ ID NO:88 and SEQ ID NO:89; SEQ ID NO:90 and SEQ ID NO:91; SEQ ID NO:92 and SEQ ID NO:93; SEQ ID NO:94 and SEQ ID NO:95; SEQ ID NO:96 and SEQ ID NO:97; SEQ ID NO:98 and SEQ ID NO:99; or SEQ ID NO:100 and SEQ ID NO:101. 【0160】 Also provided is a composition comprising the CD25-specific monoclonal antibodies disclosed herein and a pharmaceutically acceptable carrier. The disclosed CD25-specific monoclonal antibodies can also be part of a kit. 【0161】 IV. Antibody Conjugates The CD25-specific monoclonal antibodies disclosed herein can be conjugated to agents, such as photon absorbers (e.g., IR700), effector molecules, or detectable labels, using any number of suitable means. Both covalent and non-covalent binding means can be used. IR700, toxins, and radioactive agents, such as 125 I, 32 P, 14 C. 3 H and 35 A variety of photon absorbers, effector molecules and detectable markers may be used, including (but not limited to) S, as well as other labels, targeting moieties and ligands. 【0162】 In some embodiments, the present invention provides a conjugate comprising the CD25-specific monoclonal antibody disclosed herein and a photon absorber, an effector molecule or a detectable label.In some examples, the photon absorber is IR700; the effector molecule is a toxin; or the detectable label is a fluorescent label, an enzymatic label or a radioactive label. 【0163】 Also provided herein are compositions comprising the conjugates disclosed herein and a pharmaceutically acceptable carrier. 【0164】 The selection of a particular photon absorber, effector molecule, or detectable marker will depend on the particular target molecule or cell and the desired biological effect. Thus, for example, an effector molecule can be a cytotoxin used to cause the death of a particular target cell (e.g., a CD25-positive cell). 【0165】 In some embodiments, the photon absorber is IR700 (IRDye® 700DX), commercially available from Rakuten Medical (San Diego, Calif.). Amino-reactive IR700 is a relatively hydrophilic dye that can be covalently conjugated to antibodies using an NHS ester of IR700. 【0166】 The procedure for attaching a photon absorber, effector molecule, or detectable marker to an antibody varies depending on the chemical structure of the conjugate. Polypeptides typically contain various functional groups available for reaction with an appropriate functional group, such as a carboxylic acid (COOH), free amine (-NH), or sulfhydryl (-SH) group, to result in attachment of the photon absorber, effector molecule, or detectable marker. Alternatively, the antibody can be derivatized to expose or attach additional reactive functional groups. Derivatization can include attachment of any of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to link an antibody to a photon absorber, effector molecule, or detectable marker. The linker is capable of forming a covalent bond to both the antibody and the photon absorber / effector molecule / detectable marker. Suitable linkers include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and effector molecule or detectable marker are polypeptides, the linkers may be linked to the constituent amino acids through their side groups (e.g., to cysteine via a disulfide linkage) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acid. 【0167】 The antibody can be conjugated to a detectable marker, such as a marker that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic testing, and laparoscopy). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide fluorophores, and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also used. Antibodies or antigen-binding fragments can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When an antibody or antigen-binding fragment is conjugated to a detectable enzyme, it can be detected by adding an additional reagent that the enzyme uses to produce a discernible reaction product. For example, in the presence of the agent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. Antibodies or antigen-binding fragments can also be conjugated to biotin and detected through indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label. 【0168】 The antibody can be conjugated with a paramagnetic agent, such as gadolinium. Paramagnetic agents, such as superparamagnetic iron oxide, can also be used as labels. The antibody can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. The antibody can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag). 【0169】 Antibodies can also be conjugated with radiolabeled amino acids.Radiolabeling can be used for both diagnostic and therapeutic purposes.For example, radiolabeling can be used to detect CD25-expressing cells by x-ray, emission spectroscopy or other diagnostic techniques.Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I. 【0170】 Any known means for detecting a detectable marker can be used. Thus, for example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. 【0171】 The average number of photon absorbers, effector molecules, or detectable marker moieties per antibody in the conjugate can range, for example, from 1 to 20 moieties per antibody. In certain embodiments, the average number of photon absorbers, effector molecules, or detectable marker moieties per antibody in the conjugate ranges from about 1 to about 2, about 1 to about 3, about 1 to about 8, about 2 to about 6, about 3 to about 5, about 2 to about 3, or about 3 to about 4. In a specific, non-limiting example, the conjugate comprises one CD25-specific monoclonal antibody conjugated to two or three IR700 molecules. The loading of the conjugate (e.g., effector molecule / antibody ratio) can be controlled in different ways, for example, by: (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent compared to the antibody; (ii) limiting the time or temperature of the conjugation reaction; (iii) partial or limited reducing conditions for cysteine thiol modification; (iv) recombinantly manipulating the amino acid sequence of the antibody so that the number and position of cysteine residues are modified to control the number or position of linker-effector molecule bonds. 【0172】 V. Nucleic acid molecules Nucleic acid molecules (e.g., DNA, RNA, or cDNA molecules) encoding the amino acid sequences of the disclosed CD25-specific antibodies and conjugates are provided. The nucleic acids encoding these molecules can be encoded by any of the amino acid sequences provided herein (e.g., CDR sequences and V sequences). H and V L sequences), sequences available in the art (e.g., framework or constant region sequences) and the genetic code. In some embodiments, nucleic acid molecules can be readily produced using the V sequences of the disclosed antibodies. H , V L , or V H and V L In some embodiments, the nucleic acid molecule can be expressed in a host cell (e.g., a mammalian cell) to produce the disclosed antibodies. 【0173】 The genetic code encodes a variety of functionally equivalent nucleic acids, e.g., different sequences but the same antibody sequence, or V L and / or V H It can be used to construct nucleic acids that encode conjugates or fusion proteins that include the nucleic acid sequences. 【0174】 Nucleic acid sequences for the VH and VL domains (SEQ ID NOS: 102-143), exemplary leader sequences (SEQ ID NOS: 144 and 145), an exemplary human IgG4 constant region coding sequence (SEQ ID NO: 146), and an exemplary human Igκ light chain coding sequence (SEQ ID NO: 147) of the disclosed CD25-specific monoclonal antibodies are provided below. Nucleic acid sequences encoding the complete heavy and light chains of the disclosed monoclonal antibodies (SEQ ID NOS: 148-189) are further provided. 【0175】 14554-VH (SEQ ID NO: 102) [ka] 【0176】 14554-VL (SEQ ID NO: 103) [ka] 【0177】 14555-VH (SEQ ID NO: 104) [ka] 【0178】 14555-VL (SEQ ID NO: 105) [ka] 【0179】 14556-VH (SEQ ID NO: 106) [ka] 【0180】 14556-VL (SEQ ID NO: 107) [ka] 【0181】 14557-VH (SEQ ID NO: 108) [ka] 【0182】 14557-VL (SEQ ID NO: 109) [ka] 【0183】 14558-VH (SEQ ID NO: 110) [ka] 【0184】 14558-VL (SEQ ID NO: 111) [ka] [ka] 【0185】 14559-VH (SEQ ID NO: 112) [ka] 【0186】 14559-VL (SEQ ID NO: 113) [ka] 【0187】 14560-VH (SEQ ID NO: 114) [ka] 【0188】 14560-VL (SEQ ID NO: 115) [ka] 【0189】 14561-VH (SEQ ID NO: 116) [ka] 【0190】 14561-VL (SEQ ID NO: 117) [ka] 【0191】 14562-VH (SEQ ID NO: 118) [ka] 【0192】 14562-VL (SEQ ID NO: 119) [ka] 【0193】 14563-VH (SEQ ID NO: 120) [ka] 【0194】 14563-VL (SEQ ID NO: 121) [ka] 【0195】 14564-VH (SEQ ID NO: 122) [ka] 【0196】 14564-VL (SEQ ID NO: 123) [ka] 【0197】 14565-VH (SEQ ID NO: 124) [ka] 【0198】 14565-VL (SEQ ID NO: 125) [ka] 【0199】 14566-VH (SEQ ID NO: 126) [ka] 【0200】 14566-VL (SEQ ID NO: 127) [ka] 【0201】 14567-VH (SEQ ID NO: 128) [ka] 【0202】 14567-VL (SEQ ID NO: 129) [ka] 【0203】 14568-VH (SEQ ID NO: 130) [ka] [ka] 【0204】 14568-VL (SEQ ID NO: 131) [ka] 【0205】 14569-VH (SEQ ID NO: 132) [ka] 【0206】 14569-VL (SEQ ID NO: 133) [ka] 【0207】 14570-VH (SEQ ID NO: 134) [ka] 【0208】 14570-VL (SEQ ID NO: 135) [ka] 【0209】 14571-VH (SEQ ID NO: 136) [ka] 【0210】 14571-VL (SEQ ID NO: 137) [ka] 【0211】 14572-VH (SEQ ID NO: 138) [ka] 【0212】 14572-VL (SEQ ID NO: 139) [ka] 【0213】 14573-VH (SEQ ID NO: 140) [ka] 【0214】 14573-VL (SEQ ID NO: 141) [ka] 【0215】 U579-WT-VH (SEQ ID NO: 142) [ka] 【0216】 U579-WT-VL (SEQ ID NO: 143) [ka] Leader for VH (SEQ ID NO: 144): [ka] Leader for VL (SEQ ID NO: 145): [ka] 【0217】 Human IgG4 constant region (SEQ ID NO: 146): [ka] 【0218】 Human immunoglobulin kappa light chain (SEQ ID NO: 147): [ka] 【0219】 14554-VH (SEQ ID NO: 148) [ka] [ka] 【0220】 14554-VL (SEQ ID NO: 149) [ka] 【0221】 14555-VH (SEQ ID NO: 150) [ka] 【0222】 14555-VL (SEQ ID NO: 151) [ka] 【0223】 14556-VH (SEQ ID NO: 152) [ka] [ka] 【0224】 14556-VL (SEQ ID NO: 153) [ka] 【0225】 14557-VH (SEQ ID NO: 154) [ka] 【0226】 14557-VL (SEQ ID NO: 155) [ka] 【0227】 14558-VH (SEQ ID NO: 156) [ka] [ka] 【0228】 14558-VL (SEQ ID NO: 157) [ka] 【0229】 14559-VH (SEQ ID NO: 158) [ka] 【0230】 14559-VL (SEQ ID NO: 159) [ka] 【0231】 14560-VH (SEQ ID NO: 160) [ka] [ka] 【0232】 14560-VL (SEQ ID NO: 161) [ka] 【0233】 14561-VH (SEQ ID NO: 162) [ka] 【0234】 14561-VL (SEQ ID NO: 163) [ka] 【0235】 14562-VH (SEQ ID NO: 164) [ka] [ka] 【0236】 14562-VL (SEQ ID NO: 165) [ka] 【0237】 14563-VH (SEQ ID NO: 166) [ka] 【0238】 14563-VL (SEQ ID NO: 167) [ka] 【0239】 14564-VH (SEQ ID NO: 168) [ka] [ka] 【0240】 14564-VL (SEQ ID NO: 169) [ka] 【0241】 14565-VH (SEQ ID NO: 170) [ka] 【0242】 14565-VL (SEQ ID NO: 171) [ka] 【0243】 14566-VH (SEQ ID NO: 172) [ka] [ka] 【0244】 14566-VL (SEQ ID NO: 173) [ka] 【0245】 14567-VH (SEQ ID NO: 174) [ka] 【0246】 14567-VL (SEQ ID NO: 175) [ka] 【0247】 14568-VH (SEQ ID NO: 176) [ka] [ka] 【0248】 14568-VL (SEQ ID NO: 177) [ka] 【0249】 14569-VH (SEQ ID NO: 178) [ka] 【0250】 14569-VL (SEQ ID NO: 179) [ka] 【0251】 14570-VH (SEQ ID NO: 180) [ka] [ka] 【0252】 14570-VL (SEQ ID NO: 181) [ka] 【0253】 14571-VH (SEQ ID NO: 182) [ka] 【0254】 14571-VL (SEQ ID NO: 183) [ka] 【0255】 14572-VH (SEQ ID NO: 184) [ka] [ka] 【0256】 14572-VL (SEQ ID NO: 185) [ka] 【0257】 14573-VH (SEQ ID NO: 186) [ka] 【0258】 14573-VL (SEQ ID NO: 187) [ka] 【0259】 U579-WT-VH (SEQ ID NO: 188) [ka] [ka] 【0260】 U579-WT-VL (SEQ ID NO: 189) [ka] 【0261】 Thus, in some embodiments, the nucleic acid molecule encodes the VH domain, the VL domain, or both the VH and VL domains of a CD25-specific monoclonal antibody. In some examples, the nucleic acid molecule encodes the complete heavy chain, the complete light chain, or both the complete heavy chain and the complete light chain of a CD25-specific monoclonal antibody. 【0262】 In specific embodiments, the nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 102 to 143, or a degenerate variant thereof. In some examples, the nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, and 142, or a degenerate variant thereof; and the nucleotide sequence of any one of SEQ ID NOs: 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, and 143, or a degenerate variant thereof. 【0263】 In other specific embodiments, the nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 148 to 189, or a degenerate variant thereof. In some examples, the nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, and 188, or a degenerate variant thereof; and the nucleotide sequence of any one of SEQ ID NOs: 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, and 189, or a degenerate variant thereof. 【0264】 In some embodiments, the disclosed nucleic acid molecules further comprise one or more stop codons at the 3' end, eg, TGA, TAA, or TGATAA. 【0265】 In particular examples, the nucleic acid molecule comprises: the nucleotide sequence of SEQ ID NO: 122, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 123, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 122 and SEQ ID NO: 123, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 132, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 133, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 132 and SEQ ID NO: 133, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 138, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 139, or a degenerate variant thereof; the nucleotide sequences of SEQ ID NO: 138 and SEQ ID NO: 139, or a degenerate variant thereof. Degenerate variants; the nucleotide sequence of SEQ ID NO: 168, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 169, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 168 and SEQ ID NO: 169, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 178, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 179, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 178 and SEQ ID NO: 179, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 184, or a degenerate variant thereof; the nucleotide sequence of SEQ ID NO: 185, or a degenerate variant thereof; or the nucleotide sequences of SEQ ID NO: 184 and .... 【0266】 Further provided is a vector comprising one or more of the nucleic acid molecules disclosed herein.In some embodiments, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector or an adeno-associated viral vector. 【0267】 Also provided are isolated host cells comprising the disclosed nucleic acid molecules or vectors. In some aspects, the isolated host cells are eukaryotic cells, e.g., mammalian cells. In other aspects, the isolated host cells are prokaryotic cells. 【0268】 V. Kit Kits comprising the CD25-specific monoclonal antibodies, conjugates, compositions, nucleic acid molecules, vectors, and / or isolated host cells disclosed herein are also provided. In some embodiments, the kits further comprise a buffer, cell culture medium, one or more checkpoint inhibitors, one or more immunotherapies, one or more additional anti-cancer reagents (e.g., chemotherapeutic agents or biologics), one or more reducing agents, one or more transfection reagents, and / or instructional materials. 