CD25-specific antibodies and uses thereof

Abstract

Monoclonal antibodies that specifically bind to CD25, as well as conjugates of anti-CD25 antibodies, are described. The CD25-specific monoclonal antibodies and conjugates thereof do not block the binding of IL-2 to CD25 and hardly induce antibody dependent cytotoxicity (ADCC). The anti-CD25 antibodies and conjugates may be used, for example, to target photoimmunotherapy to T-regulatory (Treg) cells in a tumor bed to enhance a local host immune response to a tumor.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 357,215, filed June 30, 2022, which is incorporated herein by reference in its entirety.

[0003] field

[0004] 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 antibody-dependent cellular cytotoxicity (ADCC). The present disclosure further relates to the use of CD25-specific antibodies, for example, for targeting CD25-expressing T regulatory (Treg) cells in tumor beds.

[0005] Government Support Statement

[0006] This invention was made with government support from the National Institutes of Health under Grant ZIABC 010656. The government has certain rights in this invention.

[0007] Incorporation of Electronic Sequence Listings

[0008] The electronic sequence listing is submitted as an XML file named 4239-107471-02.xml (245,918 bytes), created on June 20, 2023, and is incorporated herein by reference in its entirety.

[0009] background

[0010] Near-infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that uses targeted monoclonal antibody-photoabsorber conjugates. After the antibody-photoabsorber conjugate is localized to the tumor cell surface antigen via the antibody, NIR light is used to induce highly selective cell lysis. NIR-PIT induces rapid necrotic cell death and produces innate immune ligands that activate dendritic cells (DCs), which is consistent with immunogenic cell death (ICD). Sato et al. described how NIR-PIT kills tumor cells (ACS Cent. Sci. 4: 1559-69, 2018). In short, after the antibody-photoabsorber conjugate binds to its target, activation by NIR light causes a physical change in the shape of the antibody-antigen complex, which causes physical stress within the cell membrane, leading to increased transmembrane water flow, and ultimately leading to cell rupture and necrotic cell death.

[0011] CD25 is a type I transmembrane protein present in activated T cells, activated B cells, certain thymocytes, bone marrow precursors and oligodendrocytes. T regulatory (Treg) cells also express CD25 and play an important role in immunosuppression by allowing tumors to escape immune surveillance. Although Treg cell depletion can lead to tumor regression, the systemic depletion of Treg cells can also induce autoimmune adverse events (Okada et al., Bioconjugate Chem 30 (10): 2624-2633, 2019). Therefore, there is still a need for a method for targeting CD25 positive Treg cells without producing negative systemic side effects, such as in a tumor bed.

[0012] Overview

[0013] Disclosed herein is a group of humanized anti-CD25 monoclonal antibodies that specifically bind to CD25 (also known as interleukin-2 receptor alpha chain – IL-2Rα). The disclosed antibodies do not significantly block the binding of IL-2 to CD25 and induce little ADCC. In some instances, the disclosed CD25 antibodies have sub-nM affinity for CD25. 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 instances, the disclosed CD25 antibodies are conjugated to IR700 (CD25 antibody-IR700 conjugates) and can be used to treat tumors, such as immunogenic tumors. In some instances, the treatment is local rather than systemic.

[0014] Provided herein are monoclonal antibodies that specifically bind to CD25. The disclosed antibodies include at least a heavy chain variable (VH) domain and a light chain variable (VL) domain. In certain aspects, the VH domain complementarity determining region 1 (CDR), CDR2, and CDR3 sequences include SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively, and the VL domain CDR1, CDR2, and CDR3 sequences include SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively. In some instances, the CDR sequences of the VH domain and the VL domain are the CDR sequences of antibodies 14564, 14569, or 14572. In certain aspects, the monoclonal antibody further includes a constant region, such as a heavy chain constant region and / or a light chain constant region. In certain aspects, the antibody does not significantly inhibit the binding of IL-2 to CD25 and / or hardly induces ADCC.

[0015] 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 certain aspects, the photon absorber is IR700.

[0016] Also provided are compositions comprising a pharmaceutically acceptable carrier and the disclosed antibodies or conjugates.

[0017] Also provided are nucleic acid molecules encoding the CD25-specific monoclonal antibodies disclosed herein or encoding the VH domain or VL domain thereof. Also provided are vectors comprising the disclosed nucleic acid molecules, and isolated cells comprising the vectors or nucleic acid molecules disclosed herein.

[0018] Also provided are kits comprising monoclonal antibodies, conjugates, compositions, nucleic acid molecules, vectors or isolated cells disclosed herein. In some aspects, the kit further includes a buffer, a cell culture medium, a transfection reagent, one or more checkpoint inhibitors, one or more immunotherapies, one or more other anticancer agents (such as chemotherapeutics or biological agents) and / or explanatory materials. In one example, the kit further includes a tumor-specific antibody-IR700 conjugate (e.g., cetuximab-IR700 or any other antibody provided in Table 1).

[0019] Further provided are methods of treating cancer in a subject. In some aspects, the method comprises administering to the subject a therapeutically effective amount of a CD25 antibody-IR700 conjugate (wherein the CD25 antibody is one disclosed herein), followed by administration of a 660 to 740 nm wavelength and at least 1 J / cm 2 or at least 4J / cm 2 In some examples, the method further comprises administering a second monoclonal antibody coupled to IR700, wherein the second monoclonal antibody specifically binds to a tumor antigen expressed by cancer cells (e.g., see Table 1).

[0020] The above and other features of the present disclosure will become more apparent from the following detailed description of several aspects with reference to the accompanying drawings.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 1 is a set of images showing the binding of antibodies 14564, 14569, and 14572 to cynomolgus monkey CD25 by surface plasmon resonance. Parental antibodies VH2+VL4 were used as a control. Antibodies 14564, 14569, and 14572 bind to CD25 with high affinity.

[0023] Figure 2 This graph shows the binding of antibodies 14564, 14569, and 14572 to CD25-expressing KIT-225 cells (a human T cell line) as analyzed by FACS. The anti-CD25 antibody basiliximab was used as a positive control. Antibodies 14564, 14569, and 14572 bind to CD25 with similar affinities to basiliximab.

[0024] Figure 3 This graph shows the binding affinity of antibodies 14564, 14569, and 14572 to CD25-expressing KIT-225 cells as analyzed by FACS. The anti-CD25 antibody basiliximab was used as a positive control. The calculated effective concentration 50 (EC50) for each antibody is also shown.

[0025] Figures 4A-4B This figure shows the results of a cytotoxicity assay to determine whether antibodies 14564, 14569, and 14572 can induce antibody-dependent cellular cytotoxicity (ADCC) on cells expressing CD25. Basiliximab was used as a positive control. Only basiliximab induced ADCC on target cells.

[0026] Figures 5A-5C Results from studies evaluating targeting photoimmunotherapy (PIT) to cells expressing CD25 are shown. Figure 5A ) Binding of anti-hCD25-IR700 to KIT-225 cells. KIT-225 cells (0.5×10 6 ) 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 before incubation with anti-hCD25-IR700. Cells were stained with a viability dye and analyzed by fluorescence-activated cell sorting (FACS). Unstained cells served as a negative control. ( Figure 5B ) Representative flow cytometry images showing live / dead staining after PIT (0 J = no NIR light; 5 J = with 5 J / cm 2 NIR light irradiation; 20J = 20J / cm 2 NIR light irradiation). ( Figure 5C ) Cell survival rate after hCD25 targeting PIT. KIT-225 cells (0.5×10 6 ) were incubated with 1 μg of anti-hCD25-IR700 conjugate and the 2 Apply 0, 5, 20 or 50 J / cm2 The cells were then stained with a viability dye and analyzed by FACS. Results are shown as the mean ± SEM of five replicates.

[0027] Figure 6 Anti-CD25 antibodies do not block the binding of IL-2 to CD25. KIT-225 cells were starved of IL-2 for five days before the proliferation assay was performed. 50,000 starved cells were pre-incubated with 0, 5, or 10 μg / ml of 14564, 14569, 14572, or parent anti-CD25 antibodies in culture medium for 30 minutes at 37°C and then cultured for 1.5 days in the presence or absence of 50 pM human IL-2. Cells cultured in the absence of antibody and in the presence or absence of IL-2 served as controls. 3 After 14 h of thymidine pulse (37 kBq), cells were harvested 4 hours later and the incorporated activity was measured using a β-counter. The results showed that antibodies 14564, 14569, and 14572 did not inhibit IL-2-induced proliferation of KIT-225 cells.

[0028] Figures 7A-7C : PIT targeting hCD25 kills human Treg cells. Splenocytes obtained from hCD34-NSG humanized mice bearing MDA-MD 231 tumors were incubated with 5 μg of IR700-conjugated anti-CD25 antibody 14564 and then irradiated at 150 mW / cm 2 Use 50J / cm 2 NIR light irradiation was performed. Splenocytes cultured without the antibody-IR700 conjugate and not irradiated were used as controls. The percentage of CD25+ Treg cells was assessed by flow cytometry. Figure 7A ) Representative dot plots showing CD25+ Treg cells in control (upper) and CD25 PIT (lower) splenocytes. ( Figure 7B ) The image shows the percentage of CD25+ Treg cells in control and CD25 PIT splenocyte samples (n=5). ( Figure 7C ) gating strategy.

[0029] Sequence Listing

[0030] The nucleic acid and amino acid sequences listed in the accompanying sequence listing use standard letter abbreviations for nucleotide bases and single-letter codes for amino acids as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand should be understood to be included in any reference to the displayed strand. In the accompanying sequence listing:

[0031] SEQ ID NOs: 1-42 are the amino acid sequences of the VH and VL domains of anti-CD25 monoclonal antibodies.

[0032] SEQ ID NOs: 43-48 are the consensus amino acid sequences of anti-CD25 monoclonal antibody CDRs.

[0033] SEQ ID NOs: 49-56 are the amino acid sequences of exemplary anti-CD25 monoclonal antibody CDRs.

[0034] SEQ ID NO: 57 is the amino acid sequence of an exemplary leader sequence.

[0035] SEQ ID NO: 58 is the amino acid sequence of a modified human IgG4 heavy chain constant region.

[0036] SEQ ID NO: 59 is the amino acid sequence of the human Ig kappa light chain constant region.

[0037] SEQ ID NOs: 60-101 are the amino acid sequences of the heavy and light chains of anti-CD25 antibodies.

[0038] SEQ ID NOs: 102-143 are nucleic acid sequences encoding the anti-CD25 VH and VL domains.

[0039] SEQ ID NOs: 144-145 are the nucleic acid sequences of exemplary leader sequences.

[0040] SEQ ID NO: 146 is a nucleic acid sequence encoding a modified human IgG4 heavy chain constant region.

[0041] SEQ ID NO: 147 is a nucleic acid sequence encoding the human Igκ light chain constant region.

[0042] SEQ ID NOs: 148-189 are nucleic acid sequences encoding the heavy and light chains of anti-CD25 monoclonal antibodies.

[0043] Details

[0044] I. Introduction

[0045] Therapeutic monoclonal antibodies (mAbs) are typically administered systemically to block a specific biological function (e.g., by binding to the ligand binding site of a specific receptor) or to attract immune cells to the target site to kill cells bound to the antibody, thereby producing antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). While these strategies have achieved some success, drug doses of mAbs can also cause unexpected and adverse side effects. The present disclosure describes the development of humanized anti-CD25 antibodies that can be used as a local delivery platform (as opposed to systemic therapy) without significant biological or cytotoxic properties.

[0046] Two anti-CD25 antibodies developed for systemic therapy have been commercialized: daclizimab and basiliximab. Both antibodies block the IL-2α receptor, thereby interfering with the activation of effector CD8 + These two antibodies were developed as immunosuppressants to combat autoimmune diseases, such as daclizumab for multiple sclerosis (MS) and basiliximab for organ transplant rejection. However, because daclizumab and basiliximab also block the proliferation of Treg cells, their potential side effect is autoimmune syndrome. In fact, daclizumab was withdrawn from the market in 2018 due to complications associated with brain inflammation. Basiliximab continues to be used as an induction therapy to reduce immune responses in organ transplants.

[0047] Photoimmunotherapy (PIT) is a cancer treatment method that is completely different from the principles of surgery, drug therapy (including immunotherapy) or radiotherapy. A specific infrared dye (such as IR700) is coupled to a target-specific antibody to form an antibody-photoabsorbent conjugate (APC). When the APC is injected systemically, it binds to the target receptor on the tumor cell membrane, a process that usually takes about 24 hours in the human body. Near-infrared (NIR) light is then applied to the tumor. This causes photochemical changes in the APC, resulting in damage to the part of the cell membrane that is bound to the APC. By causing a large number of such microperforations in the cell membrane, the cells are irreversibly damaged and die. Within a few minutes of NIR light exposure, the target cells swell, blister and rupture, leading to cell death. This type of treatment completely avoids the side effects of systemic mAb therapy.

[0048] The present disclosure describes humanized CD25-specific mAbs suitable for NIR-PIT because they do not have any specific biological function when not coupled to effector molecules (such as IR700). CD25 is an IL-2α receptor that is present on regulatory T cells (Treg cells) and activated effector T cells, as well as on other types of immune cells. CD25 antibodies can be blocking or non-blocking for IL-2, which is a cytokine that causes cell proliferation of immune cell subtypes. Non-IL-2 blocking CD25-specific antibodies bind to CD25 without interfering with the binding of IL-2, and therefore have no direct pharmacokinetic effect. Therefore, APCs directed against CD25 protein can selectively consume Treg cells within the tumor range without producing systemic effects.

[0049] The present disclosure provides humanized anti-CD25 antibodies having at least the following characteristics: (i) high-affinity binding to CD25 that is not impaired by conjugation to IR700; (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 characteristics, the disclosed anti-CD25 antibodies provide an excellent delivery platform for effector molecules, including IR700, for example, for the treatment of cancer.

[0050] II. Abbreviations

[0051] ADCC Antibody-dependent cellular cytotoxicity

[0052] CDR complementarity determining region

[0053] DC dendritic cells

[0054] FACS fluorescence-activated cell sorting

[0055] FLT fluorescence lifetime

[0056] J / cm 2 Joules / square centimeter

[0057] IL-2 Interleukin-2

[0058] IL-2R interleukin-2 receptor

[0059] LED light-emitting diode

[0060] mAb monoclonal antibody

[0061] NIR near infrared light

[0062] PIT Photoimmunotherapy

[0063] RU Response Unit

[0064] Treg T regulation

[0065] VH heavy chain variable region

[0066] VL light chain variable region

[0067] III. Summary of Terminology

[0068] Unless otherwise indicated, technical terms are used in accordance with 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 both singular and plural antigens and may be considered equivalent to the phrase "at least one antigen". As used herein, the term "comprising" means "including". It should also be understood that any and all base sizes or amino acid sizes of nucleic acids or polypeptides, and all molecular weight or molecular mass values ​​are approximate and are provided for descriptive purposes only, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described herein. In the event of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples described are illustrative only and are not intended to be limiting. All references to the present specification and all ... The sequences associated with the accession numbers are all incorporated by reference as of June 30, 2022. To facilitate reference to various aspects of this disclosure, the following explanations of specific terms are provided:

[0069] Abscopal effect: Treatment of tumors, such as metastases, in parts of the body that are not the direct target of a local treatment (e.g., NIR-PIT). For example, irradiating a specific tumor with NIR light in combination with an appropriate antibody-IR700 conjugate can reduce the size of a different tumor (e.g., metastases) that was not irradiated with NIR light. In some instances (e.g., in humans), the non-irradiated / distant tumor is at least 3 inches away from the tumor treated with NIR light, such as 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.

[0070] Administration: providing or administering an agent, such as 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 by intravenous injection. In one example, administration is by intraperitoneal administration. In one example, administration is not systemic. In one example, administration is local administration to a tumor or cancer.

[0071] Antibody: A polypeptide ligand containing at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an epitope of an antigen, such as a tumor-specific protein or a protein specifically expressed on an immune cell such as a T cell (e.g., a T regulatory cell). Mammalian immunoglobulin molecules are composed of heavy (H) chains and light (L) chains, each of which has a variable region, referred to as a heavy chain variable (V H ) region and light chain variable (V L ) area. H Area and V L The two regions are collectively responsible for binding to the antigen recognized by the antibody. Typically, naturally occurring mammalian immunoglobulins have heavy chains and light chains interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). Mammalian immunoglobulins have five major heavy chain classes (or isotypes), which 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 main antibody produced by birds and reptiles and has similar functions to 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.

[0072] Antibodies, such as those in antibody-IR700 conjugates, include intact immunoglobulins as well as variants and portions of antibodies, such as Fab fragments, Fab' fragments, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which the variable region of an immunoglobulin light chain and the variable region of an immunoglobulin heavy chain are linked together by a linker, while in dsFv, the chains are mutated to introduce disulfide bonds to stabilize the binding 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). See Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0073] The variable region of an antibody consists of a "framework" region and hypervariable regions, called "complementarity determining regions" or "CDRs." CDRs are primarily responsible for binding to antigenic epitopes. The framework region of an antibody serves to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR are readily determined by any of a number of well-known numbering schemes, including Kabat et al. (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; “Kabat” numbering scheme), Chothia et al. (see 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. (see Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40:W521-524, 2012; “Paratome The Kabat, Paratome, and IMGT databases are maintained online. In addition, the AbRSA tool can be used to determine CDR boundaries according to Kabat, IMGT, or Chothia (available online at aligncdr.labshare.cn / aligncdr / abrsa.php).

[0074] "Single domain antibody" refers to an antibody with a single domain (variable domain) that can specifically bind to an antigen or antigenic epitope in the absence of other antibody domains. Single domain antibodies include, for example, V H Domain antibodies, V NAR Antibodies, camel V H H antibody and V L Domain antibodies, etc. NAR Antibodies are produced by cartilaginous fishes such as nurse sharks, long-arm sharks, spiny dogfish, and bamboo sharks, which produce heavy chain antibodies (IgNARs). H H antibodies are produced by several species, including camels, llamas, alpacas, and guanacos, which naturally produce heavy chain antibodies without light chains.

[0075] A "monoclonal antibody" is an antibody produced by a single lymphocyte clone or by cells transfected with a single antibody coding sequence. Monoclonal antibodies are produced by known methods. Monoclonal antibodies include humanized monoclonal antibodies.

[0076] "Chimeric antibodies" have framework residues from one species, such as human, and the CDRs (typically conferring antigen binding) from another species.

[0077] "Humanized" antibodies are immunoglobulins that include human framework regions and one or more CDRs from non-human (e.g., mouse, rabbit, rat, shark, camel, or synthetic) immunoglobulins. The non-human immunoglobulin that provides the CDRs is called a "donor," and the human immunoglobulin that provides the framework is called an "acceptor." In one aspect, all CDRs in the humanized immunoglobulin are from donor immunoglobulins. The constant region does not necessarily have to be present, but if present, it is substantially identical to the human immunoglobulin constant region, i.e., at least about 85-90%, such as about 95% or higher percentages. Therefore, except for possible CDRs, all parts of the humanized immunoglobulin are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. Humanized antibodies bind to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions that have little or no effect on antigen binding or other immunoglobulin functions.

[0078] "Specific binding" of an antibody to an antigen refers to the ability of an individual antibody to specifically immunoreact with an antigen, such as a tumor-specific antigen, relative to binding to unrelated proteins, such as non-tumor proteins, such as β-actin. For example, a CD25-specific binding agent binds substantially only to the 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 the tumor-specific protein in the formulation.

[0079] Binding is a non-random binding reaction between an antibody molecule and an antigenic determinant, such as CD25. The desired binding specificity is often determined based on the antibody's ability to differentially bind to the target antigen and an unrelated antigen, thereby distinguishing between the two different antigens, particularly when the two antigens have unique epitopes. Antibodies that specifically bind to a particular epitope are called "specific antibodies."

[0080] Antibody-IR700 conjugates: Molecules comprising an antibody conjugated to IR700, such as a CD25-specific antibody or a tumor-specific antibody. In certain embodiments, the antibody is a humanized antibody that specifically binds to CD25, such as the antibodies disclosed herein. In other embodiments, the antibody is a monoclonal antibody that specifically binds to a surface protein on cancer cells, such as a tumor-specific antigen.

[0081] Antigen (Ag): A compound, composition, or substance capable of stimulating an animal to produce an antibody or T cell response, including compositions injected into or absorbed into an animal (e.g., compositions comprising a tumor-specific protein or CD25 protein). Antigens react with products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. An "epitope" or "antigenic determinant" refers to a region of an antigen to which B cells and / or T cells respond. In one aspect, T cells respond to an epitope when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed by either contiguous amino acids or by non-contiguous amino acids juxtaposed after tertiary folding of a protein. Epitopes formed by contiguous amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. Epitopes typically comprise at least three, more typically at least five, about nine, or about 8-10 amino acids, in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and nuclear magnetic resonance.

[0082] Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as antigenic determinants recognized by immune cells. In some instances, antigens include tumor-specific proteins or peptides (such as proteins or peptides found on the surface of cells such as cancer cells) or immunogenic fragments thereof. In some instances, antigens include immune cell-specific proteins or peptides (proteins or peptides found on the surface of immune cells such as T cells, such as regulatory T cells) or immunogenic fragments thereof. In one example, the antigen is CD25, such as mammalian CD25, such as mouse or human CD25.

[0083] Binding affinity: The affinity of an antibody or other antigen-binding molecule (such as an antibody-IR700 conjugate) for an antigen such as CD25 or a tumor-specific antigen. In one aspect, affinity is calculated by the modified Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. On the other hand, binding affinity is measured by antigen / antibody dissociation rate. In another aspect, binding affinity is measured by competitive radioimmunoassay. In another aspect, binding affinity is measured by ELISA. In certain aspects, binding affinity is measured using the Octet system (ForteBio) based on biolayer interferometry (BLI) technology. In other aspects, Kd is measured by surface plasmon resonance assay using BIACores-2000 or BIACores-3000 (BIAcore, Inc., Piscataway, NJ). In other aspects, antibody affinity is measured by flow cytometry. An antibody or antibody conjugate that "specifically binds" to an antigen (e.g., CD25) is one that binds to the antigen with high affinity and does not significantly bind to other unrelated antigens. In certain embodiments, an antibody (e.g., the antibody in an antibody-IR700 conjugate) specifically binds to a target (e.g., a cell surface protein such as CD25 or a tumor-specific protein) with a binding constant that is at least 10 greater than the binding constant for other molecules in the sample or subject. 3 M -1 , 10 4 M -1 or 10 5 M -1 In certain embodiments, the antibody (e.g., mAb) or fragment thereof has an equilibrium constant (Kd) of 1 nM or less. For example, the antibody binds to a target, such as CD25 or a tumor-specific protein, with an affinity of at least about 0.1×10 -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 M.

