Anti-CD25 antigen-binding proteins and uses thereof

Abstract

The present application provides antigen-binding proteins (e.g., antibodies such as single-domain antibodies) that specifically bind cluster of differentiation 25 (CD25). The application also provides fusion proteins and conjugates comprising the antigen-binding proteins, polynucleotides and recombinant vectors encoding the antigen-binding proteins, as well as host cells and methods for preparing the antigen-binding proteins. The application further provides pharmaceutical compositions comprising the antigen-binding proteins.

Description

ANTI-CD25 ANTIGEN-BINDING PROTEINS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 664,893, filed on June 27, 2024, the disclosure of which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 18, 2025, is named 260525_000074_SL.xml and is 5,270,508 bytes in size.FIELD OF THE INVENTION

[0003] The present application relates to antigen-binding proteins (e.g., antibodies such as single-domain antibodies) that specifically bind cluster of differentiation 25 (CD25), methods for their preparation, and uses thereof.BACKGROUND OF THE INVENTION

[0004] Regulatory T cells (Tregs) are a subset of T cells that play a crucial role in peripheral self-tolerance and the prevention of autoimmunity. Due to their potent immunosuppressive function, Tregs can be targeted for the treatment of autoimmunity. Current strategies seeking to Increase or modulate Tregs in autoimmune patients are based on the ex vivo expansion of Tregs prior to autologous transfer. However, a major limitation of the current strategies is their inability to stabilize Tregs phenotype to ensure long-lasting immunoregulation.

[0005] While Tregs can support immune homeostasis under normal, healthy conditions, and their activation can be beneficial in the context of autoimmune disease, during proliferative diseases (e.g., cancer), Tregs can accumulate within the tumor microenvironment where they can hamper antitumor responses mounted by infiltrating immune cells, effectively protecting the cancer cells from immune attack. Tregs are capable of suppressing most types of immune cells including CD4+ and CD8+ T cells, B cells, and antigen-presenting cells (APCs) (e.g., dendritic cells macrophages and monocytes), natural killer (NK) cells, and NKT cells. The number of Tregs is higher in tumors and peripheral blood mononuclear cells (PBMCs) of many cancer patients, and high Treg levels can be associated with poor prognosis, e.g., in solid tumors including breast, cervical, renal, melanomas, ovarian, hepatocellular, gastric and pancreatic cancers.

[0006] Cluster of differentiation 25 (CD25), also called interleukin-2 receptor subunit alpha (IL-2Rα or IL2RA), is the alpha chain component of the high-affinity heterotrimeric interleukin-2 (IL-2) receptor, a type I transmembrane protein highly expressed on the surface of the majority of Tregs. IL-2 activation of CD25 can facilitate immune tolerance in Tregs. High cell surface expression of CD25 can also occur in malignant cells, e.g., in several lymphomas and leukemias.

[0007] Accordingly, there is a need in the art to develop molecules that can effectively target and specifically bind CD25-expressing cells, e.g., Tregs.SUMMARY OF THE INVENTION

[0008] As mentioned in the background section above, there is an unmet need in the art to develop molecules that can effectively target and specifically bind cluster of differentiation 25 (CD25). This application provides compositions and methods to address this and other related needs.

[0009] ln one aspect, the present disclosure provides an antigen-binding protein that specifically binds cluster of differentiation 25 (CD25), comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence a).(A / V / S)(A / K / T)(A / G)(A / R / K)(A / G / H / N / R)(A / S)(A / G)(A / S / G)(A / Y)(A / Y)(A / P)(A / W / F / L)(A / D / E)(A / D / E)(A / Y / V); or b). (A / G)(A / G)(A / K)(A / R)(A / L)(A / G)(A / P)(M / l / A / L)(A / V)(A / H)(A / R / Q)(A / Y)(A / S)(A / L)(A / E)(A / V) (A / L)(A / T)(A / P)(A / L)(A / F)(A / L)(A / D)(A / E)(A / Y)(A / D)(A / Y).

[0010] ln some embodiments, the CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 4314, and 5211-5251.

[0011] In some embodiments, the antigen-binding portion further comprises a CDR1 comprising an amino acid sequence a). GFTFS(N / S)YA (SEQ ID NO: 40); or b). GFTLDYYA (SEQ ID NO: 2242).

[0012] ln some embodiments, the CDR1 comprises an amino acid sequence SEQ ID NO: 13 or 2242.

[0013] ln some embodiments, the antigen-binding portion further comprises a CDR2 comprising an amino acid sequence a). IYSD(G / S)SGT (SEQ ID NO: 4341); or b). ISSTDGRT (SEQ ID NO: 2248).

[0014] ln some embodiments, the CDR2 comprises an amino acid sequence SEQ ID NO: 2248, or 4335.

[0015] ln some embodiments, the antigen-binding protein comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence of (A / V / S)(A / K / T)(A / G)(A / R / K)(A / G / H / N / R)(A / S)(A / G)(A / S / G)(A / Y)(A / Y)(A / P)(A / W / F / L)(A / D / E)(A / D / E)(A / Y / V); orii). a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of (A / G)(A / G)(A / K)(A / R)(A / L)(A / G)(A / P)(M / I / A / L)(A / V)(A / H)(A / R / Q)(A / Y)(A / S)(A / L)(A / E)(A / V) (A / L)(A / T)(A / P)(A / L)(A / F)(A / L)(A / D)(A / E)(A / Y)(A / D)(A / Y).

[0016] ln some embodiments, the antigen-binding protein comprises i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5211; ii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5212; iii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5213; iv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5214; v)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5215; vi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5216; vii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5217; viii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5218; ix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5219; x)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5220; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5221; xii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5222; xiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5223;xiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5224; xv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5225; xvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5226; xvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5227; xviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5228; xix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5229; xx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5230; xxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5231; xxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5232; xxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5233; xxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5234; xxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5235; xxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5236; xxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5237; xxviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5238; xxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5239;xxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5240; xxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5241; xxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5242; xxxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5243; xxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5244; xxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5245; xxxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5246; xxxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5247; xxxviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5248; xxxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5249; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5250; or xii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5251.

[0017] ln some embodiments, the antigen-binding protein is a single-domain antibody.

[0018] ln some embodiments, the single-domain antibody is a VHH, a VNAR, or a VH domain.

[0019] ln some embodiments, the VHH is a humanized VHH.

[0020] ln some embodiments, the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5252-5292, or a sequence having at least 75% identity thereto.

[0021] ln some embodiments, the antigen-binding protein binds to human CD25.

[0022] ln some embodiments, the antigen-binding protein binds to cyno CD25.

[0023] ln some embodiments, the antigen-binding protein binds to the same epitope(s) on CD25 as IL-2.

[0024] ln some embodiments, the antigen-binding protein competes for binding to CD25 with IL-2.

[0025] ln some embodiments, the antigen-binding protein has an antagonistic effect upon binding toCD25.

[0026] ln some embodiments, the antigen-binding protein does not bind to the same epitope(s) on CD25 as IL-2.

[0027] ln some embodiments, the antigen-binding protein does not compete with binding CD25 with IL-2.

[0028] ln some embodiments, the antigen-binding protein comprises one or more modifications that reduce binding of said antigen-binding protein by pre-existing antibodies found in human blood or serum.

[0029] ln some embodiments, the single-domain antibody comprises one or more modifications at the amino-terminus and / or the carboxy-terminus.

[0030] ln some embodiments, the single-domain antibody comprises the amino acid sequence VPAG (SEQ ID NO: 4327) or VAGG (SEQ ID NO: 4697) at the carboxy-terminus starting from position 111 according to Chothia.

[0031] ln some embodiments, the single-domain antibody comprises a substitution of amino acid residue Glu with Asp (EID) at the first position of the amino-terminus.

[0032] ln another aspect, provided herein is an antigen-binding protein that specifically binds cluster of differentiation 25 (CD25), comprising a means for binding an epitope within human CD25 bound by an antibody selected from C-005Hu1.A1, C-005Hu1.A2, C-005Hu1.A3, C-005Hu1.A4, C-005Hu1.A5, C-005Hu1.A6, C-005Hu1.A7, C-005Hu1.A8, C-005Hu1.A9, C-005Hu1.A10, C-005Hu1.A11, C-005Hu1.A12, C-005Hu1.A13, C-005Hu1.A14, C-005Hu1.A15, C-010Hu1.L8.G1, C-010Hu1.L8.G2, C-010Hu1.L8.A3, C-010Hu1.L8.A4, C-010Hu1.L8.A5, C-010Hu1.L8.A6, C-010Hu1.L8.A7, C-010Hu1.A8, C-010Hu1.L8.A9, C-010Hu1.L8.A10, C-010Hu1.L8.A11, C-010Hu1.L8.A12, C-010Hu1.L8.A13, C-010Hu1.L8.A14, C-010Hu1.L8.A15, C-010Hu1.L8.A16, C-010Hu1.L8.A17, C-010Hu1.L8.A18, C-010Hu1.L8.A19, C-010Hu1.L8.A20, C-010Hu1.L8.A21, C-010Hu1.L8.A22, C-010Hu1.L8.A23, C-010Hu1.L8.A24, C-010Hu1.L8.A25, C-010Hu1.L8.A26, and C-010Hu1.L8.A27.

[0033] ln another aspect, the present disclosure provides a fusion protein that specifically binds cluster of differentiation 25 (CD25), comprising one or more of an antigen-binding protein described herein.

[0034] ln some embodiments, the fusion protein described herein may comprise two antigen-binding proteins described herein.

[0035] ln some embodiments, the fusion protein described herein may comprise four antigen-binding proteins described herein.

[0036] ln some embodiments, the one or more antigen-binding proteins bind to the same epitope on CD25.

[0037] ln some embodiments, the one or more antigen-binding proteins bind to different epitopes on CD25.

[0038] ln some embodiments, the one or more antigen-binding proteins are one or more single-domain antibodies.

[0039] ln some embodiments, one or more single-domain antibodies are one or more VHHs.

[0040] ln some embodiments, a fusion protein described herein may further comprise an immunoglobulin Fc region.

[0041] ln some embodiments, the immunoglobulin Fc region is an Fc region of a human immunoglobulin.

[0042] ln some embodiments, the immunoglobulin Fc region is an Fc region of human IgG1, lgG2, lgG3 or lgG4, or a variant thereof.

[0043] ln some embodiments, the immunoglobulin Fc region is an Fc region of human IgG1, or a variant thereof.