【0269】 For example, a kit for treating cancer in a subject is provided. The kit may include the disclosed CD25-specific monoclonal antibody, CD25 antibody-IR700 conjugate, or a composition comprising a CD25-specific monoclonal antibody or a CD25 antibody-IR700 conjugate. More than one of the disclosed CD25-specific antibody, CD25 antibody-IR700 conjugate, or composition comprising such a molecule may be included in the kit. 【0270】 In some examples, the kit further comprises one or more buffers, for example, PBS. 【0271】 In some examples, the kit further includes one or more cell culture media, such as media used for propagating prokaryotic or eukaryotic cells in vitro. Such culture media can be solid, liquid, or powder. In one example, the cell culture medium is DMEM or RPMI. In one example, the cell culture medium is a bacterial culture medium. In one example, the cell culture medium is a medium optimized for transfection. 【0272】 In some examples, the kit further comprises one or more transfection reagents, for example, liposome, lipid-rich, or non-liposome-based reagents (e.g., calcium phosphate-based). 【0273】 In some examples, the kit further comprises one or more checkpoint inhibitors, e.g., those that reduce or inhibit the function of PD-1, PD-L1, and / or CTLA-4. In one example, the kit further comprises a PD-L1 antibody, e.g., atezolizumab, avelumab, durvalumab, or cosibelimab (in some examples, such mAbs are conjugated to IR700, e.g., atezolizumab-IR700, avelumab-IR700, durvalumab-IR700, or cosibelimab-IR700). In one example, the kit further comprises a PD-1 antibody, e.g., nivolumab, pembrolizumab, cemiplimab, or dostallimab (in some examples, such mAbs are conjugated to IR700). In one example, the kit further comprises a CTLA-4 antibody, such as ipilimumab or tremelimumab (in some examples, such mAbs are conjugated to IR700). 【0274】 In some examples, the kit further comprises one or more immunotherapies, e.g., one or more immune modulators and / or immune activators listed below, e.g., IL-2, IL-15, IL-7, IL-12 and / or IL-21. 【0275】 In some examples, the kit further comprises one or more chemotherapeutic agents, such as one or more of those listed below, for example, one or more microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and / or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators. In one example, the kit further comprises one or more of carboplatin, cisplatin, paclitaxel, docetaxel, doxorubicin, epirubicin, topotecan, irinotecan, gemcitabine, tiazofurin, gemcitabine, etoposide, vinorelbine, tamoxifen, valspodar, cyclophosphamide, methotrexate, fluorouracil, mitoxantrone, Doxil (liposome-encapsulated doxorubicin), and vinorelbine. 【0276】 In some examples, the kit includes one or more biologics, such as 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, besilesomab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide, catumaxomab, CC49, cetuximab, sitatuzumab bogatox Bogatox, Cixutumumab, Crivatuzumab Tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab Ozogamicin, Girentuximab, Glembatumumab Vedotin, Ibritumomab Tiuxetan, Igovomab mab, Imciromab, Intetumumab, Inotuzumab Ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab Mertansine, Lucatumumab, Rumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab Butafenatox tafenatox), naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentane (Nofetumomabmerpentan, ofatumumab, olaratumab, oportuzumab monatox, oregovomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, robatumumab, satumomab pendetide, sibrotuzumab, sonepcizumab, tacatuzumab tetraxetan, taplitumomab paptox paptox, Tenatumomab, TGN1412, Ticilimumab (Tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab or Zalutumumab. 【0277】 In one example, the kit further comprises a tumor-specific antibody-IR700 conjugate, such as cetuximab-IR700, trastuzumab-IR700, tivozanib-IR700, or any other antibody-IR700 conjugate provided in Table 1. 【0278】 In some examples, the kit further comprises one or more reducing agents, such as one or more of L-cysteine, sodium L-ascorbate (L-NaAA), ascorbic acid (e.g., L- or R-ascorbic acid), and glutathione. In a specific example, the kit further comprises L-NaAA. 【0279】 The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed CD25-specific monoclonal antibodies, CD25 antibody-IR700 conjugates, or compositions. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). 【0280】 The label or package insert indicates that the composition is used to treat a specific condition. The label or package insert typically further includes instructions for using the antibody, conjugate, or composition included in the kit. The package insert typically includes instructions that are customarily included in the commercially available packaging of a therapeutic product, including information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such a therapeutic product. The instruction material can be written, electronic, or visual. The kit can also include additional components to facilitate the specific application for which the kit is designed. 【0281】 VI. Methods of Treating Cancer Also provided herein are methods for treating cancer in a subject. The cancer can be a solid tumor or a hematological malignancy, such as an immunogenic one (e.g., T cell-rich). These methods include administering to a subject a therapeutically effective amount of a conjugate comprising a CD25-specific monoclonal antibody and a photon absorber, or a composition thereof, disclosed herein, followed by irradiation with a wavelength of 660-740 nm and at least 1 J / cm. 2 (e.g., at least 4 J / cm 2irradiating the subject and / or irradiating cancer cells in the subject with a dose of 1000 mg / kg / day (e.g., 1000 mg / kg / day). In some aspects, the cancer is cancer of the breast, liver, lung, esophagus, stomach, colon, ovary, prostate, pancreas, brain, cervix, kidney, bone, skin, head and neck, oropharynx, or blood. In some aspects, the conjugate is administered locally at or near the site of the tumor. In other aspects, the conjugate is administered systemically, and the effect of the treatment is local to the cancer (e.g., has no significant adverse systemic effects). 【0282】 In some embodiments, the method further comprises administering a therapeutically effective amount of a second monoclonal antibody conjugated to IR700, wherein the second monoclonal antibody specifically binds to a tumor antigen expressed by cells of the cancer. In some examples, the tumor antigen is selected from the group consisting of epidermal growth factor receptor (EGFR / HER1), mesothelin, prostate-specific membrane antigen (PSMA), HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), Lewis Y, tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2 (EphA2), and glypican-1. (GPC1), glypican-2 (GPC2), glypican-3 (GPC3), gpA33, mucin, CAIX, folate-binding protein, ganglioside, integrin αVβ3, integrin α5β3,1, Erb-B2 receptor tyrosine kinase 3 (ERBB3), MET proto-oncogene, receptor tyrosine kinase (MET), insulin-like growth factor 1 receptor (IGF1R), ephrin type A receptor 3 (EPHA3), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAILR1), TRAILR2, nuclear factor kappa-B receptor activator ligand (receptor The tumor antigen is selected from: nuclear factor kappa-B ligand (RANKL), fibroblast activation protein (FAP), tenascin, BCR complex, gp72, HLA-DR 10β, HLA-DR antigen, IgE, CA242, polymorphic epithelial mucin (PEM) antigen, and SK-1 antigen. In some aspects, the method further comprises administering a therapeutically effective amount of a second monoclonal antibody conjugated to IR700, wherein the second monoclonal antibody specifically binds to a checkpoint inhibitor, e.g., programmed cell death 1 (PD-1), CTLA4, and programmed cell death ligand (PD-L1). Specific exemplary tumor antigens and checkpoint inhibitor antibodies that can be used are provided herein.In some examples, the CD25-specific conjugate and the second monoclonal antibody conjugated to IR700 are both administered prior to irradiation. In some examples, the CD25-specific conjugate and the second monoclonal antibody conjugated to IR700 are administered intravenously. 【0283】 In some aspects, the methods provided herein further comprise administering to the subject one or more additional cancer therapies, in some examples, the one or more additional cancer therapies include immune activators, chemotherapy, radiation therapy, and / or surgery. 【0284】 The immune activator includes one or more inhibitors of immune system activators and / or immune suppressor cells (e.g., in the presence of a pharmaceutically acceptable carrier, e.g., a pharmaceutically and physiologically acceptable fluid). In a specific example, the immune activator is IL-15. In another specific example, the immune activator is interferon gamma. In some examples, the one or more immune activators are administered to the subject concurrently (e.g., simultaneously or substantially simultaneously) with one or more antibody-IR700 conjugates, e.g., in the same composition. In other examples, the one or more CD25-specific conjugates and the one or more immune activators are administered to the subject sequentially (in either order), e.g., separated by at least about 1 hour (e.g., within about 5 minutes, within about 10 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 40 minutes, within about 50 minutes, within about 60 minutes, within about 2 hours, within about 12 hours, within about 24 hours, within about 48 hours, within about 3 days, within about 4 days, within about 5 days, within about 6 days, or within about 7 days) from each other. 【0285】 In some examples, the subject is further administered a therapeutically effective amount of one or more reducing agents, for example, to reduce edema that may result from NIR-PIT. In some examples, the antibody-IR700 conjugate(s) and reducing agent(s) are present in a single composition and are administered simultaneously. In other examples, the antibody-IR700 conjugate and reducing agent(s) are present in separate compositions and are administered simultaneously or contemporaneously, or sequentially (e.g., with at least 1 minute, at least 5 minutes, at least 30 minutes, at least 60 minutes, at least 12 hours, or at least 24 hours between them, e.g., antibody-IR700 conjugate(s) followed by reducing agent(s)). 【0286】 After administering the CD25 antibody-IR700 conjugate, and optionally one or more tumor antigen-targeting antibody-IR700 conjugates and / or one or more checkpoint inhibitor antibody-IR700 conjugates, the conjugate(s) accumulate in the targeted tumor or immune cells of the tumor. The tumor cells and other cells in the tumor bed (or subject with cancer) are then irradiated under conditions that allow killing of the cells to which the antibody-IR700 conjugate(s) have bound (e.g., at least 1 J / cm). 2 at a dose of 660-740 nm (e.g., 600-710 nm) at a wavelength of 4-60 J / cm 2 Dose, for example, 4-25J / cm 2 , 4~10J / cm 2 , 20~50J / cm 2 or 20-30J / cm 2 Irradiation at 680 nm or 690 nm at a dose of 1000 nm. 【0287】 In one example, there is at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, or at least about 48 hours (e.g., about 1-4 hours, 30 minutes to 1 hour, 10 minutes to 60 minutes, 30 minutes to 8 hours, 2-10 hours, 12-24 hours, 18-36 hours, or 24-48 hours) between administration of the antibody-IR700 conjugate(s) and irradiation. In one example, one or more antibody-IR700 conjugate(s) are administered (e.g., iv, ip, or intratumorally) and the tumor (or subject) is irradiated at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, or at least about 48 hours (e.g., about 1-4 hours, 30 minutes to 1 hour, 10 minutes to 60 minutes, 30 minutes to 8 hours, 2-10 hours, 12-24 hours, 18-36 hours, or 24-48 hours, e.g., about 12 hours or about 24 hours). One or more immune activators can be administered (e.g., systemically, e.g., iv or ip) before or after the one or more antibody-IR700 conjugate(s) and before or after the irradiation. In some examples, the one or more immune activators are administered before and after irradiation, e.g., at least one dose of immune activator before irradiation and at least one dose of immune activator after irradiation (e.g., 24 hours before irradiation and one or more of 24, 48, 72, 96, or more hours after irradiation). In additional examples, the doses of immune activator may also be administered on the same day as the at least one irradiation treatment. The one or more reducing agents may be administered (e.g., systemically, e.g., iv or ip) before or after the one or more antibody-IR700 conjugate(s), but before irradiation (e.g., at least 5, at least 10, at least 15, or at least 30 minutes, e.g., 5-60 minutes before irradiation). 【0288】 In some examples, multiple doses of one or more of the antibody-IR700 conjugate(s) are administered to the subject, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In some examples, multiple doses of one or more CTLA4 antibody-IR700 conjugates, one or more PD-L1 antibody-IR700 conjugates, one or more PD-1 antibody-IR700 conjugates, or combinations thereof are administered to the subject, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In some examples, the subject is administered multiple doses of one or more immune activators, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In some examples, the subject is administered multiple doses of one or more reducing agents, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In some examples, the subject is administered multiple doses of NIR irradiation, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten separate doses (or administrations). In a specific example, the subject is administered at least one dose of one or more antibody-IR700 conjugate(s), at least two doses of one or more immune activators, and at least two separate administrations of NIR irradiation. 【0289】 NIR excitation light wavelengths allow penetration into tissues of at least several centimeters. For example, by using a fiber-coupled laser diode with a diffuser tip, NIR light can be delivered within several centimeters of otherwise inaccessible tumors located deep relative to the body surface. In addition to treating solid cancers, circulating tumor cells (including, but not limited to, hematological malignancies) can be targeted because they can be excited when crossing superficial blood vessels (e.g., using the wearable NIR LED device described herein). 