[0084] Cancer: A malignant tumor characterized by abnormal or uncontrolled cell growth. Other characteristics commonly associated with cancer include metastasis, interference with the normal function of neighboring cells, abnormal release of cytokines or other secretory products, suppression or exacerbation of inflammatory or immune responses, and invasion of surrounding or distant tissues or organs, such as lymph nodes. "Metastatic disease" refers to the process by which cancer cells leave the initial tumor site and metastasize to other parts of the body, for example, via the blood or lymphatic system. In one embodiment, the cells killed by the disclosed compositions and methods are cancer cells.

[0085] CD25 (IL-2 receptor alpha chain): (e.g., OMIM 147730) A type I transmembrane protein present on activated T cells, activated B cells, certain thymocytes, myeloid precursors, and oligodendrocytes. 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 tumors, some acute non-lymphocytic leukemias, neuroblastoma, mastocytosis, and tumor infiltrating lymphocytes. It functions as a receptor for HTLV-1 and is therefore expressed on tumor cells in adult T cell lymphomas / leukemias. Exemplary CD25 sequences can be found in database (e.g., Accession No. CAA44297.1, NP_000408.1, and NP_001295171.1).

[0086] Complementarity Determining Region (CDR): A region of hypervariable amino acid sequence that defines an antibody's binding affinity and specificity. Mammalian immunoglobulins have three CDRs each in their light and heavy chains, designated L-CDR1, L-CDR2, and L-CDR3, and H-CDR1, H-CDR2, and H-CDR3, respectively.

[0087] Conjugate: In the present 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.

[0088] Conservative variants: In the present disclosure, "conservative" amino acid substitutions refer to substitutions that do not substantially affect or reduce the affinity of a protein (e.g., a CD25 antibody). As an example, a monoclonal antibody that specifically binds to CD25 may include up to about 1, up to about 2, up to about 5, up to about 10, up to about 15, up to about 20, or up to about 25 conservative substitutions and specifically bind 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 refer to substitutions that reduce the activity (e.g., affinity) of a protein.

[0089] Conservative amino acid substitution tables that provide functionally similar amino acids are well known. The following six groups of amino acids are examples of amino acids that are considered conservative substitutions for each other:

[0090] 1) Alanine (A), serine (S), threonine (T);

[0091] 2) Aspartic acid (D), glutamic acid (E);

[0092] 3) Asparagine (N), glutamine (Q);

[0093] 4) Arginine (R), Lysine (K);

[0094] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0095] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0096] In certain aspects herein, amino acid sequences are provided that comprise 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 relative to any amino acid sequence disclosed herein.

[0097] Contacting: Placing in direct physical association, including solid and liquid forms. Contacting can occur in vitro, for example with isolated cells, such as tumor cells, or in vivo by administration to a subject, such as a subject having a tumor, such as cancer.

[0098] Effector molecule: The portion of an antibody conjugate that is designed to produce a desired effect on the cells targeted by the conjugate. The effector molecule can be, for example, a detectable label, a photon absorber (such as IR700), or a toxin. Effector molecules are also referred to 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 crosslinking with single-stranded or double-stranded DNA, and oligonucleotides that form triplexes. In addition, the effector molecule can be contained in an encapsulation system coupled to the antibody, such as a nanoparticle, liposome, or micelle. Encapsulation can prevent direct exposure of the effector molecule to the circulatory system. Methods for preparing liposomes linked to antibodies are well known to those skilled in the art (e.g., see U.S. Patent No. 4,957,735; and Connor et al., Pharm Ther 28:341-365, 1985). Diagnostic agents or molecules include radioisotopes and other detectable labels (such as 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 and 125 I.

[0099] Framework region: The amino acid sequence between the CDRs. The framework region includes both the light chain variable and heavy chain variable framework regions. The framework region serves to maintain the CDRs in the proper orientation for antigen binding.

[0100] Heterologous: Derived from a different genetic source or species.

[0101] Host cell: A cell in which a vector can replicate and express its DNA. The cell can be prokaryotic or eukaryotic. In some instances, a prokaryotic cell is an Escherichia coli (E. coli) cell. In some instances, 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 differ from the parent cell due to mutations that may occur during replication. However, when the term "host cell" is used, these progeny are included.

[0102] Immune response: A response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In one aspect, the response is specific for a particular antigen (an "antigen-specific response"). In one aspect, the immune response is a T cell response, such as a CD4 + response or CD8 + On the other hand, the response is a B cell response and results in the production of specific antibodies.

[0103] Immunomodulators: Immunomodulators are substances that alter (e.g., increase or decrease) one or more functions of the immune system. In certain instances, immunomodulators activate the immune system. In other instances, immunomodulators inhibit the activity of or kill immunosuppressive cells. In some instances, the immunomodulator is a mAb, which can be linked to IR700 to form mAb-IR700, which can be used in the disclosed methods to target cells expressing the protein recognized by the mAb. Exemplary immunomodulators include the immune activators IL-15 and IFN-γ.

[0104] IR700( 700DX): a dye having the formula:

[0105]

[0106] Amine-reactive IR700 is a relatively hydrophilic dye that can be covalently coupled to antibodies via the NHS ester of IR700. The extinction coefficient of IR700 (at a maximum absorption of 689 nm) is 2.1×10 5 M -1 cm -1 ) are traditional photosensitizers such as hematoporphyrin derivatives (1.2×10 at 630 nm 3 M -1 cm -1 ), m-tetrahydroxyphenyl chloride;

[0107] (2.2×10 at 652nm 4 M -1 cm -1 ) and mono-L-aspartylchlorin e6;

[0108] NPe6 / (4.0×10 at 654 nm 4 M -1 cm -1 ) is more than 5 times.

[0109] Label (or detectable label): A detectable compound or composition that is coupled directly or indirectly to another molecule, such as an antibody or protein, to facilitate detection of the molecule. Specific, non-limiting examples of labels include fluorescent tags, enzyme conjugates, and radioactive isotopes. In one example, "labeled antibody" refers to the incorporation of another molecule into the antibody. For example, a label is a detectable marker, such as the incorporation of a radiolabeled amino acid, or the attachment of a biotin molecule to a polypeptide, which can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity, which can be detected optically or colorimetrically). Various methods of labeling polypeptides and glycoproteins are known and can be used. Examples of polypeptide labels include, but are not limited to, the following: radioisotopes or radionucleotides (such as 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 phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotin groups, predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In certain aspects, the labels are attached via spacer arms of varying lengths to reduce potential steric hindrance.

[0110] Pharmaceutical composition: A chemical compound or composition capable of producing a desired therapeutic or prophylactic effect when properly administered to a subject. Pharmaceutical compositions may include therapeutic agents, such as one or more antibodies, antibody-IR700 conjugates, and / or one or more immunostimulants. A therapeutic agent or pharmaceutical agent is an agent that, alone or in combination with other compounds, induces a desired response (e.g., a therapeutic or prophylactic effect when administered to a subject). In certain embodiments, a pharmaceutical composition includes a therapeutically effective amount of at least one antibody-IR700 conjugate.

[0111] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers (vehicles) that can be used in the present 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 drug delivery of one or more therapeutic compounds, such as one or more antibodies or antibody-IR700 conjugates disclosed herein.

[0112] In general, the character of the carrier depends on the specific mode of administration adopted. For example, parenteral formulations generally include injectable liquids, including pharmaceutically and physiologically acceptable liquids such as water, physiological saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. as carriers. For solid compositions (e.g., powders, pills, tablets, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain a small amount of non-toxic auxiliary substances such as wetting agents or emulsifiers, preservatives, pH buffers, etc., such as sodium acetate or sorbitol monolaurate.

[0113] Photoimmunotherapy (PIT): A molecular targeted therapy that utilizes a target-specific photosensitizer based on the near-infrared (NIR) phthalocyanine dye IRDye700DX (IR700) coupled to a monoclonal antibody (mAb) that targets a cellular protein (e.g., a cell surface protein, such as CD25 on the surface of Treg cells, or a tumor-specific protein). In one example, the cell surface protein is a specific protein found on cancer cells or immune cells, so PIT can be used to kill these cells. When the antibody-IR700 conjugate binds to the cell and the cell is irradiated with NIR, cell death occurs, while cells that do not express the cell surface protein recognized by the antibody-IR700 conjugate are not killed in significant amounts.

[0114] Reducing agent: An element or compound that loses (or "donates") electrons to an electron acceptor (oxidant) in a redox chemical reaction. Thus, a reducing agent is oxidized when it loses electrons in a redox reaction. The reducing agent "reduces" the oxidant (or is "oxidized" by the oxidant). Therefore, one or more reducing agents can be used in conjunction with NIR-PIT in vitro and in vivo to reduce unwanted nonspecific tissue damage caused by reactive oxygen species generated by IR700 following exposure to NIR light. For example, reducing agents can inactivate unbound reactive oxygen species or those within tumor areas and reduce 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 (such as L- or R-ascorbic acid), and glutathione. In certain instances, sodium azide is not used in vivo due to its toxicity. Therefore, in certain instances, 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.

[0115] Sequence identity: Similarity between amino acid or nucleic acid sequences is expressed as sequence similarity, also known as sequence identity. Sequence identity is often measured as a percentage of 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 will have relatively high sequence identity when aligned using standard methods.

[0116] Alignment methods for comparing sequences 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, provides a detailed introduction to sequence alignment methods and homology calculations.

[0117] NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.

[0118] 215:403, 1990) is available from a number of sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, and can be used with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions for determining sequence identity using this program are available on the NCBI website on the Internet.

[0119] Homologs and variants of antibodies that specifically bind to CD25 are typically characterized by having at least about 75%, such as at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, calculated using NCBI Blast 2.0, gapped blastp, set to default parameters, over the entire length of the antibody amino acid sequence. For comparisons of amino acid sequences greater than about 30 amino acids, the Blast 2 sequences function is used, using the default BLOSUM62 matrix, with default parameters (gap existence cost of 11, per residue gap cost of 1). When aligning short peptides (less than about 30 amino acids), the Blast 2 sequences function is used, using the PAM30 matrix, with default parameters (open gap 9, extension gap 1 penalty). When assessed using this method, proteins that are more similar to a reference sequence will display increasing percentage identities, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When comparing sequences that are less than their full length for sequence identity, homologs and variants will typically have at least 80% sequence identity, and may have at least 85%, at least 90%, or at least 95% sequence identity, over a short window of 10-20 amino acids, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the internet at the NCBI website. These sequence identity ranges are provided for guidance only; it is entirely possible to obtain strong, significant homologs outside the ranges provided.

[0120] Subject or patient: A term encompassing both humans and non-human mammals. In one example, the subject is a human or a veterinary subject, such as a mouse, rat, dog, cat, or non-human primate. In certain examples, the subject is a mammal (e.g., a human) that has cancer or is being treated for cancer.

[0121] Therapeutically effective amount: an amount of a composition (such as a CD25-specific antibody-IR700 conjugate) used alone or in combination with other therapeutic agents (such as anti-tumor antigen-specific antibody-IR700 conjugates, anti-CTLA4-IR700 molecules, anti-PD-L1-IR700 molecules, immunomodulators, checkpoint inhibitors and / or reducing agents) that is sufficient to achieve the desired effect in a subject or cell treated with the formulation. The effective amount of an agent (such as an antibody-IR700 conjugate, used alone or in combination with other agents) depends on a variety of factors, including but not limited to the subject or cell being treated, the specific therapeutic agent, and the mode of administration of the therapeutic composition. In one example, the therapeutically effective amount or concentration of the antibody-IR700 conjugate is an amount or concentration sufficient to prevent disease worsening (such as metastasis), delay disease progression, or cause disease regression, or to reduce symptoms caused by a disease such as cancer. In one example, the therapeutically effective amount or concentration of the antibody-IR700 conjugate is an amount or concentration sufficient to prolong the survival time of a tumor patient. In one example, a therapeutically effective amount or concentration of a reducing agent is an amount or concentration sufficient to reduce edema, acute inflammatory response, or both in a subject being treated with NIR-PIT.

[0122] In one example, the desired reaction is to reduce or suppress one or more symptoms associated with cancer. It is not necessary for the composition to be effective to completely eliminate one or more symptoms. For example, in some instances, compared with the size of the tumor when not treating (or using only a subset of the treatment provided herein), the size of the tumor (such as the volume or weight of the tumor or the metastasis of the tumor) is reduced by treating provided herein, for example, by reducing at least 20%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100%. In a specific example, compared with the cell killing when not treating (or using only a subset of the treatment provided herein), the cell (such as cancer cell) group is treated by provided herein, for example, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100% is killed. In a specific example, treatment with a modality provided herein increases the survival time of a tumor patient (or a patient whose tumor has recently been removed) compared to the survival time without treatment (or using only a subset of the treatments provided herein), for example, by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, 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, for example, by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at least 200%, or at least 500%, compared to the amount of memory T cells without treatment (or using only a subset of the treatments provided herein). In some examples, treatment with the methods provided herein reduces the number of Treg cells (e.g., FOXP3 Tregs) in the targeted tumor / tumor bed compared to the number of Treg cells in the targeted tumor / tumor bed without treatment (or with only a subset of the treatments provided herein). + CD25 + CD4 + In some instances, the number of Treg cells) is increased 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%. In some instances, compared to the CD8+ / Treg ratio when there is no treatment (or only a subset of the treatment provided herein), the CD8+ / Treg ratio of the treated subject is increased by treatment provided herein, for example, by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at least 200% or at least 500%. In some instances, the combination of these effects is achieved by the disclosed method.

[0123] The effective amount of a therapeutic agent administered to a human or veterinary subject depends on a variety of factors related to the subject, such as the subject's overall health. The effective amount of the agent can be determined by varying the dosage of the composition and measuring the resulting therapeutic response, such as tumor regression. The effective amount can also be determined by various in vitro, in vivo, or in situ immunoassays. The disclosed agents can be administered in a single dose or multiple doses as needed to obtain the desired response. However, the effective amount may depend on the treatment method used, the subject being treated, the severity and type of the condition being treated, and the mode of administration.

[0124] In certain examples, a therapeutically effective dose of an antibody-IR700 conjugate is at least 0.5 mg / 60 kg (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, at least 225 mg / 60 kg, at least 250 mg / 60 kg.

[0014] In some embodiments, the therapeutically effective dose of an antibody-IR700 conjugate, such as 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 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, e.g., when administered intratumorally or by intraperitoneal injection. In one example, a therapeutically effective dose of the antibody-IR700 conjugate is at least 1 μg / ml, such as at least 500 μg / ml, such as between 20 μg / ml and 100 μg / ml, such as 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, administered as a topical solution. However, one skilled in the art will recognize that higher or lower doses may also be used, for example, depending on the specific antibody-IR700 conjugate. In certain examples, such daily doses are administered in one or more divided doses (e.g., 2, 3 or 4 doses) or in a single formulation. The disclosed antibody-IR700 conjugates can be administered alone, in the presence of a pharmaceutically acceptable carrier, or in the presence of other therapeutic agents (e.g., other anti-tumor agents and / or reducing agents).

[0125] Generally, a suitable irradiation dose after administration of one or more antibody-IR700 conjugates (and in some instances also a reducing agent) is at least 1 J / cm2 at a wavelength of 660-740 nm. 2 , for example, at least 4 J / cm at a wavelength of 660-740 nm 2 , 660-740nm wavelength at least 10J / cm 2 , 660-740nm wavelength at least 20J / cm 2 , 660-740nm wavelength at least 25J / cm 2 , 660-740nm wavelength at least 50J / cm 2 , or at least 100 J / cm at a wavelength of 660-740 nm 2 For example, 660-740 nm wavelength 1 to 500 J / cm 2 , 660-740nm wavelength 10 to 100J / cm 2 , or 20 to 60 J / cm at 660-740 nm wavelength 2 In certain embodiments, the wavelength is 660-710 nm, such as 680 nm or 690 nm. In a specific embodiment, a suitable radiation dose after administration of the antibody-IR700 conjugate is at least 1 J / cm2 at a wavelength of 680 or 690 nm. 2 , for example, at least 4 J / cm at 680 or 690 nm 2 , 680 or 690 nm wavelength at least 10 J / cm 2 , 680 or 690 nm wavelength at least 20 J / cm 2 , 680 or 690 nm wavelength at least 25 J / cm 2 , 680 or 690 nm wavelength at least 50 J / cm 2 , or at least 100 J / cm at 680 or 690 nm 2 , such as 680 or 690 nm wavelength 4 to 100 J / cm 2 , 4 to 50 J / cm 2 or 10 to 25 J / cm 2 In one non-limiting example, a suitable radiation dose after administration of an antibody-IR700 conjugate is 50 J / cm at a wavelength of 690 nm. 2 In certain examples, multiple irradiations (e.g., at least 2, at least 3, or at least 4 irradiations, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate administrations) are performed after administration of one or more antibody-IR700 conjugates (and in certain examples, also 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).

[0126] Treatment: When used to refer to treating cells or tissues with a therapeutic agent, the term includes contacting or incubating one or more agents (such as one or more CD25 antibody-IR700 conjugates, and in some instances, 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 cells or tissues, and / or administering one or more agents to a subject, such as a cancer subject. Treated cells are cells that have been contacted with a desired composition in an amount sufficient to produce a desired response 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 to bind the antibody to a surface protein of the cell, optionally in contact with a reducing agent, and irradiated with NIR light until sufficient cell killing is achieved. In other examples, the subject being treated is one who has been administered one or more antibody-IR700 conjugates under conditions sufficient to allow the antibody to bind to a surface protein of a cell, optionally administered one or more reducing agents, and irradiated with NIR light until sufficient cell killing is achieved.

[0127] Tumor, neoplasia, malignancy or cancer: A neoplasm is an abnormal growth of tissue or cells caused by excessive cell division. Neoplastic growth can give rise to a tumor. The number of tumors in an individual is the "tumor burden" and can be measured as the number, volume or weight of the tumors. Tumors that do not metastasize are called "benign." Tumors that invade surrounding tissues and / or are able to metastasize are called "malignant." "Non-cancerous tissue" is tissue from the same organ in which the malignant neoplasm forms, but does not have the pathological characteristics of the neoplasm. Generally, non-cancerous tissue appears normal histologically. "Normal tissue" refers to tissue from an organ that has not been affected by cancer or another disease or condition of that organ. "Cancer-free" subjects have not been diagnosed with cancer of that organ and have no detectable cancer.

[0128] Tumors include original (primary) tumors, recurrent tumors, and metastatic (secondary) tumors. Tumor recurrence is when a tumor returns in the same location as the original (primary) tumor, for example, after it has been removed by surgery, medication, or other treatment, or has otherwise disappeared. Metastasis is when a tumor spreads from one part of the body to another. Tumors formed from spread cells are called secondary tumors and contain cells similar to those in the original (primary) tumor. Either a primary tumor or a metastatic tumor can recur.

[0129] Exemplary tumors that can be treated with the disclosed methods, such as cancers, include solid tumors, such as breast cancer (e.g., lobular carcinoma and ductal carcinoma), sarcoma, lung cancer (e.g., non-small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma), lung mesothelioma, colorectal adenocarcinoma, head and neck cancer (e.g., adenocarcinoma, squamous cell carcinoma, metastatic squamous cell carcinoma, such as cancer caused by HPV or Epstein-Barr virus, such as HPV16; and can 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 tumors, testicular cancer and germ cell tumors, pancreatic adenocarcinoma, biliary tract adenocarcinoma, hepatocellular carcinoma, bladder cancer (e.g., In one example, the tumor is adenocarcinoma, adenocarcinoma, and squamous cell carcinoma, including transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma, renal cell adenocarcinoma, endometrial cancer (including, for example, adenocarcinoma and mixed Mullerian tumors (carcinosarcoma)), cancer of the endocervix, endocervix, and vagina (such as adenocarcinoma and squamous cell carcinoma of each thereof), skin tumors (such as squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin appendage tumors, Kaposi's sarcoma, cutaneous lymphoma, skin appendage tumors, and various sarcomas and Merkel cell carcinoma), esophageal cancer, nasopharyngeal cancer, and oropharyngeal cancer (including squamous cell carcinomas and adenocarcinomas 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 bone and chondrosarcomas and lymphoid tumors (including B-cell and T-cell malignant lymphomas). In one example, the tumor is adenocarcinoma. In one example, the tumor is oropharyngeal cancer. In one example, the tumor is squamous cell carcinoma, such as squamous cell carcinoma of the head and neck or skin.

[0130] These methods can also be used to treat liquid tumors (e.g., hematological malignancies), such as lymphatic, leukocyte, 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 myeloma.

[0131] Under conditions sufficient to: This phrase is used to describe any circumstances that allow for a desired activity. In one example, "under conditions sufficient to" includes administering an antibody-IR700 conjugate to a subject in an environment sufficient to allow the antibody-IR700 conjugate to bind to a cell surface protein to which it is targeted (e.g., a tumor-specific antigen or an immune cell-specific antigen, such as CD25). In certain aspects, the desired activity is killing cells to which the antibody-IR700 conjugate binds following therapeutic irradiation of the cells.

[0132] Untreated cells: cells that have not been exposed to a therapeutic agent, such as an antibody-IR700 conjugate and / or irradiation. In one example, an untreated cell is a cell that has not received a vector for delivering a therapeutic agent. Similarly, an untreated subject is a subject that has not been administered a therapeutic agent, such as an antibody-IR700 conjugate and / or irradiation. In one example, an untreated subject is a subject that has not received a vector for delivering a therapeutic agent.

[0133] Vector: A nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may include nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements. In certain instances, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

[0134] The disclosure of certain specific embodiments is not intended to exclude other aspects. In addition, any treatment method described herein does not necessarily exclude other treatments, but can be combined with other biologically active agents or treatment modalities.