[0044] ln some embodiments, the Fc region of human IgG1 comprises one or more mutations selected from L234A, L235A, G237A, M252Y, S254T,T256E, D265A, N297A, and / or P329A according to EU numbering.

[0045] ln some embodiments, the Fc region of human IgG1 comprises a set of mutations selected from 1).L234A and L235A;2).L234A, L235A, and P329A;3).D265A, N297A and P329A;4).L234A, L235A, and G237A; and5).M252Y, S254T, and T256E.

[0046] ln some embodiments, the Fc region of human IgG1 comprises L234A, L235A, and P329A.

[0047] ln some embodiments, the Fc region of human IgG1 comprises M252Y, S254T, and T256E.

[0048] ln some embodiments, the immunoglobulin Fc region is an Fc region of human lgG4, or a variant thereof.

[0049] ln some embodiments, the Fc region of human lgG4 comprises one or more mutations selected from S228P, L235E, L235A, and / or F234A according to EU numbering.

[0050] ln some embodiments, the Fc region of human lgG4 comprises a set of mutations selected from1). S228P and L235E;2). S228P and L235A;3). S228P, F234A, and L235E; and4). S228P, F234A, and L235A.

[0051] ln another aspect, the present disclosure provides a conjugate comprising an antigen-binding protein described herein or a fusion protein described herein, wherein the antigen-binding protein or the fusion protein is conjugated to a second moiety.

[0052] ln some embodiments, the second moiety is selected from a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

[0053] ln another aspect, the present disclosure provides a polynucleotide molecule encoding an antigen-binding protein described herein or a fusion protein described herein. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence selected from SEQ ID Nos: 5295-5336.

[0054] ln another aspect, the present disclosure provides a recombinant vector comprising a polynucleotide molecule described herein.

[0055] ln another aspect, the present disclosure provides a host cell comprising a polynucleotide molecule described herein, or an expression vector described herein.

[0056] ln another aspect, the present disclosure provides a kit comprising an antigen-binding protein described herein, a fusion protein described, a conjugate described herein, a polynucleotide molecule described herein, a recombinant vector described herein, or the host cell described herein, and optionally, instructions and / or packaging for the same.

[0057] ln another aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding protein described herein, a fusion protein described herein, a conjugate described herein, a polynucleotide molecule described herein, or a recombinant vector described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0058] ln another aspect, the present disclosure provides a method for preparing an antigen-binding protein or a fusion protein that specifically binds cluster of differentiation 25 (CD25), comprising the steps of:(a) culturing a host cell described herein in a culture medium under conditions suitable for expression of the antigen-binding protein or fusion protein, and(b) isolating the antigen-binding protein or fusion protein from the host cell and / or culture medium.

[0059] ln another aspect, the present disclosure provides a method for targeting a cell expressing CD25 comprising contacting the cell with an antigen-binding protein described herein, a fusion protein described herein, or a conjugate described herein.

[0060] In some embodiments, the cell is a regulatory T cell (Treg).

[0061] ln some embodiments, contacting of a cell may occur in vitro.

[0062] I n some embodiments, contacting of a cell may occur in vivo.

[0063] ln some embodiments, a method described herein may further comprise administering the antigen-binding protein, the fusion protein, or the conjugate into a subject in need thereof.

[0064] ln another aspect, the present disclosure provides a method of treating or preventing a disease or disorder in a subject In need thereof, and the method may comprise administering to the subject an antigen-binding protein described herein, a fusion protein described herein, or a conjugate described herein.

[0065] ln some embodiments, the disease or disorder is an immunological disease, inflammatory disease, cancer, cardiovascular disease, or an infertility and pregnancy-associated disease.

[0066] ln some embodiments, the immunological disease is selected from an autoimmune disease, a neurological condition, an allergy, asthma, macular degeneration, muscular atrophy, a disease related to miscarriage, atherosclerosis, bone loss, a musculoskeletal disease, obesity, a graft-versus-host disease, and an allograft rejection.

[0067] ln some embodiments, the autoimmune disease is selected from lupus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis. Goodpastures disease, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosis, lgG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disease, pemphigus vulgaris or related blistering skin disease, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, premature ovarian failure, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, primary ovarian insufficiency, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthr'rtis, stiff-man syndrome, type I diabetes, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (Granulomatosis with polyangiitis) or other immune vasculitis.

[0068] In some embodiments, the lupus is systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, neonatal lupus, or drug-induced lupus.

[0069] ln some embodiments, the cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DLE), or subacute cutaneous lupus erythematosus.

[0070] ln some embodiments, the neurological condition is selected from a brain tumor, a brain metastasis, a spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease, and stroke.

[0071] ln some embodiments, the allergy is selected from food allergy, seasonal allergy, pet allergy, hives, hay fever, allergic conjunctivitis, poison ivy allergy oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, and chemical allergy.

[0072] ln some embodiments, the allograft rejection is selected from skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, and organ graft rejection.

[0073] ln some embodiments, the ligament graft rejection is selected from cricothyroid ligament graft rejection, caudal cruciate ligament graft rejection, periodontal ligament graft rejection, suspensory ligament of the lens graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, ulnar collateral ligament graft rejection, radial collateral ligament graft rejection, suspensory ligament of the breast graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection, lateral collateral ligament graft rejection, posterior cruciate ligament graft rejection, medial collateral ligament graft rejection, cranial cruciate ligament graft rejection, and patellar ligament graft rejection.

[0074] ln some embodiments, the organ graft rejection is selected from heart graft rejection, lung graft rejection, kidney graft rejection, liver graft rejection, pancreas graft rejection, intestine graft rejection, and thymus graft rejection.

[0075] ln some embodiments, the graft-versus-host disease arises from a bone marrow transplant or one or more blood cells selected from B-cells, T-cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.

[0076] ln some embodiments, the inflammatory disease is acute or chronic inflammation.

[0077] ln some embodiments, the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovarian syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.

[0078] ln some embodiments, the cancer is selected from adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith- Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia and hairy cell leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, hodgkin lymphoma, non-hodgkin lymphoma, lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), small bowel cancer, stomach cancer,testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma), Werner syndrome, Wilms tumor, or xeroderma pigmentosum.

[0079] ln some embodiments, a cardiovascular disease described herein may be selected from atherosclerosis, heart failure, left heart failure with reduced ejection fraction, left heart failure with preserved ejection fraction, right ventricular failure, congestive heart failure, restrictive cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, idiopathic cardiomyopathy, and hypertension.

[0080] ln some embodiments, the infertility and pregnancy-associated diseases is selected from recurrent pregnancy loss, pre-eclampsia, preterm labor, fetal growth restriction, or Intrauterine growth restriction.

[0081] In another aspect, the present disclosure provides a method of regenerating a tissue or organ comprising one or more CD25+ cells, and the method may comprise contacting the tissue or organ with an effective amount of an antigen-binding protein described herein, a fusion protein described herein, or a conjugate described herein.

[0082] ln some embodiments, a tissue or organ described herein may be selected from pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerve, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structures, embryo, and testes tissue.

[0083] ln some embodiments, the contacting of a tissue or organ occurs in vitro.

[0084] ln some embodiments, the contacting of a tissue or organ occurs in vivo.

[0085] ln some embodiments, a method disclosed herein may further comprises administering the antigen-binding protein, the fusion protein, or the conjugate into a subject in need thereof.

[0086] ln another aspect, the present disclosure provides a method for inducing tolerance to a foreign agent and / or preventing or reducing immune response to a foreign agent in a subject in need thereof, and the method may comprise administering to the subject an antigen-binding protein described herein, a fusion protein described herein, or a conjugate described herein.

[0087] ln some embodiments, the foreign agent is a therapeutic protein or peptide, a viral vector, a bacterial vector, a fungal vector, a biochemical vector, a lipid, carbohydrate, a nucleic acid, a sperm, an oocyte, or an embryo.

[0088] ln some embodiments, the viral vector is a DNA or RNA vector.

[0089] ln some embodiments, the subject is a mammal.

[0090] ln some embodiments, the mammal is human.BRIEF DESCRIPTION OF DRAWINGS

[0091] Flgure 1 depicts an exemplary general panning strategy for isolation of CD25-specific variable domain of heavy chain (VHH) antibodies, also referred to herein as V-bodies (Vbs). Binders to human and rodent CD25 were enriched from VHH immune libraries by two rounds of phage display. BM, bone marrow.

[0092] Flgure 2 shows VHH immune library selection for next-generation sequencing (NGS) across the phage display process. Three initial libraries, 12 samples of the first panning round, and 36 samples of the second panning round, were sequenced with 20 million, 2 million, and 2 million reads, respectively. Comparison of V-body enrichment from the initial library to the first and second round of panning enabled identification of potential V-body candidates.

[0093] Figure 3 shows a schematic diagram of an exemplary NGS workflow. Following phage display, the VHH region of the phage eluate was amplified via polymerase chain reaction (PCR). Unique and samplespecific barcodes were then fused, and NGS was subsequently performed using the Illumina NovaSeq platform (Genewiz). The raw data were de-multiplexed, and then processed by the NGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions and the VHHs, including complementarity determining regions (CDRs) were annotated. Based on CDR3 identity, V-body sequences were clustered, thereby allowing for detailed analysis of, e.g., V-body enrichment during phage display, sequence diversity, CDR3 length distribution, and cluster abundance. Based on such analyses, up to ~300 candidates were selected for DNA synthesis (Twist) and further characterization.

[0094] Figure 4 illustrates human CD25 (hCD25) V-body binding validation at a fixed concentration of 100 nM V-body. The bar histogram shows the mean fluorescence intensity (MFI) of Alexa488-positive cells for V-bodies C-004 and C-006 versus an anti-His only control condition.

[0095] Figure 5 illustrates V-body binding to cynomolgus (cCD25) (left panel) and mouse CD25 (mCD25) (right panel) at a fixed concentration of 100 nM V-body. The bar histograms show the mean fluorescence intensity (MFI) of Alexa488-positive cells for tested-bodies C-004 and C-006 versus an anti- His only control condition.

[0096] Figures 6A-6B shows testing of human CD25 V-body binding across a range of concentrations for V-bodies C-004 and C-006. V-bodies were tested at molar concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, and 0.390625 nM (shown from left to right). The bar histogram in Figure 6A shows the percentage of Alexa488 positive cells for C-004 and C-006. The barhistogram in figure SB shows the mean fluorescent intensity (MFI) of Alexa488 positive cells for C-004 and C-006.