【0290】 In some examples, the disclosed methods kill at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98% or more of the treated target cells (e.g., cancer cells expressing a tumor-specific antigen, or CD25+ immune cells in the tumor bed, e.g., Tregs in the tumor bed), e.g., compared to the absence of treatment with the disclosed methods. 【0291】 In some examples, the disclosed methods reduce the weight, volume, or size of metastases, e.g., by at least 10%, at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100%, compared to, for example, the absence of treatment with the disclosed methods. In some examples, the disclosed methods have an abscopal effect, reducing the metastasis weight, volume, or size by at least 1 Jcm at wavelengths of 660-740 nm. -2 at a dose of (e.g., at least 4 J / cm -2 , at least 10Jcm -2 For example, 4 to 50 Jcm -2 , 1~10Jcm -2 or 1 to 5 Jcm -2or a reduction in the weight, volume, or size of metastases located remotely from the irradiated area of the tumor or lesion that are not themselves irradiated with a dose of 1000 mg / kg or more (e.g., 100 mg / kg or more). In some cases, the weight, volume, or size of non-irradiated metastases is reduced by at least 10%, at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% upon treatment with the disclosed methods. 【0292】 Treg cells compared to other immune cells (e.g., CD4 + Foxp3 + By selectively killing Tregs, these methods can kill irradiated Treg cells more effectively than other immune cells, such as Tregs that are not in the tumor bed. In one example, the disclosed methods can kill Tregs (e.g., FOXP3 Tregs) that are in the tumor bed but not in other parts of the body, such as the spleen or regional lymph nodes. + CD4 + Treg cells). For example, the disclosed methods may reduce the number of Treg cells in the tumor bed by at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or more, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation). In some examples, the disclosed methods do not substantially reduce the number of Treg cells in the spleen or tumor-draining lymph nodes (e.g., a reduction of 10% or less, 5% or less, 1% or less, or 0.5% or less), compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation). 【0293】 In some examples, the disclosed methods reduce intratumoral blood perfusion by at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98% or more compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation). 【0294】 In some examples, the disclosed methods reduce one or more symptoms associated with tumors, recurrence, and / or metastatic tumors. In one example, the disclosed methods slow tumor growth, e.g., by at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, or more, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation). In one example, the disclosed methods reduce or eliminate tumor recurrence, e.g., by at least 10%, e.g., at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100%, compared to the absence of treatment with the disclosed methods (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation). 【0295】 In some examples, the disclosed methods increase the survival time of a subject (e.g., a subject having a tumor or a subject from whom a tumor has previously been removed) compared to, for example, the absence of treatment with a disclosed method (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation). In some examples, the survival time of a subject is increased by at least 20%, at least 25%, at least 40%, at least 50%, at least 80%, at least 90% or more compared to, for example, the absence of treatment with a disclosed method (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation). In some examples, the subject's survival time is increased by at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or longer, e.g., compared to the absence of treatment with a disclosed method (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation). For example, the disclosed methods, in some examples, increase a subject's progression-free or disease-free survival time (e.g., lack of recurrence of a primary tumor or lack of metastasis) by at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, at least 60 months, or longer, compared to the average survival time in the absence of treatment with a disclosed method (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation). 【0296】 In some examples, the use of one or more reducing agents in combination with NIR-PIT reduces edema in a treated subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or even at least 95% compared to the amount of edema without the use of one or more reducing agents in combination with NIR-PIT. In some examples, the use of one or more reducing agents in combination with NIR-PIT reduces an acute inflammatory response in a treated subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or even at least 95% compared to the amount of acute inflammatory response without the use of one or more reducing agents in combination with NIR-PIT. 【0297】 In one example, a combination of the above-listed advantages is achieved using the disclosed method. 【0298】 The disclosed methods can be used to treat fixed tumors in the body as well as circulating hematological malignancies and / or tumors (e.g., leukemia cells, metastases, and / or circulating tumor cells). However, circulating cells, by their nature, cannot be exposed to light for very long. Therefore, when the cells to be killed are cells circulating throughout the body, these methods can be achieved by using a device that can be worn or that covers a body part. For example, such devices can be worn for extended periods of time. Wearable everyday items (e.g., wristwatches, jewelry (e.g., necklaces or bracelets), blankets, clothing (e.g., underwear, socks, and shoe insoles), and other wearable everyday items) incorporating NIR-emitting light-emitting diodes (LEDs) and battery packs can be used. Such devices generate light to the skin underlying the device for an extended period of time, resulting in continuous exposure of superficial blood vessels to light for an extended period of time. Circulating tumor cells are exposed to light as they pass through the area underlying the device. For example, a wristwatch or bracelet version of this device could include a series of NIR LEDs with a battery power pack that would be worn for most of the day. Following administration of one or more CD25 antibody-IR700 conjugates (e.g., systemically, e.g., intravenously and / or intraperitoneally, in combination with one or more tumor-specific Ab-IR700 conjugates or checkpoint inhibitor Ab-IR700 conjugates (e.g., CTLA4 antibody-IR700 conjugates, PD-L1 antibody-IR700 conjugates, PD-L1 antibody-IR700 conjugates), one or more reducing agents, one or more immune activators, or a combination thereof), circulating cells bind to the antibody-IR700 conjugates and become susceptible to killing by PIT. As these cells flow within blood vessels adjacent to the LEDs present in the daily wearable item (e.g., bracelet or wristwatch), they are exposed to NIR light, which sensitizes them to cell killing. The dose of light can be adjusted according to the diagnosis and cell type. 【0299】 In some examples, the method further includes monitoring the treatment, for example, tumor cell death, Treg death, or both. In such examples, the subject is administered one or more antibody-IR700 conjugates (e.g., CD25-specific antibody-IR700 conjugates and, optionally, tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitor Ab-IR700 conjugates), for example, in combination with one or more reducing agents, one or more immune activators, or a combination thereof, and irradiated as described herein. However, lower doses (doses) of antibody-IR700 conjugates and NIR light can be used for monitoring (because cell death may not be necessary, and only treatment is monitored). In one example, the amount of antibody-IR700 conjugate administered for monitoring is at most half (e.g., at most one-third, one-quarter, one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, or one-tenth of the therapeutic dose). In one example, the amount of antibody-IR700 conjugate administered for monitoring is at least 20% or at least 25% less than the therapeutic dose. In one example, the amount of NIR light used for monitoring is at least 1 / 1000 or at least 1 / 10,000 of the therapeutic dose. This allows for the detection of cells being treated. For example, by using such a method, the size of tumors and metastases can be monitored. 【0300】 In some cases, this method is useful during surgery, for example, endoscopic procedures. For example, for example, antibody-IR700 conjugates (e.g., CD25-specific antibody-IR700 conjugates and, if necessary, tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitor Ab-IR700 conjugates) are administered in combination with one or more reducing agents, one or more immune activators, or a combination thereof, and cells / subjects are irradiated as described above. This not only causes cell death, but also allows surgeons or other medical providers to visualize the tumor margins, helping to ensure that tumor (e.g., skin, breast, lung, colon, head and neck, or prostate tumor) resection is complete and the margins are clear. In some cases, a lower dose, for example, at most half (e.g., at most one-third, one-fourth, one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, or one-tenth of the therapeutic dose) of antibody-IR700 conjugates can be used for visualization. 【0301】 One or more antibody-IR700 conjugates (e.g., a CD25-specific antibody-IR700 conjugate and, optionally, a tumor-specific antibody-IR700 conjugate and / or a checkpoint inhibitor Ab-IR700 conjugate), one or more reducing agents, and one or more immune activators can be administered locally or systemically, for example, to a subject having a tumor, e.g., cancer, or a subject from whom a tumor has previously been removed (e.g., via surgery). While specific examples are provided, those skilled in the art will understand that alternative methods of administering the disclosed therapeutic agents can be used. Such methods can include, for example, the use of a catheter or implantable pump to provide continuous infusion over a period of several hours to several days into a subject in need of treatment. 【0302】 In one example, one or more antibody-IR700 conjugates (e.g., a CD25-specific antibody-IR700 conjugate and, optionally, a tumor-specific antibody-IR700 conjugate and / or a checkpoint inhibitor Ab-IR700 conjugate), one or more reducing agents, and one or more immune activators are administered by parenteral means, including direct injection or infusion into a tumor (intratumorally) or an organ (e.g., the prostate). In some examples, one or more antibody-IR700 conjugates (e.g., a CD25-specific antibody-IR700 conjugate and, optionally, a tumor-specific antibody-IR700 conjugate and / or a checkpoint inhibitor Ab-IR700 conjugate), one or more reducing agents, and one or more immune activators are administered to a tumor by applying the agent to the tumor, for example, by local injection of the therapeutic agent, by bathing the tumor in a solution containing the therapeutic agent, or by pouring the therapeutic agent onto the tumor. 【0303】 Additionally or alternatively, the disclosed compositions can be administered systemically to a subject with a tumor (e.g., cancer), for example, intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, or orally. One or more antibody-IR700 conjugates (e.g., CD25-specific antibody-IR700 conjugates and, optionally, tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitor Ab-IR700 conjugates), one or more reducing agents, and one or more immune activators can be administered by the same or different routes. In one example, one or more antibody-IR700 conjugates are administered intravenously, and one or more reducing agents are delivered intraperitoneally. In one example, one or more antibody-IR700 conjugates and one or more immune activators are administered intravenously and / or intratumorally, and one or more reducing agents are delivered intraperitoneally. In another example, one or more antibody-IR700 conjugates (e.g., a CD25-specific antibody-IR700 conjugate and, optionally, a tumor-specific antibody-IR700 conjugate and / or a checkpoint inhibitor Ab-IR700 conjugate), one or more reducing agents, and one or more immune activators are administered systemically (e.g., intravenously or intraperitoneally). In one example, one or more CD25-specific antibody-IR700 conjugates and, optionally, a tumor-specific antibody-IR700 conjugate and / or a checkpoint inhibitor Ab-IR700 conjugate, one or more reducing agents, and one or more immune activators are administered intraperitoneally. In one example, one or more CD25-specific antibody-IR700 conjugates, one or more tumor-specific antibody-IR700 conjugates, one or more immune activators, and one or more reducing agents are administered intravenously. 【0304】 The dosage of the therapeutic agents provided herein administered to a subject is not subject to absolute limitations, but may depend on the nature of the composition, its active ingredient and its potential undesirable side effects (e.g., immune response to antibodies), the subject being treated, and the type of condition being treated, and the mode of administration. Generally, the dose will be a therapeutically effective amount, e.g., an amount sufficient to achieve a desired biological effect, e.g., an amount effective to reduce the size (e.g., volume and / or weight) of a tumor, or to attenuate further growth of a tumor, or to reduce undesirable symptoms of a tumor. 【0305】 For intravenous administration of an antibody-IR700 conjugate, an exemplary dosage for administration to a subject for a single treatment can be in the range of 0.5 to 200 mg / 60 kg body weight, 1 to 100 mg / 60 kg body weight, 1 to 50 mg / 60 kg body weight, or 1 to 20 mg / 60 kg body weight, e.g., about 1 or 2 mg / kg body weight. In yet another example, a therapeutically effective amount of an antibody-IR700 conjugate administered intraperitoneally or intratumorally is 10 μg to 5000 μg of antibody-IR700 conjugate per kg body weight, e.g., 10 μg / kg to 1000 μg / kg, 10 μg / kg to 500 μg / kg, or 100 μg / kg to 1000 μg / kg. In one example, the dose of antibody-IR700 conjugate administered to a human patient is at least 50 mg, such as at least 100 mg, at least 300 mg, at least 500 mg, at least 750 mg, or even 1 g. 