[0135] IV. CD25-specific monoclonal antibodies

[0136] Described herein are monoclonal antibodies that bind to human and cynomolgus monkey CD25 (interleukin-2 receptor alpha; IL-2Rα) with high affinity. The disclosed antibodies specifically bind to CD25 but do not inhibit the binding of CD25 to IL-2, nor do the CD25-specific antibodies induce ADCC. In some examples, the CD25 antibodies provided herein, including variants thereof, have sub-nM affinity for CD25, such as human, cynomolgus monkey, or mouse CD25. The amino acid sequences of the VH and VL domains of 21 exemplary CD25-specific monoclonal antibodies are shown in SEQ ID NOs: 1-42. The position of each CDR determined by IMGT is underlined in the sequences below. 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.

[0137] 14554-VH (SEQ ID NO: 1)

[0138] EVQLVESGGGLVQPGGSLRLSCAASGFWFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0139] WYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYTNSYYYAHDY

[0140] WGQGTLVTVSS

[0141] 14554-VL(SEQ ID NO:2)

[0142] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATANLAS GVP

[0143] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0144] 14555-VH(SEQ ID NO:3)

[0145] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SWGMS WVRQAPGKGLELVS TINGYGDT

[0146] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYTNSYYYAHDY W

[0147] GQGTLVTVSS

[0148] 14555-VL(SEQ ID NO:4)

[0149] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATANLAS GVP

[0150] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0151] 14556-VH(SEQ ID NO:5)

[0152] EVQLVESGGGLVQPGGSLRLSCAASGYTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0153] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYSNSYYYALDY W

[0154] GQGTLVTVSS

[0155] 14556-VL(SEQ ID NO:6)

[0156] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATANLAS GVP

[0157] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0158] 14557-VH(SEQ ID NO:7)

[0159] EVQLVESGGGLVQPGGSLRLSCAASGYTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0160] WYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYSNSYYYAHDY W

[0161] GQGTLVTVSS

[0162] 14557-VL(SEQ ID NO:8)

[0163] EIVLTQSPATLSLSPGERATLSC RASSWVSFMH WLQQKPGQAPRPWIY ATQNLAS GVP

[0164] ARFIGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0165] 14558-VH(SEQ ID NO:9)

[0166] EVQLVESGGGLVQPGGSLRLSCAASGYWFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0167] TYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYTNSYYYAHDY W

[0168] GQGTLVTVSS

[0169] 14558-VL(SEQ ID NO:10)

[0170] EIVLTQSPATLSLSPGERATLSC RASSSVSFMH WLQQKPGQAPRPWIY ATMNLAS GVP

[0171] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0172] 14559-VH(SEQ ID NO:11)

[0173] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0174] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYSNSYYYAHDY W

[0175] GQGTLVTVSS

[0176] 14559-VL(SEQ ID NO:12)

[0177] EIVLTQSPATLSLSPGERATLSC RASSWVSFMH WLQQKPGQAPRPWIY ATQNLAS GVP

[0178] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0179] 14560-VH(SEQ ID NO:13)

[0180] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0181] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYTNSYYYAHDY W

[0182] GQGTLVTVSS

[0183] 14560-VL(SEQ ID NO:14)

[0184] EIVLTQSPATLSLSPGERATLSC RASSSVSFMH WLQQKPGQAPRPWIY ATQNLAS GVPA

[0185] RFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0186] 14561-VH(SEQ ID NO:15)

[0187] EVQLVESGGGLVQPGGSLRLSCAASGYTFS SWGMS WVRQAPGKGLELVS TINGYGDT

[0188] WYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYSNSYYYAHDY W

[0189] GQGTLVTVSS

[0190] 14561-VL(SEQ ID NO:16)

[0191] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATMNLAS GVP

[0192] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0193] 14562-VH(SEQ ID NO:17)

[0194] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0195] WYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYSNSYYYAHDY W

[0196] GQGTLVTVSS

[0197] 14562-VL(SEQ ID NO:18)

[0198] EIVLTQSPATLSLSPGERATLSC RASSSVSFMH WLQQKPGQAPRPWIY ATMNLAS GVP

[0199] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0200] 14563-VH(SEQ ID NO:19)

[0201] EVQLVESGGGLVQPGGSLRLSCAASGYTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0202] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYGNSYYYAHDY W

[0203] GQGTLVTVSS

[0204] 14563-VL(SEQ ID NO:20)

[0205] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATMNLAS GVP

[0206] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0207] 14564-VH(SEQ ID NO:21)

[0208] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SWGMS WVRQAPGKGLELVS TINGYGDT

[0209] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYSNSYYYAHDY W

[0210] GQGTLVTVSS

[0211] 14564-VL(SEQ ID NO:22)

[0212] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATANLAS GVP

[0213] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0214] 14565-VH(SEQ ID NO:23)

[0215] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0216] WYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYGNSYYYAHDY

[0217] WGQGTLVTVSS

[0218] 14565-VL(SEQ ID NO:24)

[0219] EIVLTQSPATLSLSPGERATLSC RASSWVSFMH WLQQKPGQAPRPWIY ATQNLAS GVP

[0220] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0221] 14566-VH(SEQ ID NO:25)

[0222] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0223] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYGNSYYYAHDY W

[0224] GQGTLVTVSS

[0225] 14566-VL(SEQ ID NO:26)

[0226] EIVLTQSPATLSLSPGERATLSC RASSWVSFMH WLQQKPGQAPRPWIY ATMNLAS GVP

[0227] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0228] 14567-VH(SEQ ID NO:27)

[0229] EVQLVESGGGLVQPGGSLRLSCAASGFWFS SWGMS WVRQAPGKGLELVS TINGYGDT

[0230] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYTNSYYYAHDY W

[0231] GQGTLVTVSS

[0232] 14567-VL(SEQ ID NO:28)

[0233] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATQNLAS GVP

[0234] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0235] 14568-VH(SEQ ID NO:29)

[0236] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0237] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYTNSYYYAHDY W

[0238] GQGTLVTVSS

[0239] 14568-VL(SEQ ID NO:30)

[0240] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATMNLAS GVP

[0241] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0242] 14569-VH(SEQ ID NO:31)

[0243] EVQLVESGGGLVQPGGSLRLSCAASGYWFS SWGMS WVRQAPGKGLELVS TINGYGD

[0244] TTYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYSNSYYYAHDY

[0245] WGQGTLVTVSS

[0246] 14569-VL(SEQ ID NO:32)

[0247] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATMNLAS GVP

[0248] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0249] 14570-VH(SEQ ID NO:33)

[0250] EVQLVESGGGLVQPGGSLRLSCAASGFWFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0251] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYTNSYYYAHDY W

[0252] GQGTLVTVSS

[0253] 14570-VL(SEQ ID NO:34)

[0254] EIVLTQSPATLSLSPGERATLSC RASSSVSFMH WLQQKPGQAPRPWIY ATANLAS GVPA

[0255] RFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0256] 14571-VH(SEQ ID NO:35)

[0257] EVQLVESGGGLVQPGGSLRLSCAASGYTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0258] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYGNSYYYAHDY W

[0259] GQGTLVTVSS

[0260] 14571-VL(SEQ ID NO:36)

[0261] EIVLTQSPATLSLSPGERATLSC RASSWVSFMH WLQQKPGQAPRPWIY ATMNLAS GVP

[0262] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0263] 14572-VH(SEQ ID NO:37)

[0264] EVQLVESGGGLVQPGGSLRLSCAASGYWFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0265] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYTNSYYYAHDY W

[0266] GQGTLVTVSS

[0267] 14572-VL(SEQ ID NO:38)

[0268] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATMNLAS GVP

[0269] ARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0270] 14573-VH(SEQ ID NO:39)

[0271] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SWGMS WVRQAPGKGLELVS TINGYGDT

[0272] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYTNSYYYAHDY W

[0273] GQGTLVTVSS

[0274] 14573-VL(SEQ ID NO:40)

[0275] EIVLTQSPATLSLSPGERATLSC RASSYVSFMH WLQQKPGQAPRPWIY ATSNLAS GVPA

[0276] RFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK U579-WT-VH(SEQ ID NO:41)

[0277] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLELVS TINGYGDT

[0278] TYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDYGNSYYYALDY W

[0279] GQGTLVTVSS

[0280] U579-WT-VL(SEQ ID NO:42)

[0281] EIVLTQSPATLSLSPGERATLSC RASSSVSFMH WLQQKPGQAPRPWIY ATSNLAS GVPAR

[0282] FSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPA FGGGTKVEIK

[0283] The following is the consensus amino acid sequence of each VH domain CDR (HCDR) and each VL domain CDR (LCDR) derived from the alignment of 21 antibody sequences.

[0284] Consensus HCDR1: SXGMS, wherein X is W or Y (SEQ ID NO: 43)

[0285] Consensus HCDR2: TINGYGDTXYYPDSVKG, wherein X is W or T (SEQ ID NO: 44)

[0286] Consensus HCDR3: DRDYX1NSYYYAX2DY, wherein X1 is S or G; X2 is L or H (SEQ ID NO: 45)

[0287] Consensus LCDR1: RASSXVSFMH, wherein X is S, W, or Y (SEQ ID NO: 46)

[0288] Consensus LCDR2: ATXNLAS, wherein X is S, A, Q, or M (SEQ ID NO: 47)

[0289] Consensus LCDR3: QQWSSNPPA (SEQ ID NO: 48)

[0290] Below are the specific amino acid sequences of exemplary VH domain CDRs (HCDRs) and exemplary VL domain CDRs (LCDRs).

[0291] Exemplary HCDR1: SWGMS (SEQ ID NO: 49) or SYGMS (SEQ ID NO: 50)

[0292] Exemplary HCDR2: TINGYGDTTYYPDSVKG (SEQ ID NO: 51)

[0293] Exemplary HCDR3: DRDYSNSYYYAHDY (SEQ ID NO: 52) or

[0294] DRDYTNSYYYAHDY (SEQ ID NO: 53)

[0295] Exemplary LCDR1: RASSYVSFMH (SEQ ID NO: 54)

[0296] Exemplary LCDR2: ATANLAS (SEQ ID NO: 55) or ATMNLAS (SEQ ID NO: 56)

[0297] Exemplary LCDR3: QQWSSNPPA (SEQ ID NO: 48)

[0298] Provided herein are monoclonal antibodies that specifically bind to human CD25. These monoclonal antibodies comprise a heavy chain variable (VH) domain and a light chain variable (VL) domain. In certain aspects, the VH domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 comprise SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively; and / or the VL domain comprises CDR1, CDR2, and

[0299] CDR3, and the CDR1, CDR2 and CDR3 amino acid sequences include SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively.

[0300] In some examples, 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; and / or the amino acid sequences of CDR1, CDR2, and CDR3 of the VL domain include SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 48, respectively. In a specific example, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 21, and the VH domain

[0301] CDR1, CDR2 and CDR3 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 VL domain CDR1, CDR2 and CDR3 comprise SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:48, respectively.

[0302] 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 a specific example, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 31, and the VH domain CDR1, CDR2, and / or CDR3 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 VL domain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 48, respectively.

[0303] 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 a specific example, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 37, and the VH domain CDR1, CDR2, and CDR3 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 VL domain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 48, respectively.

[0304] In some aspects, the monoclonal antibody comprises at least a portion of the amino acid sequence of any one of SEQ ID NOs: 1-42, e.g., one or more (e.g., all three) CDR sequences of any one of SEQ ID NOs: 1-42. In some instances, the CDR positions are determined by IMGT, Kabat, Paratome, Chothia, or a combination of one or more thereof.

[0305] 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 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 NO: 14 CDR sequence; 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

[0306] CDR sequences; the VH domain of the antibody comprises the CDR sequences of SEQ ID NO: 27 and the VL domain of the antibody comprises the CDR sequences of SEQ ID NO: 28; the VH domain of the antibody comprises the CDR sequences of SEQ ID NO: 29 and the VL domain of the antibody comprises the CDR sequences of SEQ ID NO: 30; the VH domain of the antibody comprises the CDR sequences of SEQ ID NO: 31 and the VL domain of the antibody comprises the CDR sequences of SEQ ID NO: 32; the VH domain of the antibody comprises the CDR sequences of SEQ ID NO: 33 and the VL domain of the antibody comprises the CDR sequences of SEQ ID NO: 34.

[0307] CDR sequences; the VH domain of the antibody comprises the CDR sequences of SEQ ID NO: 35 and the VL domain of the antibody comprises the CDR sequences of SEQ ID NO: 36; the VH domain of the antibody comprises the CDR sequences of SEQ ID NO: 37 and the VL domain of the antibody comprises the CDR sequences of SEQ ID NO: 38; the VH domain of the antibody comprises the CDR sequences of SEQ ID NO: 39 and the VL domain of the antibody comprises the CDR sequences of SEQ ID NO: 40; or the VH domain of the antibody comprises the CDR sequences of SEQ ID NO: 41 and the VL domain of the antibody comprises the CDR sequences of SEQ ID NO: 42. In certain examples, the VH domain CDR1, CDR2 and CDR3 are located at positions 31-35, 50-66 and 99-112 of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41, respectively; and / or the VL domain CDR1, CDR2 and CDR3 are located at positions 24-33, 49-55 and 88-96 of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and 42, respectively.

[0308] 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

[0309] SEQ ID NO: 6 or consisting thereof; the amino acid sequence of the VH domain comprises SEQ ID NO: 7 or consisting thereof, and the amino acid sequence of the VL domain comprises SEQ ID NO: 8 or consisting thereof; the amino acid sequence of the VH domain comprises SEQ ID NO: 9 or consisting thereof, and the amino acid sequence of the VL domain comprises SEQ ID NO: 10 or consisting thereof; the amino acid sequence of the VH domain comprises SEQ ID NO: 11 or consisting thereof, and the amino acid sequence of the VL domain comprises SEQ ID NO: 12 or consisting thereof; the amino acid sequence of the VH domain comprises SEQ ID NO: 13 or consisting thereof, and the amino acid sequence of the VL domain comprises SEQ ID NO: 14 or consisting thereof; the amino acid sequence of the VH domain comprises SEQ ID NO: 15 or consisting thereof, and the amino acid sequence of the VL domain comprises SEQ ID NO: 16 or consisting thereof; the amino acid sequence of the VH domain comprises SEQ ID NO: 17 or consisting thereof, and the amino acid sequence of the VL domain comprises SEQ ID NO: NO: 18 or consists of it; the amino acid sequence of the VH domain comprises SEQ ID NO: 19 or consists of it, and the amino acid sequence of the VL domain comprises SEQ ID NO: 20 or consists of it; the amino acid sequence of the VH domain comprises SEQ ID NO: 21 or consists of it, and the amino acid sequence of the VL domain comprises SEQ ID NO: 22 or consists of it; the amino acid sequence of the VH domain comprises SEQ ID NO: 23 or consists of it, and the amino acid sequence of the VL domain comprises SEQ ID NO: 24 or consists of it; the amino acid sequence of the VH domain comprises SEQ ID NO: 25 or consists of it, and the amino acid sequence of the VL domain comprises SEQ ID NO: 26 or consists of it; the amino acid sequence of the VH domain comprises SEQ ID NO: 27 or consists of it, and the amino acid sequence of the VL domain comprises SEQ ID NO: 28 or consists of it; the amino acid sequence of the VH domain comprises SEQ ID NO: 29 or consists of it, and the amino acid sequence of the VL domain comprises SEQ ID NO: NO: 30 or consisting thereof; 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.

[0310] In certain aspects, the 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 refers to 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) relative to the binding of IL-2 to CD25 in the absence of the antibody. In some instances, the CD25-specific monoclonal antibody does not prevent the maturation or proliferation of T cells, such as CD8+ T cells.

[0311] In certain aspects, the CD25-specific monoclonal antibodies induce little ADCC. In the context of the present disclosure, an antibody that induces little ADCC refers to an antibody that induces 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 cell death detected) of cell death of target cells in a cytotoxicity assay. In certain instances, the CD25-specific monoclonal antibodies induce less than 2% cell death of target cells, such as less than 1.5%, 1%, 0.5% or 0.25%. Cytotoxicity assays that can be used to evaluate antibody ADCC activity are well known, and an exemplary assay is described in Example 3. Suitable target cells are cells expressing human CD25, such as KIT-225 cells.

[0312] In certain aspects 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).

[0313] In certain aspects, the monoclonal antibody is an IgG, such as IgG4. In other aspects, the monoclonal antibody is an IgA, IgD, IgE, or IgM.

[0314] In certain aspects, the monoclonal antibody is a humanized, chimeric, or synthetic monoclonal antibody.

[0315] 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 certain aspects, the heavy and / or light chain amino acid sequences comprise a leader sequence, such as MGWSCIILFLVATATGVHS (SEQ ID NO: 57). In certain aspects, the heavy chain comprises a modified human IgG4 constant region as set forth in SEQ ID NO: 58. In certain aspects, the light chain comprises a human Ig kappa constant region as set forth in SEQ ID NO: 49. In other aspects, the heavy chain constant region is a constant region from a different isotype, such as IgG1, but modified to prevent induction of ADCC.

[0316] Human (modified) IgG4 constant region (SEQ ID NO: 58)

[0317] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0318] GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPS

[0319] VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF

[0320] NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS

[0321] QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD

[0322] KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0323] Human Ig kappa constant region (SEQ ID NO: 59)

[0324] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE

[0325] QDSKDSTYSLSSTLLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0326] The heavy and light chain sequences of each CD25-specific antibody are provided below, including the leader sequence (SEQ ID NO: 57), VH or VL domain (underlined), and a modified human IgG4 constant region (SEQ ID NO: 58; for the heavy chain sequence) or human Ig kappa constant region (SEQ ID NO: 59; for the light chain sequence).

[0327] 14554-VH (SEQ ID NO: 60)

[0328] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFWFSSYGMSWVR

[0329] QAPGKGLELVSTINGYGDTWYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0330] CARDRDYTNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0331] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0332] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0333] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0334] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0335] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0336] KSLSLSLGK

[0337] 14554-VL (SEQ ID NO: 61)

[0338] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0339] QAPRPWIYATANLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0340] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0341] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0342] 14555-VH(SEQ ID NO:62)

[0343] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSWGMSWVR

[0344] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0345] CARDRDYTNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0346] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0347] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0348] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0349] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0350] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0351] KSLSLSLGK

[0352] 14555-VL(SEQ ID NO:63)

[0353] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0354] QAPRPWIYATANLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0355] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0356] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0357] 14556-VH(SEQ ID NO:64)

[0358] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYGMSWVR

[0359] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0360] CARDRDYSNSYYYALDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVK

[0361] DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK

[0362] PSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

[0363] QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0364] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0365] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0366] KSLSLSLGK

[0367] 14556-VL(SEQ ID NO:65)

[0368] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0369] QAPRPWIYATANLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0370] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0371] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0372] 14557-VH(SEQ ID NO:66)

[0373] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYGMSWVR

[0374] QAPGKGLELVSTINGYGDTWYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0375] CARDRDYSNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0376] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0377] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0378] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0379] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0380] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0381] KSLSLSLGK

[0382] 14557-VL(SEQ ID NO:67)

[0383] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSWVSFMHWLQQKP

[0384] GQAPRPWIYATQNLASGVPARFIGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0385] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0386] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0387] 14558-VH(SEQ ID NO:68)

[0388] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGYWFSSYGMSWVR

[0389] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0390] CARDRDYTNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0391] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0392] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0393] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0394] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0395] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0396] KSLSLSLGK

[0397] 14558-VL(SEQ ID NO:69)

[0398] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSSVSFMHWLQQKPG

[0399] QAPRPWIYATMNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0400] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0401] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0402] 14559-VH(SEQ ID NO:70)

[0403] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR

[0404] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0405] CARDRDYSNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0406] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0407] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0408] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0409] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0410] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0411] KSLSLSLGK

[0412] 14559-VL(SEQ ID NO:71)

[0413] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSWVSFMHWLQQKP

[0414] GQAPRPWIYATQNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAF

[0415] GGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

[0416] GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0417] 14560-VH(SEQ ID NO:72)

[0418] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR

[0419] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0420] CARDRDYTNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0421] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0422] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0423] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0424] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0425] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0426] KSLSLSLGK

[0427] 14560-VL(SEQ ID NO:73)

[0428] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSSVSFMHWLQQKPG

[0429] QAPRPWIYATQNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0430] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0431] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0432] 14561-VH(SEQ ID NO:74)

[0433] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGYTFSSWGMSWVR

[0434] QAPGKGLELVSTINGYGDTWYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0435] CARDRDYSNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0436] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0437] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0438] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0439] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0440] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0441] KSLSLSLGK

[0442] 14561-VL(SEQ ID NO:75)

[0443] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0444] QAPRPWIYATMNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0445] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0446] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0447] 14562-VH(SEQ ID NO:76)

[0448] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR

[0449] QAPGKGLELVSTINGYGDTWYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0450] CARDRDYSNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0451] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0452] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0453] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0454] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0455] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0456] KSLSLSLGK

[0457] 14562-VL(SEQ ID NO:77)

[0458] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSSVSFMHWLQQKPG

[0459] QAPRPWIYATMNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0460] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0461] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0462] 14563-VH(SEQ ID NO:78)

[0463] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYGMSWVR

[0464] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0465] CARDRDYGNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0466] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0467] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0468] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0469] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0470] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0471] KSLSLSLGK

[0472] 14563-VL(SEQ ID NO:79)

[0473] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0474] QAPRPWIYATMNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0475] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0476] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0477] 14564-VH(SEQ ID NO:80)

[0478] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSWGMSWVR

[0479] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0480] CARDRDYSNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0481] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0482] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0483] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0484] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0485] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0486] KSLSLSLGK

[0487] 14564-VL(SEQ ID NO:81)

[0488] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0489] QAPRPWIYATANLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0490] GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0491] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0492] 14565-VH(SEQ ID NO:82)

[0493] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR

[0494] QAPGKGLELVSTINGYGDTWYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0495] CARDRDYGNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0496] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0497] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0498] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0499] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0500] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0501] KSLSLSLGK

[0502] 14565-VL(SEQ ID NO:83)

[0503] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSWVSFMHWLQQKP

[0504] GQAPRPWIYATQNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAF

[0505] GGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

[0506] GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0507] 14566-VH(SEQ ID NO:84)