[0097] Figure 7 shows a schematic diagram of an exemplary experimental setup for determination of binding affinities of the V-bodies for their respective target via surface plasmon resonance (SPR). Figure discloses "HHHHHH" as SEQ ID NO: 4325.

[0098] Figures 8A-8C depict surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-004 and C-006. Fitted binding curves and calculated dissociation constants (KD) are included. Data corresponding to an anti-CD25 IgG (aCD25 IgG) control condition are also included (Figure 8C). Figure discloses "HHHHHH" as SEQ ID NO: 4325.

[0099] Flgure 9 shows a summary of binding affinities of two candidate anti-CD25 V-bodles to human, cynomolgus and mouse CD25. Data corresponding to an anti-CD25 IgG (aCD25 IgG) control condition are also included. NB, no binding.

[0100] Figures 10A-10B demonstrate that some humanized anti-CD25 V-bodies targeted the epitope recognized by IL-2. Data are shown for a first experiment 1 (Expl) and second experiment 2 (Exp2) performed using V-bodies C-004 (Figure 10A) and C-006 (Figure MB).[O10ljFigures 11A-11B demonstrate humanized anti-CD25 V-bodies C-0Olhul, C-002Hu1, and C-003 are non-competitive binders. Data are shown for a first experiment 1 (Figure 11A) and second experiment 2 (Figure 11B).

[0102] Flgures 12A-12C depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-008Hu1, C-010Hu1, and C-009Hu1. Fitted binding curves and calculated dissociation constants (KD) are included.

[0103] Flgures 13A-13C illustrate ligand (IL-2) competition by SPR. Each panel represents a sensorgram overlay plot for a single V-body captured onto a discrete spot. The sensorgrams display IL-2-Fc competition: association of the human CD25-extracellular domain (CD25-ECD) tothe V-body was followed either by additional binding by IL2-Fc, indicating an unoccupied epitope (non-overlapping epitopes), or no IL2-Fc binding, indicating epitope blocking (overlapping epitopes), and a buffer control, association and dissociation of human CD25-ECD in the absence of IL2-Fc.

[0104] Flgure 14 shows binding of His-tagged anti-CD25 VHHs to Human Embryonic Kidney (HEK) cells transfected with human or cyno CD25 detected by flow cytometry using a fluorescently-labelled secondary anti-His antibody. Binding is expressed as mean fluorescent intensity.

[0105] Flgures 15A-15C depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-010Hu1 A8, C-010Hu1.L8, and C-0O9Hu1.L8. Fitted binding curves and calculated dissociation constants (KD) are included.

[0106] Flgures 16A-16C depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-011Hu1, C-012Hu1, C-013Hu1, C-014Hu1, C-015Hu1, C-005Hu1, C-016Hu1, C- 017Hu1, and C-018Hu1. Fitted binding curves and calculated dissociation constants (KD) are included.

[0107] Figures 17A-17C depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-031Hu1, C-032Hu1, C-033Hu1, C-034Hu1, C-035Hu1, C-036Hu1, C-037Hu1, C-038Hu1, C-039Hu1, C-040Hu1, and C-041Hu1. Fitted binding curves and calculated dissociation constants (KD) are included.

[0108] Flgure 18 depicts SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-019Hu1, C-020Hu1, and C-022Hu1. Fitted binding curves and calculated dissociation constants ( KD) are included.

[0109] Flgure 19 depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-023Hu1 and C-024Hu1. Fitted binding curves and calculated dissociation constants (KD) are included.

[0110] Figures 20A-20B depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-025Hu1, C-026Hu1, C-027Hu1, C-028Hu1, C-029Hu1, and C-030Hu1. Fitted binding curves and calculated dissociation constants (KD) are included.

[0111] Figures 21A-21D depict binding of His-tagged VHHs to CD25+ human embryonic kidney (HEK) cells analyzed via flow cytometry. Figures 21A-21B show binding of His-tagged VHH to CD25+ HEK cells detected after washing with secondary anti-His antibody. Figure 21C-21D show binding measured by Incubation of pre-incubated VHH / antl-Hls antibody complex to CD25+ HEK cells.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0112] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications and other publications are incorporated by reference in theirentirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

[0113] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 5%. For example, as used herein, the expression "about 100" includes 95 and 105 and all values in between (e.g., 96, 97, 98, 99, etc.).

[0114] The term "antigen" encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. In various embodiments of the present disclosure, the antigen described herein is CD25, including human, cynomolgus, and / or mouse CD25.

[0115] The term "epitope" can refer to an antigenic determinant on the surface of an antigen to which an antibody molecule binds. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects (e.g., agnostic or antagonistic effects). Epitopes may be either conformational or linear. A conformational epitope is formed by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include non-peptidic moieties on the antigen, such as saccharides, phosphoryl groups, or sulfonyl groups.

[0116] The term "antigen-binding protein" refers in its broadest sense to a protein that specifically binds an antigen (e.g., CD25). In certain embodiments, an antigen-binding protein is an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single chain antibody (e.g., single chain fragment variable (scFv)); a dlabody; a triabody; a tetrabody; a Fab fragment; a F(ab') 2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an lgG2 antibody; an lgG3 antibody; or an lgG4 antibody, and fragments thereof. The term "antigen-binding protein" also encompasses, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. In addition, peptide antibody mimetics can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components (e.g., fibronectin type III domain (FN3)) as a scaffold.

[0117] The term "CD25", or "cluster of differentiation 25", or "interleukin 2 receptor alpha chain", or "interleukin 2 receptor alpha subunit", or "IL2Rα", or "IL2RA", or the like, are used interchangeably herein and can refer to any isoform(s), variant(s), and / or species homolog(s) of CD25 from any source, e.g., mammals including primates (e.g., humans and monkeys) and rodents (e.g., rats and mice). The term encompasses naturally-occurring variants of CD25 such as but not limited to allelic variants and splice variants. The term also encompasses "full-length" or unprocessed CD25 in addition to any form of CD25 that can result from processing such as that which may occur within a cell. In some embodiments, CD25 is human CD25. As an example, without limitation, CD25 can be expressed by activated lymphocytes (e.g., activated T lymphocytes and / or activated B lymphocytes). The majority of regulatory T cells (Tregs) can express CD25. A heterotrimeric complex comprising IL2Rα, IL2R (beta) β (also called CD122), and IL2R (gramma) y (also called CD132) can form a high-affinity IL2R. IL2Rα and IL2Rβ can form a pseudo-high affinity receptor.

[0118] The term "interleukin-2", or "IL-2", or "IL2", or the like, are used interchangeably herein and can refer to any isoform(s), variant(s), and / or species homolog(s) of IL-2 from any source, e.g., mammals including primates (e.g., humans and monkeys) and rodents (e.g., rats and mice). The term encompasses naturally-occurring variants of IL-2 such as but not limited to allelic variants and splice variants. The term also encompasses "full-length" or unprocessed IL-2 in addition to any form of IL-2 that can result from processing such as that which may occur within a cell.

[0119] The term "antibody" and "immunoglobulin" or "ig" are used interchangeably herein, and is used in the broadest sense and encompasses, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, muhlspeclflc antibodies (e.g., bispecific antibodies), single-domain antibodies (e.g., VHH), single chain antibodies, intrabodies, anti-idiotypic (anti-ld) antibodies, and antigen-binding fragments of antibodies, as described below. An antibody can be human, humanized, camelized, recombinantly produced, chimeric, synthetic, affinity de-matured and / or affinity matured as well as an antibody from other species, for example mouse, camel, llama, rabbit, etc. In specific embodiments, the specific target antigen that can be bound by an antibody provided herein includes a CD25 polypeptide, CD25 fragment or CD25 epitope. An "antigen-binding fragment" generally refers a portion of an antibody heavy and / or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of antigen-binding fragments include single-domain antibody (e.g., VHH), single-chain Fvs (scFv), Fab fragments, F(ab')fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody, or a chemically modified derivative thereof. In particular, antibodies provided herein include immunoglobulin molecules and molecules that contain immunologically active portion(s) of an immunoglobulin molecule, for example, one or more complementarity determining regions (CDRs) of an antibody that binds to CD25. Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189- 224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgG1, lgG2, lgG3, lgG4, IgA1 and lgA2), or any subclass (e.g., lgG2a and lgG2b) of immunoglobulin molecule.

[0120] The term "single-domain antibody" or "sdAb" as used herein, refers to an antibody or antibody fragment containing a single antibody variable domain that is able to bind to a specific antigen alone, without the requirement of another antibody variable domain. The complementarity determining regions (CDRs) of a single-domain antibody are part of a single antibody variable domain. Examples of single-domain antibodies include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, variable domains derived from the aforementioned antibodies, and single domain scaffolds other than those derived from antibodies. Single-domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, shark, goat, rabbit, and / or bovine. In some embodiments, a single-domain antibody as used herein is a naturally occurring singledomain antibody known as heavy chain antibody devoid of light chains. For clarity reasons, the variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four-chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, e.g., camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain, which are also within the scope of the invention. For example, cartilaginous fishes such as sharks can produce immunoglobulin-like structures known as VNAR. In some embodiments, a single-domain antibody may be obtained from a Camelidae VH domain. In some embodiments, a single-domain antibody may be obtained from human VH by camelization. See Saerens et al., Current Opinion inPharmacology, 2008, 8:600-608, the disclosure of which being incorporated by reference, for review of single-domain antibodies.

[0121] The term "specifically binds" as used herein means that an antigen-binding protein forms a complex with a target antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a dissociation constant (KD) of about 1x10-6M or less (e.g., less than 10-6M, less than 5x10- 7M, less than 10-7M, less than 5x10* M, less than 10-8M, less than 5x10-9M, less than 10-9M, or less than 10- 10M). Methods for determining the binding affinity of an antigen-binding protein, e.g., an antibody or an antibody fragment, to a target antigen are well known in the art and include, e.g., surface plasmon resonance (e.g., BIACORE* assays), bio-layer interferometry, ligand binding assays (e.g., enzyme-linked immunosorbent assay (ELISA)), equilibrium dialysis, fluorescent-activated cell sorting (FACS), or flow cytometry-based binding assays and the like. Specific binding to a particular target antigen from a certain species does not exclude that the antigen-binding protein can also specifically bind to the analogous target from a different species. For example, specific binding to human CD25 does not exclude that the antigen-binding protein can also specifically bind to CD25 from cynomolgus monkeys ("cyno") or mouse.