【0306】 For intravenous administration of a reducing agent, exemplary dosages for administration to a subject for a single treatment can be in the range of 0.5 to 300 g per 60 kg of body weight, 1 to 300 g per 60 kg of body weight, 1 to 50 g per 60 kg of body weight, 1 to 20 g per 60 kg of body weight, 1 to 10 g per 60 kg of body weight, or 10 to 300 g per 60 kg of body weight, e.g., 1, 2, 5, 10, 20, 50, 100, 200, or 300 g per 60 kg of body weight. In yet another example, a therapeutically effective amount of a reducing agent administered intraperitoneally or intratumorally is 10 mg to 5000 mg of reducing agent per kg of body weight, e.g., 10 mg / kg to 1000 mg / kg, 10 mg / kg to 500 mg / kg, or 100 mg / kg to 1000 mg / kg. In one example, the dose of reducing agent administered to a human patient is at least 1 g, at least 10 g, at least 20 g, at least 50 g, at least 100 g, at least 200 g, at least 300 g, for example, 1, 2, 5, 10, 20, 50, 100, 200 or 300 g. 【0307】 Treatment with the disclosed antibody-IR700 conjugates (including CD25-specific antibody-IR700 conjugates, checkpoint inhibitor-specific antibody-IR700 conjugates, and / or tumor-specific antibody-IR700 conjugates), one or more reducing agents, and / or one or more immune activators can be completed on a single day or can be repeated on multiple days at the same or different dosages. Repeated treatments can be performed on the same day, consecutively, or every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or even at longer intervals. In some examples, the antibody-IR700 conjugates, one or more reducing agents, and / or one or more immune activators are administered on the same day. In other examples, the antibody-IR700 conjugate, one or more reducing agents, and / or one or more immune activators are administered on different days, e.g., one or more immune activators are administered the day before the antibody-IR700 conjugate, or the antibody-IR700 conjugate is administered the day before the one or more reducing agents. In one non-limiting example, one or more antibody-IR700 conjugates and one or more immune activators are administered to a subject on the same day, and repeat doses (at the same or different dosage levels) of one or more immune activators (e.g., one, two, three, four, five, or more additional doses of one or more immune activators) are administered to the subject daily, or every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or even at longer intervals. In some examples, the amount of the repeat doses of one or more immune activators is reduced (e.g., reduced by 50%) compared to the initial dose. 【0308】 In additional aspects, these methods also include administering one or more additional therapeutic agents to the subject. Irradiation (e.g., at a wavelength of 660-710 nm and at least 10 J / cm ) can be administered as described in International Patent Application Publication No. WO 2013 / 009475, which is incorporated herein by reference in its entirety. 2 , at least 20 J / cm 2 , at least 30 J / cm2 , at least 40J / cm 2 , at least 50J / cm 2 , at least 70J / cm 2 , at least 80J / cm 2 or at least 100J / cm 2 , e.g., at least 10-100 J / cm 2 After irradiation (at a dose of 100 nm or more), there is an approximately 8-hour window during which uptake of an additional agent (e.g., a nano-sized agent, e.g., about at least 1 nm in diameter, at least 10 nm in diameter, at least 100 nm in diameter, or at least 200 nm in diameter, e.g., 1-500 nm in diameter) by PIT-treated cells is enhanced. Accordingly, one or more additional therapeutic agents can be further administered to the subject concurrently with or sequentially with PIT. In one example, the additional therapeutic agent is administered after irradiation, e.g., about 0-8 hours after irradiation of the cells (e.g., at least 10 minutes, at least 30 minutes, at least 60 minutes, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, or at least 7 hours after irradiation, e.g., not more than 10 hours, not more than 9 hours, or not more than 8 hours after irradiation, e.g., 1 hour to 10 hours, 1 hour to 9 hours, 1 hour to 8 hours, 2 hours to 8 hours, or 4 hours to 8 hours after irradiation). In another example, the additional therapeutic agent is administered immediately prior to irradiation (e.g., about 10 to 120 minutes prior to irradiation, e.g., 10 to 60 minutes or 10 to 30 minutes prior to irradiation). Additional therapeutic agents that may be used are discussed below. 【0309】 In additional embodiments, methods are provided that allow for the detection or monitoring of cell death in real time. Such methods are useful, for example, to ensure that a sufficient amount of antibody-IR700 conjugate (including a CD25-specific antibody-IR700 conjugate, a tumor-specific antibody-IR700 conjugate, and / or a CTLA4-, PD-1, and PD-L1-antibody-IR700 conjugate), one or more reducing agents, and / or one or more immune activators, or a sufficient amount of radiation has been delivered to cells or tumors to promote cell death. These methods allow for the detection of cell death before morphological changes are evident. In one example, these methods include contacting cells having a cell surface protein with a therapeutically effective amount of one or more antibody-IR700 conjugates (including a CD25-specific antibody-IR700 conjugate, a tumor-specific antibody-IR700 conjugate, and / or a checkpoint inhibitor-antibody-IR700 conjugate), one or more reducing agents, and / or one or more immune activators; at a wavelength of 660-740 nm and at least 4 J / cm 2 or at least 10J / cm 2and detecting the cells using fluorescence lifetime (FLT) imaging approximately 0-48 hours after irradiation (e.g., at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours after irradiation, e.g., 1 minute to 30 minutes, 10 minutes to 30 minutes, 10 minutes to 1 hour, 1 hour to 8 hours, 6 hours to 24 hours, or 6 hours to 48 hours after irradiation), thereby detecting cell death in real time. A shortening of FLT serves as an indicator of acute membrane damage induced by PIT. Thus, cells are irradiated under conditions sufficient to shorten the IR700 FLT by at least 25%, e.g., at least 40%, at least 50%, at least 60%, or at least 75%. In one example, the cells are exposed to a wavelength of 660 nm to 740 nm (e.g., 680 nm to 700 nm, e.g., 680 or 690 nm) and at least 1 J / cm 2 , at least 4J / cm 2 , at least 10 J / cm 2 , at least 20 J / cm 2 , at least 30 J / cm 2 , at least 40J / cm 2 , at least 50J / cm 2 or at least 60J / cm 2 , e.g., 10 to 60 J / cm 2 , 20~50J / cm 2 , 20~25J / cm 2 , 30~50J / cm 2 , 4~50J / cm 2 or 1 to 10 J / cm 2 It is irradiated with a dose of 【0310】 In some examples, the method for detecting cell death in real time includes contacting the cells with one or more additional therapeutic agents, for example, about 0 to 8 hours after irradiating the cells. Real-time imaging can be performed before or after contacting the cells with one or more additional therapeutic agents. For example, if insufficient cell death occurs after administration of one or more antibody-IR700 conjugates (including a CD25-specific antibody-IR700 conjugate, a tumor-specific antibody-IR700 conjugate, and / or a checkpoint inhibitor-antibody-IR700 conjugate), one or more reducing agents, and / or one or more immune activators, as determined by real-time imaging, the cells can be contacted with one or more additional therapeutic agents. However, in some examples, the cells are contacted with an antibody-IR700 conjugate (including a CD25-specific antibody-IR700 conjugate, a tumor-specific antibody-IR700 conjugate and / or a checkpoint inhibitor-antibody-IR700 conjugate), one or more reducing agents and / or one or more immune activators, and an additional therapeutic agent prior to detecting cell death in real time. 【0311】 Exemplary cells The target cell can be an unwanted cell or cell whose growth is undesired, such as a cancer cell (e.g., a tumor cell) or an immune cell (e.g., a T cell, e.g., a Treg). The cell can be present in the mammal being treated, e.g., a subject (e.g., a human or veterinary subject) with cancer. Any target cell can be treated with the claimed methods. In some embodiments, the target cell is a CD25+ Treg cell. In some examples, the target cell expresses a cell surface protein that is substantially not found on the surface of other normal (desired) cells, and antibodies that specifically bind to such proteins, and antibody-IR700 conjugates generated against the protein, can be selected. In one example, the cell surface protein is a tumor-specific protein (e.g., an antigen), such as EGFR. In one non-limiting example, the cell surface protein is CTLA4 (e.g., CD4 in the tumor bed).+ Foxp3 + (To target Tregs.) In one non-limiting example, the cell surface protein is PD-L1, PD-1, or CTLA4. 【0312】 In some examples where a second antibody-IR700 conjugate (e.g., a tumor antigen-specific antibody IR700 conjugate) is administered to a subject, the tumor cells are cancer cells, e.g., cells in a patient with cancer. Exemplary cells that can be killed by the disclosed methods include cells of the following tumors: hematological malignancies, e.g., leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, and heavy chain disease). In another example, the cell is a solid tumor cell, e.g., a sarcoma and carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, squamous cell carcinoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), basal cell carcinoma, adenocarcinoma (e.g., pancreatic, colon, ovarian, lung, breast, stomach, , prostate, cervical or esophageal adenocarcinoma), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder cancer, and CNS cancer (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In one example, the tumor is oropharyngeal cancer. 【0313】 In a specific example, the cell is a lung cancer cell. 【0314】 In a specific example, the cell is a breast cancer cell. 【0315】 In a specific example, the cell is a colon cancer cell. 【0316】 In a specific example, the cells are gastric cancer cells. 【0317】 In a specific example, the cells are kidney cancer cells. 【0318】 In a specific example, the cells are head and neck cancer cells. 【0319】 In a specific example, the cells are prostate cancer cells. 【0320】 In a specific example, the cell is an oropharyngeal cancer cell. 【0321】 In a specific example, the cell is an esophageal cancer cell. 【0322】 In a specific example, the cell is a squamous cell carcinoma cancer cell, for example, a head and neck or skin cancer cell. 【0323】 In some cases, these methods may have an abscopal effect, so the cancer or cancer cells being treated should be those at a site remote from the irradiated tumor, e.g., at wavelengths of 660-740 nm and at least 1 J / cm. 2 distant metastases not receiving a dose of radiation, e.g., 1 mm, 1 inch, 2 inches, 3 inches, 4 inches, 6 inches, 12 inches, or more. 【0324】 In some cases, the cancer or cancer cells to be treated are moderately or highly immunogenic, i.e., can induce an immune response. In some cases, the NIR-PIT method comprising the administration of CTLA4 antibody-IR700, PD-1 antibody-IR700 and / or PD-L1 antibody-IR700 is used to treat moderately or highly immunogenic tumors. Examples of moderately or highly immunogenic cancers include melanoma, lung cancer (e.g., NSCLC), colon cancer and renal cell carcinoma. In one example, the highly immunogenic cancer is prostate cancer. In some cases, the cancer or cancer cells to be treated are not highly immunogenic, i.e., cannot induce an immune response. In some examples, NIR-PIT in combination with CTLA4 antibody-IR700, PD-1 antibody-IR700 and / or PD-L1 antibody-IR700 is used in combination with anti-tumor IL700 (e.g., anti-EGFR-IR700 or anti-PSA antigen-IR700) to treat low-immunogenic tumors. Examples of low-immunogenic cancers include oral squamous cell carcinoma, breast cancer, pancreatic cancer and multiple myeloma. In some examples, tumor immunogenicity score is used to determine whether a cancer is low-immunogenic, moderately immunogenic, or highly immunogenic (see, for example, Wang et al., eLife, 8:e49020, 2019). 【0325】 Exemplary Subjects In some examples, the disclosed methods are used to treat subjects with cancer or tumors, such as those described herein. In some examples, the tumor has previously been treated, for example, surgically or chemically removed, and the disclosed methods are subsequently used to kill any remaining unwanted tumor cells that may remain in the patient and / or to reduce tumor recurrence or metastasis. In some examples, the subjects treated with the disclosed CD25 antibody-IR700 conjugate have highly or moderately immunogenic cancers. 【0326】 The disclosed methods can be used to treat any mammalian subject (e.g., a human or veterinary subject, e.g., a dog or cat), e.g., a human with or having previously removed or treated a tumor, e.g., cancer. Subjects in need of the disclosed treatments can include human subjects with cancer, e.g., cancers expressing tumor-specific proteins on the cell surface that can specifically bind to a tumor-specific antibody-IR700 conjugate, or cancers infiltrated with immune cells that can bind to immune cell-specific antibody-IR700 molecules (e.g., CTLA4 antibody-IR700). For example, the disclosed methods can be used as initial treatments for cancer, either alone or in combination with radiation or other chemotherapy or surgery. The disclosed methods can also be used in patients where previous radiation or chemotherapy has failed. Thus, in some cases, the subject is a subject who has received other treatments, but the other treatments have not provided the desired therapeutic response. The disclosed methods can also be used in patients with localized and / or metastatic cancer and / or recurrence of the primary tumor. 