[0508] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR

[0509] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0510] CARDRDYGNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0511] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0512] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0513] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0514] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0515] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0516] KSLSLSLGK

[0517] 14566-VL(SEQ ID NO:85)

[0518] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSWVSFMHWLQQKP

[0519] GQAPRPWIYATMNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAF

[0520] GGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

[0521] GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0522] 14567-VH(SEQ ID NO:86)

[0523] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFWFSSWGMSWVR

[0524] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0525] CARDRDYTNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0526] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0527] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0528] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0529] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0530] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0531] KSLSLSLGK

[0532] 14567-VL(SEQ ID NO:87)

[0533] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0534] QAPRPWIYATQNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0535] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0536] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0537] 14568-VH(SEQ ID NO:88)

[0538] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR

[0539] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0540] CARDRDYTNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0541] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0542] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0543] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0544] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0545] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0546] KSLSLSLGK

[0547] 14568-VL(SEQ ID NO:89)

[0548] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0549] QAPRPWIYATMNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0550] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0551] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0552] 14569-VH(SEQ ID NO:90)

[0553] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGYWFSSWGMSWV

[0554] RQAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY

[0555] YCARDRDYSNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCL

[0556] VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD

[0557] HKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD

[0558] VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC

[0559] KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE

[0560] WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT

[0561] QKSLSLSLGK

[0562] 14569-VL(SEQ ID NO:91)

[0563] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0564] QAPRPWIYATMNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0565] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0566] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0567] 14570-VH(SEQ ID NO:92)

[0568] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFWFSSYGMSWVR

[0569] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0570] CARDRDYTNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0571] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0572] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0573] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0574] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0575] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0576] KSLSLSLGK

[0577] 14570-VL(SEQ ID NO:93)

[0578] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSSVSFMHWLQQKPG

[0579] QAPRPWIYATANLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0580] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0581] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0582] 14571-VH(SEQ ID NO:94)

[0583] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYGMSWVR

[0584] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0585] CARDRDYGNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0586] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0587] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0588] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0589] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0590] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0591] KSLSLSLGK

[0592] 14571-VL(SEQ ID NO:95)

[0593] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSWVSFMHWLQQKP

[0594] GQAPRPWIYATMNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAF

[0595] GGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

[0596] GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0597] 14572-VH(SEQ ID NO:96)

[0598] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGYWFSSYGMSWVR

[0599] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0600] CARDRDYTNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0601] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0602] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0603] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0604] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0605] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0606] KSLSLSLGK

[0607] 14572-VL(SEQ ID NO:97)

[0608] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0609] QAPRPWIYATMNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFG

[0610] GGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0611] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0612] 14573-VH(SEQ ID NO:98)

[0613] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSWGMSWVR

[0614] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0615] CARDRDYTNSYYYAHDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0616] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0617] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0618] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0619] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0620] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0621] KSLSLSLGK

[0622] 14573-VL(SEQ ID NO:99)

[0623] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSYVSFMHWLQQKPG

[0624] QAPRPWIYATSNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFGG

[0625] GTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN

[0626] SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0627] U579-WT-VH(SEQ ID NO:100)

[0628] MGWSCIILFLVATATGVHS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR

[0629] QAPGKGLELVSTINGYGDTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0630] CARDRDYGNSYYYALDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLV

[0631] KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0632] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0633] SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK

[0634] VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW

[0635] ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ

[0636] KSLSLSLGK

[0637] U579-WT-VL(SEQ ID NO:101)

[0638] MGWSCIILFLVATATGVHS EIVLTQSPATLSLSPGERATLSCRASSSVSFMHWLQQKPGQ

[0639] APRPWIYATSNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPAFGGGT

[0640] KVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

[0641] ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0642] In some aspects disclosed herein, the monoclonal antibody further includes a constant region, e.g., a constant region comprising at least one modification to increase the half-life, stability, and / or function of the monoclonal antibody. In some instances, the constant region includes a heavy chain constant region and / or a light chain constant region. In a specific instance, the heavy chain constant region is a human IgG4 constant region, e.g., a human IgG4 constant region having an amino acid sequence of SEQ ID NO: 58, and / or the light chain constant region is a human Ig κ light chain, e.g., a human Ig κ light chain having an amino acid sequence of SEQ ID NO: 59.

[0643] In a specific, non-limiting example, the monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise or consist of the amino acid sequence set forth below: 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; 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.

[0644] Also provided are compositions 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.

[0645] IV. Antibody Conjugates

[0646] The CD25-specific monoclonal antibodies disclosed herein can be coupled to agents such as photon absorbers (e.g., IR700), effector molecules, or detectable labels in a variety of suitable ways. Both covalent and non-covalent attachment methods can be used. Various photon absorbers, effector molecules, and detectable labels can be used, including but not limited to IR700, toxins, and radioactive agents, such as 125 I. 32 P. 14 C. 3 H and 35 S as well as other labels, targeting moieties and ligands.

[0647] In certain aspects, the conjugates provided herein include a CD25-specific monoclonal antibody disclosed herein and a photon absorber, an effector molecule, or a detectable label. In some instances, the photon absorber is IR700; the effector molecule is a toxin; or the detectable label is a fluorescent, enzymatic, or radioactive label.

[0648] Also provided herein are compositions comprising a conjugate disclosed herein and a pharmaceutically acceptable carrier.

[0649] The choice of a particular photon absorber, effector molecule or detectable label depends on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin that is used to cause the death of a specific target cell (such as a CD25 positive cell).

[0650] In certain aspects, the photon absorber is IR700 ( IR700DX) is commercially available from Rakuten Medical (San Diego, CA). The amino-reactive IR700 is a relatively hydrophilic dye that can be covalently coupled to antibodies via the NHS ester of IR700.

[0651] The step of attaching a photon absorber, effector molecule or detectable label to the antibody varies depending on the chemical structure of the conjugate. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which can react with appropriate functional groups on the polypeptide to result in the binding of the photon absorber, effector molecule or detectable label. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization may involve attaching any one of many known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to connect the antibody to a photon absorber, effector molecule or detectable label. The linker can form a covalent bond with the antibody and the photon absorber / effector molecule / detectable label. Suitable linkers include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers or peptide linkers. When the antibody and effector molecule or detectable label are polypeptides, the linker can be attached to the constituent amino acids via its side chain groups (e.g., via a disulfide bond to cysteine), or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0652] The antibody can be linked to a detectable label; for example, a detectable label that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic inspection, and laparoscopy. Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent labels are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). The antibody or antigen-binding fragment can also be coupled to an enzyme that can be used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When the antibody or antigen-binding fragment is coupled to a detectable enzyme, detection can be achieved by adding another reagent that the enzyme uses to produce a recognizable reaction product. For example, when the horseradish peroxidase reagent is present, the addition of hydrogen peroxide and diaminobenzidine will produce a colored reaction product that can be visually detected. The antibody or antigen-binding fragment can also be coupled to biotin, and detection can be achieved by indirect detection of the binding of avidin or streptavidin. It should be noted that avidin itself can be coupled to an enzyme or a fluorescent label.

[0653] Antibodies can be conjugated to paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be conjugated to lanthanides (such as europium and dysprosium) and manganese. Antibodies can also be labeled with a predetermined polypeptide epitope (such as a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag) that is recognized by a secondary reporter molecule.

[0654] Antibodies can also be coupled to radiolabeled amino acids. Radiolabels can be used for diagnostic and therapeutic purposes. For example, radiolabels can be used to detect cells expressing CD25 by x-ray, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labels 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.

[0655] Detectable labels can be detected using any known means. Thus, for example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme 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, while colorimetric labels are detected by simply visualizing the colored label.

[0656] The average number of photon absorbers, effector molecules, or detectable label moieties per antibody in the conjugate can be, for example, 1 to 20 moieties per antibody. In certain aspects, the average number of photon absorbers, effector molecules, or detectable label 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., the effector molecule / antibody ratio) can be controlled in various ways, for example, by: (i) limiting the molar excess of the effector molecule-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reducing conditions for cysteine ​​thiol modification, (iv) modifying the amino acid sequence of the antibody by recombinant technology to change the number and position of cysteine ​​residues to control the number or position of linker-effector molecule linkages.

[0657] V. Nucleic Acid Molecules

[0658] 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. Nucleic acids encoding these molecules can be readily modified using the amino acid sequences (e.g., CDR sequences and V H and V L sequences), sequences available in the art (such as framework or constant region sequences), and genetic code generation. In several aspects, the nucleic acid molecule can encode the V H 、V L or V H and V L Both (eg, in a bicistronic expression vector). In several aspects, the nucleic acid molecules can be expressed in a host cell (eg, a mammalian cell) to produce the disclosed antibodies.

[0659] The genetic code can be used to construct various functionally equivalent nucleic acids, such as nucleic acids with different sequences but encoding the same antibody sequence, or nucleic acids encoding V L and / or V H A conjugate of a nucleic acid sequence or a nucleic acid fusion protein.

[0660] Provided below are nucleic acid sequences of the VH and VL domains of the disclosed CD25-specific monoclonal antibodies (SEQ ID NOs: 102-143), exemplary leader sequences (SEQ ID NOs: 144 and 145), exemplary human IgG4 constant region encoding sequences (SEQ ID NO: 146), and exemplary human Igκ light chain encoding sequences (SEQ ID NO: 147). Further provided are nucleic acid sequences encoding the complete heavy and light chains of the disclosed monoclonal antibodies (SEQ ID NOs: 148-189).

[0661] 14554-VH (SEQ ID NO: 102)

[0662] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0663] GCTGCGAGCGGTTTCTGGTTCAGCCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0664] TGGAACTGGTGTCCACCATCAACGGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCCG

[0665] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0666] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATT

[0667] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0668] 14554-VL(SEQ ID NO:103)

[0669] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0670] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0671] GTGGATTTATGCGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0672] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0673] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0674] 14555-VH(SEQ ID NO:104)

[0675] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0676] GCTGCGAGCGGTTTCACCTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0677] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0678] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0679] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATT

[0680] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0681] 14555-VL(SEQ ID NO:105)

[0682] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0683] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0684] GTGGATTTATGCGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0685] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0686] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0687] 14556-VH(SEQ ID NO:106)

[0688] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0689] GCTGCGAGCGGTTACACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0690] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0691] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0692] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCTGGATTA

[0693] CTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0694] 14556-VL(SEQ ID NO:107)

[0695] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0696] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0697] GTGGATTTATGCGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0698] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0699] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0700] 14557-VH(SEQ ID NO:108)

[0701] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0702] GCTGCGAGCGGTTACACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0703] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCCG

[0704] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0705] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATT

[0706] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0707] 14557-VL(SEQ ID NO:109)

[0708] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0709] GTCGTGCCAGCTCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0710] GTGGATTTATGCGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCATCGGCAGCGGCTCTGGC

[0711] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0712] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0713] 14558-VH(SEQ ID NO:110)

[0714] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0715] GCTGCGAGCGGTTACTGGTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0716] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0717] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0718] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATT

[0719] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0720] 14558-VL(SEQ ID NO:111)

[0721] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0722] GTCGTGCCAGCTCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0723] GTGGATTTATGCGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0724] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0725] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0726] 14559-VH(SEQ ID NO:112)

[0727] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0728] GCTGCGAGCGGTTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0729] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0730] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0731] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATT

[0732] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0733] 14559-VL(SEQ ID NO:113)

[0734] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0735] GTCGTGCCAGCTCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0736] GTGGATTTATGCGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0737] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0738] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0739] 14560-VH(SEQ ID NO:114)

[0740] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0741] GCTGCGAGCGGTTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0742] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0743] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0744] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATT

[0745] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0746] 14560-VL(SEQ ID NO:115)

[0747] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0748] GTCGTGCCAGCTCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0749] GTGGATTTATGCGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0750] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0751] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0752] 14561-VH(SEQ ID NO:116)

[0753] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0754] GCTGCGAGCGGTTACACCTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGC

[0755] CTGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCC

[0756] GTTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGA

[0757] GGACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGAT

[0758] TACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0759] 14561-VL(SEQ ID NO:117)

[0760] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0761] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0762] GTGGATTTATGCGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0763] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0764] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0765] 14562-VH(SEQ ID NO:118)

[0766] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0767] GCTGCGAGCGGTTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0768] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCCG

[0769] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0770] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATT

[0771] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0772] 14562-VL(SEQ ID NO:119)

[0773] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0774] GTCGTGCCAGCTCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0775] GTGGATTTATGCGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0776] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0777] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0778] 14563-VH(SEQ ID NO:120)

[0779] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0780] GCTGCGAGCGGTTACACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0781] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0782] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0783] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCACGATT

[0784] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0785] 14563-VL(SEQ ID NO:121)

[0786] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0787] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0788] GTGGATTTATGCGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0789] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0790] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0791] 14564-VH(SEQ ID NO:122)

[0792] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0793] GCTGCGAGCGGTTTCACCTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0794] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0795] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0796] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATT

[0797] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0798] 14564-VL(SEQ ID NO:123)

[0799] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0800] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0801] GTGGATTTATGCGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0802] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0803] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0804] 14565-VH(SEQ ID NO:124)

[0805] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0806] GCTGCGAGCGGTTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0807] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCCG

[0808] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0809] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCACGATT

[0810] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0811] 14565-VL(SEQ ID NO:125)

[0812] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0813] GTCGTGCCAGCTCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0814] GTGGATTTATGCGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0815] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0816] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0817] 14566-VH(SEQ ID NO:126)

[0818] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0819] GCTGCGAGCGGTTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0820] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0821] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0822] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCACGATT

[0823] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0824] 14566-VL(SEQ ID NO:127)

[0825] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0826] GTCGTGCCAGCTCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0827] GTGGATTTATGCGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0828] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0829] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0830] 14567-VH(SEQ ID NO:128)

[0831] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0832] GCTGCGAGCGGTTTCTGGTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0833] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0834] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0835] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATT

[0836] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0837] 14567-VL(SEQ ID NO:129)

[0838] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0839] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0840] GTGGATTTATGCGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0841] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0842] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0843] 14568-VH(SEQ ID NO:130)

[0844] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0845] GCTGCGAGCGGTTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0846] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0847] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0848] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATT

[0849] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0850] 14568-VL(SEQ ID NO:131)

[0851] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0852] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0853] GTGGATTTATGCGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0854] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0855] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0856] 14569-VH(SEQ ID NO:132)

[0857] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0858] GCTGCGAGCGGTTACTGGTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGC

[0859] CTGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCC

[0860] GTTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGA

[0861] GGACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGAT

[0862] TACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0863] 14569-VL(SEQ ID NO:133)

[0864] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0865] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0866] GTGGATTTATGCGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0867] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0868] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0869] 14570-VH(SEQ ID NO:134)

[0870] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0871] GCTGCGAGCGGTTTCTGGTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0872] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0873] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0874] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATT

[0875] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0876] 14570-VL(SEQ ID NO:135)

[0877] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0878] GTCGTGCCAGCTCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0879] GTGGATTTATGCGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0880] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0881] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0882] 14571-VH(SEQ ID NO:136)

[0883] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0884] GCTGCGAGCGGTTACACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0885] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0886] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0887] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCACGATT

[0888] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG14571-VL(SEQ ID NO:137)

[0889] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0890] GTCGTGCCAGCTCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0891] GTGGATTTATGCGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0892] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0893] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0894] 14572-VH(SEQ ID NO:138)

[0895] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0896] GCTGCGAGCGGTTACTGGTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0897] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0898] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0899] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATT

[0900] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0901] 14572-VL(SEQ ID NO:139)

[0902] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0903] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0904] GTGGATTTATGCGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0905] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0906] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0907] 14573-VH(SEQ ID NO:140)

[0908] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0909] GCTGCGAGCGGTTTCACCTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0910] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0911] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0912] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATT

[0913] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0914] 14573-VL(SEQ ID NO:141)

[0915] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0916] GTCGTGCCAGCTCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0917] GTGGATTTATGCGACGAGCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0918] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0919] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0920] U579-WT-VH(SEQ ID NO:142)

[0921] GAGGTTCAACTAGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGT

[0922] GCTGCGAGCGGTTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCC

[0923] TGGAACTGGTGTCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCG

[0924] TTTTACCATTAGCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAG

[0925] GACACTGCCGTTTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCTGGATT

[0926] ACTGGGGTCAAGGTACGTTGGTCACCGTGTCTTCG

[0927] U579-WT-VL (SEQ ID NO:143)

[0928] GAAATAGTACTAACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTT

[0929] GTCGTGCCAGCTCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCC

[0930] GTGGATTTATGCGACGAGCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGC

[0931] ACTGACTACACCTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAAT

[0932] GGAGCTCCAACCCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG

[0933] For VH leader sequence (SEQ ID NO:144):

[0934] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT

[0935] For VL leader sequence (SEQ ID NO:145):

[0936] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT

[0937] Human IgG4 constant region (SEQ ID NO:146):

[0938] GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTCCCGGTCCACATCTGAGAGCACCG

[0939] CCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTGACCGTGAGCTGGAACAGCGGCG

[0940] CCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGCTCCGGCCTGTACTCCCTGTCTAG

[0941] CGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATACCTGTAACGTGGACCACAAGCCA

[0942] AGCAATACCAAGGTGGAATAGCGGGTGGAGTCTAAGTACGGCCCTCCCTTGCCCTCCATGTCCTGCTC

[0943] CAGAGTTTCTGGGCGGCCTTCCGTGTTCCTGTTTCCACCCAAACCAAAGGACACACTGATGATCTCT

[0944] AGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGATCCCGAGGTGCAGTTCAAC

[0945] TGGTACGTGGATGGCTGGAGGTGCACAATGCCAAGACCAAGCCAAGAGAGGAGCAGTTTAACTCT

[0946] ACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGATTGGCTCAACGGCAAGGAGTATAAG

[0947] TGCAAGGTGTCCAATAAGGGCCTGCCTCCTCTATCGAGAAGACAATCTCTAAGGCTAAGGGCCAGC

[0948] CAAGAGAGCCTCAGGTGACACCCTGCCTCCAAGCCAGGAGGAGATGACAAAGACCAGGTGTCCC

[0949] TGACATGTCTGGTGAAGGGCTTCTATCCTCCGACATCGCCGTGGAGTGGGAGTCTAATGGCCAGCC

[0950] TGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTGATGGCAGCTTCTTTCTGTATTCCAGG

[0951] CTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTTCAGCTGCTCTGTGATGCACGAAGCCC

[0952] TGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTGGGAAAG

[0953] Human immunoglobulin kappa light chain (SEQ ID NO:147):

[0954] AGGACAGTGGCCGCCCCAAGCGTGTTCATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCG

[0955] CCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATA

[0956] ACGCCCTGCAGTCTGGCAATAGCCAGGAGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATT

[0957] CCCTGTCTAGCACCCTGACACTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAG

[0958] TCACCCATCAGGGGCTGTCATCACCCGTCACTAAGTCATTCAATCGCGGAGAATGC

[0959] 14554-VH(SEQ ID NO:148)

[0960] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[0961] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[0962] TTTCTGGTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[0963] TCCACCATCAACGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[0964] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[0965] TTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[0966] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[0967] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[0968] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[0969] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[0970] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[0971] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[0972] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[0973] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[0974] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[0975] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[0976] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[0977] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[0978] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[0979] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[0980] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[0981] GGAAAG

[0982] 14554-VL(SEQ ID NO:149)

[0983] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[0984] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[0985] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[0986] CGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[0987] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[0988] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[0989] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[0990] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[0991] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[0992] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[0993] ACTAAGTCATTCAATCGCGGAGAATGC

[0994] 14555-VH(SEQ ID NO:150)

[0995] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[0996] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[0997] TTTCACCTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[0998] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[0999] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1000] TTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1001] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1002] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1003] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1004] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1005] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1006] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1007] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1008] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1009] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1010] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1011] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1012] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1013] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1014] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1015] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1016] GGAAAG

[1017] 14555-VL(SEQ ID NO:151)

[1018] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1019] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1020] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1021] CGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1022] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1023] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1024] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1025] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1026] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1027] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1028] ACTAAGTCATTCAATCGCGGAGAATGC

[1029] 14556-VH(SEQ ID NO:152)

[1030] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1031] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1032] TTACACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1033] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1034] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1035] TTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCTGGATTACTGGGGTCAA

[1036] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1037] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1038] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1039] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1040] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1041] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1042] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1043] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1044] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1045] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1046] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1047] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1048] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1049] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1050] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1051] GGAAAG

[1052] 14556-VL(SEQ ID NO:153)

[1053] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1054] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1055] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1056] CGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1057] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1058] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1059] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1060] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1061] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1062] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1063] ACTAAGTCATTCAATCGCGGAGAATGC

[1064] 14557-VH(SEQ ID NO:154)

[1065] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1066] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1067] TTACACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1068] TCCACCATCAACGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1069] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1070] TTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1071] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1072] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1073] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1074] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1075] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1076] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1077] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1078] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1079] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1080] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1081] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1082] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1083] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1084] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1085] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1086] GGAAAG

[1087] 14557-VL(SEQ ID NO:155)

[1088] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1089] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1090] TCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1091] CGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCATCGGCAGCGGCTCTGGCACTGACTACAC

[1092] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1093] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1094] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1095] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1096] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1097] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1098] ACTAAGTCATTCAATCGCGGAGAATGC

[1099] 14558-VH(SEQ ID NO:156)

[1100] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1101] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1102] TTACTGGTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1103] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1104] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1105] TTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1106] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1107] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1108] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1109] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1110] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1111] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1112] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1113] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1114] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1115] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1116] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1117] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1118] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1119] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1120] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1121] GGAAAG

[1122] 14558-VL(SEQ ID NO:157)

[1123] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1124] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1125] TCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1126] CGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1127] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1128] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1129] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1130] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1131] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1132] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1133] ACTAAGTCATTCAATCGCGGAGAATGC

[1134] 14559-VH(SEQ ID NO:158)

[1135] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1136] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1137] TTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1138] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1139] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1140] TTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1141] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1142] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1143] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1144] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1145] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1146] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1147] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1148] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1149] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1150] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1151] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1152] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1153] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1154] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1155] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1156] GGAAAG

[1157] 14559-VL(SEQ ID NO:159)