[0122] The term "isolated" when used in the context of antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides, and vectors, means the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides and vectors are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antigen-binding protein may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term "isolated" is not intended to refer to a complete absence of such biological molecules (e.g., minor or insignificant amounts of impurity may remain) or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies).

[0123] The term "operably linked" as used herein can refer to a functional relationship between two or more regions of a polypeptide chain in which the two or more regions are linked so as to produce a functional polypeptide.

[0124] As used herein, the term "variant", "derivative" or "derived from" in the context of proteins or polypeptides (e.g., antigen-binding proteins or domains thereof) refer to: (a) a polypeptide that has atleast 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polypeptide it is a variant or derivative of; (b) a polypeptide encoded by a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a nucleotide sequence encoding the polypeptide it is a variant or derivative of; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and / or substitutions) relative to the polypeptide it is a variant or derivative of; (d) a polypeptide encoded by nucleic acids can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acids encoding the polypeptide it is a variant or derivative of; (e) a polypeptide encoded by a nucleotide sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleotide sequence encoding a fragment of the polypeptide, it is a variant or derivative of, of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of the polypeptide it is a variant or derivative of. The terms also encompass a fusion protein or polypeptide comprising the polypeptide it is a variant or derivative of.

[0125] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, In certain Instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0126] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence Identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differfrom each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur- containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leuclne-isoleuclne, phenylalanlne-tyroslne, lysine-arginine, alanine-vallne, glutamateaspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443- 1445, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0127] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program In GCG Version 6.1. FASTA (e.g., FA5TA2 and FA5TA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.

[0128] The terms "enhance" or "promote," or "increase," or "expand," or "improve" refer generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a controlmolecule / composition. A measurable physiological response may include an increase in immune cell expansion, activation, effector function, persistence, and / or an increase in tumor cell death killing ability, among others apparent from the understanding in the art and the description herein. In certain embodiments, an "increased" or "enhanced" amount can be a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.

[0129] The terms "decrease" or "lower," or "lessen," or "reduce," or "abate", or "attenuate", or "suppress" refer generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. In certain embodiments, a "decrease" or "reduced" amount can be a "statistically significant" amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle or a control composition.

[0130] The terms "treat" or "treatment" of a state, disease, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub- clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disease, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disease, disorder or condition; or (2) inhibiting the state, disease, disorder or condition, e.g., arresting, reducing or delaying the development of the state, disease, disorder, or condition or a relapse thereof or at least one clinical or sub-clinical symptom of the state, disease, disorder, or condition; or (3) relieving the state, disease, disorder, or condition, e.g., causing regression of the state, disease, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0131] The terms "effective amount" or "therapeutically effective amount" refer to a quantity and / or concentration of a composition containing an active ingredient (e.g., anti-CD25 antigen-binding protein) that when administered into a patient either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a significant decrease in the progression of the state, disease, disorder, or condition, as, for example, by ameliorating or eliminating symptoms and / or the cause of the state, disease, disorder, or condition. An effective amount may be an amount that relieves, lessens, oralleviates at least one symptom or biological response or effect associated with a state, disease, disorder, or condition, prevents progression of the state, disease, disorder, or condition, or improves physical functioning of the patient. A therapeutically effective amount of a composition containing an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic and / or prophylactic result.

[0132] The terms "individual”, "subject”, and "patient" are used interchangeably herein to refer to an animal, for example a mammal. The terms include human and veterinary subjects. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, a subject can be a subject in need of treatment for a disease or disorder. In particular embodiments, the subject is a human.Antl-CD25 Antigen-binding Proteins

[0133] The present disclosure provides antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) that bind to CD25.

[0134] Cluster of differentiation 25 (CD25), also called interleukin-2 receptor subunit alpha (IL-2Rα or IL2RA) is the alpha chain component of the heterotrimeric interleukin-2 receptor complex. IL-2Rα is a single pass type-1 transmembrane protein with a total length of 251 amino acids. The receptor subunit consists of two sushi or elbow domains that are connected via an unordered loop region (Wang et al., Science 310, 1159-1163. 2005). The C-terminal domain of the protein is a long, disordered region that is needed to allow CD25 forming a cap like structure in the IL-2 receptor complex but still being anchored in the membrane. The actual structure and positioning of the loop has not been resolved in any of the available crystal structures. The sushi domains of CD25 form five stranded beta sheet sandwiches that are related to each other in a pseudo-2-fold symmetry. Sushi domain 1 accounts for most of the interactions with IL-2 (82%) while Sushi domain 2 contributes significantly less (Stauber et al., Proc NatlAcad Sci U S A 103, 2788-2793. 2006). The structure of CD25 is stabilized by several intradomain and two interdomain disulfide bonds. In addition, CD25 carries several glycans with one N-glycosylation located at the C-terminus of Sushi domain 2 and four O-glycans located in the C-terminal unordered region.

[0135] CD25 interacts with IL-2 in a tight manner. It is postulated that the IL-2 receptor complex forms in a stepwise manner starting with IL-2 binding to CD25 / IL-2Rα, then engaging subunit β and finally interacting with the y receptor subunit (Stauber et al., Proc Natl Acad Sci U S A 103, 2788-2793. 2006). Interestingly, it has been reported that CD25 can present IL-2 in cis and in trans (Liao et al., Immunity 38, 13-25. 2013; Wuest et al., Nat Med 17, 604-609. 2011), both resulting in IL-2 receptor complex assembly. The CD25 / IL-2Rα has the largest Interface with IL-2 within the complex, which is reflected in the very high affinity between IL-2 and CD25 (Liao et al., 2013). However, in the complex itself CD25 / IL- 2Rα makes no direct contact with the other two subunits β or y. Deglycosylation experiments of the individual subunits were found to impact the complex formation with the γ subunit aggregating, while the subunits α and β were still able to bind to IL-2 (Stauber et al.. Proc Natl Acad Sci U S A 103, 2788- 2793. 2006). Hence, the glycosylation on CD25 / IL-2Rα is not essential for the interaction with IL-2. IL-2- Rγ and IL-2Rβ are also part of other interleukin receptor complexes while CD25 / IL-2Rα is exclusively found in the IL-2 receptor complex (Liao et al., Immunity 38, 13-25. 2013).

[0136] Besides the membrane anchored version of CD25, it has been reported that soluble CD25 can be found in the human serum (Pedersen and Lauritsen, Scand J Immunol 70, 40-43. 2009). This soluble form of CD25 can result from a shedding event of the membrane anchored protein, producing a truncated CD25 with a molecular weight (MW) of ~20kDa.

[0137] ln some embodiments, antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) described herein bind to human CD25. In some embodiments, the human CD25 protein Is encoded by the human interleukin-2 receptor subunit alpha (IL2RA) gene (NCBI Gene ID: 3559) and has the amino acid sequence ofMDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIK5GSLYMLCTGNSSHSSWD NQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQC VQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAAT METSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (UniProtKB Accession No. P01589) (SEQ ID NO: 2210)

[0138] ln some embodiments, antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) described herein bind to cynomolgus monkey ("cyno”) CD25. In some embodiments, thecyno CD25 protein is encoded by the cyno interleukin-2 receptor subunit alpha (IL2RA) gene (NCBI Gene ID: 102123605) and has the amino acid sequence ofMDPYLLMWGLLTFITVPGCQAELCDDDPPKITHATFKAVAYKEGTMLNCECKRGFRRIKSGSPYMLCTGNSSHSSWDN QCQCTSSAARNTTKQVTPQPEEQKERKTTEMQ5QMQLADQVSLPGHCREPPPWENEATERIYHFWGQTVYYQCVQ GYRALHRGPAESICKMTHGKTRWTQPQLICTGETEPSQFPGEEEPQASPDGLPESETSRLVTTTDFRIQTEVAATMETFI FTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKNRRTI (GenBank Accession No. EHH64536.1) (SEQ ID NO: 2211)

[0139] ln some embodiments, antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) described herein bind to mouse CD25. In some embodiments, the mouse CD25 protein is encoded by the mouse interleukin 2 receptor subunit alpha (H2ra) gene (NCBI Gene ID: 16184) and has the amino acid sequence ofMEPRLLMLGFLSLTIVPSCRAELCLYDPPEVPNATFKALSYKNGTILNCECKRGFRRLKELVYMRCLGNSWSSNCQCTSN SHDKSRKQVTAQLEHQKEQQTTTDMQKPTQSMHQENLTGHCREPPPWKHEDSKRIYHFVEGQSVHYECIPGYKALQ RGPAISICKMKCGKTGWTQPQLTCVDEREHHRFLASEESQGSRNSSPESETSCPITTTDFPQPTETTAMTETFVLTMEYK VAVASCLFLLISILLLSGLTWQHRWRKSRRTI (UniProtKB Accession No. P01590) (SEQ ID NO: 2212)

[0140] ln some embodiments, antigen-binding proteins of the present disclosure upon binding to CD25 do not impair the binding of its cognate ligand, interleukin-2 (IL-2), to CD25. In some embodiments, antigen-binding proteins of the present disclosure do not have overlapping epitopes with IL-2.

[0141] ln some embodiments, antigen-binding proteins of the present disclosure upon binding to CD25 may impair the binding of IL-2 to CD25. In some embodiments, antigen-binding proteins of the present disclosure may have overlapping epitopes with IL-2. In some embodiments, when the antigen-binding proteins have overlapping epitopes with IL-2, the antigen-binding proteins may impair IL-2 binding to CD25. In some embodiments, when the antigen-binding proteins have overlapping epitopes with IL-2, the antigen-binding proteins may compete for binding to CD25 with IL-2.

[0142] ln various embodiments, antigen-binding proteins of the present disclosure may have an antagonistic effect (e.g., a blocking effect) upon binding to CD25. An antagonistic CD25 binder can block or decrease activation of CD25 and / or attenuate one or more signal transduction pathways mediated by CD25. Antagonistic CD25 binders may block or decrease CD25 activation by binding CD25, e.g., to induce a conformational change that renders the receptor biologically inactive. For example, antagonistic CD25 binders may prevent the trimerization of an IL-2 receptor complex as can occur due to the interaction between CD25 and its cognate ligand, IL-2, thus impairing CD25-mediated signaling.

[0143] ln some embodiments, when the antigen binding proteins of the present disclosure have overlapping epitopes with IL-2, such antigen-binding proteins may have an antagonistic effect upon binding to CD25.