【0327】 In some examples, the method includes selecting a subject who will benefit from the disclosed treatment, e.g., selecting a subject having a tumor that expresses a cell surface protein (e.g., a tumor-specific protein) that can specifically bind to an antibody-IR700 conjugate. For example, if the subject is determined to have HER2-expressing breast cancer, the subject may be selected to be treated with an anti-HER2-IR700 conjugate, e.g., trastuzumab-IR700, in combination with a CD25-specific antibody-IR700 conjugate, e.g., in combination with a CTLA4 antibody-IR700 conjugate, a PD-1 antibody-IR700 conjugate, a PD-L1 antibody-IR700 conjugate, one or more reducing agents, one or more immune activators, or a combination thereof, and the subject is subsequently irradiated as described herein. 【0328】 Exemplary Cell Surface Proteins In some embodiments of the disclosed methods, a CD25-specific antibody-IR700 conjugate is administered in combination with an antibody-IR700 conjugate targeted to a protein on the cell surface of tumor or cancer cells. In one example, the protein on the cell surface of the target cells to be killed is not present in significant amounts on other cells. For example, the cell surface protein may be a receptor found only on the target cell type. 【0329】 In a specific example, the cell surface protein is a cancer-specific or tumor-specific protein (also called a tumor-specific antigen or tumor-associated antigen), such as members of the EGF receptor family (e.g., HER1, 2, 3, and 4) and cytokine receptors (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.). Thus, in some examples, the cell surface protein is an antigen expressed on the cell membrane of a tumor cell. A tumor-specific protein is a protein that is unique to cancer cells or is much more abundant on cancer cells than on other cells, e.g., normal cells. For example, HER2 is primarily found in breast cancer, while HER1 is primarily found in adenocarcinoma, which can be found in many organs, such as the pancreas, breast, prostate, and colon. 【0330】 Exemplary tumor-specific proteins that may be found on target cells (and antibodies specific for which proteins may be used to formulate antibody-IR700 molecules) include, but are not limited to, any of the various MAGEs (melanoma-associated antigen E), including MAGE1 (e.g., GenBank Accession Nos. M77481 and AAA03229), MAGE2 (e.g., GenBank Accession Nos. L18920 and AAA17729), MAGE3 (e.g., GenBank Accession Nos. U03735 and AAA17446), MAGE4 (e.g., GenBank Accession Nos. D32075 and A06841.1), and the like; any of the various tyrosinases (e.g., GenBank Accession Nos. U01873 and AAB60319); mutant ras; mutant p53 (e.g., GenBank Accession Nos. Nos. X54156, CAA38095, and AA494311; p97 melanoma antigen (e.g., GenBank accession numbers M12154 and AAA59992); human milk fat globule (HMFG) associated with breast tumors (e.g., GenBank accession numbers S56151 and AAB19771); any of the various BAGE (human B melanoma-associated antigen E), including BAGE1 (e.g., GenBank accession number Q13072) and BAGE2 (e.g., GenBank accession numbers NM_182482 and NP_872288), any of the various GAGE (G antigen), including GAGE1 (e.g., GenBank accession number Q13065) or any of GAGE2-6; various gangliosides, CD25 (e.g., GenBank accession numbers NP_000408.1 and NM_000417.2). 【0331】 Other tumor-specific antigens include HPV16 / 18 and E6 / E7 antigens associated with cervical cancer (e.g., GenBank accession numbers NC_001526, FJ952142.1, ADB94605, ADB94606, and U89349), mucin (MUC1)-KLH antigen associated with breast cancer (e.g., GenBank accession numbers J03651 and AAA35756), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank accession numbers X98311 and CAA66955), gp100 associated with melanoma (e.g., GenBank accession numbers S73003 and AAC60634), MART1 antigen associated with melanoma (e.g., GenBank accession number NP_005502), and ovarian and other cancers. cancer antigen 125 (CA125, also known as mucin 16 or MUC16) associated with liver cancer (e.g., GenBank Accession Nos. NM_024690 and NP_078966); alpha-fetoprotein (AFP) associated with liver cancer (e.g., GenBank Accession Nos. NM_001134 and NP_001125); Lewis Y antigen associated with colorectal, biliary tract, breast, small cell lung, and other cancers; tumor-associated glycoprotein 72 (TAG72) associated with adenocarcinoma; glypican 1 (GPC1 associated with pancreatic cancer, glioma, and breast cancer), glypican 2 (associated with neuroblastoma), and glypican 3 (associated with hepatocellular carcinoma), and PSA antigen associated with prostate cancer (e.g., GenBank Accession Nos. X14810 and CAA32915). 【0332】 Other exemplary tumor-specific proteins include PMSA (prostate membrane-specific antigen; e.g., GenBank Accession Nos. AAA60209 and AAB81971.1), which is associated with solid tumor angiogenesis, as well as prostate cancer; HER-2 (human epidermal growth factor receptor 2; e.g., GenBank Accession Nos. M16789.1, M16790.1, M16791.1, M16792.1, and AAA58637), which is associated with breast, ovarian, gastric, and uterine cancer; HER-1 (e.g., GenBank Accession Nos. NM_005228 and NP_005219), which is associated with lung, anal, and glioblastoma and adenocarcinoma; NY-ESO-1 (e.g., GenBank Accession Nos. U87459 and AAB49693), which is associated with melanoma, sarcoma, testicular, and other cancers; hTERT (telomerase also known as NP_937983 (variant 1), NM_198255 and NP_937986 (variant 2)); proteinase 3 (e.g., GenBank accession numbers M29142, M75154, M96839, X55668, NM00277, M96628, X56606, CAA39943, and AAA36 342), and Wilms' tumor 1 (WT-1, e.g., GenBank accession numbers NM_000378 and NP_000369 (variant A), NM_024424 and NP_077742 (variant B), NM_024425 and NP_077743 (variant C), and NM_024426 and NP_077744 (variant D)). 【0333】 In one example, the tumor-specific protein is EGFR, and in some examples, the EGFR antibody-IR700 is or includes panitumumab or cetuximab. 【0334】 In one example, the tumor-specific protein is PD-L1, and in some examples, the PD-L1 antibody-IR700 is or includes atezolizumab, avelumab, durvalumab, or cosibelimab. 【0335】 In one example, the tumor-specific protein is CD52, associated with chronic lymphocytic leukemia (e.g., GenBank accession numbers AAH27495.1 and CAI15846.1); CD33, associated with acute myeloid leukemia (e.g., GenBank accession numbers NM_023068 and CAD36509.1); or CD20, associated with non-Hodgkin's lymphoma (e.g., GenBank accession numbers NP_068769, NP_031667). 【0336】 In a specific example, the tumor-specific protein is CD44 (e.g., OMIM107269, GenBank accession number ACI46596.1 and NP_000601.3).CD44 is a marker of cancer stem-like cells and various types of cancer, and is involved in cell-cell adhesion, cell migration, cell spatial orientation, and promoting matrix-derived survival signals.High expression of CD44 on the plasma membrane of tumors can be associated with tumor aggressiveness and poor outcome. 【0337】 Thus, the disclosed methods can be used to treat any cancer that expresses a tumor-specific protein. 【0338】 Exemplary Antibody-IR700 Conjugates Because cell surface protein sequences are publicly available (e.g., as described above), it is possible to generate or purchase antibodies specific to such proteins (or other small molecules that can be conjugated to IR700). For example, if the tumor-specific protein HER2 is selected as the target, an antibody specific to HER2 (e.g., trastuzumab) can be purchased or generated and conjugated to the IR700 dye. Other specific examples are provided in Table 1 and elsewhere herein. In one example, the antibody conjugated to IR700 is a humanized monoclonal antibody. 【0339】 In one example, the antibody-IR700 conjugate is an EGFR-antibody-IR700 molecule, such as panitumumab-IR700 or cetuximab-IR700. 【0340】 Antibody-IR700 molecules can be produced using methods such as those described in WO2013 / 009475, which is incorporated herein by reference in its entirety. Table 1. Exemplary tumor-specific antigens and antibodies [Table 1-1] [Table 1-2] [Table 1-3] 【0341】 Exemplary antibodies that can be conjugated to IR700 and used in the disclosed methods include 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, altumomab pentetate, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, besilesomab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide, and catuzumab. Maxomab, CC49, cetuximab, sitatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, detumomab, ecromeximab, eculizumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, galiximab, gemtuzumab ozogamicin, girentuximab, glenbatumumab vedotin, ibritumomab tiuxetan, igovomab, imciromab, intetumumab, inotuzumab Buozogamicin, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab mertansine, lucatumumab, rumiliximab, mapatumumab, matuzumab, mepolizumab, metelimumab, milatuzumab, mitumomab, morolimumab, nacolomab butafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentanone, ofatumumab, olaratumumab, oportuzumab monatox, oregovomab, panitumumab, pe These include mutumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, lobatumumab, satumomab pendetide, sibrotuzumab, sonepcizumab, tacatuzumab tetraxetan, taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, and zalutumumab. 【0342】 In some examples, the cell surface protein recognized by the antibody conjugated to IR700 is a protein found on immune cells, such as Tregs. In one example, the protein is CTLA4. In one example, the antibody-IR700 molecule is a CTLA4-antibody-IR700 molecule, such as ipilimumab-IR700 or tremelimumab-IR700. 【0343】 In one example, a patient is treated with at least two different antibody-IR700 conjugates specific for cancer cell surface antigens. In one example, the two different antibody-IR700 conjugates are specific for the same protein (e.g., HER-2) but for different epitopes of the protein (e.g., epitope 1 and epitope 2 of HER-2). In another example, the two different antibody-IR700 conjugates are specific for two different proteins or antigens. For example, anti-HER1-IR700 and anti-HER2-IR700 can be injected together as a cocktail to facilitate the killing of cells bearing either HER1 or HER2. 【0344】 Immune modulator / immune activator Immune activators used in the disclosed methods include agents or compositions that activate the immune system and / or inhibit immune suppressor cells (also referred to herein as suppressor cells). Inhibition of immune suppressor cells and / or activation of the immune response can increase tumor cell killing and lead to the production of memory T cells, which can provide a "vaccine" effect against recurrence and / or distant tumors or metastasis. In some examples, one or more immune activators are used in combination with the CD25-specific antibody-IR700 conjugates disclosed herein, one or more tumor-specific antibody-IR700 molecules, and / or one or more reducing agents. 【0345】 In some embodiments, the immune activator is an inhibitor of immune suppressor cells, e.g., an agent that inhibits or reduces the activity of immune suppressor cells. In some cases, the immune modulator kills immune suppressor cells. In some examples, the immune suppressor cells are regulatory T (Treg) cells. In some examples, not all of the suppressor cells are killed in vivo (because this can lead to the development of autoimmunity). Thus, in some examples, the method reduces the activity or number of immune suppressor cells in an area of a subject, e.g., in the area of a tumor or a previously tumor-bearing area, by at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or at least 95%. In some examples, the method reduces the total number of suppressor cells in the subject by at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or at least 95%. 【0346】 Inhibitors of immune suppressor cells include tyrosine kinase inhibitors (e.g., sorafenib, sunitinib, and imatinib), or chemotherapeutic agents (e.g., cyclophosphamide or interleukin-toxin fusions, such as denileukin difitox (an IL2-diphtheria toxin fusion)), or other antibodies that bind to suppressor cell surface proteins (e.g., those described below). In another example, inhibitors of immune suppressor cells include immune checkpoint inhibitors, such as anti-PD-L1 antagonist antibodies, which prevent PD-L1 from binding to PD-1 (referred to herein as PD-1 / PD-L1 mAb-mediated immune checkpoint blockade (ICB)). Thus, in some examples, the immunomodulator is a PD-L1 antagonist antibody, such as atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (envafolimab), BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737 (or other examples provided herein). In some examples, the immunomodulator is a PD-1 antagonist antibody, such as nivolumab, pembrolizumab, cemiplimab, or dostarlimab. Checkpoint inhibitors also include CTLA-4 antibodies, including ipilimumab and tremelimumab. The inhibitor of immune suppressor cells may also be a LAG-3 or B7-H3 antagonist, such as BMS-986016 and MGA271. In some examples, two or more of the inhibitors of immune suppressor cells may be administered to the subject. In one non-limiting example, the subject is administered an anti-PD1 antibody and an anti-LAG-3 antibody. 【0347】 In some examples, the agent that inhibits or reduces the activity of suppressor cells includes one or more antibody-IR700 conjugates, wherein the antibody binds to a suppressor cell surface protein, such as CD4, C-X-C chemokine receptor 4 (CXCR4), C-C chemokine receptor 4 (CCR4), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), glucocorticoid-inducible TNF receptor (GITR), OX40, folate receptor 4 (FR4), CD16, C Specifically binds to D56, CD8, CD122, CD23, CD163, CD206, CD11b, Gr-1, CD14, interleukin-4 receptor alpha chain (IL-4Ra), interleukin-1 receptor alpha (IL-1Ra), interleukin-1 decoy receptor, CD103, fibroblast activation protein (FAP), CXCR2, CD33, CD66b, or V-domain immunoglobulin suppressor of T-cell activation (VISTA). 【0348】 In other embodiments, the immune modulator is an immune system activator (immunoactivator). In some examples, the immune system activator stimulates (activates) one or more T cells and / or natural killer (NK) cells. In one example, the immune system activator includes one or more interleukins (ILs), such as IL-2, IL-15, IL-7, IL-12, and / or IL-21. In a non-limiting example, the immune modulator includes IL-15. In a non-limiting example, the immune modulator includes IL-2 and IL-15. In a non-limiting example, the immune modulator includes interferon gamma. In another example, the immune system activator includes one or more agonists for costimulatory receptors, such as 4-1BB, OX40, or GITR. In a non-limiting example, the immune modulator includes stimulatory anti-4-1BB, anti-OX40, and / or anti-GITR antibodies. 