[1158] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1159] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1160] TCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1161] CGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1162] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1163] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1164] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1165] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1166] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1167] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1168] ACTAAGTCATTCAATCGCGGAGAATGC

[1169] 14560-VH(SEQ ID NO:160)

[1170] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1171] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1172] TTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1173] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1174] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1175] TTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1176] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1177] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1178] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1179] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1180] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1181] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1182] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1183] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1184] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1185] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1186] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1187] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1188] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1189] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1190] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1191] GGAAAG

[1192] 14560-VL(SEQ ID NO:161)

[1193] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1194] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1195] TCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1196] CGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1197] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1198] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1199] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1200] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1201] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1202] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1203] ACTAAGTCATTCAATCGCGGAGAATGC

[1204] 14561-VH(SEQ ID NO:162)

[1205] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1206] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1207] TTACACCTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1208] TCCACCATCAACGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1209] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1210] TTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1211] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1212] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1213] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1214] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1215] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1216] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1217] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1218] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1219] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1220] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1221] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1222] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1223] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1224] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1225] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1226] GGAAAG

[1227] 14561-VL(SEQ ID NO:163)

[1228] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1229] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1230] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1231] CGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1232] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1233] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1234] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1235] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1236] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1237] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1238] ACTAAGTCATTCAATCGCGGAGAATGC

[1239] 14562-VH(SEQ ID NO:164)

[1240] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1241] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1242] TTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1243] TCCACCATCAACGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1244] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1245] TTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1246] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1247] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1248] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1249] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1250] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1251] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1252] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1253] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1254] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1255] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1256] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1257] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1258] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1259] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1260] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1261] GGAAAG

[1262] 14562-VL(SEQ ID NO:165)

[1263] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1264] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1265] TCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1266] CGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1267] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1268] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1269] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1270] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1271] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1272] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1273] ACTAAGTCATTCAATCGCGGAGAATGC

[1274] 14563-VH(SEQ ID NO:166)

[1275] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1276] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1277] TTACACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1278] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1279] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1280] TTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1281] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1282] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1283] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1284] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1285] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1286] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1287] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1288] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1289] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1290] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1291] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1292] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1293] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1294] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1295] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1296] GGAAAG

[1297] 14563-VL(SEQ ID NO:167)

[1298] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1299] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1300] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1301] CGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1302] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1303] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1304] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1305] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1306] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1307] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1308] ACTAAGTCATTCAATCGCGGAGAATGC

[1309] 14564-VH(SEQ ID NO:168)

[1310] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1311] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1312] TTTCACCTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1313] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1314] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1315] TTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1316] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1317] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1318] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1319] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1320] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1321] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1322] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1323] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1324] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1325] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1326] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1327] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1328] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1329] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1330] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1331] GGAAAG

[1332] 14564-VL(SEQ ID NO:169)

[1333] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1334] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1335] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1336] CGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1337] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1338] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1339] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1340] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1341] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1342] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1343] ACTAAGTCATTCAATCGCGGAGAATGC

[1344] 14565-VH(SEQ ID NO:170)

[1345] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1346] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1347] TTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1348] TCCACCATCAACGGTTATGGCGATACGTGGTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1349] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1350] TTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1351] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1352] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1353] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1354] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1355] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1356] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1357] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1358] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1359] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1360] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1361] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1362] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1363] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1364] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1365] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1366] GGAAAG

[1367] 14565-VL(SEQ ID NO:171)

[1368] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1369] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1370] TCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1371] CGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1372] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1373] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1374] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1375] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1376] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1377] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1378] ACTAAGTCATTCAATCGCGGAGAATGC

[1379] 14566-VH(SEQ ID NO:172)

[1380] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1381] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1382] TTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1383] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1384] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1385] TTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1386] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1387] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1388] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1389] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1390] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1391] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1392] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1393] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1394] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1395] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1396] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1397] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1398] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1399] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1400] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1401] GGAAAG

[1402] 14566-VL(SEQ ID NO:173)

[1403] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1404] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1405] TCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1406] CGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1407] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1408] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1409] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1410] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1411] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1412] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1413] ACTAAGTCATTCAATCGCGGAGAATGC

[1414] 14567-VH(SEQ ID NO:174)

[1415] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1416] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1417] TTTCTGGTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1418] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1419] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1420] TTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1421] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1422] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1423] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1424] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1425] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1426] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1427] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1428] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1429] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1430] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1431] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1432] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1433] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1434] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1435] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1436] GGAAAG

[1437] 14567-VL(SEQ ID NO:175)

[1438] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1439] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1440] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1441] CGACGCAGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1442] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1443] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1444] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1445] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1446] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1447] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1448] ACTAAGTCATTCAATCGCGGAGAATGC

[1449] 14568-VH(SEQ ID NO:176)

[1450] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1451] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1452] TTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1453] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1454] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1455] TTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1456] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1457] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1458] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1459] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1460] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1461] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1462] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1463] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1464] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1465] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1466] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1467] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1468] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1469] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1470] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1471] GGAAAG

[1472] 14568-VL(SEQ ID NO:177)

[1473] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1474] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1475] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1476] CGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1477] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1478] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1479] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1480] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1481] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1482] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1483] ACTAAGTCATTCAATCGCGGAGAATGC

[1484] 14569-VH(SEQ ID NO:178)

[1485] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1486] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1487] TTACTGGTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1488] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1489] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1490] TTATTACTGCGCGCGTGACAGAGACTACTCTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1491] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1492] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1493] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1494] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1495] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1496] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1497] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1498] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1499] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1500] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1501] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1502] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1503] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1504] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1505] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1506] GGAAAG

[1507] 14569-VL(SEQ ID NO:179)

[1508] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1509] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1510] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1511] CGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1512] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1513] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1514] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1515] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1516] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1517] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1518] ACTAAGTCATTCAATCGCGGAGAATGC

[1519] 14570-VH(SEQ ID NO:180)

[1520] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1521] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1522] TTTCTGGTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1523] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1524] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1525] TTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1526] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1527] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1528] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1529] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1530] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1531] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1532] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1533] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1534] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1535] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1536] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1537] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1538] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1539] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1540] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1541] GGAAAG

[1542] 14570-VL(SEQ ID NO:181)

[1543] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1544] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1545] TCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1546] CGACGGCCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1547] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1548] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1549] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1550] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1551] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1552] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1553] ACTAAGTCATTCAATCGCGGAGAATGC

[1554] 14571-VH(SEQ ID NO:182)

[1555] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1556] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1557] TTACACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1558] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1559] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1560] TTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1561] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1562] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1563] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1564] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1565] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1566] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1567] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1568] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1569] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1570] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1571] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1572] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1573] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1574] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1575] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1576] GGAAAG

[1577] 14571-VL(SEQ ID NO:183)

[1578] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1579] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1580] TCCTGGGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1581] CGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1582] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1583] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1584] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1585] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1586] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1587] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1588] ACTAAGTCATTCAATCGCGGAGAATGC

[1589] 14572-VH(SEQ ID NO:184)

[1590] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1591] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1592] TTACTGGTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1593] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1594] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1595] TTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1596] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1597] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1598] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1599] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1600] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1601] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1602] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1603] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1604] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1605] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1606] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1607] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1608] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1609] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1610] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1611] GGAAAG

[1612] 14572-VL(SEQ ID NO:185)

[1613] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1614] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1615] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1616] CGACGATGAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1617] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1618] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1619] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1620] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1621] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1622] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1623] ACTAAGTCATTCAATCGCGGAGAATGC

[1624] 14573-VH(SEQ ID NO:186)

[1625] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1626] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1627] TTTCACCTTCAGCTCCTGGGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1628] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1629] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1630] TTATTACTGCGCGCGTGACAGAGACTACACTAATTCCTATTACTACGCGCACGATTACTGGGGTCAA

[1631] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1632] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1633] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1634] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1635] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1636] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1637] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1638] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1639] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1640] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1641] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1642] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1643] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1644] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1645] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1646] GGAAAG

[1647] 14573-VL(SEQ ID NO:187)

[1648] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1649] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1650] TCCTACGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1651] CGACGAGCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1652] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1653] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1654] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1655] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1656] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1657] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1658] ACTAAGTCATTCAATCGCGGAGAATGC

[1659] U579-WT-VH(SEQ ID NO:188)

[1660] ATGGGCTGGTCTTGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGCGTGCATTCT GAGGTTCAACT

[1661] AGTAGAATCAGGTGGAGGGTTGGTGCAGCCGGGTGGTAGCCTCCGCCTGAGCTGTGCTGCGAGCGG

[1662] TTTCACCTTCAGCTCCTACGGCATGAGCTGGGTTCGTCAGGCACCGGGCAAAGGCCTGGAACTGGTG

[1663] TCCACCATCAACGGTTATGGCGATACGACCTATTACCCGGATAGCGTTAAAGGCCGTTTTACCATTA

[1664] GCCGTGACAACTCTAAGAACACCTTATATCTGCAAATGAATTCACTGCGCGCAGAGGACACTGCCGT

[1665] TTATTACTGCGCGCGTGACAGAGACTACGGTAATTCCTATTACTACGCGCTGGATTACTGGGGTCAA

[1666] GGTACGTTGGTCACCGTGTCTTCG GCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTC

[1667] CCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTG

[1668] ACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGC

[1669] TCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATA

[1670] CCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCC

[1671] CTCCTTGCCCTCCATGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAA

[1672] CCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAG

[1673] GAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAG

[1674] CCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAT

[1675] TGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAG

[1676] ACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAG

[1677] GAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCG

[1678] TGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG

[1679] ATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTT

[1680] CAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTG

[1681] GGAAAG

[1682] U579-WT-VL(SEQ ID NO:189)

[1683] ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGT GAAATAGTACT

[1684] AACACAATCACCCGCTACTTTGTCACTCTCGCCGGGTGAGCGCGCGACCCTGTCTTGTCGTGCCAGC

[1685] TCCTCCGTGAGCTTTATGCACTGGCTGCAGCAAAAACCGGGTCAGGCACCGCGTCCGTGGATTTATG

[1686] CGACGAGCAATCTGGCTAGCGGTGTTCCGGCGCGTTTCAGCGGCAGCGGCTCTGGCACTGACTACAC

[1687] CTTGACCATCAGCTCTCTGGAACCGGAAGATTTCGCTGTGTATTACTGCCAGCAATGGAGCTCCAAC

[1688] CCACCGGCGTTTGGTGGTGGCACCAAAGTTGAGATCAAG AGGACAGTGGCCGCCCCAAGCGTGTTC

[1689] ATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACT

[1690] TCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGG

[1691] AGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAA

[1692] GGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTC

[1693] ACTAAGTCATTCAATCGCGGAGAATGC

[1694] Thus, in certain aspects, the nucleic acid molecule encodes the VH domain, VL domain, or both the VH domain and the VL domain of a CD25-specific monoclonal antibody. In certain examples, the nucleic acid molecule encodes the entire heavy chain, the entire light chain, or both the entire heavy chain and the entire light chain of a CD25-specific monoclonal antibody.

[1695] In certain aspects, the nucleic acid molecule comprises a nucleotide sequence of any one of SEQ ID NOs: 102-143, or a degenerate variant thereof. In some examples, the nucleic acid molecule comprises a 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 a 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.

[1696] In other specific aspects, the nucleic acid molecule comprises a nucleotide sequence of any one of SEQ ID NOs: 148-189, or a degenerate variant thereof. In some examples, the nucleic acid molecule comprises a 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 SEQ ID NOs: 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,

[1697] The nucleotide sequence of any one of 185, 187 and 189, or a degenerate variant thereof.

[1698] In certain aspects, the disclosed nucleic acid molecules further comprise one or more stop codons at the 3' end, such as TGA, TAA, or TGATAA.

[1699] In a specific example, the nucleic acid molecule includes: a nucleotide sequence of SEQ ID NO: 122 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 123 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 122 and SEQ ID NO: 123 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 132 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 133 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 132 and SEQ ID NO: 133 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 138 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 139 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 138 and SEQ ID NO: 139 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 168 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 169 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 168 and SEQ ID NO: 169 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 178 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 179 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 180 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 181 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 182 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 183 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 184 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 185 or a degenerate variant thereof; a nucleotide sequence of SEQ ID NO: 186 or a degenerate nucleotide sequence of SEQ ID NO: 179 or a degenerate variant thereof; nucleotide sequence of SEQ ID NO: 178 and SEQ ID NO: 179 or a degenerate variant thereof; nucleotide sequence of SEQ ID NO: 184 or a degenerate variant thereof; nucleotide sequence of SEQ ID NO: 185 or a degenerate variant thereof; or nucleotide sequence of SEQ ID NO: 184 and SEQ ID NO: 185 or a degenerate variant thereof.

[1700] Further provided are vectors comprising one or more nucleic acid molecules disclosed herein.In certain aspects, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[1701] Also provided are isolated host cells comprising the disclosed nucleic acid molecules or vectors. In some aspects, the isolated host cells are eukaryotic cells, such as mammalian cells. In other aspects, the isolated host cells are prokaryotic cells.

[1702] V. Kit

[1703] In some aspects, a kit comprising a CD25-specific monoclonal antibody, conjugate, composition, nucleic acid molecule, vector and / or isolated host cell disclosed herein is also provided. In some aspects, the kit further comprises a buffer, a cell culture medium, one or more checkpoint inhibitors, one or more immunotherapies, one or more other anti-cancer agents (such as chemotherapeutic agents or biological agents), one or more reducing agents, one or more transfection reagents and / or instructional materials.

[1704] For example, kits for treating cancer in a subject are provided. The kits can include a disclosed CD25-specific monoclonal antibody, CD25 antibody-IR700 conjugate, or a composition comprising a CD25-specific monoclonal antibody or CD25 antibody-IR700 conjugate. The kits can contain more than one disclosed CD25-specific antibody, CD25 antibody-IR700 conjugate, or a composition comprising such molecules.

[1705] In some examples, the kit further includes one or more buffers, such as PBS.

[1706] In certain instances, the kit further comprises one or more cell culture media, such as a culture medium for in vitro propagation of prokaryotic or eukaryotic cells. Such culture media can be solid, liquid, or powdered. In one example, the cell culture media is DMEM or RPMI. In one example, the cell culture media is a bacterial culture medium. In one example, the cell culture media is an optimized transfection medium.

[1707] In certain examples, the kits further comprise one or more transfection reagents, such as liposomes, high lipid-based, or non-liposomal-based (eg, calcium phosphate-based) transfection reagents.

[1708] In some instances, the kit further includes one or more checkpoint inhibitors, such as inhibitors that reduce or inhibit PD-1, PD-L1 and / or CTLA-4 function. In one example, the kit further includes a PD-L1 antibody, such as atezolizumab, avelumab, durvalumab or cosibelimab (in some instances, these monoclonal antibodies are coupled to IR700, such as atezolizumab-IR700, avelumab-IR700, durvalumab-IR700 or cosibelimab-IR700). In one example, the kit further includes a PD-1 antibody, such as nivolumab, pembrolizumab, cemiplimab or dostarlimab (in some instances, these monoclonal antibodies are coupled to IR700). In one example, the kit further comprises a CTLA-4 antibody, such as ipilimumab or tremelimumab (in some examples, these monoclonal antibodies are conjugated to IR700).

[1709] In certain examples, the kit further comprises one or more immunotherapies, such as one or more immunomodulators and / or immune activators listed below, such as IL-2, IL-15, IL-7, IL-12, and / or IL-21.

[1710] In some instances, the kit further includes one or more chemotherapeutic agents, such as one or more of the following listed, such as one or more microtubule binding agents, DNA intercalators or cross-linking agents, DNA synthesis inhibitors, DNA and / or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators. In one example, the kit further includes carboplatin, cisplatin, paclitaxel, docetaxel, adriamycin, epirubicin, topotecan, irinotecan, gemcitabine, iazofurine, gemcitabine, etoposide, vinorelbine, tamoxifen, valspodar, cyclophosphamide, methotrexate, fluorouracil, mitoxantrone, doxorubicin liposomes (Doxil) (liposome-encapsulated adriamycin) and vinorelbine. One or more.

[1711] In some examples, the kit further includes one or more biologics, such as 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Bavituximab, Betumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab

[1712] Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab

[1713] Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicin), Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin), Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumabmertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomab estafenatox), Necitumumab, Nimotuzumab, Nofetumomabmerpentan), Ofatumumab, Olaratumab, Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab One or more of tetraxetan, taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, or zalutumumab.

[1714] In one example, the kit further includes a tumor-specific antibody-IR700 conjugate, such as cetuximab-IR700, trastuzumab-IR700, tivozanib-IR700, or any other antibody-IR700 conjugate provided in Table 1.

[1715] In certain instances, the kit further comprises one or more reducing agents, such as one or more of L-cysteine, L-sodium ascorbate (L-NaAA), ascorbic acid (such as L- or R-ascorbic acid), and glutathione. In a specific example, the kit further comprises L-NaAA.

[1716] The kit can include a container and a label or package insert on or associated with the container. Suitable containers include bottles, vials, syringes, and the like. The container can be made of 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 several aspects, the container can have a sterile access port (e.g., the container can be an intravenous fluid bag or a vial with a stopper pierceable by a hypodermic needle).

[1717] The label or package insert indicates that the composition is used to treat a specific condition. The label or package insert typically also includes instructions for use of the antibody, conjugate, or composition contained in the kit. The package insert typically also includes instructions customarily included in commercial packaging of therapeutic products, including information regarding the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. These instructions may be in written, electronic, or visual form. The kit may also include other components to facilitate the specific application for which the kit is designed.

[1718] VI. Methods of Treating Cancer

[1719] Also provided herein is a method for treating cancer in a subject. The cancer can be a solid tumor or a hematological malignancy, such as an immunogenic cancer (e.g., a T-cell-rich cancer). The method comprises administering to the subject a therapeutically effective amount of a conjugate or a composition thereof, the conjugate comprising a CD25-specific monoclonal antibody disclosed herein and a photon absorber, followed by a photon absorber at a wavelength of 660 to 740 nm and at least 1 J / cm 2 (e.g. at least 4 J / cm 2 ) dose to irradiate the subject and / or irradiate cancer cells in the subject. In certain aspects, the cancer is breast cancer, liver cancer, lung cancer, esophageal cancer, stomach cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer, cervical cancer, kidney cancer, bone cancer, skin cancer, head and neck cancer, oropharyngeal cancer, or blood cancer. In certain aspects, the conjugate is administered locally at or near the tumor site. In other aspects, the conjugate is administered systemically and the therapeutic effect is local to the cancer (e.g., without significant systemic adverse reactions).

[1720] In certain 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 tumor antigen expressed by the cancer cell. In some embodiments, the tumor antigen is selected from 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), vascular endothelial growth factor receptor (VEGFR), epithelial cell adhesion molecule (EpCAM), ephrin-A type 2 receptor (EphA2), glypican-1 (GPC1), glypican-2 (GPC2), glypican-3 (GPC3), gpA33, mucin (a 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, receptor activator of nuclear factor kappa-B ligand (RANKL), fibroblast activation protein (FAP), tenascin, BCR complex, gp72, HLA-DR10β, HLA-DR antigen, IgE, CA242, polymorphic epithelial mucin (PEM) antigen, SK-1 antigen. In certain aspects, the method further comprises administering a therapeutically effective amount of a second monoclonal antibody coupled to IR700, wherein the second monoclonal antibody specifically binds to a checkpoint inhibitor, such as programmed death 1 (PD-1), CTLA4, and programmed death ligand (PD-L1). Specific exemplary tumor antigens and checkpoint inhibitor antibodies are provided herein. In some instances, the CD25-specific conjugate and the second monoclonal antibody coupled to IR700 are both administered prior to irradiation. In some instances, the CD25-specific conjugate and the second monoclonal antibody coupled to IR700 are administered intravenously.

[1721] In certain aspects, the methods provided herein further comprise administering to the subject one or more additional cancer therapies. In some instances, the one or more additional cancer therapies comprise immune activators, chemotherapy, radiation therapy, and / or surgery.

[1722] Immune activators include immune system activators and / or inhibitors of one or more immunosuppressive cells (e.g., in the presence of a pharmaceutically acceptable carrier, such as a pharmaceutically and physiologically acceptable liquid). In a specific example, the immune activator is IL-15. In another specific example, the immune activator is interferon gamma. In some examples, one or more immune activators are administered to the subject in parallel (e.g., simultaneously or substantially simultaneously) with one or more antibody-IR700 conjugates, e.g., in the same composition. In other examples, one or more CD25-specific conjugates and one or more immune activators are administered sequentially (in any order) to the subject, e.g., at least about 1 hour apart from each other (e.g., within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 2 hours, about 12 hours, about 24 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days).

[1723] In some instances, the subject is further administered a therapeutically effective amount of one or more reducing agents, e.g., to reduce edema that may be caused by NIR-PIT. In some instances, the antibody-IR700 conjugate and the reducing agent are present in a single composition and administered simultaneously. In other instances, the antibody-IR700 conjugate and the reducing agent are present in separate compositions and administered simultaneously, contemporaneously, or sequentially (e.g., the antibody-IR700 conjugate is administered first, followed by the reducing agent).

[1724] The reducing agent is then administered again, for example, 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 apart).

[1725] 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 conjugates are allowed to accumulate in the target tumor or tumor immune cells. Then, the tumor cells and other cells in the tumor bed (or cancer-bearing subject) are irradiated under conditions that allow killing of cells bound to the antibody-IR700 conjugate, for example, at a wavelength of 660 to 740 nm (e.g., 600 to 710 nm) and at least 1 J / cm 2 The dose is irradiated (for example, the wavelength is 680nm or 690nm and the dose is 4 to 60J / cm 2 , such as a dose of 4 to 25 J / cm 2 , 4 to 10 J / cm 2 , 20 to 50 J / cm 2 or 20 to 30 J / cm 2 ).