[0144] ln various embodiments, antigen-binding proteins of the present disclosure may have an agonistic effect (e.g., a stimulatory effect) upon binding to CD25. An agonistic CD25 binder can stimulate or enhance activation of CD25 and / or strengthen one or more signal transduction pathways mediated by CD25. Agonistic CD25 binders may stimulate or enhance CD25 activation by binding CD25, e.g., to induce a conformational change that renders the receptor biologically active. For example, agonistic CD25 binders may promote the trimerization of an IL-2 receptor complex as can occur due to the interaction between CD25 and its cognate ligand, IL-2, thus promoting CD25-mediated signaling.

[0145] ln some embodiments, when the antigen binding proteins of the present disclosure have overlapping epitopes with IL-2, such antigen-binding proteins may have an agonistic effect upon binding to CD25.

[0146] ln some embodiments, antigen-binding proteins of the present disclosure bind to human CD25. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human CD25 with a KDof less than about 1x10-6M, for example, less than about 5x10-7M, less than about 3x10-7M, less than about 1x10-7M, less than about 8x10-9M, less than about 5x10-8M, less than about 3x10- 8M, less than about 1x10-8M, less than about 8x10- 9M, less than about 5x10-9M, less than about 3x10- 5M, less than about 1x10-9M, about 1x10-10to 1x10- 5M, about 1x10-10to 5x10-9M, about 1x10-10to 1x10-8M, about 1x10-10to 5x10-8M, about 1x10-9to 1x10- 5M, about 1x10-9to 5x10- 5M, about 1x10- 5to 1x10-7M, or about 1x10-8to 1x10-7M.

[0147] ln one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 1.6 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 7.6 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 9.4 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to human CD25 with a KDof about 10 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 11 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 12 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 13 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 14 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 17 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 18 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 19 nM. In one embodiment, an antigen-binding protein of the presentdisclosure binds to human CD25 with a KDof about 20 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 21 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 22 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 26 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 31 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 35 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 49 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 50 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 58 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 61 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 62 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 66 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 73 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 76 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 97 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 102 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 107 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 149 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 241 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a KDof about 348 nM.

[0148] ln some embodiments, antigen-binding proteins of the present disclosure bind to cynomolgus monkey ("cyno") CD25. In some embodiments, antigen-binding proteins (e.g., antibodies such as singledomain antibodies) of the present disclosure may bind to cyno CD25 with a KDof less than about 1x10-6M, for example, less than about 5x10-7M, less than about 3x10-7M, less than about 1x10-7M, less than about 8x10-8M, less than about 5x10-9M, less than about 3x10-8M, less than about 1x10-8M, less than about 8x10-9M, less than about 5x10-9M, less than about 3x10-9M, less than about 1x10-9M, about 1x10-10to 1x10-9M, about 1x10-10to 5x10-9M, about 1x10-10to 1x10-8M, about 1x10-10to 5x10-8M, about 1x10-9to 1x10-8M, about 1x10-9to 5x10-8M, about 1x10-9to 1x10-7M, about 1x10-9to 2x10-7M, about 1x10- 9to 5x10-7M, about 1x10- 6to 1x10- 7M, about 1x10-6to 2x10-7M, about 1x10-8to 5x10- 7M, or about 1x10-8to 1x10-6M.

[0149] ln one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 706 pM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 793 pM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno OD25 with a Ko of about 1.5 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a Ko of about 73 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 34 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 48 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 49 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 52 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 57 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 70 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 79 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 97 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 107 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 112 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 115 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to cyno CD25 with a KDof about 117 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 119 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 121 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 131 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 136 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to cyno CD25 with a KDof about 142 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 146 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 148 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 149 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 162 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 163 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 186 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 191 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 211 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 235 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to cyno CD25 with a KDof about 283 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 339 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 380 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 411 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a KDof about 956 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to cyno CD25 with a KDof about 2.1 μM.

[0150] In some embodiments, antigen-binding proteins of the present disclosure bind to mouse CD25. In some embodiments, antigen-binding proteins of the present disclosure may bind to mouse CD25 with a KD of less than about 1x10-6M, for example, less than about 5x10-7M, less than about 3x10- 7M, less than about 1x10-7M, less than about 8x10-6M, less than about 5x10-8M, less than about 3x10-8M, less than about 1x10-8M, less than about 8x10-9M, less than about 5x10-9M, less than about 3x10-9M, less than about 1x10-9M, about 1x10-10to 1x10- 9M, about 1x10- 10to 5x10-9M, about 1x10- 10to 1x10-8M, about 1x10- 10to 5x10sM, about 1x10-9to 1x10-8M, about 1x10- 9to 5x10-8M, about 1x10- 9to 1x10- 7M, about 1x10-9to 2x10-7M, about 1x10-9to 5x10- 7M, about 1x10-8to 1x10-7M, about 1x10-8to 2x10-7M, about 1x10-8to 5x10- 7M, or about 1x10-8to 1x10- 6M. In some embodiments, antigenbinding proteins of the present disclosure do not bind to mouse CD25.

[0151] ln one embodiment, an antigen-binding protein of the present disclosure binds to mouse CD25 with a KD of about 420 nM.

[0152] Binding affinity of a molecular interaction between two molecules can be measured via various techniques, such as surface plasmon resonance (SPR), bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescent-activated cell sorting (FACS), or flow cytometry binding assays and the like. Surface plasmon resonance is a biosensor technique that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule underflow conditions (see e.g., Ober et al. 2001,Intern. Immunology 13: 1551-1559). SPR can for example be performed using the BIACORE®system or Carterra LSA system. Another biosensor technique that can be used to determine affinities of biomolecular interactions is bio-layer interferometry (BLI) (see e.g., Abdiche et al. 2008, Anal. Biochem. 377: 209-217). Bio-layer Interferometry is a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and affinities can be determined. BU can for example be performed using the Octet* Systems. Alternatively, affinities can be measured in Kinetic Exclusion Assay (KinExA) (see e.g., Drake et al. 2004, Anal.Biochem., 328: 35-43), which is a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules. Equilibrated solutions of an antibody / antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).

[0153] Antigen-binding proteins of the present disclosure can include an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single chain antibody (e.g., single chain fragment variable (scFv)); a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab') 2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an lgG2 antibody; an lgG3 antibody; or an lgG4 antibody, and fragments thereof.

[0154] ln some embodiments, an antigen-binding protein that binds to CD25 Is a single-domain antibody (also termed as "sdAb"). The single-domain antibodies of the present disclosure can be derived from numerous sources, including but not limited to VHH, VNAR, or VH domains (naturally occurring or engineered VH domains). VHHs can be generated from camelid heavy chain only antibodies and libraries thereof. VNARs can be generated from cartilaginous fish heavy chain only antibodies and libraries thereof. Various methods have been implemented to generate monomeric sdAbs from conventionally heterodimeric VH and VL domains, including interface engineering and selection of specific germline families. In some embodiments, sdAbs of the present invention are human or humanized.

[0155] ln some embodiments, a single-domain antibody described herein is a VHH fragment (also known as a nanobody). VHH fragments are also referred to as "V-bodies" in the present disclosure. In someembodiments, the VHH is a camelid VHH, a humanized VHH or, a camelized VH. In some embodiments, a single-domain antibody described herein is a VH domain. In some embodiments, a single-domain antibody described herein is a naturally occurring VH domain or engineered VH domain.

[0156] The variable domain of an antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises at least three complementarity determining regions (CDRs) which determine its binding specificity. Preferably, in a variable domain, the CDRs are distributed between framework regions (FRs). The variable domain typically contains 4 framework regions interspaced by 3 CDR regions, resulting in the following typical antibody variable domain structure: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs and / or FRs of the single-domain antibody of the present disclosure may be fragments or derivatives from a naturally occurring antibody variable domain or may be synthetic.

[0157] Sequence identifiers corresponding to exemplary anti-CD25 VHH antibodies provided herein are listed in Table 1-1. Table 1-1 sets forth the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DNA sequences of the full-length camelid VHH antibodies, as well as amino acid sequences of corresponding humanized VHH antibodies. Amino acid sequences of additional exemplary anti-CD25 VHH antibodies and corresponding humanized VHH antibodies are provided in Table 1-2. In the present disclosure, "Hu1” indicates a humanized variant of the parental VHH antibody. "I8”, "A8", or "L8" indicate that the 8thposition in the CDR3 of the parental VHH antibody has been mutated to the amino acid residue isoleucine, alanine, or leucine, respectively.Table 1-1. Sequence Identifiers for exemplary anti-CD25 VHH antibodiesTable 1-2. Sequence identifiers for additional exemplary VHH antibodies and humanized VHH antibodies

[0158] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position, and indicates an amino acid residue is absent at the particular position) a). GR(K / R / S)FSTLI (SEQID NO: 37); b). GFTFS(N / S)YA (SEQ ID NO: 40); c). GRTF(A / S)(S / W / D)(F / N / Y)G (SEQ ID NO: 5209); d). GFTLDYYA (SEQ ID NO: 2242); and e). G(I / M)P(F / -)(A / -)L(P / V / Y)A (SEQ ID NO: 2266).

[0159] ln some embodiments, when an anti-CD25 antigen-binding protein described herein may comprise a CDR1 comprising the sequence GR(K / R / S)FSTLI (SEQ ID NO: 37), the CDR1 may comprise, e.g., the sequence GR(S / K)FSTLI (SEQ ID NO: 32).

[0160] ln some embodiments, when an anti-CD25 antigen-binding protein described herein may comprise a CDR1 comprising the sequence GR(K / R / S)FSTLI (SEQ ID NO: 37), the CDR1 may comprise, e.g., the sequence GRSFSTLI (SEQ ID NO: 5).

[0161] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position, and indicates an amino acid residue is absent at the particular position) a). GRSFSTLI (SEQ ID NO: 5); b). GR(S / K)FSTLI (SEQ ID NO: 32); c) GR(K / R / S)FSTLI (SEQ ID NO: 37); d). GFTFS(N / S)YA (SEQ ID NO: 40); e). GRTFS(S / W)(F / N / Y)G (SEQID NO: 42); f). GFTLDYYA (SEQ ID NO: 2242); and g). G(I / M)P(F / -)(A / -)L(P / V / Y)A (SEQID NO: 2266).

[0162] ln some embodiments, an anti-CD25 antigen-binding protein described herein comprises a CDR1 comprising the sequence GFTFS(N / S)YA (SEQ ID NO: 40) or GFTLDYYA (SEQ ID NO: 2242).