【0349】 In some examples, one or more (e.g., 1, 2, 3, 4, 5, or more) doses of an immune modulator are administered to a subject. Thus, administration of the immune modulator may be completed on a single day or may be repeated on multiple days with the same or different dosages (e.g., administration at least two different times, three different times, four different times, five different times, or ten different times). In some examples, the repeated administrations are the same dose. In other examples, the repeated administrations are different doses (e.g., subsequent doses higher than the preceding dose, or subsequent doses lower than the preceding dose). Repeated administration of an immune modulator may be performed on the same day, consecutively, every other day, every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or even at longer intervals. In some examples, at least one dose of the immune modulator is administered before irradiation and at least one dose of the immune modulator is administered after irradiation. 【0350】 irradiation After administering one or more antibody-IR700 conjugates to a subject, the subject (or a tumor in the subject) is irradiated. Because only cells expressing the target protein (e.g., CD25) are recognized by the antibody, only those cells have a sufficient amount of antibody-IR700 conjugate associated therewith to kill the cells. Because irradiation kills only cells to which the antibody-IR700 conjugate is bound, and not other cells, this reduces the possibility of undesired side effects, such as the killing of non-tumor cells. 【0351】 The subject (e.g., a tumor in the subject) is irradiated with a therapeutic dose of radiation at a wavelength of 660-740 nm, e.g., 660-710 nm, 660-700 nm, 680-700 nm, 670-690 nm, e.g., 680 nm or 690 nm. In particular examples, the cell, tumor, or subject is irradiated with a therapeutic dose of at least 1 J / cm. 2 , e.g., at least 4 J / cm 2 , at least 10 J / cm 2 , at least 20 J / cm 2 , at least 25J / cm2 , at least 30 J / cm 2 , at least 50J / cm 2 , at least 100 J / cm 2 or at least 500J / cm 2 , e.g., 1 to 1000 J / cm 2 , 1~500J / cm 2 , 1~100J / cm 2 , 4~50J / cm 2 , 4~25J / cm 2 , 30~50J / cm 2 , 10~100J / cm 2 , 20~30J / cm 2 , 1 to 10 J / cm 2 , 4~10J / cm 2 , 20~50J / cm 2 or 10 to 50 J / cm 2 In a particular example, the subject (or a tumor in the subject) is irradiated with a wavelength of 690 nm and a dose of 50 J / cm. 2 A dose of is irradiated. 【0352】 A subject may be irradiated one or more times. Thus, irradiation may be completed on a single day, or may be repeatedly performed on multiple days with the same or different dosages (e.g., irradiation at least two different times, three different times, four different times, five different times, or ten different times). In some cases, the repeated irradiation is the same dose. In other cases, the repeated irradiation is at a different dose (e.g., a subsequent dose higher than the preceding dose, or a subsequent dose lower than the preceding dose). Repeated irradiation may be performed on the same day, every day, every other day, every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or even at longer intervals. In one example, the first irradiation is 50 J / cm 2 and the second irradiation is 100 J / cm 2 and these doses are on consecutive days (e.g., about 24 hours apart). In one example, the first dose is 10-50 J / cm 2 and the second irradiation is 10 to 50 J / cm 2 and these irradiations are on consecutive days (e.g., about 24 hours apart). 【0353】 In some examples, the illumination is provided by a wearable device incorporating an NIR LED. In other examples, another type of device that can be used with the disclosed methods is a flashlight-like device with an NIR LED. Such a device can be used for localized treatment of a lesion during surgery or can be incorporated into an endoscope to apply NIR light to a body surface after administration of one or more PIT agents. Such a device can be used by a physician or qualified medical professional to direct treatment to a specific target on the body. 【0354】 Treatment using wearable NIR LEDs As described herein, the disclosed methods are highly specific to cancer cells and / or Tregs in the tumor bed. However, to kill cells circulating throughout the body or present on the skin, patients can wear devices incorporating NIR LEDs. In some cases, patients use at least two devices, for example, one item of clothing or jewelry during the day and a blanket at night. In some cases, patients use at least two devices, for example, two items of clothing, simultaneously. These devices allow patients to be exposed to NIR light using portable everyday items of clothing and jewelry, so that the treatment remains unobtrusive and does not interfere with daily activities. In some cases, the devices can be worn discreetly during the day for PIT therapy. Exemplary devices incorporating NIR LEDs are disclosed in International Patent Application Publication No. WO2013 / 009475 (incorporated herein by reference). 【0355】 In one example, a patient is administered one or more antibody-IR700 molecules, e.g., in combination with one or more reducing agents and / or one or more immune activators, using the methods described herein. The patient is then fitted with a device incorporating NIR LEDs, which allows for long-term therapy and treatment of tumor cells present in the blood or lymph or on the skin. In some examples, the dose is at least at least 1 J / cm. 2 , at least at least 4 J / cm 2 , at least 10 J / cm 2 , at least 20 J / cm 2 , at least 30 J / cm 2 , at least 40J / cm 2 or at least 50J / cm 2 , e.g., 10 to 100 J / cm 2 , 10~50J / cm 2 , e.g., 20 J / cm 2 or 30 J / cm 2 In some examples, administration of one or more antibody-IR700 conjugates, e.g., in combination with one or more reducing agents and / or one or more immune activators, is repeated over a period of time (e.g., bi-weekly or monthly) to ensure therapeutic levels are present in the body. 【0356】 In some cases, the patient wears or uses the device or combination of devices for at least one week, for example, at least two weeks, at least four weeks, at least eight weeks, at least twelve weeks, at least four months, at least six months, or even at least one year. In some cases, the patient wears or uses the device or combination of devices for at least four hours per day, for example, at least 12 hours per day, at least 16 hours per day, at least 18 hours per day, or 24 hours per day. It is entirely possible that multiple devices of similar "daily" nature (blankets, bracelets, necklaces, underwear, socks, shoe insoles) can be worn by the same patient during the treatment period. At night, the patient can use a NIR LED blanket or other covering. 【0357】 Administration of additional treatment As discussed above, the subject can receive one or more other therapies before, during, or after administration of one or more antibody-IR700 conjugates, e.g., in combination with one or more reducing agents and / or one or more immune activators, and irradiation. In one example, the subject receives one or more treatments to remove or reduce tumors, e.g., before administration of one or more antibody-IR700 conjugates, e.g., in combination with one or more reducing agents and / or one or more immune activators, and irradiation. In other examples, the additional treatment or therapeutic agent (e.g., an anti-neoplastic agent) can be administered to the subject to be treated, for example, after irradiation, e.g., about 0 to 8 hours after irradiating the cells (e.g., at least 10 minutes, at least 30 minutes, at least 60 minutes, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, or at least 7 hours after irradiation, e.g., not more than 10 hours, not more than 9 hours, or not more than 8 hours after irradiation, e.g., 1 to 10 hours, 1 to 9 hours, 1 to 8 hours, 2 to 8 hours, or 4 to 8 hours after irradiation). In another example, the additional therapeutic agent is administered immediately before irradiation (e.g., about 10 to 120 minutes before irradiation, e.g., 10 to 60 minutes or 10 to 30 minutes before irradiation). 【0358】 Examples of treatments that can be used in combination with the disclosed methods, which in some cases enhance tumor accessibility to additional therapeutic agents for approximately 8 hours after PIT, include, but are not limited to, surgical procedures for tumor removal or reduction (e.g., surgical resection, cryotherapy, or chemoembolization), and antitumor pharmaceutical treatments that can include radiotherapeutic agents, anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents. In some cases, the additional therapeutic agent is conjugated to nanoparticles. Specific examples of additional therapeutic agents that can be used include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and / or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators. These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination. 【0359】 "Microtubule-binding agent" refers to an agent that interacts with tubulin to stabilize or destabilize microtubule formation, thereby inhibiting cell division. Examples of microtubule-binding agents that can be used in conjunction with the disclosed methods include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), epothilone, colchicine, dolastatin 15, nocodazole, podophyllotoxin, and rhizoxin. Analogs and derivatives of such compounds can also be used. For example, suitable epothilones and epothilone analogs are described in International Publication No. WO2004 / 018478. Taxoids, such as paclitaxel and docetaxel, and paclitaxel analogs taught in U.S. Patent Nos. 6,610,860; 5,530,020; and 5,912,264, can be used. 【0360】 The following classes of compounds may be used with the methods disclosed herein: Suitable DNA and / or RNA transcription regulators, including, without limitation, actinomycin D, daunorubicin, doxorubicin, and their derivatives and analogs, are also suitable for use in combination with the disclosed treatments. DNA intercalators and cross-linking agents that may be administered to a subject include, without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide, and their derivatives and analogs. DNA synthesis inhibitors suitable for use as therapeutic agents include, without limitation, methotrexate, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, and their analogs. Examples of suitable enzyme inhibitors include, without limitation, camptothecin, etoposide, formestane, trichostatin, and their derivatives and analogs. Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone, and their derivatives and analogs. Kinase inhibitors include Gleevec® (imatinib), Iressa® (gefitinib), and Tarceva® (erlotinib), which prevent the phosphorylation and activation of growth factors. 【0361】 Non-limiting examples of antiangiogenic agents include molecules such as proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, and recombinant vectors, as well as small molecules that function to reduce or even inhibit blood vessel growth. Examples of suitable angiogenesis inhibitors include, without limitation, angiostatin K1-3, staurosporine, genistein, fumagillin, medroxyprogesterone, suramin, interferon-alpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, thromobospondin, endostatin, thalidomide, and their derivatives and analogs. For example, in some embodiments, the antiangiogenic agent is an antibody that specifically binds to VEGF (e.g., Avastin, Roche) or an antibody that specifically binds to VEGF receptor (e.g., VEGFR2 antibody). In one example, the anti-angiogenic agent comprises VEGFR2 antibody or DMXAA (also known as vadimezan or ASA404; for example, commercially available from Sigma Corp., St. Louis, MO) or both.The anti-angiogenic agent can be bevacizumab, sunitinib, anti-angiogenic tyrosine kinase inhibitor (TKI), for example, sunitinib, axitinib and dasatinib.These can be used individually or in any combination. 【0362】 Other therapeutic agents, such as anti-tumor agents that may or may not fall into one or more of the above categories, are also suitable for administration in combination with the disclosed methods. By way of example, such agents include adriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof. 【0363】 In some examples, subjects receiving a therapeutic antibody-IR700 conjugate(s) are also administered interleukin-2 (IL-2), for example, via intravenous administration. In a particular example, IL-2 (Chiron Corp., Emeryville, CA) is administered at a dose of at least 500,000 IU / kg as an intravenous bolus over a 15-minute period every 8 hours, beginning the day following administration of the antibody-IR700 conjugate(s) and continuing for up to 5 days. Doses may be skipped depending on the subject's tolerance. 【0364】 Exemplary additional therapeutic agents include anti-neoplastic agents, e.g., chemotherapeutic agents or treatments, and anti-angiogenic agents or treatments, e.g., radiation therapy. In one example, the agent is a chemotherapeutic immunosuppressant (e.g., rituximab, steroids) or a cytokine (e.g., GM-CSF). Exemplary chemotherapeutic agents are provided, for example, in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc.; Baltzer and Berkery (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer Knobf, and Durivage (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. 【0365】 Exemplary chemotherapeutic agents that can be used with the methods provided herein include, but are not limited to, carboplatin, cisplatin, paclitaxel, docetaxel, doxorubicin, epirubicin, topotecan, irinotecan, gemcitabine, tiazofurin, gemcitabine, etoposide, vinorelbine, tamoxifen, valspodar, cyclophosphamide, methotrexate, fluorouracil, mitoxantrone, Doxil (liposome-encapsulated doxorubicin), and vinorelbine. Additional examples of chemotherapeutic agents that can be used include alkylating agents, antimetabolites, natural products, or hormones and their antagonists. Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, or dacarbazine). Specific, non-limiting examples of alkylating agents are temozolomide and dacarbazine. Examples of antimetabolites include folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine. Examples of natural products include vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (e.g., L-asparaginase). Examples of various pharmaceutical agents include platinum coordination complexes (e.g., cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenocrotical suppressants (e.g., mitotane and aminoglutethimide).Examples of hormones and antagonists include corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., tamoxifen), and androgens (e.g., testosterone propionate and fluoxymesterone). 