[1726] In one example, the interval between administration of the antibody-IR700 conjugate and irradiation 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 to 4 hours, 30 minutes to 1 hour, 10 minutes to 60 minutes, 30 minutes to 8 hours, 2 to 10 hours, 12 to 24 hours, 18 to 36 hours, or 24 to 48 hours). In one example, one or more antibody-IR700 conjugates are administered (e.g., intravenously, intraperitoneally, 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 to 4 hours, 30 minutes to 1 hour, 10 minutes to 60 minutes, 30 minutes to 8 hours, 2 hours to 10 hours, 12 hours to 24 hours, 18 hours to 36 hours, or 24 hours to 48 hours, such as about 12 hours or about 24 hours). One or more immune activators can be administered (e.g., systemically, such as by intravenous or intraperitoneal injection) before or after the one or more antibody-IR700 conjugates and before or after irradiation. In some examples, one or more immune activators are administered before and after irradiation, for example, at least one dose of the immune activator is administered before irradiation and at least one dose of the immune activator is administered after irradiation (e.g., one or more of 24 hours before irradiation and 24, 48, 72, 96 or more hours after irradiation). In other examples, the immune activator dose can also be administered on the same day as at least one irradiation treatment. One or more reducing agents can be administered (e.g., systemically, such as intravenously or intraperitoneally) before or after the one or more antibody-IR700 conjugates, but before irradiation (e.g., at least 5 minutes, at least 10 minutes, at least 15 minutes, or at least 30 minutes before irradiation, for example, 5 to 60 minutes before irradiation).

[1727] In some instances, multiple doses of one or more antibody-IR700 conjugates are administered to the subject, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 separate doses (or administrations). In some instances, 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 a combination thereof are administered to the subject, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 separate doses (or administrations). In some instances, multiple doses of one or more immune activators are administered to the subject, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 separate doses (or administrations). In some instances, multiple doses of one or more reducing agents are administered to a subject, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 separate doses (or administrations). In some instances, multiple doses of NIR irradiation are administered to a subject, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 separate doses (or administrations). In a specific example, at least one dose of one or more antibody-IR700 conjugates, at least two doses of one or more immune activators, and at least two separate NIR irradiations are administered to a subject.

[1728] The wavelength of NIR excitation light can penetrate tissue for at least several centimeters. For example, by using a fiber-coupled laser diode with a diffuser tip, NIR light can be delivered to within several centimeters of an inaccessible tumor located deep within the body surface. In addition to treating solid cancers, circulating tumor cells (including but not limited to hematological malignancies) can also be targeted because they can be excited as they pass through superficial blood vessels (for example, using the NIR LED wearable device described herein).

[1729] 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 tumor-specific antigens, or CD25+ immune cells in the tumor bed, e.g., Tregs in the tumor bed), e.g., relative to not being treated with the disclosed methods.

[1730] In some examples, the disclosed methods can reduce the weight, volume, or size of metastases by, for example, 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% relative to treatment without the disclosed methods. In some examples, the disclosed methods have abscopal effects that can reduce the risk of metastases in patients who have not received a dose of at least 1 J cm-1 at a wavelength of 660 to 740 nm. -2 (e.g. a dose of at least 4 J cm -2 , at least 10 J cm-2, for example, a dose of 4-150 J cm -2 、1-10J cm -2 or 1-5 J cm -2 ) and the weight, volume, or size of metastases located away from the irradiated area of ​​the tumor or lesion. In some examples, 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% by treatment using the disclosed methods.

[1731] By selectively killing Treg cells (such as CD4 + Foxp3 + Treg), these methods can more effectively kill irradiated Treg cells than other immune cells, such as Treg that are not in the tumor bed. In one embodiment, the disclosed method reduces Treg (such as FOXP3 + CD4 + Treg cells, but not FOXP3 in other parts of the body, such as the spleen or regional lymph nodes + CD4 + Treg cells). For example, the disclosed methods can reduce the number of Treg cells in the tumor bed by at least 10%, for example, at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98% or more, relative to treatment without the disclosed methods (e.g., without administering one or more antibody-IR700 conjugates and / or without NIR irradiation). In some examples, the disclosed methods do not significantly reduce the number of Treg cells in the tumor spleen or regional lymph nodes, for example, by no more than 10%, no more than 5%, no more than 1%, or no more than 0.5%, relative to treatment without the disclosed methods (e.g., without administering one or more antibody-IR700 conjugates and / or without NIR irradiation).

[1732] 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, relative to not being treated with the disclosed methods (e.g., not administering one or more antibody-IR700 conjugates and / or not performing NIR irradiation).

[1733] In some instances, the disclosed methods reduce one or more symptoms associated with tumors, recurrences, and / or metastatic tumors. In one example, the disclosed methods slow the growth of the tumor, for example, by at least 10%, for example, by at least 20%, at least 40%, at least 50%, at least 80%, at least 90% or more, relative to treatment without the disclosed methods (e.g., without administering one or more antibody-IR700 conjugates and / or without NIR irradiation). In one example, the disclosed methods reduce or eliminate tumor recurrence, for example, by at least 10%, for example, by 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%, relative to treatment without the disclosed methods (e.g., without administering one or more antibody-IR700 conjugates and / or without NIR irradiation).

[1734] In some instances, the disclosed methods increase the survival time of a subject (e.g., a subject having a tumor or a previously removed tumor) relative to treatment without the disclosed methods (e.g., without administering one or more antibody-IR700 conjugates and / or without NIR irradiation). In some instances, for example, relative to treatment without the disclosed methods (e.g., without administering one or more antibody-IR700 conjugates and / or without NIR irradiation), the survival time of the subject is increased by at least 20%, at least 25%, at least 40%, at least 50%, at least 80%, at least 90% or more. In some instances, for example, relative to treatment without the disclosed methods (e.g., without administering one or more antibody-IR700 conjugates and / or without NIR irradiation), the survival time of the subject 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 more. For example, the methods disclosed in certain examples extend a subject's progression-free survival or disease-free survival (e.g., no recurrence or metastasis of the primary tumor) 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, relative to the average survival time without treatment using the disclosed methods (e.g., without administration of one or more antibody-IR700 conjugates and / or without NIR irradiation).

[1735] In some examples, the use of one or more reducing agents in combination with NIR-PIT reduces the amount of edema in the 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 in the absence of the one or more reducing agents and NIR-PIT. In some examples, the use of one or more reducing agents in combination with NIR-PIT reduces the acute inflammatory response in the 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 in the absence of the one or more reducing agents and NIR-PIT.

[1736] In one example, the disclosed method can achieve a combination of the above effects.

[1737] The disclosed method can be used to treat fixed tumors in the body as well as hematological malignancies and / or circulating tumors (such as leukemia cells, metastases and / or circulating tumor cells). However, due to their characteristics, circulating cells cannot be exposed to light for a long time. Therefore, if the cells to be killed are cells circulating in the body, the above method can be implemented by using a wearable or body-covering device. For example, such a device can be worn for a long time. Everyday wearable items (such as watches, jewelry (such as necklaces or bracelets), blankets, clothing (such as underwear, socks and insoles) and other everyday wearable items) containing NIR light emitting diodes (LEDs) and battery packs can be used. Such a device will generate light on the skin under the device for a long time, causing the superficial blood vessels to be continuously exposed to light for a long time. Circulating tumor cells are exposed to light when passing through the area under the device. For example, a watch or bracelet version of such a device may include a series of NIR LEDs equipped with a battery power pack and can be worn for most of the day. After administration of one or more CD25 antibody-IR700 conjugates (e.g., with one or more tumor-specific antibody-IR700 conjugates or checkpoint inhibitor antibody-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, systemically, such as by intravenous and / or intraperitoneal injection), circulating cells bind to the antibody-IR700 conjugates and become susceptible to killing by PIT. When these cells flow within blood vessels adjacent to LEDs in everyday wearable devices such as bracelets or watches, they are exposed to NIR light and are thus susceptible to killing. The dose of light can be adjusted depending on the diagnosis and cell type.

[1738] In some instances, the method also includes monitoring treatment, such as killing tumor cells, killing Treg or both. In these examples, one or more antibody-IR700 conjugates (such as CD25 specific antibody-IR700 conjugates and optionally tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitor antibody-IR700 conjugates) are administered to the subject, for example, co-administered with one or more reducing agents, one or more immune activators or a combination thereof, and irradiated as described herein. However, lower doses of antibody-IR700 conjugates and NIR light can also be used for monitoring (because it may not be necessary to kill cells, only monitoring treatment is required). In one example, the amount of the antibody-IR700 conjugate used for monitoring is at least 2 times less (such as at least 3, 4, 5, 6, 7, 8, 9 or 10 times less than the therapeutic dose). In one example, the amount of the antibody-IR700 conjugate used for monitoring is at least 20% or at least 25% less than the therapeutic dose. In one embodiment, 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 detection of cells being treated. For example, tumor size and metastasis can be monitored using this method.

[1739] In some instances, the method is useful during surgery, such as endoscopic surgery. For example, after administering an antibody-IR700 conjugate (such as a CD25-specific antibody-IR700 conjugate and optionally a tumor-specific antibody-IR700 conjugate and / or a checkpoint inhibitor antibody-IR700 conjugate), for example, in combination with one or more reducing agents, one or more immune activators, or a combination thereof, and irradiating the cells / subjects as described above, this not only results in cell death, but also allows surgeons or other healthcare providers to see the margins of the tumor, helping to ensure that the tumor (such as skin, breast, lung, colon, head and neck, or prostate tumor) is completely removed and has clear margins. In some instances, a lower dose of antibody-IR700 conjugate can be used for visualization, for example, at least 2 times less (such as at least 3, 4, 5, 6, 7, 8, 9, or 10 times less than the therapeutic dose).

[1740] One or more antibody-IR700 conjugates (e.g., CD25-specific antibody-IR700 conjugates and optional tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitor antibody-IR700 conjugates), 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, such as cancer, or a subject who has previously had a tumor removed (e.g., by surgical resection). Although specific examples are provided, it will be understood by those skilled in the art that other methods of administering the disclosed therapeutic agents can be used. These methods may include, for example, providing continuous infusion to a subject in need of treatment over a period of hours to days using a catheter or implantable pump.

[1741] In one example, one or more antibody-IR700 conjugates (e.g., CD25-specific antibody-IR700 conjugates and optionally tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitor antibody-IR700 conjugates), one or more reducing agents, and one or more immune activators are administered parenterally, including direct injection or infusion into a tumor (intratumorally) or an organ (e.g., prostate). In some examples, one or more antibody-IR700 conjugates (e.g., CD25-specific antibody-IR700 conjugates and optionally tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitor antibody-IR700 conjugates), 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, soaking the tumor with a solution containing the therapeutic agent, or pouring the therapeutic agent onto the tumor.

[1742] In addition, or alternatively, the disclosed composition can also be systemically administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, subcutaneously or orally administered to a subject with a tumor (such as cancer). One or more antibody-IR700 conjugates (e.g., CD25 specific antibody-IR700 conjugates and alternatively tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitor antibody-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., CD25-specific antibody-IR700 conjugates and optionally tumor-specific antibody-IR700 conjugates and / or check point inhibitor antibody-IR700 conjugates), 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 tumor-specific antibody-IR700 conjugates and / or check point inhibitor antibody-IR700 conjugates, 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.

[1743] 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 ingredients and their potential undesirable side effects (such as immune responses to the antibodies), the subject receiving treatment and the type of treatment condition, and the mode of administration. In general, the dosage is a therapeutically effective dose, such as an amount sufficient to achieve the desired biological effect, such as an amount that effectively reduces the size of a tumor (such as volume and / or weight), or reduces further growth of a tumor, or reduces adverse symptoms of a tumor.

[1744] For intravenous administration of the antibody-IR700 conjugate, an exemplary dosage for a single treatment of a subject can be 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, 1 to 20 mg / 60 kg body weight, for example, about 1 or 2 mg / kg body weight. In another example, a therapeutically effective amount of the antibody-IR700 conjugate administered intraperitoneally or intratumorally is 10 μg to 5000 μg of the antibody-IR700 conjugate per 1 kg body weight, for example, 10 μg / kg to 1000 μg / kg, 10 μg / kg to 500 μg / kg, or 100 μg / kg to 1000 μg / kg. In one example, the dose of the 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.

[1745] For intravenous administration of the reducing agent, an exemplary single-treatment dosage can be 0.5 to 300 g / 60 kg body weight, 1 to 300 g / 60 kg body weight, 1 to 50 g / 60 kg body weight, 1 to 20 g / 60 kg body weight, 1 to 10 g / 60 kg body weight, 10 to 300 g / 60 kg body weight, such as 1, 2, 5, 10, 20, 50, 100, 200, or 300 g / 60 kg body weight. In another example, the therapeutically effective amount of the reducing agent administered intraperitoneally or intratumorally is 10 mg to 5000 mg of the reducing agent per 1 kg body weight, such as 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, e.g., 1, 2, 5, 10, 20, 50, 100, 200, or 300 g.

[1746] 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 in one day or repeated at the same or different doses over multiple days. Repeated treatments can be performed on the same day, for several consecutive days, or every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months or longer. In certain instances, the antibody-IR700 conjugate, one or more reducing agents and / or one or more immune activators are administered on the same day. In other instances, the antibody-IR700 conjugate, one or more reducing agents and / or one or more immune activators are administered on different days, for example, 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 repeated doses of the one or more immune activators (at the same or different dosing levels) are administered to the subject on consecutive days, or every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1 to 2 months, or even longer intervals (e.g., 1, 2, 3, 4, 5 or more additional doses of one or more immune activators). In certain instances, the amount of the repeated doses of the one or more immune activators is reduced (e.g., reduced by 50%) compared to the initial dose.

[1747] In other aspects, the methods further comprise administering to the subject one or more additional therapeutic agents. As described in International Patent Application Publication No. WO 2013 / 009475 (the entire contents of which are incorporated herein by reference), after irradiation (e.g., irradiation at a wavelength of 660 to 710 nm at a dose of at least 10 J / cm 2 , at least 20J / cm 2 , or at least 30J / cm 2 , at least 40J / cm 2 , at least 50J / cm 2 , at least 70J / cm 2 , at least 80J / cm 2 , or at least 100 J / cm 2 , for example 10 to 100 J / cm 2) has a window of approximately 8 hours during which the uptake of an additional agent (e.g., a nanoscale agent, e.g., having a diameter of about at least 1 nm, a diameter of at least 10 nm, a diameter of at least 100 nm, or a diameter of at least 200 nm, e.g., a diameter of 1 to 500 nm) by cells treated with PIT is enhanced. Thus, 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 to 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., no more than 10 hours, no more than 9 hours, or no 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 just prior to irradiation (e.g., about 10 minutes to 120 minutes before irradiation, such as 10 minutes to 60 minutes or 10 minutes to 30 minutes before irradiation). Additional therapeutic agents that can be used are discussed below.

[1748] In other aspects, methods are provided that allow real-time detection or monitoring of cell killing. For example, these methods can be used, for example, to ensure that a sufficient amount of antibody-IR700 conjugates (including CD25-specific antibody-IR700 conjugates, tumor-specific antibody-IR700 conjugates and / or CTLA4-, PD-1 and PD-L1-antibody-IR700 conjugates), one or more reducing agents and / or one or more immune activators or sufficient amounts of irradiation are delivered to cells or tumors to promote cell killing. These methods allow cell killing to be detected before morphological changes are apparent. In one example, these methods include contacting cells with cell surface proteins with a therapeutically effective amount of one or more antibody-IR700 conjugates (including CD25-specific antibody-IR700 conjugates, tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitors-antibody-IR700 conjugates), one or more reducing agents and / or one or more immune activators; at a wavelength of 660 to 740 nm and at least 4 J / cm 2 or at least 10J / cm 2The cells are irradiated at a dose sufficient to shorten the IR700 FLT by at least 25%, such as at least 40%, at least 50%, at least 60%, or at least 75%. In one example, the cells are irradiated at a wavelength of 660 nm to 740 nm (such as 680 nm to 700 nm, such as 680 or 690 nm) at a rate of at least 1 J / cm 2 , at least 4J / cm 2 , at least 10J / cm 2 , at least 20J / cm 2 , at least 30J / cm 2 , at least 40J / cm 2 , at least 50J / cm 2 or at least 60 J / cm 2 , for example 10 to 60 J / cm 2 , 20 to 50 J / cm 2 , 20 to 25 J / cm 2 , 30 to 50 J / cm 2 , 4 to 50 J / cm 2 or 1 to 10 J / cm 2 dose of radiation.

[1749] In some instances, the method for detecting cell killing 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 the cells are contacted with one or more additional therapeutic agents. For example, if insufficient cell killing occurs after administration of one or more antibody-IR700 conjugates (including CD25-specific antibody-IR700 conjugates, tumor-specific antibody-IR700 conjugates and / or checkpoint inhibitors-antibody-IR700 conjugates), one or more reducing agents and / or one or more immune activators, as determined by real-time imaging, the cells may be contacted with one or more additional therapeutic agents. However, in certain instances, 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, as well as other therapeutic agents, prior to real-time detection of cell killing.

[1750] Exemplary cells

[1751] The target cells can be cells that are undesirable or whose growth is not desired, such as cancer cells (such as tumor cells) or immune cells (such as T cells, such as Treg). These cells can be present in the body of a mammal to be treated, such as a subject with cancer (such as a human or animal subject). Any target cell can be treated with the claimed method. In some aspects, the target cell is a CD25+Treg cell. In some instances, the target cell expresses a cell surface protein that is not substantially found on the surface of other normal (desirable) cells, and antibodies that specifically bind to this protein, as well as antibody-IR700 conjugates generated against this protein, can be selected. In one example, the cell surface protein is a tumor-specific protein (such as an antigen), such as EGFR. In a non-limiting example, the cell surface protein is CTLA4 (for example, targeting CD4 in the tumor bed). + Foxp3 + In one non-limiting example, the cell surface protein is PD-L1, PD-1 or CTLA4.

[1752] 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 cell is a cancer cell, such as a cell from a cancer patient. Exemplary cells that can be killed using the disclosed methods include cells from the following tumors: hematological malignancies, such as leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia, and myeloid, promyelocytic, myelomonocytic, monocytic, and erythroleukemias), chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease). In another example, the cell is a solid tumor cell, the cell is a solid tumor cell, for example, a cell from a sarcoma and carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovial sarcoma, mesothelioma, Ewing's sarcoma, 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, In one embodiment, the tumor is a carcinoma of the oropharynx, e.g., adenocarcinoma of the breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, bladder cancer, and central nervous system cancers (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In one embodiment, the tumor is oropharyngeal cancer.

[1753] In one embodiment, the cell is a lung cancer cell.

[1754] In one embodiment, the cell is a breast cancer cell.

[1755] In one embodiment, the cell is a colon cancer cell.

[1756] In one embodiment, the cell is a gastric cancer cell.

[1757] In one embodiment, the cell is a renal cancer cell.

[1758] In one embodiment, the cell is a head and neck cancer cell.

[1759] In one embodiment, the cell is a prostate cancer cell.

[1760] In one embodiment, the cell is an oropharyngeal cancer cell.

[1761] In one embodiment, the cell is an esophageal cancer cell. In one embodiment, the cell is a squamous cell carcinoma cancer cell, such as a head and neck cancer or a skin cancer cell.

[1762] In some embodiments, the cancer or cancer cells being treated are located at a site distant from the irradiated tumor, such as a site not receiving radiation at a wavelength of 660 to 740 nm and a dose of at least 1 J / cm 2 Distant metastases, such as 1 mm away, 1 inch away, 2 inches away, 3 inches away, 4 inches away, 6 inches away, 12 inches away, or more, can be irradiated with abscopal effects.

[1763] In some instances, the cancer or cancer cell being treated has moderate or high immunogenicity and can cause an immune response. In some instances, the NIR-PIT method is used, including administering CTLA4 antibody-IR700, PD-1 antibody-IR700 and / or PD-L1 antibody-IR700, to treat moderate or high immunogenic tumors. Examples of moderate or high immunogenic cancers include melanoma, lung cancer (such as NSCLC), colon cancer and renal cell carcinoma. In one example, a highly immunogenic cancer is prostate cancer. In some instances, the cancer or cancer cell being treated is not highly immunogenic and cannot cause an immune response. In some instances, NIR-PIT and CTLA4 antibody-IR700, PD-1 antibody-IR700 and / or PD-L1 antibody-IR700 are used and used in combination with anti-tumor-IL700 (such as 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 instances, a tumor immunogenicity score is used to determine whether a cancer is low-immunogenic, moderately immunogenic, or highly immunogenic (e.g., see Wang et al., eLife, 8:e49020, 2019).

[1764] Exemplary Subjects

[1765] In some examples, the disclosed methods are used to treat a subject having cancer or a subject having a tumor, such as a tumor described herein. In some examples, the tumor has been previously treated, such as by surgical resection or chemical removal, and the disclosed methods are subsequently used to kill any remaining unwanted tumor cells that may remain in the patient and / or to reduce the recurrence or metastasis of the tumor. In some examples, the subject treated with the disclosed CD25 antibody-IR700 conjugates has a highly or moderately immunogenic cancer.

[1766] The disclosed methods can be used to treat any mammalian subject (such as a human or animal subject, such as a dog or cat), such as a person who has a tumor such as cancer or has previously been removed or treated. Subjects in need of the disclosed therapy can include human subjects with cancer, such as cancers that express 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 (such as CTLA4 antibody-IR700). For example, the disclosed methods can be used alone as an initial treatment for cancer or in combination with radiotherapy or other chemotherapy or surgery. The disclosed methods can also be used for patients who have previously failed radiotherapy or chemotherapy. Therefore, in some instances, the subject has received other therapies, but these other therapies did not provide the desired therapeutic response. The disclosed methods can also be used for patients with localized and / or metastatic cancers and / or recurrence of primary tumors.

[1767] In some examples, the method includes selecting a subject who will benefit from the disclosed therapy, such as selecting a subject having a tumor that expresses a cell surface protein (such as a tumor-specific protein) that can specifically bind to an antibody-IR700 conjugate. For example, if the subject is determined to have breast cancer that expresses HER2, the subject is selected to receive a combination of an anti-HER2-IR700 conjugate, such as trastuzumab-IR700, and a CD25-specific antibody-IR700 conjugate, for example, 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 then irradiating the subject as described herein.

[1768] Exemplary cell surface proteins

[1769] In certain aspects of the disclosed methods, a CD25-specific antibody-IR700 conjugate is administered in combination with an antibody-IR700 conjugate that targets a cell surface protein of a tumor or cancer cell. In one example, the target cell to be killed has a cell surface protein that 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.