[0163] ln some embodiments, an anti-CD25 antigen-binding protein described herein comprises a CDR1 comprising the sequence GFTFS(N / S)YA (SEQ ID NO: 40).

[0164] ln some embodiments, an anti-CD25 antigen-binding protein described herein comprises a CDR1 comprising the sequence GFTLDYYA (SEQ ID NO: 2242).

[0165] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a). (I / V)(D / E)R(D / G)(D / G)T(A / P / T); b). IYSD(G / S)SGT (SEQ ID NO: 4341); c). IS(Q / R / G)(S / G)GGRT (SEQ ID NO: 5210); d) IS(R / S)(D / S)G(D / G)ST (SEQ ID NO: 2264); e). ISSGGNT (SEQ ID NO: 2246); and f). ISSTDGRT (SEQ ID NO: 2248).

[0166] ln some embodiments, when an anti-CD25 antigen-binding protein described herein may comprise a CDR2 comprising the sequence (l / V)(D / E)R(D / G)(D / G)T(A / P / T), the CDR2 may comprise, e.g., the sequence (l / V)(D / E)R(D / G)GT(A / P / T).

[0167] ln some embodiments, when an anti-CD25 antigen-binding protein described herein may comprise a CDR2 comprising the sequence (l / V)(D / E)R(D / G)(D / G)T(A / P / T), the CDR2 may comprise, e.g., the sequence l(D / E)RDGT(T / P) (SEQ ID NO: 35).

[0168] ln some embodiments, when an anti-CD25 antigen-binding protein described herein may comprise a CDR2 comprising the sequence (l / V)(D / E)R(D / G)(D / G)T(A / P / T), the CDR2 may comprise, e.g., the sequence l(D / E)R(D / G)(D / G)T(P / T).

[0169] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR2 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a). (I / V)(D / E)R(D / G)GT(A / P / T); b). I(D / E)RDGT(T / P) (SEQ ID NO: 35); c). I(D / E)R(D / G)(D / G)T(P / T); d). IYSDGSGT (SEQ ID NO: 14); e). ISQSGGRT (SEQ ID NO: 18); f). IS(R / S)(D / S)G(D / G)ST (SEQ ID NO: 2264); g). ISSGGNT (SEQ ID NO: 2246); andh). ISSTDGRT (SEQ ID NO: 2248)

[0170] ln some embodiments, an anti-CD25 antigen-binding protein described herein comprises a CDR2 comprising the sequence IYSD(G / 5)SGT (SEQ ID NO: 4341) or ISSTDGRT (SEQ ID NO: 2248).

[0171] ln some embodiments, an anti-CD25 antigen-binding protein described herein comprises a CDR2 comprising the sequence IYSD(G / S)SGT (SEQ ID NO: 4341).

[0172] ln some embodiments, an anti-CD25 antigen-binding protein described herein comprises a CDR2 comprising the sequence ISSTDGRT (SEQ ID NO: 2248).

[0173] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a). NAL(G / L / P / Q / W)Y (SEQ ID NO: 31); b). NALR(D / H / N / F) (SEQ ID NO: 34); c). (K / S / T)TLRY (SEQ ID NO: 36); d). (A / V / S)(K / T)G(R / A / K)(G / H / N / R)SG(S / G)YYP(W / F / L)(D / E)(D / E)(Y / V) (SEQ ID NO: 5119); and e). AA(S / T)(D / N / Y / K)(F / V)(L / P)(I / L)A(T / I / A)(T / S / A)IS(A / G)(Y / H)DY (SEQ ID NO: 5208); f). AAYVYPDYYCS(D / E)YVLL(K / R)YDY (SEQ ID NO: 2263); g). NIYR(P / S)QVP(P / S / T)TRYS (SEQ ID NO: 2265); and h). AAKRLGP(M / I / A / L)VH(Q / R)YSLEVLTPLFLDEYDY (SEQ ID NO: 4323), wherein one or more non-alanine residues in the CDR3 sequence is optionally replaced with an alanine, and / or one or more alanine residues in the CDR3 sequence is optionally replaced with a glycine.

[0174] ln certain embodiments, when an anti-CD25 antigen-binding protein described herein may comprise a CDR3 comprising an amino acid sequence wherein one or more non-alanine residues In the CDR3 sequence is replaced with an alanine, and / or one or more alanine residues in the CDR3 sequence is replaced with a glycine, the CDR3 may comprise an amino acid sequence selected from a).(A / V / S)(A / K / T)(A / G)(A / R / K)(A / G / H / N / R)(A / S)(A / G)(A / S / G)(A / Y)(A / Y)(A / P)(A / W / F / L)(A / D / E)(A / D / E)(A / Y / V); and b). (A / G)(A / G)(A / K)(A / R)(A / L)(A / G)(A / P)(M / I / A / L)(A / V)(A / H)(A / R / Q)(A / Y)(A / S)(A / L)(A / E)(A / V) (A / L)(A / T)(A / P)(A / L)(A / F)(A / L)(A / D)(A / E)(A / Y)(A / D)(A / Y).

[0175] ln some embodiments, an anti-CD25 antigen-binding protein described herein comprises a CDR3 comprising the sequence(A / V / S)(A / K / T)(A / G)(A / R / K)(A / G / H / N / R)(A / S)(A / G)(A / S / G)(A / Y)(A / Y)(A / P)(A / W / F / L)(A / D / E)(A / D / E)(A / Y / V).

[0176] ln some embodiments, an anti-CD25 antigen-binding protein described herein comprises a CDR3 comprising the sequence (A / G)(A / G)(A / K)(A / R)(A / L)(A / G)(A / P)(M / I / A / L)(A / V)(A / H)(A / R / Q)(A / Y)(A / S)(A / L)(A / E)(A / V) (A / L)(A / T)(A / P)(A / L)(A / F)(A / L)(A / D)(A / E)(A / Y)(A / D)(A / Y).

[0177] I n some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR3 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a). NAL(G / L / P / Q / W)Y (SEQ ID NO: 31); b). NALR(D / H / N / F) (SEQ ID NO: 34); c). (K / S / T)TLRY (SEQ ID NO: 36); d). AKGR(H / N)SGSYYPWD(D / E)Y (SEQ ID NO: 39); e). (A / V)KGR(G / H / N)SGSYYP(W / F)D(D / E)Y (SEQ ID NO: 4340); f). AA(S / T)(D / N / Y)FL(I / L)ATTI5(A / G)YDY (SEQ ID NO: 41); g). AAYVYPDYYCS(D / E)YVLL(K / R)YDY (SEQ ID NO: 2263); h). NIYR(P / S)QVP(P / S / T)TRYS (SEQ ID NO: 2265); and i). AAKRLGPMVH(Q / R)YSLEVLTPLFLDEYDY (SEQ ID NO: 2267), wherein one or more non-alanine residues in the CDR3 sequence is optionally replaced with an alanine, and / or one or more alanine residues in the CDR3 sequence is optionally replaced with a glycine.

[0178] Provlded herein are anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (l.e., CDR1-CDR2-CDR3) comprising amino acid sequences selected from any of the above-described CDR1, CDR2, and CDR3 amino acid sequences. In certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQID NO: 31; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQID NO: 34;iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQID NO: 36; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 39; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 42, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2263; vli) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2246, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2265; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2267; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence of (A / V / S)(A / K / T)(A / G)(A / R / K)(A / G / H / N / R)(A / S)(A / G)(A / S / G)(A / Y)(A / Y)(A / P)(A / W / F / L)(A / D / E)(A / D / EKA / Y / V); or x). a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of (A / G)(A / G)(A / K)(A / R)(A / L)(A / G)(A / P)(M / I / A / L)(A / V)(A / H)(A / R / Q)(A / Y)(A / S)(A / L)(A / E)(A / V) (A / L)(A / T)(A / P)(A / L)(A / F)(A / L)(A / D)(A / E)(A / Y)(A / D)(A / Y).

[0179] ln certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)GT(A / P / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 31; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of SEQ ID NO: 35, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of l(D / E)R(D / G)(D / G)T(P / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 36;iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 39; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 42, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2263; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2246, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2265; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2267; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence of (A / V / S)(A / K / T)(A / G)(A / R / K)(A / G / H / N / R)(A / S)(A / G)(A / S / G)(A / Y)(A / Y)(A / P)(A / W / F / L)(A / D / E)(A / D / E)(A / Y / V); or x). a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of (A / G)(A / G)(A / K)(A / R)(A / L)(A / G)(A / P)(M / I / A / L)(A / V)(A / H)(A / R / Q)(A / Y)(A / S)(A / L)(A / E)(A / V) (A / L)(A / T)(A / P)(A / L)(A / F)(A / L)(A / D)(A / E)(A / Y)(A / D)(A / Y).

[0180] ln certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 31; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQID NO: 36; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 39;v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 42, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2263; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2246, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2265; or viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:2267.

[0181] 1 n certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4311; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4312; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4313; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4314; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4315; or vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4316.

[0182] ln certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQID NO: 31; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34;iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQID NO: 36; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)GT(A / P / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 31; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of SEQ ID NO: 35, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of l(D / E)R(D / G)(D / G)T(P / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 36; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 31; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)(D / G)T(A / P / T), and a CDR3 comprising an amino acid sequence of SEQID NO: 34; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of (l / V)(D / E)R(D / G)GT(A / P / T), and a CDR3 comprising an amino acid sequence of SEQ IDNO: 31; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 35, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; xl) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5119; xii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4340; xiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 39; xiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5209, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5210, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5208;xv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 42, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41; xvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence of SEQ ID NO:2263; xvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2246, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2265; xviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4323; xix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2267; xx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence of (A / V / S)(A / K / T)(A / G)(A / R / K)(A / G / H / N / R)(A / S)(A / G)(A / S / GKA / Y)(A / Y)(A / P)(A / W / F / L)(A / D / E)(A / D / E)(A / Y / V); or xxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of (A / G)(A / G)(A / K)(A / R)(A / L)(A / G)(A / P)(M / I / A / L)(A / V)(A / H)(A / R / Q)(A / Y)(A / S)(A / L)(A / E)(A / V) (A / L)(A / T)(A / P)(A / L)(A / F)(A / L)(A / D)(A / E)(A / Y)(A / D)(A / Y).