【0366】 Exemplary chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-Fluorouracil (5-FU), Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Drugs that may be used include benzodiazepines (BMPs), ... Non-limiting examples of immune activators that may be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (from Imreg, New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech). 【0367】 In some examples, the additional therapeutic agent is conjugated to (or otherwise associated with) a nanoparticle, e.g., at least 1 nm in diameter (e.g., at least 10 nm in diameter, at least 30 nm in diameter, at least 100 nm in diameter, at least 200 nm in diameter, at least 300 nm in diameter, at least 500 nm in diameter, or at least 750 nm in diameter, e.g., 1 nm to 500 nm, 1 nm to 300 nm, 1 nm to 100 nm, 10 nm to 500 nm, or 10 nm to 300 nm in diameter). 【0368】 In one example, at least a portion of a tumor (e.g., a metastatic tumor) is surgically removed (e.g., via surgical resection and / or cryotherapy), irradiated (e.g., administration of radioactive material or energy (e.g., external beam therapy) to the tumor site to help eradicate or shrink the tumor), chemically treated (e.g., via chemoembolization), or a combination thereof, prior to administration of a disclosed treatment (e.g., administration of one or more antibody-IR700 conjugates, e.g., in combination with one or more reducing agents and / or one or more immune activators, and irradiation). For example, a subject with a metastatic tumor can have all or part of the tumor surgically removed prior to administration of a disclosed treatment. In one example, one or more chemotherapeutic agents are administered after treatment with one or more antibody-IR700 conjugates, e.g., in combination with one or more reducing agents and / or one or more immune activators, and irradiation. In another specific example, the subject has a metastatic tumor and is administered radiation therapy, chemoembolization therapy, or both concurrently with the administration of the disclosed treatments. 【0369】 In some examples, the additional therapeutic agent administered is a monoclonal antibody, e.g., 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, altumomab pentetate, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, besilesomab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide, catumaxomab, CC4 9. Cetuximab, sitatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, detumomab, ecromeximab, eculizumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, galiximab, gemtuzumab ozogamicin, girentuximab, glenbatumumab vedotin, ibritumomab tiuxetan, igovomab, imciromab, intetumumab, inotuzumab ozogamicin , ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab mertansine, lucatumumab, rumiliximab, mapatumumab, matuzumab, mepolizumab, metelimumab, milatuzumab, mitumomab, morolimumab, nacolomab butafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentane, ofatumumab, olaratumumab, oportuzumab monatox, oregovomab, panitumumab, pemtumomab, pel tuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, lobatumumab, satumomab pendetide, sibrotuzumab, sonepcizumab, tacatuzumab tetraxetan, taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, zalutumumab, or a combination thereof. 【0370】 Exemplary Reducing Agents Exemplary reducing agents that can be used in the methods provided herein include agents that lose electrons (or "donate" electrons to an electron acceptor (oxidizing agent)) in a redox chemical reaction. Exemplary reducing agents that can be used in the methods provided herein include L-cysteine, sodium L-ascorbate (L-NaAA), ascorbic acid (e.g., L- or R-ascorbic acid), and glutathione. In some examples, the reducing agent used in the disclosed methods is not sodium azide. In some examples, the reducing agent used in the disclosed methods is not L-cysteine. In one example, the reducing agent is L-NaAA (e.g., 5-50 g ip). 【0371】 The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described. [Example] 【0372】 Example 1 Binding affinity measured by surface plasmon resonance This example describes the affinity of antibodies 14564, 14569 and 14572 for CD25 as measured using BIACORE™ 8K. sample [Table 3] Equipment and reagents [Table 4-1] [Table 4-2] 【0373】 Affinity measurement The running buffer was prepared by diluting 1 volume of 10x buffer with 9 volumes of degassed, filtered MilliQ water. The regeneration buffer (10 mM glycine) was prepared by dissolving glycine in MilliQ water and adjusting the pH to approximately 1.5-1.7. The assay was performed at 25°C using HBS-EP+ as the running buffer. The antibody was injected as capture on a Series S Sensor Chip Protein A. The antigen was diluted to multiple concentrations and injected over the surface of flow cells 1 and 2 as the association phase, followed by running buffer as the dissociation phase. Running Configuration [Table 5] 【0374】 result All data were processed using BIACORE™ 8K Evaluation software version 3.0. Flow cell 1 and blank injections of buffer at each cycle were used as double references for response unit (RU) subtraction. The affinity of each antibody is listed in Table 2, and the sensorgrams are shown in Figure 1. Antibodies 14564, 14569, and 14572 showed high affinity (KD = 1.74 x 10, respectively). -8 M, 1.85 x 10 -8 M and 1.96 × 10 -8 VH2+VL4 is the parent antibody (before affinity maturation). Table 2. Measured affinity of antibodies to CD25 (sIL-2 receptor α) [Table 2] 【0375】 Example 2 Binding of CD25-specific antibodies to CD25-positive cells This example describes the binding of antibodies 14564, 14569, and 14572 to CD25-expressing KIT-225 cells by fluorescence-activated cell sorting (FACS). KIT-225 cells are an IL-2-dependent human T cell line. FACS buffer: 1x PBS with 1% FBS (1x PBS, Accugene catalog number 51225, lot number 8MB103) Serial antibody dilutions [Table 6] 【0376】 FACS procedure KIT-225 cells were washed once with FACS buffer, counted, and diluted to 1 × 10 in FACS buffer. 6 The cells were resuspended in 5 mL (5 × 10 6 To the cells, 25 μL of viability dye (Thermo Fisher, L34974, equivalent to a 1:200 dilution) was added and mixed thoroughly. After a 30-minute incubation at 4°C, the cells were washed twice with FACS buffer (200 × g, 5 minutes each). The cells were resuspended in 5 mL of FACS buffer, and 100 μL was added per well, followed by 100 μL of diluted antibody. The solution was mixed by pipetting up and down and then incubated for 60 minutes at 4°C. The cells were then washed twice with FACS buffer (200 × g, 5 minutes each). The cells were resuspended in 100 μL of FACS buffer. 50 μL of secondary antibody was diluted in 5 mL of FACS buffer (equivalent to a 1:100 dilution), and 100 μL was added to each well and mixed by pipetting up and down. After 30 minutes of incubation at 4°C, the cells were washed twice with FACS buffer (200 x g, 5 minutes each) and resuspended in approximately 250 μL of FACS buffer. FACS analysis was performed using an Attune flow cytometer using a tube reading module. 【0377】 result The results of the FACS analysis are shown in Figures 2 and 3. Antibodies 14564, 14569, and 14572 showed strong binding to CD25-positive KIT-225 cells, with binding affinity similar to that of basiliximab (also known as SIMULECT®), a chimeric mouse-human monoclonal antibody that binds to CD25 (Kahan et al., Transplantation 67(2):276-284, 1999). 【0378】 Example 3 ADCC This example describes studies to determine whether antibodies 14564, 14569 and 14572 induce ADCC. 【0379】 cell line Jurkat / CD16.V175 cells (ADCC responder cells) were grown in RPMI-1640 with 10% FBS, 1 mg / mL Geneticin, and 200 μg / mL hygromycin. KIT-225 cells (target cells) were grown in RPMI-1640 with 10% FBS and 50 μM β-mercaptoethanol. The ADCC assay medium was RPMI-1640 with 10% FBS. 【0380】 Antibody working solution preparation Basiliximab and three anti-CD25 mAbs: 14564, 14569 and 14572 were diluted to 50 μg / mL as shown below. [Table 7] 【0381】 The antibodies were serially diluted to 16.7 μg / mL, 5.5 μg / mL, 1.85 μg / mL, 0.6 μg / mL, and 0.2 μg / mL (120 μL previous + 240 μL medium). On the day of the assay, KIT-225 cells were harvested from culture. The cells were washed twice with medium to remove any IL-2. The cells were diluted to 4.2×10 in assay medium. 5The cells were resuspended to a concentration of 100 cells / mL (minimum 6 mL). [Table 8] 【0382】 KIT-225 cells (30 μL, equivalent to 12,500 cells) were added to a white, clear-bottom, 96-well microplate. 30 μL of mAb working solution was added to each well of the plate and mixed. Cells were incubated with antibody at 37°C for 1 hour. 【0383】 ADCC responder cells (Jurkat / CD16.V175) were harvested, washed once with assay medium, and then cultured at 1.9 × 10 6 The solution was resuspended to a concentration of 1 / mL (minimum 8 mL). [Table 9] 【0384】 ADCC responder cells (40 μL, equivalent to 75,000 cells) were added to each well of the plate. Each treatment was tested in triplicate. After approximately 5-6 hours of incubation, 100 μL of One-Step Luciferase Reagent was added per well, and the plate was gently rocked at room temperature for 30 minutes. Luminescence was measured using a luminometer. 【0385】 As shown in Figures 4A-4B, basiliximab possesses ADCC activity, whereas none of the antibodies 14564, 14569, and 14572 were able to induce ADCC activity. 【0386】 Example 4 Anti-CD25 antibody-IR700 conjugates are capable of binding to and inducing cell death of CD25-expressing cells This example describes a study to evaluate the targeting of photoimmunotherapy (PIT) to CD25-expressing cells. First, binding of anti-hCD25-IR700 to CD25-positive KIT-225 cells (a human T cell line) was tested. KIT-225 cells (0.5 x 106 ) were incubated with 1 μg of anti-hCD25 antibody (14564) conjugated to IR700 (anti-hCD25-IR700). As a blocking control, cells were incubated with 10 μg of unconjugated antibody prior to anti-hCD25-IR700 incubation. Cells were stained with a viability dye and analyzed by fluorescence-activated cell sorting (FACS). Unstained cells were used as a negative control. The results demonstrate that conjugation of IR700 to the anti-CD25 antibody did not diminish the binding of the antibody to CD25-expressing cells (Figure 5A). 【0387】 Cell viability after hCD25-targeted PIT was also assessed. KIT-225 cells (0.5 × 10 6 ) were incubated with 1 μg of anti-hCD25-IR700 conjugate, followed by 0, 5, 20 or 50 J / cm 2 of NIR light at 150mW / cm 2 Cells were then stained with a viability dye and analyzed by FACS. As shown in Figure 5C, cells were incubated with anti-hCD25-IR700 conjugate and applied at 5, 20, or 50 J / cm. 2 Exposure to NIR light of 0, 5, or 20 J / cm resulted in significant CD25-positive cell death. 2 Representative flow cytometry plots of live / dead staining after PIT using NIR light are shown. 【0388】 Example 5 Anti-CD25 antibodies do not block IL-2 binding to CD25 A proliferation assay was performed to test whether antibodies 14564, 14569, and 14572 block IL-2 binding to CD25. KIT-225 cells were IL-2 starved for 5 days before performing the proliferation assay. Fifty thousand starved cells in culture medium were preincubated with 0, 5, or 10 μg / ml of 14564, 14569, 14572, or parental anti-CD25 antibodies for 30 minutes at 37°C, and then cultured with or without 50 pM human IL-2 for 1.5 days. Cells cultured without antibody and with or without IL-2 were included as controls. 3 After pulsing with 3H-thymidine (37 kBq), the cells were harvested 4 hours later and the incorporated radioactivity was measured using a β-counter. As shown in Figure 6, antibodies 14564, 14569, and 14572 did not inhibit IL-2-induced proliferation of KIT-225, demonstrating that these antibodies do not block IL-2 binding to CD25. 【0389】 Example 6 Human CD25-targeting PIT This example describes a study performed to determine whether hCD25-targeted PIT can kill human Treg cells. Splenocytes obtained from MDA-MD231 tumor-bearing hCD34-NSG humanized mice were incubated with 5 μg of IR700-conjugated anti-CD25 antibody 14564, followed by 50 J / cm2. 2 of NIR light at 150mW / cm 2 The cells were irradiated with IR700. Non-irradiated splenocytes cultured in the absence of antibody-IR700 conjugates served as a control. The percentage of CD25+ Treg cells was assessed by flow cytometry. The gating strategy for this study is shown in Figure 7C, and representative dot plots of CD25+ Treg cells in control and CD25 PIT splenocyte samples are shown in Figure 7A. As shown in Figure 7B, the percentage of CD25+ Treg cells present in the CD25 PIT splenocyte sample was significantly reduced compared to the control splenocytes, indicating that hCD25-targeted PIT is effective in depleting human CD25-positive Treg cells. 【0390】 Example 7 Depletion of CD25-expressing Treg cells in a humanized mouse model This example describes an exemplary method for testing the ability of anti-hCD25-IR700 NIR-PIT to deplete CD25-positive Treg cells in vivo using a mouse model with a human immune system (e.g., the humanized mouse models described in WO2019 / 199799; US Patent Application Publication No. 2019 / 0320633; Yin et al., Am J Cancer Res 10(12):4568-4584, 2020; or Serr et al., Front Immunol 12:643544, 2021). 【0391】 The humanized mice are administered (e.g., by intravenous, intratumoral, intraperitoneal, or intrapleural administration) an anti-hCD25-IR700 conjugate (e.g., a conjugate in which the antibody comprises the CDR sequences or VH / VL domain sequences of antibody 14564, 14569, or 14572) at a dose of 5 μg (or another suitable dose described herein). The humanized mice are then exposed to NIR light at a wavelength of about 660 to about 740 nm (e.g., 50 J / cm). 2 The mice are exposed to 1000 uM of NIR light (or another suitable dose as described herein). The depletion of Treg cells in treated and untreated mice is assessed by flow cytometry. Treatment with anti-hCD25-IR700 NIR-PIT is expected to result in a significant reduction in the number of Treg cells compared to untreated control mice. 【0392】 It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of this disclosure, and we claim all such modifications and variations that come within the scope and spirit of the following claims.