[1770] In specific examples, cell surface proteins are cancer or tumor-specific proteins (also known as tumor-specific antigens or tumor-associated antigens), such as members of the EGF receptor family (such as HER1, 2, 3, and 4) and cytokine receptors (such as CD20, CD25, IL-13R, CD5, CD52, etc.). Thus, in certain examples, cell surface proteins are antigens expressed on the cell membrane of tumor cells. Tumor-specific proteins are proteins that are unique to cancer cells, or are present in much higher amounts on cancer cells than on other cells, such as normal cells. For example, HER2 is primarily found in breast cancer, while HER1 is primarily found in adenocarcinomas, which can be found in many organs, such as the pancreas, breast, prostate, and colon.

[1771] Exemplary tumor-specific proteins that can be found on target cells (and for which antibodies specific for the proteins can be used to formulate antibody-IR700 molecules) include, but are not limited to: any of the various MAGE (melanoma-associated antigen E) proteins, 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), etc.; any of the various tyrosinases (e.g., GenBank Accession Nos. U01873 and AAB60319); mutant ras; mutant p53 (e.g., GenBank Accession Nos. X54156, CAA3 8095 and AA494311); p97 melanoma antigen (e.g., GenBank Accession Nos. M12154 and AAA59992); human milk fat globules (HMFG) associated with breast tumors (e.g., GenBank Accession Nos. S56151 and AAB19771); any of various BAGEs (human B melanoma-associated antigens E), including BAGE1 (e.g., GenBank Accession No. Q13072) and BAGE2 (e.g., GenBank Accession Nos. NM_182482 and NP_872288); any of various GAGEs (G antigens), including GAGE1 (e.g., GenBank Accession No. Q13065) or any of GAGE2-6; various gangliosides, CD25 (e.g., GenBank Accession Nos. NP_000408.1 and NM_000417.2).

[1772] Other tumor-specific antigens include HPV16 / 18 and E6 / E7 antigens associated with cervical cancer (e.g., GenBank Accession Nos. NC_001526, FJ952142.1, ADB94605, ADB94606, and U89349), mucin (MUC1)-KLH antigen associated with breast cancer (e.g., GenBank Accession Nos. J03651 and AAA35756), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession Nos. X98311 and CAA66955), gp100 associated with, for example, melanoma (e.g., GenBank Accession Nos. NC_001526, FJ952142.1, ADB94605, ADB94606, and U89349), and glycans (e.g., GenBank Accession Nos. AAA35756). ank accession numbers S73003 and AAC60634), MART1 antigen associated with melanoma (e.g., GenBank accession number NP_005502), cancer antigen 125 (CA125, also known as mucin 16 or MUC16) associated with ovarian and other cancers (e.g., GenBank accession numbers NM_024690 and NP_078966); α-ferritin (AFP) associated with liver cancer (e.g., GenBank accession numbers NM_001134 and NP_001125); Lewis antigen associated with colorectal cancer, biliary tract cancer, breast cancer, small cell lung cancer, and other cancers. Y antigen; tumor-associated glycoprotein 72 (TAG72), which is associated with adenocarcinoma; glypican 1 (GPC1, which is associated with pancreatic cancer, glioma, and breast cancer), glypican 2 (which is associated with neuroblastoma), and glypican 3 (which is associated with hepatocellular carcinoma), and PSA antigen, which is associated with prostate cancer (e.g., GenBank accession numbers X14810 and CAA32915).

[1773] Other exemplary tumor-specific proteins include, but are not limited to, PMSA (prostate membrane-specific antigen; e.g., GenBank Accession Nos. AAA60209 and AAB81971.1), which is associated with neovascularization of solid tumors and 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 cancers; HER-1 (e.g., GenBank Accession Nos. NM_005228 and NP_005219), which is associated with lung, anal, glioblastoma, and adenocarcinoma; NY-ESO-1 (e.g., GenBank Accession Nos. U87459 and AAB4969), which is associated with melanoma, sarcoma, testicular, and other cancers. 3); hTERT (also known as telomerase) (e.g., GenBank Accession Nos. NM_198253 and NP_937983 (variant 1), NM_198255 and NP_937986 (variant 2)); Protease 3 (e.g., GenBank Accession Nos. M29142, M75154, M96839, X55668, NM00277, M96628, X56606, CAA39943 and AAA36342), 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)).

[1774] In one example, the tumor-specific protein is EGFR. In certain examples, the EGFR antibody-IR700 is or includes panitumumab or cetuximab.

[1775] In one example, the tumor-specific protein is PD-L1. In some examples, the PD-L1 antibody-IR700 is or includes atezolizumab, avelumab, durvalumab, or cosibelimab.

[1776] In one example, the tumor-specific protein is CD52, which is associated with chronic lymphocytic leukemia (e.g., GenBank Accession Nos. AAH27495.1 and CAI15846.1); CD33, which is associated with acute myeloid leukemia (e.g., GenBank Accession Nos. NM_023068 and CAD36509.1); or CD20, which is associated with non-Hodgkin's lymphoma (e.g., GenBank Accession Nos. NP_068769 and NP_031667).

[1777] In one embodiment, the tumor-specific protein is CD44 (e.g., OMIM 107269, GenBank accession numbers ACI46596.1 and NP_000601.3). CD44 is a marker for cancer stem-like cells and various types of cancer, and is associated with the promotion of cell-cell adhesion, cell migration, cell spatial orientation, and matrix-derived survival signals. High expression of CD44 on the tumor plasma membrane is associated with tumor aggressiveness and poor prognosis.

[1778] Thus, the disclosed methods can be used to treat any cancer that expresses a tumor-specific protein.

[1779] Exemplary Antibody-IR700 Conjugates

[1780] Since cell surface protein sequences are publicly available (e.g., as described above), it is possible to manufacture or purchase specific antibodies against these proteins (or other small molecules that can be coupled to IR700). For example, if the tumor-specific protein HER2 is selected as a target, a specific antibody against HER2 (e.g., trastuzumab) can be purchased or produced and attached to the IR700 dye. Other specific examples are provided in Table 1 and elsewhere herein. In one embodiment, the antibody coupled to IR700 is a humanized monoclonal antibody.

[1781] In one example, the antibody-IR700 conjugate is an EGFR-antibody-IR700 molecule, such as panitumumab-IR700 or cetuximab-IR700.

[1782] Antibody-IR700 molecules can be produced by methods such as those described in WO2013 / 009475 (incorporated herein by reference in its entirety).

[1783] Table 1. Exemplary tumor-specific antigens and antibodies

[1784]

[1785]

[1786]

[1787]

[1788] 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, Betumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, and Blinatumomab.

[1789] Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab

[1790] Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicin), Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin), Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumabmertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomab estafenatox), Necitumumab, Nimotuzumab, Nofetumomabmerpentan), Ofatumumab, Olaratumab, Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab The following are some of the drugs listed in the Table: tetraxetan, taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, and zalutumumab.

[1791] In some examples, the cell surface protein recognized by the antibody conjugated to IR700 is 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.

[1792] In one example, a patient receives treatment 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 are specific 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 kill cells carrying HER1 or HER2.

[1793] Immunomodulators / immune activators

[1794] The immune activators used in the disclosed methods include agents or compositions that activate the immune system and / or suppress immunosuppressive cells (also referred to herein as suppressor cells). Suppressing immunosuppressive cells and / or activating an immune response can increase the killing of tumor cells and lead to the generation of memory T cells, thereby providing a "vaccine" effect against recurrent and / or distant tumors or metastases. In some instances, 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.

[1795] In some aspects, immunoactivator is an inhibitor of immunosuppressive cells, for example, an agent that suppresses or reduces the activity of immunosuppressive cells. In some cases, immunomodulator kills immunosuppressive cells. In some instances, immunosuppressive cells are regulatory T (Treg) cells. In some instances, not all suppressive cells in the body are killed because this may lead to the generation of autoimmunity. Therefore, in some instances, the method reduces the activity or quantity of immunosuppressive cells in the subject's area (such as tumor area or the area where tumors were once present) by at least 50%, at least 60%, at least 75%, at least 80%, at least 90% or at least 95%. In some instances, the method reduces the total number of suppressive cells in the subject's body by at least 50%, at least 60%, at least 75%, at least 80%, at least 90% or at least 95%.

[1796] Inhibitors of immunosuppressive cells include tyrosine kinase inhibitors (such as sorafenib, sunitinib, and imatinib), or chemotherapy drugs (such as cyclophosphamide or interleukin-toxin fusion agents, such as interleukin-2 toxin).

[1797] (denileukin difitox) (IL2-diphtheria toxin fusion agent)), or other antibodies that bind to inhibitory cell surface proteins (such as the antibodies described below). In other examples, inhibitors of immunosuppressive cells include immune checkpoint inhibitors, such as anti-PD-L1 antagonistic antibodies, thereby preventing PD-L1 from binding to PD-1 (referred to herein as PD-1 / PD-L1mAb-mediated immune checkpoint blockade (ICB)). Therefore, in some examples, the immunomodulator is a PD-L1 antagonistic 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 instances, the immunomodulator is a PD-1 antagonist antibody, such as nivolumab, pembrolizumab, cemiplizumab, or dostalimumab. Checkpoint inhibitors also include CTLA-4 antibodies, including ipilimumab and tremelimumab. The immunosuppressive cell inhibitor can also be a LAG-3 or B7-H3 antagonist, such as BMS-986016 and MGA271. In some instances, two or more immunosuppressive cell inhibitors can be administered to the subject. In one non-limiting example, anti-PD1 and anti-LAG-3 antibodies are administered to the subject.

[1798] In some examples, the agent that inhibits or reduces the activity of an inhibitory cell comprises one or more antibody-IR700 conjugates, wherein the antibody specifically binds to an inhibitory cell surface protein, such as CD4, CXC chemokine receptor type 4 (CXCR4), CC chemokine receptor type 4 (CCR4), cytotoxic T lymphocyte-associated protein 4 (CTLA4), glucocorticoid-induced TNF receptor (GITR), OX40, folate receptor 4 (FR4), CD16, CD56, 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 inhibitor of T cell activation (VISTA).

[1799] In other aspects, immunomodulator is immune system activator (immune activator). In some instances, immune system activator stimulates (activates) one or more T cells and / or natural killer (NK) cells. In one example, immune system activator includes one or more interleukins (IL), such as IL-2, IL-15, IL-7, IL-12 and / or IL-21. In a non-limiting example, immunomodulator includes IL-15 or consists of it. In a non-limiting example, immunomodulator includes IL-2 and IL-15. In a non-limiting example, immunomodulator includes interferon gamma or consists of it. In another example, immune system activator includes one or more agonists of costimulatory receptors, such as 4-1BB, OX40 or GITR. In a non-limiting example, immunomodulator includes irritating anti-4-1BB, anti-OX40 and / or anti-GITR antibodies.

[1800] In some instances, one or more (such as 1, 2, 3, 4, 5 or more) doses of an immunomodulator are administered to the subject. Therefore, the administration of the immunomodulator can be completed in one day, or it can be repeatedly administered with the same or different doses over multiple days (for example, at least 2, 3, 4, 5 or 10 times). In some instances, the dosage of repeated administration is the same. In other instances, the dosage of repeated administration is different (such as a subsequent dose is higher than the previous dose or a subsequent dose is lower than the previous dose). Repeated administration of the immunomodulator can be performed on the same day, for several consecutive days, every other day, every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months or at longer time intervals. In some instances, at least one dose of immunomodulator is administered before irradiation, and at least one dose of immunomodulator is administered after irradiation.

[1801] irradiation

[1802] After administering one or more antibody-IR700 conjugates to a subject, the subject (or the subject's tumor) is irradiated. Because only cells expressing the target protein (e.g., CD25) are recognized by the antibody, only these cells will have sufficient antibody-IR700 conjugate bound to them and killed. This reduces the possibility of undesirable side effects, such as killing non-tumor cells, because irradiation only kills cells bound to the antibody-IR700 conjugate and not other cells.

[1803] The subject (e.g., a tumor in the subject) is irradiated with a therapeutic dose of radiation having a wavelength of 660-740 nm, such as 660-710 nm, 660-700 nm, 680-700 nm, 670-690 nm, such as 680 nm or 690 nm. In certain embodiments, the cell, tumor, or subject receives a dose of at least 1 J / cm 2, for example at least 4 J / cm 2 , at least 10J / cm 2 , at least 20J / cm 2 , at least 25J / cm 2 , at least 30J / cm 2 , at least 50J / cm 2 , at least 100J / cm 2 , or at least 500 J / cm 2 , for example, 1-1000 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 , 1-10J / cm 2 , 4-10J / cm 2 , 20-50J / cm 2 or 10-50J / cm 2 In a specific example, the subject (or a tumor in the subject) receives radiation at a wavelength of 690 nm and a dose of 50 J / cm 2 of irradiation.

[1804] The subject may receive one or more irradiations. Thus, the irradiation may be performed in one day or repeated over multiple days at the same or different doses (e.g., at least 2, 3, 4, 5, or 10 irradiations). In some instances, the doses of the repeated irradiations are the same. In other instances, the doses of the repeated irradiations are different (e.g., the subsequent dose is higher than the previous dose or the subsequent dose is lower than the previous dose). Repeated irradiations may be performed on the same day, on consecutive days, every other day, every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or at longer intervals. In one instance, the first irradiation is 50 J / cm 2 The second irradiation is 100 J / cm 2 , the irradiation time is several consecutive days (for example, about 24 hours apart). In one embodiment, the first irradiation is 10-50J / cm 2 The second irradiation is 10-50J / cm 2 , irradiation is performed on consecutive days (eg, about 24 hours apart).

[1805] In some examples, illumination is provided via a wearable device containing NIR LEDs. In other examples, another device that can be used with the disclosed methods is a flashlight-like device with NIR LEDs. Such a device can be used to treat lesions during surgery or in conjunction with an endoscope to apply NIR light to the body surface after administering one or more PIT agents. A physician or qualified medical professional can use this device to direct treatment to specific targets in the body.

[1806] Using wearable NIR LEDs for therapy

[1807] As described herein, the disclosed methods are highly specific for cancer cells and / or Tregs in the tumor bed. However, in order to kill cells circulating in the body or cells present on the skin, the patient can wear a device comprising a NIR LED. In some instances, the patient uses at least two devices, such as a piece of clothing or jewelry during the day and a blanket at night. In certain instances, the patient uses at least two devices, such as two pieces of clothing, at the same time. These devices can expose the patient to NIR light using portable everyday clothing and jewelry, thereby keeping the treatment private and not interfering with daily activities. In certain instances, the device can be worn discreetly during the day for PIT treatment. Exemplary devices comprising NIR LEDs are disclosed in International Patent Application Publication No. WO2013 / 009475 (incorporated herein by reference).

[1808] In one embodiment, one or more antibody-IR700 molecules are administered to a patient using the methods described herein, for example in combination with one or more reducing agents and / or one or more immune activators. The patient then wears a device containing NIR LEDs, which allows for long-term treatment of tumor cells present in the blood, lymph, or skin. In some embodiments, the dose is at least 1 J / cm 2 , at least 4J / cm 2 , at least 10J / cm 2 , at least 20J / cm 2 , at least 30J / cm 2 , at least 40J / cm 2 or at least 50 J / cm 2 , such as 10 to 100 J / cm 2 , 10 to 50 J / cm 2 , such as 20J / cm 2 or 30J / cm 2 In some examples, administration of one or more antibody-IR700 conjugates, for example in combination with one or more reducing agents and / or one or more immune activators, is repeated over a period of time (e.g., biweekly or monthly) to ensure that therapeutic levels are maintained in vivo.

[1809] In some instances, the patient wears or uses the device or combination of devices for at least 1 week, such as at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 4 months, at least 6 months, or even at least 1 year. In certain instances, the patient wears or uses the device or combination of devices for at least 4 hours a day, such as at least 12 hours a day, at least 16 hours a day, at least 18 hours a day, or 24 hours a day. During treatment, it is entirely possible that the same patient wears multiple devices with similar "everyday" properties (blankets, bracelets, necklaces, underwear, socks, insoles). At night, the patient can use a NIR LED blanket or other covering.

[1810] Administer additional therapy

[1811] As described above, before, during or after administering 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, the subject may receive one or more other therapies. In one example, before administering one or more antibody-IR700 conjugates (e.g., in combination with one or more reducing agents and / or one or more immune activators) and irradiating, the subject receives one or more treatments to eliminate or reduce the tumor. In other examples, the subject may be administered additional treatment or therapeutic agents (e.g., anti-tumor agents) to the subject to be treated, for example, after irradiation, for example, about 0 to 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, e.g., no more than 10 hours, no more than 9 hours, or no more than 8 hours, 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). In another example, the additional therapeutic agent is administered just prior to irradiation (eg, about 10 minutes to 120 minutes prior to irradiation, eg, 10 minutes to 60 minutes or 10 minutes to 30 minutes prior to irradiation).

[1812] Examples of such therapies that can be used in combination with the disclosed methods, which in some instances enhance tumor accessibility to additional therapeutic agents for about 8 hours after PIT, include but are not limited to surgical treatments for tumor removal or reduction (such as surgical resection, cryotherapy, or chemoembolization), and anti-tumor drug treatments, which may include radiotherapeutic agents, anti-tumor chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents. In some instances, the additional therapeutic agent is coupled to the nanoparticles. Specific examples of additional therapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linking agents, DNA synthesis inhibitors, DNA and / or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene modulators. These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination.

[1813] "Microtubule-binding agents" refer to agents that interact with tubulin to stabilize or disrupt microtubule formation, thereby inhibiting cell division. Examples of microtubule-binding agents that can be used in combination with the disclosed methods include, but are not limited to, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (Navelbine), epothilones, 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. Taxanes, such as paclitaxel and docetaxel, and paclitaxel analogs described in U.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264 can also be used.

[1814] The following classes of compounds can be used together with the methods disclosed herein: suitable DNA and / or RNA transcription regulators, including but not limited to actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof, are also suitable for use in combination with disclosed therapies. DNA chimeras and cross-linking agents that can be applied to subjects include but are not limited to cisplatin, carboplatin, oxaliplatin, mitomycin (such as mitomycin C), bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof. DNA synthesis inhibitors suitable for use as therapeutic agents include but are not limited to methotrexate, 5-fluoro-5'-deoxyuridine, 5-fluorouracil and analogs thereof. Suitable enzyme inhibitors include but are not limited to camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof. Suitable compounds affecting gene regulation include agents that cause one or more gene expression increases or decreases, such as raloxifene, 5-azacytosine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof. Kinase inhibitors include those that block the phosphorylation and activation of growth factors (imatinib), (gefitinib) and (erlotinib).

[1815] Non-limiting examples of anti-angiogenic agents include molecules such as proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA and recombinant vectors, as well as small molecules that have the function of reducing or even inhibiting blood vessel growth. Suitable angiogenesis inhibitors include, but are not limited to, angiostatin K1-3, staurosporine, genistein, fumagillin, medroxyprogesterone, suramin, interferon-α, metalloproteinase inhibitors, platelet factor 4, somatostatin, thrombospondin, endostatin, thalidomide and its derivatives and analogs. For example, in certain aspects, the anti-angiogenic agent is an antibody that specifically binds to VEGF (such as Avastin, Roche) or a VEGF receptor (such as a VEGFR2 antibody). In one example, the anti-angiogenic agent includes a VEGFR2 antibody, or DMXAA (also known as Vadimezan or ASA404; commercially available from, for example, SigmaCorp., St. Louis, MO), or both. The anti-angiogenic agent may be bevacizumab, sunitinib, or an anti-angiogenic tyrosine kinase inhibitor (TKI), such as sunitinib, citinib, and dasatinib. These may be used alone or in any combination.

[1816] Other therapeutic agents, such as anti-tumor agents, which may or may not belong to one or more of the above categories, are also suitable for use in combination with the disclosed methods. For example, such agents include doxorubicin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.

[1817] In some examples, subjects receiving the therapeutic antibody-IR700 conjugate are also administered interleukin-2 (IL-2), for example, by intravenous injection. In a specific example, IL-2 (Chiron Corp., Emeryville, CA) is administered at a dose of at least 500,000 IU / kg, starting on the second day of administration of the antibody-IR700 conjugate, as a 15-minute intravenous bolus every 8 hours for up to 5 days. Doses may be skipped depending on the subject's tolerance.

[1818] Exemplary additional therapeutic agents include anti-tumor agents, such as chemotherapy and anti-angiogenic agents or therapies, such as radiation therapy. In one example, the agent is a chemotherapeutic immunosuppressant (such as rituximab, steroids) or a cytokine (such as GM-CSF). Exemplary chemotherapeutic agents are provided in, for example, 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 refers to the use of more than one agent to treat cancer.

[1819] 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, azauridine, etoposide, vinorelbine, tamoxifen, valspodar, cyclophosphamide, methotrexate, fluorouracil, mitoxantrone, liposomal doxorubicin (Doxil, i.e., liposome-encapsulated doxorubicin) and vinorelbine. Other examples of chemotherapeutic agents that can be used include alkylating agents, antimetabolites, natural products, hormones and their antagonists. Examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozotocin or dacarbazine). Specific non-limiting examples of alkylating agents are temozolomide and dacarbazine. Examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine) and purine analogs such as mercaptopurine or thioguanine. Examples of natural products include vinca alkaloids (such as vinblastine, vincristine or vindesine), podophyllotoxin derivatives (such as etoposide or teniposide), antibiotics (such as actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin or mitomycin C) and enzymes (such as L-asparaginase). Examples of other types of agents include platinum coordination complexes (such as cis-diamine dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine) and adrenaline inhibitors (such as mitotane and aminoglutethimide). Examples of hormones and their antagonists include adrenocortical hormones (such as prednisone), progestogens (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone).

[1820] Exemplary chemotherapy drugs include doxorubicin, Alkeran, cytarabine (Ara-C), carmustine (BiCNU), busulfan, lomustine (CCNU), carboplatin, cisplatin, cyclophosphamide, daunorubicin, dacarbazine (DTIC), 5-fluorouracil (5-FU), fludarabine, hydroxyurea (Hydrea), idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes such as docetaxel), Velban, vincristine, VP-16, gemcitabine (Gemzar), Herceptin, irinotecan (Capto, CPT-11), leustatin, Navelbine, rituximab STI-571, Taxotere, topotecan (Hemisild), Xeloda (capecitabine), Zevelin, and calcitriol. Non-limiting examples of immune activators that can be used include AS-101 (Wyeth-Ayerst Labs.), brompirine (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, Louisiana), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[1821] In some instances, the additional therapeutic agent is coupled to (or otherwise associated with) a nanoparticle, e.g., a nanoparticle of 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, such as between 1 nm and 500 nm, between 1 nm and 300 nm, between 1 nm and 100 nm, between 10 nm and 500 nm, or between 10 nm and 300 nm in diameter).