[0183] ln certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5211; ii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5212; iii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5213; iv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5214;v)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5215; vi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5216; vii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5217; viii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5218; ix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5219; x)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5220; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5221; xii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5222; xiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5223; xiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5224; xv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5225; xvl)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5226; xvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5227; xviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5228; xix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5229;xx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5230; xxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5231; xxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5232; xxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:4314; xx"iv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5233; xxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5234; xxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5235; xxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5236; xxvill)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5237; xxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5238; xxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5239;xxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5240; xxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5241; xxxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5242; xxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5243; xxxv)a ODR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5244; xxxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5245; xxxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5246; xxxviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5247; xxxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5248; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5249; xii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5250; orxlii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5251.

[0184] Provided herein are anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a CDR1 comprising an amino acid sequence selected from any of the CDR1 amino acid sequences listed in Table 1-1, Table 5, Table 6, or Table 7, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0185] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos: 1, 5, 9, 13, 17, 626-930, 2242, 2245, 2831-3126, and 4560-4670, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence Identity.

[0186] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos: 1, 5, 9, 13, 17, 32, 42, 805, 809, 818, 2242, and 2245, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0187] Provlded herein are anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a CDR2 comprising an amino acid sequence selected from any of the CDR2 amino acid sequences listed in Table 1-1, Table 5, Table 6, or Table 7, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0188] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as singledomain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 18, 931-1235, 2243, 2246, 2248, 3127-3422, 4335, and 4671-4780, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0189] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as singledomain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 18, 942, 946, 959, 967, 992, 1114, 1115, 1116, 1117, 2243, 2246, 2248, and 4335, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0190] Provided herein are anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a CDR3 comprising an amino acid sequence selected from any of the CDR3 amino acid sequences listed in Table 1-1, Table 5, Table 6, or Table 7, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0191] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as singledomain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 3, 7,11, 15, 19, 1236-1540, 2244, 2247, 2249, 2250, 3423-3718, 43114316, 4336, 47814891, and 5211-5251, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0192] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as singledomain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID NOs:, 3, 7, 11, 15, 19, 1237, 1239, 1271, 1275, 1298, 1301, 1331, 1415, 1419, 1421, 1428, 1432, 1442, 1444,1445, 1447, 1448, 2244, 2247, 2249, 2250, 4311-4316, 4336, 4787, 4866, 4875, 4878, 4879, 4880, and 5211-5251, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0193] ln some embodiments, an anti-CD25 antigen-binding protein comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NO: 13; a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs:5211-5225.

[0194] ln some embodiments, an anti-CD25 antigen-binding protein comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NO: 2242; a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs:5226-5251.

[0195] ln some embodiments, an anti-CD25 antigen-binding protein comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 626-930, 2831-3126, and 45604670; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 931-1235, 3127-3422, and 46714780; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1236-1540, 3423-3718, and 4781-4891.

[0196] ln some embodiments, an anti-CD25 antigen-binding protein comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 626-930, 2831-3126, and 4560-4670; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 931-1235, 3127-3422, and 4671-4780; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1236- 1540, 3423-3718, and 4781-4891.

[0197] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 626-658; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 931-963; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1236-1268.

[0198] ln one aspect, provided herein is an antigen-binding protein that specifically bindsCD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 626-658; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 931-963; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1236-1268.

[0199] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 659-685; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 964-990; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1269-1295.

[0200] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 659-685; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 964-990; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1269-1295.

[0201] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 686-691; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 991-996; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1296-1301.

[0202] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 686-691; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 991-996; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1296-1301.

[0203] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 692-804 and 4560-4670; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 997- 1109 and 4671-4780; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1302-1414 and 4781-4891.

[0204] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ, ID NOs: 692-804 and 4560-4670; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 997- 1109 and 4671-4780; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1302-1414 and 47814891.

[0205] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 805-930; aCDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1110-1235; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1415-1540.

[0206] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 805-930; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1110-1235; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1415-1540.

[0207] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 2831- 3020; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3127-3316; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3423-3612.

[0208] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 2831- 3020; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3127-3316; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3423-3612.

[0209] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3021- 3124; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3317-3420; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3613-3716.

[0210] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3021- 3124; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3317-3420; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3613-3716.

[0211] ln one aspect, provided herein Is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3125- 3126; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3421-3422; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3717-3718.

[0212] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3125- 3126; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3421-3422; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3717-3718.

[0213] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 13; a CDR2 comprising an aminoacid sequence of SEQ ID NO: 4335; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5211-5225.

[0214] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 13; a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5211-5225.

[0215] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242; a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248; and / or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4314 and 5226-5251.

[0216] ln one aspect, provided herein is an antigen-binding protein that specifically binds CD25, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242; a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4314 and 5226-5251.

[0217] Provided herein are anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within any of the exemplary anti-CD25 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 6, or Table 7. In certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6, a CDR3 comprising an amino acid sequence of SEQ ID NO: 7; ill) a CDR1 comprising an amino acid sequence of SEQ ID NO: 9, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10, a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, a CDR3 comprising an amino acid sequence of SEQ ID NO: 15; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 17, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 19; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2243, a CDR3 comprising an amino acid sequence of SEQ ID NO: 2244;vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2245, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2246, a CDR3 comprising an amino acid sequence of SEQ ID NO: 2247; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 2249; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 2250; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4311; xi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4312; xii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4313; xiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4314; xiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4315; xv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4316; xvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4875; xvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1331; xviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4787; xix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4866; xx) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4336; xxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4878; xxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4879;xxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4880; xxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1239; xxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 946, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1239; xxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 959, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1237; xxvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 946, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1237; xxviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 967, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1271; xxix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 946, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1275; xxx) a CDR1 comprising an amino acid sequence of SEQ ID NO: 9, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10, a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; xxxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 992, a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; xxxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 992, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1298; xxxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 942, a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; xxxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 959, a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; xxxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 942, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1301; xxxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1415; xxxvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 809, a CDR2 comprising an amino acid sequence of SEQ ID NO: 1114, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1419; xxxviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 1116, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1421;xxxix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 809, a CDR2 comprising an amino acid sequence of SEQ ID NO: 1117, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1419; xl) a CDR1 comprising an amino acid sequence of SEQ ID NO: 818, a CDR2 comprising an amino acid sequence of SEQ ID NO: 1115, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1428; xli) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1432; xlii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1442; xliii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1444; xliv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1445; xlv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1447; xlvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1448; xlvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5211; xlviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5212; xlix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5213;I) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5214; li) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5215; lii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5216; liii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5217; liv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5218;Iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5219;Ivi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5220;Ivii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5221;Iviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5222; lix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5223; lx) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5224;Ixi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5225;Ixii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5226;Ixiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5227;Ixiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5228;Ixv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5229;Ixvl) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5230;Ixvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5231;Ixviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5232;Ixix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4314;Ixx) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5233;Ixxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5234;Ixxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5235;Ixxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5236;Ixxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5237;Ixxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5238;Ixxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5239;Ixxvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5240;Ixxviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5241;Ixxix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5242;Ixxx) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5243;Ixxxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5244;Ixxxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5245;Ixxxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5246;Ixxxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5247;Ixxxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5248;Ixxxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5249;Ixxxvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5250; orIxxxviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5251.

[0218] ln one embodiment, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5238.

[0219] ln one embodiment, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5241.

[0220] ln a related embodiment, provided herein are anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within a VHH amino acid sequence as defined by any of the exemplary anti-CD25 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 6, or Table 7. For example, provided herein are antibodies, or antigen-binding fragments thereof, comprising the set of CDR1-CDR2-CDR3 amino acid sequences contained within a VHH amino acid sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 26-30, 43-625, 1541-1845, 2251-2254, 2259-2262, 2268-2830, 3719-4014, 4317-4322, 4337, 4339, 4342-4559, 4892-5002, 5114-5176, and 5252-5292.

[0221] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure can include a), a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4; b). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 8; c). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 12; d). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 16;e). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 20; f). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2251; g). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2252; h). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2253; i). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2254; or j). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4337.

[0222] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure can include a), a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 26; b). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 27; c). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 28; d). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 29; e). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 30; f). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2259; g). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2260; h). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2261; i). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2262;j). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4317; k). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4318; l), a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4319; m). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4320; n). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4321; o). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4322; p). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4339; q).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5252; r).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5253; s).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5254; t).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5255; u).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5256; v).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5257; w).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5258; x).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5259; y).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5260;z).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5261; aa).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5262; bb).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5263; cc).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5264; dd).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5265; ee).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5266; ff).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5267; gg).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5268; hh).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5269; ii).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5270; jj).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5271; kk).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5272; ll).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5273; mm).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5274; nn).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5275; oo).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5276;pp).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5277; qq).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5278; rr).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5279; ss).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5280; tt).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5281; uu).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5282; vv).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5283; ww).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5284; xx).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5285; yy).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5286; zz).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5287; aaa).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5288; bbb).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5289; ccc).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5290; ddd).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5291; or eee).a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5292.

[0223] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5279.

[0224] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5282.

[0225] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected fromSEQ ID NOs: 4, 8, 12, 16, 20, 43-342, 1541-1845, 2251-2254, 2268-2559, 3719-4014, 4337, 4342-4451,4892-5002, and 5146-5176, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0226] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 2251-2254, 4337, and 5146-5176, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0227] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NOs: 26-30, 343-625, 2259-2262, 2560-2830, 4317-4322, 4339, 5114-5145, 4452- 4559, and 5252-5292, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0228] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NOs: 26-30, 2259-2262, 4317-4322, 4339, 5114-5145, and 5252-5292, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity.

[0229] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 26, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity.

[0230] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 27, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity.

[0231] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 28, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity.

[0232] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 29, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity.

[0233] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 30, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity.

[0234] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 2259, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0235] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 2260, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0236] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 2261, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0237] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 2262, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0238] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 4317, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0239] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 4318, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0240] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 4319, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0241] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 4320, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0242] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:4321, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0243] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 4322, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0244] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 4339, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0245] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5114, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0246] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5115, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence Identity.

[0247] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5116, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0248] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5117, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0249] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5118, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0250] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5120, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0251] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5121, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0252] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5122, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0253] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5123, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0254] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5124, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0255] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5125, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0256] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5126, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0257] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5127, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0258] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5128, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0259] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5129, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0260] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5130, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence Identity.

[0261] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:5131, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0262] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:5132, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0263] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:5133, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0264] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5134, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0265] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5135, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence Identity.