Claims

[Claim 1] A monoclonal antibody that specifically binds to CD25, comprising a variable weight (VH) domain and a variable light (VL) domain, The VH domain comprises complementarity-determining regions 1 (CDR1), 2, and 3, and the amino acid sequences of CDR1, 2, and 3 comprise SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively; A monoclonal antibody in which the VL domain comprises CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 each comprise SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively. [Claim 2] The amino acid sequences of CDR1, CDR2, and CDR3 of the VH domain include SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 52, respectively; The monoclonal antibody according to claim 1, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the VL domain each include SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 48, respectively. [Claim 3] The amino acid sequence of the VH domain is at least 90% identical to that of SEQ ID NO: 21, and CDR1, CDR2, and CDR3 of the VH domain include SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 52, respectively; and / or The monoclonal antibody according to claim 2, wherein the amino acid sequence of the VL domain is at least 90% identical to that of SEQ ID NO: 22, and CDR1, CDR2, and CDR3 of the VL domain each contain SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 48, respectively. [Claim 4] The amino acid sequences of CDR1, CDR2, and CDR3 of the VH domain include SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 52, respectively; The monoclonal antibody according to claim 1, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the VL domain each include SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 48, respectively. [Claim 5] The amino acid sequence of the VH domain is at least 90% identical to that of SEQ ID NO: 31, and CDR1, CDR2, and CDR3 of the VH domain include SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 52, respectively; and / or The monoclonal antibody according to claim 4, wherein the amino acid sequence of the VL domain is at least 90% identical to that of SEQ ID NO: 32, and CDR1, CDR2, and CDR3 of the VL domain each contain SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 48, respectively. [Claim 6] The amino acid sequences of CDR1, CDR2, and CDR3 of the VH domain include SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 53, respectively; The monoclonal antibody according to claim 1, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the VL domain each include SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 48, respectively. [Claim 7] The amino acid sequence of the VH domain is at least 90% identical to that of SEQ ID NO: 37, and CDR1, CDR2, and CDR3 of the VH domain include SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 53, respectively; and / or The monoclonal antibody according to claim 4, wherein the amino acid sequence of the VL domain is at least 90% identical to that of SEQ ID NO: 38, and CDR1, CDR2, and CDR3 of the VL domain each contain SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 48, respectively. [Claim 8] The amino acid sequence of the VH domain includes SEQ ID NO: 21, and the amino acid sequence of the VL domain includes SEQ ID NO: 22; The amino acid sequence of the VH domain includes SEQ ID NO: 31, and the amino acid sequence of the VL domain includes SEQ ID NO: 32; The amino acid sequence of the VH domain includes SEQ ID NO: 37, and the amino acid sequence of the VL domain includes SEQ ID NO: 38; The amino acid sequence of the VH domain includes SEQ ID NO: 1, and the amino acid sequence of the VL domain includes SEQ ID NO: 2; The amino acid sequence of the VH domain includes SEQ ID NO: 3, and the amino acid sequence of the VL domain includes SEQ ID NO: 4; The amino acid sequence of the VH domain includes SEQ ID NO: 5, and the amino acid sequence of the VL domain includes SEQ ID NO: 6; The amino acid sequence of the VH domain includes SEQ ID NO: 7, and the amino acid sequence of the VL domain includes SEQ ID NO: 8; The amino acid sequence of the VH domain includes SEQ ID NO: 9, and the amino acid sequence of the VL domain includes SEQ ID NO: 10; The amino acid sequence of the VH domain includes SEQ ID NO: 11, and the amino acid sequence of the VL domain includes SEQ ID NO: 12; The amino acid sequence of the VH domain includes SEQ ID NO: 13, and the amino acid sequence of the VL domain includes SEQ ID NO: 14; The amino acid sequence of the VH domain includes SEQ ID NO: 15, and the amino acid sequence of the VL domain includes SEQ ID NO: 16; The amino acid sequence of the VH domain includes SEQ ID NO: 17, and the amino acid sequence of the VL domain includes SEQ ID NO: 18; The amino acid sequence of the VH domain includes SEQ ID NO: 19, and the amino acid sequence of the VL domain includes SEQ ID NO: 20; The amino acid sequence of the VH domain includes SEQ ID NO: 23, and the amino acid sequence of the VL domain includes SEQ ID NO: 24; The amino acid sequence of the VH domain includes SEQ ID NO: 25, and the amino acid sequence of the VL domain includes SEQ ID NO: 26; The amino acid sequence of the VH domain includes SEQ ID NO: 27, and the amino acid sequence of the VL domain includes SEQ ID NO: 28; The amino acid sequence of the VH domain includes SEQ ID NO: 29, and the amino acid sequence of the VL domain includes SEQ ID NO: 30; The amino acid sequence of the VH domain includes SEQ ID NO: 33, and the amino acid sequence of the VL domain includes SEQ ID NO: 34; The amino acid sequence of the VH domain includes SEQ ID NO: 35, and the amino acid sequence of the VL domain includes SEQ ID NO: 36; The amino acid sequence of the VH domain includes SEQ ID NO: 39, and the amino acid sequence of the VL domain includes SEQ ID NO: 40; or The monoclonal antibody according to claim 1, wherein the amino acid sequence of the VH domain includes SEQ ID NO: 41 and the amino acid sequence of the VL domain includes SEQ ID NO:

42. [Claim 9] Fab fragment, Fab' fragment, F(ab)' ２ The monoclonal antibody according to claim 1, wherein the antigen-binding fragment is selected from a fragment, a single-chain variable fragment (scFv), and a disulfide-stabilized variable fragment (dsFv). [Claim 10] The monoclonal antibody according to claim 1, further comprising a constant region. [Claim 11] The monoclonal antibody according to claim 10, wherein the constant region includes at least one modification that increases the half-life, stability and / or function of the monoclonal antibody. [Claim 12] The monoclonal antibody according to claim 10, wherein the constant region includes a heavy chain constant region and / or a light chain constant region. [Claim 13] The monoclonal antibody according to claim 12, wherein the heavy chain constant region is a human IgG4 constant region containing SEQ ID NO:

58. [Claim 14] The monoclonal antibody according to claim 12, wherein the constant light chain region is a human Igκ light chain containing SEQ ID NO:

59. [Claim 15] Sequence IDs 80 and 81; Sequence IDs 90 and 91; Sequence IDs 96 and 97; Sequence IDs 60 and 61; Sequence IDs 62 and 63; Sequence IDs 64 and 65; Sequence IDs 66 and 67; Sequence IDs 68 and 69; Sequence IDs 70 and 71; Sequence IDs 72 and 73; Sequence IDs 74 and 75; Sequence IDs 76 and 77; Sequence IDs 78 and 79; Sequence IDs 82 and 83; Sequence IDs 84 and 85; Sequence IDs 86 and 87; Sequence IDs 88 and 89; Sequence IDs 92 and 93; Sequence IDs 94 and 95; Sequence IDs 98 and 99; or Sequence IDs 100 and 101 A monoclonal antibody according to claim 1, comprising: [Claim 16] (i) It does not significantly inhibit the binding of interleukin-2 (IL-2) to CD25; (ii) Does little to no induce antibody-dependent cell-mediated cytotoxicity (ATCC); or (iii) The monoclonal antibody according to claim 1, which is both (i) and (ii). [Claim 17] A monoclonal antibody according to any one of claims 1 to 16; and Photon absorber, effector molecule, or detectable label A conjugate that includes this. [Claim 18] The conjugate according to claim 17, wherein the photon absorber is IR700. [Claim 19] The conjugate according to claim 17, wherein the effector molecule is a toxin. [Claim 20] The conjugate according to claim 17, wherein the detectable label is a fluorescent label, an enzymatic label, or a radioactive label. [Claim 21] A monoclonal antibody according to any one of claims 1 to 16; and Pharmacologically acceptable carriers A composition containing the following: [Claim 22] A nucleic acid molecule encoding a monoclonal antibody according to any one of claims 1 to 16, or encoding its VH domain or VL domain. [Claim 23] The nucleotide sequence of sequence number 122, or its degenerate variant; The nucleotide sequence of sequence number 123, or its degenerate variant; The nucleotide sequences of SEQ ID NOs. 122 and 123, or their degenerate variants; The nucleotide sequence of sequence number 132, or its degenerate variant; The nucleotide sequence of sequence number 133, or its degenerate variant; The nucleotide sequences of SEQ ID NOs. 132 and 133, or their degenerate variants; The nucleotide sequence of sequence number 138, or its degenerate variant; The nucleotide sequence of sequence number 139, or its degenerate variant; The nucleotide sequences of SEQ ID NOs. 138 and 139, or their degenerate variants; The nucleotide sequence of sequence number 168, or its degenerate variant; The nucleotide sequence of sequence number 169, or its degenerate variant; The nucleotide sequences of SEQ ID NOs. 168 and 169, or their degenerate variants; The nucleotide sequence of sequence number 178, or its degenerate variant; The nucleotide sequence of sequence number 179, or its degenerate variant; The nucleotide sequences of SEQ ID NOs. 178 and 179, or their degenerate variants; The nucleotide sequence of sequence number 184, or its degenerate variant; The nucleotide sequence of sequence number 185, or its degenerate variant; or The nucleotide sequences of SEQ ID NOs. 184 and 185, or their degenerate variants. A nucleic acid molecule according to claim 22, comprising: [Claim 24] A vector comprising the nucleic acid molecule described in claim 22. [Claim 25] An isolated host cell comprising the vector according to claim 24. [Claim 26] (i) the monoclonal antibody according to any one of claims 1 to 16; and (ii) Buffers, cell culture media, one or more checkpoint inhibitors, one or more immunotherapies, one or more additional anticancer reagents, one or more reducing agents, one or more transfection reagents and / or explanatory materials A kit that includes this. [Claim 27] A composition for use in a method of treating cancer in a subject, comprising the conjugate described in claim 18, wherein the method is The step of administering the composition to the subject; and Next, wavelengths of 660–740 nm and at least 1 J / cm ２ The steps of irradiating the target with the dose and / or irradiating cancer cells in the target. A composition comprising, thereby treating cancer in the subject. [Claim 28] The composition according to claim 27, characterized in that the conjugate is administered to the site of the cancer or its vicinity. [Claim 29] The composition according to claim 27, wherein the administration of the conjugate is not systemic. [Claim 30] The composition according to claim 27, wherein the administration of the conjugate is systemic. [Claim 31] The composition according to claim 27, further comprising the step of administering a second monoclonal antibody conjugated to IR700, wherein the second monoclonal antibody specifically binds to a tumor antigen expressed by the cancer cells. [Claim 32] The tumor antigens include epidermal growth factor receptor (EGFR / HER1), mesothelin, prostate-specific membrane antigen (PSMA), HER2 / ERBB2, CD3, CD18, CD20, CD25 (IL-2Rα receptor), CD30, CD33, CD44, CD52, CD133, CD206, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), Lewis Y, tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 2 (EphA2), glypican-1 (GPC1), glypican-2 (GPC2), and glypican-2. Pican-3 (GPC3), gpA33, mucin, CAIX, folate-binding protein, ganglioside, integrin αVβ3, integrin α5β3,1, Erb-B2 receptor tyrosine kinase 3 (ERBB3), MET proto-oncogene, receptor tyrosine kinase (MET), insulin-like growth factor 1 receptor (IGF1R), ephrin type A receptor 3 (EPHA3), tumor necrosis factor-associated apoptosis-inducing ligand receptor 1 (TRAILR1), TRAILR2, nuclear factor kappa-B receptor activator ligand (RANKL), fibroblast-activating protein (FAP), tenascin, BCR complex, gp72, HLA-DR The composition according to claim 31, selected from 10β, HLA-DR antigen, IgE, CA242, polymorphic epithelial mucin (PEM) antigen, SK-1 antigen, programmed cell death 1 (PD-1), and programmed cell death ligand 2 (PD-L2). [Claim 33] The composition according to claim 31, characterized in that the conjugate and the second monoclonal antibody conjugated to IR700 are both administered before irradiation. [Claim 34] The composition according to claim 27, further comprising the step of administering one or more additional cancer treatments to the subject. [Claim 35] The composition according to claim 34, wherein the one or more additional cancer treatments include an anti-CTLA4-IR700 conjugate, an anti-PD-L1-IR700 conjugate, an immune activator, a checkpoint inhibitor, chemotherapy, radiotherapy, and / or surgery. [Claim 36] The composition according to claim 31, characterized in that the conjugate and the second monoclonal antibody conjugated to IR700 are administered intravenously. [Claim 37] The composition according to claim 27, wherein the cancer is a cancer of the breast, liver, lung, esophagus, stomach, colon, ovary, prostate, pancreas, brain, cervix, kidney, bone, skin, head and neck, oropharynx, or blood. [Claim 38] A monoclonal antibody that specifically binds to CD25, (i) It does not significantly inhibit the binding of interleukin-2 (IL-2) to CD25; (ii) Does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) at all or in very little; or (iii) A monoclonal antibody that is both (i) and (ii). [Claim 39] The monoclonal antibody according to claim 38; and Photon absorber, effector molecule, or detectable label A conjugate that includes this. [Claim 40] The conjugate according to claim 39, wherein the photon absorber is IR700.

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