[1822] In one example, prior to administration of the disclosed therapy (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), at least a portion of a tumor (e.g., a metastatic tumor) is surgically removed (e.g., by surgical resection and / or cryotherapy), irradiated (e.g., administration of a radioactive substance or energy (e.g., external beam radiotherapy) to the tumor site to help eradicate or shrink the tumor), chemotherapy (e.g., by chemoembolization), or a combination thereof. For example, a subject with a metastatic tumor may have all or part of the tumor surgically removed prior to administration of the disclosed therapy. In one example, following 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, one or more chemotherapeutic drugs are administered, and irradiation is performed. In another specific example, the subject has a metastatic tumor and is administered radiation therapy, chemoembolization, or both concurrently with the administration of the disclosed therapy.

[1823] In some examples, the additional therapeutic agent administered is a monoclonal antibody, such as 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Bavituximab, Betumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab

[1824] Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab

[1825] Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicin), Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin), Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumabmertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomab estafenatox), Necitumumab, Nimotuzumab, Nofetumomabmerpentan), Ofatumumab, Olaratumab, Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab

[00135] The present invention relates to an agent that is administered intravenously to patients with relapses or intolerances, or to an agent that is administered intravenously to patients with relapses or intolerances. The invention also relates to an agent that is administered intravenously to patients with relapses or intolerances, or ...

[1826] Example reducing agents

[1827] Exemplary reducing agents that can be used in the methods provided herein include reagents that release (or "donate") electrons to electron acceptors (oxidants) in redox chemical reactions. Exemplary reducing agents that can be used in the methods provided herein include L-cysteine, sodium L-ascorbate (L-NaAA), ascorbic acid (such as L- or R-ascorbic acid), and glutathione. In some instances, the reducing agent used in the disclosed methods is not sodium azide. In some instances, the reducing agent used in the disclosed methods is not L-cysteine. In one example, the reducing agent is L-NaAA (e.g., 5 to 50 g intraperitoneally).

[1828] The following examples are provided to illustrate certain specific features and / or aspects. These examples should not be construed as limiting the disclosure to the specific features or aspects described. Example

[1829] Example 1: Measuring Binding Affinity Using Surface Plasmon Resonance

[1830] This example describes the use of BIACORE TM Affinity of antibodies 14564, 14569, and 14572 for CD25 measured by 8K.

[1831] sample

[1832]

[1833]

[1834] Instruments and reagents

[1835]

[1836] Affinity determination

[1837] Running buffer was prepared by diluting one volume of 10× buffer with nine volumes of degassed, filtered MilliQ water. Regeneration buffer (10 mM glycine) was prepared by dissolving glycine in MilliQ water and adjusting the pH to approximately 1.5-1.7. HBS-EP+ was used as the running buffer and the assay was performed at 25°C. Antibodies were injected as capture molecules onto the S-Series Sensor Chip Protein A. Antigens were diluted to various concentrations and injected onto the surface of flow cells 1 and 2 as the binding phase, followed by injection of running buffer as the dissociation phase.

[1838] Run Settings

[1839]

[1840]

[1841] result

[1842] All data were collected using BIACORE TM The data were processed using 8K Evaluation Software version 3.0. A blank injection of flow cell 1 and buffer in each cycle was used as a double reference for subtraction of response units (RU). Table 2 lists the affinity of each antibody. Figure 1 The sensorgrams are shown. Antibodies 14564, 14569, and 14572 bind to CD25 with high affinity (KD of 1.74 × 10 -8 M, 1.85×10 -8 M and 1.96×10 -8 M). VH2+VL4 are parental antibodies (before affinity maturation).

[1843] Table 2. Affinity determination of CD25 (sIL-2 receptor α) antibodies

[1844]

[1845] Example 2: Binding of CD25-specific antibodies to CD25-positive cells

[1846] This example describes the detection of 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: 1× PBS containing 1% FBS (1× PBS, Accugene Cat. No. 51225, Lot No.

[1847] 8MB103)

[1848] Serial dilution of antibodies

[1849]

[1850] FACS procedures

[1851] KIT-225 cells were washed once with FACS buffer, counted, and analyzed at 1 × 10 6 / mL concentration was resuspended in FACS buffer. 6 To each well, add 25 μL of viability dye (Thermo Fisher, L34974, equivalent to a 1:200 dilution) and mix thoroughly. After incubation at 4°C for 30 minutes, wash the cells twice with FACS buffer (200 × g, 5 minutes each time). Resuspend the cells in 5 mL of FACS buffer, add 100 μL to each well, and then add 100 μL of diluted antibody. Move the mixed solution up and down with a pipette, then incubate at 4°C for 60 minutes, after which the cells are washed twice with FACS buffer (200 × g, 5 minutes each). Resuspend the cells in 100 μL of FACS buffer. Dilute 50 μL of secondary antibody (equivalent to a 1:100 dilution) with 5 mL of FACS buffer, then add 100 μL of secondary antibody to each well and mix by moving up and down with a pipette. After incubation at 4°C for 30 minutes, wash the cells twice with FACS buffer (200 × g, 5 minutes each), and then resuspend the cells with approximately 250 μL of FACS buffer. FACS analysis was performed using an Attune flow cytometer with a tube reader module.

[1852] result

[1853] FACS analysis results Figure 2 and Figure 3Antibodies 14564, 14569, and 14572 showed strong binding to CD25-positive KIT-225 cells, with binding affinities comparable to those of basiliximab (also known as ), basiliximab is a mouse-human chimeric monoclonal antibody that binds to CD25 (Kahan et al., Transplantation 67(2):276-284, 1999).

[1854] Example 3: ADCC

[1855] This example describes studies to determine whether antibodies 14564, 14569, and 14572 induce ADCC.

[1856] cell lines

[1857] Jurkat / CD16.V175 cells (ADCC responder cells) were cultured in RPMI-1640 medium supplemented with 10% FBS, 1 mg / mL Geneticin, and 200 μg / mL Hygromycin. KIT-225 cells (target cells) were cultured in RPMI-1640 medium supplemented with 10% FBS and 50 μM β-mercaptoethanol. ADCC assay medium consisted of RPMI-1640 medium supplemented with 10% FBS.

[1858] Prepare antibody working solution

[1859] Basiliximab and three anti-CD25 monoclonal antibodies 14564, 14569, and 14572 were diluted to 50 μg / mL as shown below.

[1860]

[1861] The antibody was serially diluted to 16.7 μg / mL, 5.5 μg / mL, 1.85 μg / mL, 0.6 μg / mL, and 0.2 μg / mL (previous 120 μL + 240 μL culture medium). On the day of the assay, KIT-225 cells were harvested from the culture medium. The cells were washed twice with culture medium to remove any IL-2. The cells were resuspended in assay medium to 4.2 × 10 5 cells / mL (minimum 6 mL).

[1862]

[1863] 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. The cells and antibodies were incubated at 37°C for 1 hour.

[1864] ADCC responder cells (Jurkat / CD16.V175) were collected, washed once with assay medium, and resuspended to 1.9×10 6 / mL (minimum 8mL).

[1865]

[1866] 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 incubation for approximately 5-6 hours, 100 μL of the One-Step luciferase reagent was added to each well, and the plate was gently shaken at room temperature for 30 minutes. Fluorescence was measured using a fluorimeter.

[1867] like Figures 4A - 4B As shown, basiliximab has ADCC activity, but antibodies 14564, 14569 and 14572 cannot induce ADCC activity.

[1868] Example 4: Anti-CD25 antibody-IR700 conjugate can bind to cells expressing CD25 and induce cell killing

[1869] This example describes a study evaluating targeting photoimmunotherapy (PIT) to cells expressing CD25. First, anti-hCD25-IR700 was tested for binding to CD25-positive KIT-225 cells (a human T cell line). KIT-225 cells (0.5×10 6 ) 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 before incubation with anti-hCD25-IR700. 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 showed that conjugation of IR700 to the anti-CD25 antibody did not reduce the binding of the antibody to cells expressing CD25 ( ).

[1870] Cell survival after hCD25-targeted PIT was also assessed. KIT-225 cells (0.5 × 10 6 ), then at 150 mW / cm 2 Power applied 0, 5, 20 or 50 J / cm 2 The cells were then stained with a viability dye and analyzed by FACS. [[ID=z12]]Figure 5AAs shown, when cells were incubated with anti-hCD25-IR700 conjugate and exposed to 5, 20, or 50 J / cm 2 Significant CD25-positive cell death occurred when exposed to NIR light. Figure 5C Displays the use of 0, 5 or 20 J / cm 2 Representative flow cytometric plots of live / dead staining after PIT with NIR light.

[1871] Example 5: Anti-CD25 antibodies do not block the binding of IL-2 to CD25

[1872] To test whether antibodies 14564, 14569, and 14572 block IL-2 binding to CD25, a proliferation assay was performed. KIT-225 cells were starved of IL-2 for five days prior to the proliferation assay. 50,000 starved cells were preincubated in culture medium with 0, 5, or 10 μg / ml of 14564, 14569, 14572, or the parent anti-CD25 antibody for 30 minutes at 37°C and then cultured for 1.5 days in the presence or absence of 50 pM human IL-2. Cells cultured in the absence of antibody and with or without IL-2 served as controls. 3 After 4 h of H-thymidine pulse (37 kBq), cells were harvested and the incorporated activity was measured using a β-counter. Figure 5B As shown, antibodies 14564, 14569, and 14572 did not inhibit IL-2-induced proliferation of KIT-225, indicating that these antibodies do not block the binding of IL-2 to CD25.

[1873] Example 6: PIT targeting human CD25

[1874] The study described in this example was designed to determine whether hCD25-targeted PIT can kill human Treg cells. Splenocytes obtained from hCD34-NSG humanized mice bearing MDA-MD231 tumors were incubated with 5 μg of IR700-conjugated anti-CD25 antibody 14564 and then irradiated with 150 mW / cm 2 50J / cm 2 NIR light irradiation. Splenocytes cultured without antibody-IR700 conjugate and not irradiated were used as controls. The percentage of CD25+ Treg cells was assessed by flow cytometry. The gating strategy of this study was as follows Figure 6 Representative dot plots of CD25+ Treg cells in control and CD25 PIT splenocyte samples are shown. Figure 7C As shown. Figure 7A Figure 7BAs shown in Figure 3, the percentage of CD25+ Treg cells in CD25 PIT splenocyte samples was significantly reduced compared with control splenocytes, indicating that hCD25-targeted PIT can effectively deplete human CD25-positive Treg cells.

[1875] Example 7: Depletion of CD25-expressing Treg cells in a humanized mouse model

[1876] 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., a humanized mouse model described in WO2019 / 199799; U.S. Patent Application No. 2019 / 0320633; Yin et al., Am J Cancer Res 10(12):4568-4584, 2020; or Serr et al., Front Immunol 12:643544, 2021).

[1877] The humanized mice are administered (e.g., intravenously, intratumorally, intraperitoneally, or intrapleurally) an anti-hCD25-IR700 conjugate (e.g., a conjugate wherein the antibody comprises the CDR sequences or VH / VL domain sequences of antibodies 14564, 14569, or 14572) at a dose of 5 μg (or another suitable dose as described herein). The humanized mice are then exposed to NIR light of a wavelength of about 660 to about 740 nm (e.g., 50 J / cm 2 The mice were treated with NIR light or another suitable dose as described herein. Treg cell depletion was assessed by flow cytometry in treated and untreated mice. It is expected that treatment with anti-hCD25-IR700 NIR-PIT will significantly reduce the number of Treg cells compared to untreated control mice.

[1878] Obviously, the precise details of the methods or compositions may be changed or modified without departing from the spirit of the described aspects of the present disclosure. We intend all such modifications and variations to come within the scope and spirit of the following claims.

Claims

1. A monoclonal antibody that specifically binds to CD25, comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein: The VH domain comprises a complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein the amino acid sequences of CDR1, CDR2 and CDR3 comprise SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively; and The VL domain comprises CDR1, CDR2 and CDR3, and the amino acid sequences of CDR1, CDR2 and CDR3 comprise SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, respectively.

2. The monoclonal antibody according to claim 1, wherein: The amino acid sequences of the VH domain CDR1, CDR2 and CDR3 comprise SEQ ID NO:49, SEQ ID NO:51 and SEQ ID NO:52, respectively; and The amino acid sequences of the VL domain CDR1, CDR2 and CDR3 comprise SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:48, respectively.

3. The monoclonal antibody according to claim 2, wherein: The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 21, and the VH domain CDR1, CDR2 and CDR3 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 VL domain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 48, respectively.

4. The monoclonal antibody according to claim 1, wherein: The amino acid sequences of the VH domain CDR1, CDR2 and CDR3 comprise SEQ ID NO:49, SEQ ID NO:51 and SEQ ID NO:52, respectively; and The amino acid sequences of the VL domain CDR1, CDR2 and CDR3 comprise SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:48, respectively.

5. The monoclonal antibody according to claim 4, wherein: The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:31, and the VH domain CDR1, CDR2 and CDR3 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 VL domain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 48, respectively.

6. The monoclonal antibody according to claim 1, wherein: The amino acid sequences of the VH domain CDR1, CDR2 and CDR3 comprise SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:53, respectively; and The amino acid sequences of the VL domain CDR1, CDR2 and CDR3 comprise SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:48, respectively.

7. The monoclonal antibody according to claim 4, wherein: The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 37, and the VH domain CDR1, CDR2 and CDR3 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 VL domain CDR1, CDR2 and CDR3 comprise SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 48, respectively.

8. The monoclonal antibody according to claim 1, wherein: The amino acid sequence of the VH domain comprises SEQ ID NO: 21 and the amino acid sequence of the VL domain comprises SEQ ID NO: 22; The amino acid sequence of the VH domain comprises SEQ ID NO:31 and the amino acid sequence of the VL domain comprises SEQ ID NO:32; the amino acid sequence of the VH domain comprises SEQ ID NO: 37 and the amino acid sequence of the VL domain comprises SEQ ID NO: 38; The amino acid sequence of the VH domain comprises SEQ ID NO: 1 and the amino acid sequence of the VL domain comprises SEQ ID NO: 2; The amino acid sequence of the VH domain comprises SEQ ID NO: 3 and the amino acid sequence of the VL domain comprises SEQ ID NO: 4; The amino acid sequence of the VH domain comprises SEQ ID NO: 5 and the amino acid sequence of the VL domain comprises SEQ ID NO: 6; The amino acid sequence of the VH domain comprises SEQ ID NO:7 and the amino acid sequence of the VL domain comprises SEQ ID NO:8; The amino acid sequence of the VH domain comprises SEQ ID NO:9 and the amino acid sequence of the VL domain comprises SEQ ID NO:10; The amino acid sequence of the VH domain comprises SEQ ID NO: 11 and the amino acid sequence of the VL domain comprises SEQ ID NO: 12; The amino acid sequence of the VH domain comprises SEQ ID NO: 13 and the amino acid sequence of the VL domain comprises SEQ ID NO: 14; The amino acid sequence of the VH domain comprises SEQ ID NO: 15 and the amino acid sequence of the VL domain comprises SEQ ID NO: 16; The amino acid sequence of the VH domain comprises SEQ ID NO: 17 and the amino acid sequence of the VL domain comprises SEQ ID NO: 18; The amino acid sequence of the VH domain comprises SEQ ID NO: 19 and the amino acid sequence of the VL domain comprises SEQ ID NO: 20; The amino acid sequence of the VH domain comprises SEQ ID NO: 23 and the amino acid sequence of the VL domain comprises SEQ ID NO: 24; The amino acid sequence of the VH domain comprises SEQ ID NO: 25 and the amino acid sequence of the VL domain comprises SEQ ID NO: 26; the amino acid sequence of the VH domain comprises SEQ ID NO: 27 and the amino acid sequence of the VL domain comprises SEQ ID NO: 28; the amino acid sequence of the VH domain comprises SEQ ID NO:29 and the amino acid sequence of the VL domain comprises SEQ ID NO:30; The amino acid sequence of the VH domain comprises SEQ ID NO: 33 and the amino acid sequence of the VL domain comprises SEQ ID NO: 34; the amino acid sequence of the VH domain comprises SEQ ID NO:35 and the amino acid sequence of the VL domain comprises SEQ ID NO:36; the amino acid sequence of the VH domain comprises SEQ ID NO:39 and the amino acid sequence of the VL domain comprises SEQ ID NO:40; Alternatively, the amino acid sequence of the VH domain comprises SEQ ID NO:41 and the amino acid sequence of the VL domain comprises SEQ ID NO:

42.

9. The monoclonal antibody according to any one of claims 1 to 8, wherein 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).

10. The monoclonal antibody according to any one of claims 1 to 8, further comprising a constant region.

11. The monoclonal antibody according to claim 10, wherein the constant region comprises at least one modification to increase the half-life, stability and / or function of the monoclonal antibody.

12. The monoclonal antibody according to claim 10 or 11, wherein the constant region comprises a heavy chain constant region and / or a light chain constant region.

13. The monoclonal antibody of claim 12, wherein the heavy chain constant region is a human IgG4 constant region comprising SEQ ID NO:

58.

14. The monoclonal antibody according to claim 12 or 13, wherein the light chain constant region is a human Ig kappa light chain comprising SEQ ID NO:

59.

15. The monoclonal antibody according to any one of claims 1 to 14, comprising: SEQ ID NO:80 and SEQ ID NO:81; SEQ ID NO:90 and SEQ ID NO:91; SEQ ID NO:96 and SEQ ID NO:97; 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: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:92 and SEQ ID NO:93; SEQ ID NO:94 and SEQ ID NO:95; SEQ ID NO:98 and SEQ ID NO:99; or SEQ ID NO:100 and SEQ ID NO:

101.

16. The monoclonal antibody according to any one of claims 1 to 15, wherein: (i) The antibody does not significantly inhibit the binding of interleukin-2 (IL-2) to CD25; (ii) the antibody induces little antibody-dependent cellular cytotoxicity (ATCC); or (iii) Both (i) and (ii).

17. A conjugate comprising: The monoclonal antibody according to any one of claims 1 to 16; and Photon absorbers, effector molecules or detectable labels.

18. The conjugate according to claim 17, wherein the photon absorber is IR700.

19. The conjugate of claim 17, wherein the effector molecule is a toxin.

20. The conjugate of claim 17, wherein the detectable label is a fluorescent label, an enzyme label or a radioactive label.

21. A composition comprising: The monoclonal antibody according to any one of claims 1 to 16 or the conjugate according to any one of claims 16 to 20; and a pharmaceutically acceptable carrier.

22. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 16, or encoding the VH domain or VL domain thereof.

23. The nucleic acid molecule according to claim 22, comprising: 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 sequences of SEQ ID NO:122 and SEQ ID NO:123, or degenerate variants 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 sequences of SEQ ID NO:132 and SEQ ID NO:133, or degenerate variants 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 degenerate variants thereof; 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 SEQ ID NO: 185, or degenerate variants thereof.

24. A vector comprising the nucleic acid molecule of claim 22 or 23.

25. An isolated host cell comprising the nucleic acid molecule of claim 22 or 23, or the vector of claim 24.

26. A kit comprising: (i) the monoclonal antibody of any one of claims 1 to 16, the conjugate of any one of claims 16 to 19, the composition of claim 20, the nucleic acid molecule of claim 22 or 23, the vector of claim 24, or the isolated cell of claim 25; and (ii) buffers, cell culture media, one or more checkpoint inhibitors, one or more immunotherapies, one or more additional anti-cancer agents, one or more reducing agents, one or more transfection reagents, and / or instructional materials.

27. A method for treating cancer in a subject, comprising: Administering a therapeutically effective amount of the conjugate of claim 18 to a subject; and Then at a wavelength of 660 to 740 nm and at least 1 J / cm 2 irradiating the subject and / or irradiating cancer cells in the subject at a dose of The subject's cancer is thereby treated.

28. The method of claim 27, wherein the conjugate is administered at or near the site of cancer.

29. The method of claim 27, wherein administration of the conjugate is not systemic.

30. The method of claim 27, wherein administration of the conjugate is systemic.

31. The method of any one of claims 27-30, further comprising administering a second monoclonal antibody conjugated to IR700, wherein the second monoclonal antibody specifically binds to a tumor antigen expressed by a cancer cell.

32. The method of claim 31, wherein 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), glypican-1 (GPC1), glypican-2 (GPC2), glypican-3 (GPC3), gpA33, mucin, CAIX, folate binding protein, ganglioside, integrin αVβ3, integrin α5β3, Erb1, -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, receptor activator of 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, SK-1 antigen, programmed death 1 (PD-1), and programmed death ligand 2 (PD-L2).

33. The method of claim 31 or 32, wherein the conjugate and the second monoclonal antibody conjugated to IR700 are both administered prior to irradiation.

34. The method of any one of claims 27-33, further comprising administering to the subject one or more additional cancer therapies.

35. The method of claim 34, wherein the one or more additional cancer therapies comprise an anti-CTLA4-IR700 conjugate, an anti-PD-L1-IR700 conjugate, an immune activator, a checkpoint inhibitor, chemotherapy, radiotherapy, and / or surgery.

36. The method of any one of claims 27-35, wherein the conjugate and the second monoclonal antibody conjugated to IR700 are administered intravenously.

37. The method of any one of claims 27-36, wherein the cancer is of the breast, liver, lung, esophagus, stomach, colon, ovary, prostate, pancreas, brain, cervix, kidney, bone, skin, head and neck, oropharynx, or blood.

38. A monoclonal antibody that specifically binds to CD25, wherein: (i) the antibody does not significantly inhibit the binding of interleukin-2 (IL-2) to CD25; (ii) the antibody hardly induces antibody-dependent cellular cytotoxicity (ADCC); or (iii) Both (i) and (ii).

39. A conjugate comprising: The monoclonal antibody of claim 38; and Photon absorbers, effector molecules or detectable labels.

40. The conjugate of claim 39, wherein the photon absorber is IR700.

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