[0266] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5136, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0267] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5137, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0268] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5138, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0269] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5139, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0270] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5140, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0271] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5141, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0272] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5142, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0273] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5143, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0274] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5144, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0275] In an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5145, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0276] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5252, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0277] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5253, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0278] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5254, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0279] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5255, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0280] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:5256, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0281] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:5257, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0282] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:5258, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0283] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5259, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0284] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5260, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence Identity.

[0285] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5261, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0286] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5262, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0287] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5263, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0288] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5264, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0289] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5265, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0290] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5266, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0291] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5267, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0292] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5268, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0293] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5269, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0294] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5270, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0295] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5271, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0296] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5272, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0297] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5273, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0298] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5274, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0299] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:5275, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0300] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:5276, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0301] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:5277, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0302] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5278, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0303] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5279, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence Identity.

[0304] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5280, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0305] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5281, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0306] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5282, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0307] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5283, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0308] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5284, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0309] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5285, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0310] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5286, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0311] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5287, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0312] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5288, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0313] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5289, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0314] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5290, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0315] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5291, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0316] ln an embodiment provided herein, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5292, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0317] ln some embodiments, the present disclosure also provides an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) that competes for binding to CD25 with any one of the exemplary anti-CD25 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 6, or Table 7.

[0318] In some embodiments, the present disclosure also provides an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) that binds to the same epitope on CD25 as any one of the exemplary anti-CD25 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 6, or Table 7.Single-domain antibodies

[0319] A single-domain antibody (e.g., VHH) can be obtained by immunization of dromedaries, camels, llamas, alpacas, or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens. By reverse transcription and polymerase chain reaction (PCR), a gene library of single-domain antibodies containing several million clones can be produced. Screening techniques such as phage display, yeast display, and ribosome display help to identify the clones binding the antigen. Methods generation of heavy-chain antibody fragments are described in e.g., WO 94 / 04678; Hamers-Casterman et al. 1993; Muyldermans et al. 2001; and Arbabi Ghahroudi, M. et al. (1997). FEBS Letters 414 (3): 521-526, each of which is incorporated herein by reference in its entirety.

[0320] A different method may use gene libraries from animals that have not been previously immunized. Such naive libraries usually contain only antibodies with low affinity to the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step. See e.g., Saerens, D.; et al. (2008). "Single-domain antibodies as building blocks for novel therapeutics". Current Opinion in Pharmacology 8 (5): 600-608.

[0321] Affinity maturation strategies can be categorized as either targeted / rational approaches or untargeted / random approaches. For targeted approaches information about the VHH of interest is needed, such as hot spots for affinity maturation or structural information on the VHH:antigen complex, whereas for untargeted approaches no prior information is needed. Targeted approaches that may be applied for affinity maturation of VHHs include site-directed in-vitro mutagenesis and in- silico / computational approaches. Common untargeted approaches used for affinity maturation of VHHs include random in-vitro mutagenesis, CDR swapping and autonomous hypermutation yeast surface display, with the latter two being novel, emerging and very time efficient techniques. Most of these strategies have in common, that after applying a certain randomization strategy to generate a mutational library, the resulting library can be screened by employing standard display techniques such as yeast, phage or ribosome display to select for the best binders. The choice of the display system is often guided by the library size to be displayed, with yeast display being able to handle library sizes of ~107- 109, phage display ~108-1010and ribosome display ~1012-1013(Chan and Groves, 2021). Notably, during affinity maturation the number of highly interactive residues such as aromatic amino acids usually increase in the CDR regions. The selected affinity matured clones may be further evaluated by adevelopability assessment to test for undesired properties, such as unspecific binding to off-targets or VHH instability.

[0322] For targeted in vitro mutagenesis, a set of selected residues within the CDRs of a VHH may be mutated (Tiller et al„ 2017; Yau et al., 2005). Pre-selection of these residues can be either performed using alanine scanning to identify hot spot residues for mutation or by using structural data of the antigen:VHH complex to identify positions to be mutated. These sites can then be either submitted to saturating mutagenesis to substitute a specific site with all possible amino acids or specific amino acid substitutions yielding several smaller libraries. After mutagenesis binders can be displayed to select the best matured candidate. Usually, several rounds of targeted mutagenesis are performed with separate sub-libraries to obtain combinations of Individual mutations that cooperatively result in increased binding affinity.

[0323] Computer-aided / m silico methods are often used to guide targeted in vitro mutagenesis. Using homology modeling of the target:VHH complex or docking, hotspots for mutations can be identified that are then submitted to in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et al., 2019; Inoue et al., 2013; Mahajan et al., 2018). Further, in silico methods can search all designed variants in a virtual library (~1040members) in a rather short amount of time to identify a feasible number of promising candidates to be tested experimentally. These techniques can be especially valuable if structural data on the drugtarget interaction are available.

[0324] Untargeted / random affinity maturation strategies that can be applied to affinity mature VHHs include random in vitro mutagenesis, CDR shuffling / swapping and in vivo affinity maturation via yeast display. For random in vitro mutagenesis the sequence of either the entire VHH or only the CDRs are mutated randomly (Chen et al., 2021; Ye et al., 2021; Zupancic et al., 2021). The most commonly used technique Is error prone PCR employing a DNA polymerase that lacks proof reading activity and PCR conditions that increase the polymerase error rate even further. This technique can be applied without further structural knowledge or information on the importance of residues that contribute to antigen:VHH interaction. The resulting mutational library can then be displayed to select the best matured candidate. This technique may also be combined with NGS sequencing of the display elutions to get an in-depth readout of all obtained candidates, enabling the identification of low abundant but still promising clones (Chen et al., 2021).

[0325] ln some embodiments, CDR shuffling or swapping is applied for VHH affinity maturation, such as described in Zupancic et al., 2021. For CDR swapping, enriched libraries can be used as input material for a PCR reaction to individually amplify the CDR of the VHHs. The PCR products can then be mixed andreassembled using overlapping PCR to generate the entire plasmid for further rounds of display to select for the best matured binder. One limitation of this approach is that it can only be used for VHHs comprising the same framework as it is the case for synthetic libraries.

[0326] ln some embodiments, in vivo affinity maturation via yeast display is applied for VHH affinity maturation, such as described in Wellner et al., 2021. The method is based on an autonomous hypermutation yeast surface display (AHEAD), which imitates somatic hypermutation during VHH selection using engineered yeast strains. The yeast's error prone orthogonal DNA replication system can generate new variants during plasmid replication by randomly introducing mutations. The new variants can then be displayed and selected using yeast surface display to identify the best binders. This enables the production of high affinity clones in very little time (about 2 weeks), which is significantly faster than classical affinity maturation procedures. The method can be applied using synthetic or immune libraries using unenriched libraries enriched libraries or a subset of preselected clones.

[0327] ln case binders with medium affinity are required, as it is the case for the anti-CD25 V-bodies and the affinity of the identified candidates need to be decreased, very similar techniques can be applied. For example, mutations that are aiming at lowering the affinity can be introduced using the same targeted or untargeted approaches as described for the affinity maturation. The selection afterwards can be adapted accordingly. If larger libraries are generated that need to be screened via a display technique, the selection strategy can be adapted to enrich medium affinity binders while excluding high affinity candidates. This could, for example be a pre-panning in phage display with low antigen concentration to remove all higher affinity candidates, followed by a selection with high antigen concentration to obtain medium affinity VHHs. For library sizes of up to 1000 candidates a kinetic off- rate characterization can be used to get immediate information about the kinetic behavior of the candidates.

[0328] When the most patent clones have been identified, their DNA sequence can be optimized, for example to improve their stability towards enzymes. Another goal is humanization to prevent immunological reactions of the human organism against the antibody. Humanization can be achieved based on the homology between camelid VHH and human VH fragments, which is described in further detail below. Finally, the optimized single-domain antibody can be translated and expressed in suitable organisms such as E. coli or Saccharomyces cerevisiae.

[0329] Single-domain antibodies can also be derived from conventional antibodies. In some embodiments, single-domain antibodies can be made from conventional murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and displaytechniques for identification of the most specific antigens. However, the binding region of a conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity. Monomerization can be accomplished by replacing lipophilic by hydrophilic amino acids. (See e.g., Borrebaeck, C. A. K.; Ohlin, M. (2002). "Antibody evolution beyond Nature". Nature Biotechnology 20 (12): 1189-90.) If affinity can be retained after monomerization, the single-domain antibodies can likewise be produced in E. coll, S. cerevisiae or other suitable organisms.

[0330] A "humanized antibody" refers to a chimeric, genetically engineered, antibody in which the amino acid sequences (typically CDRs) from an antibody (donor antibody), e.g., a camelid antibody, are grafted onto a human antibody (acceptor antibody). Thus, a humanized antibody typically comprises CDRs from a donor antibody and variable region framework and constant regions, when present, from a human antibody. Accordingly, a "humanized VHH" comprises CDRs that corresponds to the CDRs of a naturally occurring VHH domain (e.g., a camelid VHH), but that has been "humanized". Humanized VHH may be prepared by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain human antibody. Such humanized VHHs can be obtained in any suitable manner known to a skilled person in the art and thus not strictly limited to methods described herein.

[0331] Humanization of VHHs can achieved using resurfacing or CDR grafting. Resurfacing strategies have been described in e.g., Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et al., 2018; Vincke et al., 2009 J Biol Chem, and CDR grafting strategies have been described in e.g., ben Abderrazek et al., 2011; van Faassen et al., 2020 FASEB; Li et al., 2018; Vaneycken et al., 2010; Vincke et al., 2009 J Biol Chem; and Yu et al., 2017, each of which is incorporated herein by reference in its entirety.

[0332] To humanize a camelid VHH using a resurfacing approach, a human germllne reference that Is most similar to the camelid germline sequence of the selected VHH may be identified. Most of the isolated camelid VHHs in literature belong to the camelid IGHV3 subfamily 2 (Nguyen et al., 2000, EMBO J) with DP-47 / VH3-23 from the IGHV3 family commonly used as human reference. The framework of the camelid VHH can then be compared to the human reference sequence. Surface exposed residues are substituted to their human counterpart as it is assumed that their contribution to protein stability is rather low. Buried residues however remain of camelid origin, as they likely contribute to the overall VHH stability. Humanization of framework regions 1, 3 and 4 usually does not impact the physicochemical properties of the VHHs, whereas a general humanization of framework 2 would significantly increase local hydrophobicity. Residues H37, H44, H45 and H47 (Chothia numbering) inframework 2, the so called tetrade or hallmark residues, have a rather hydrophobic nature in human VHs (VGLW) as they are partially buried and involved in VH / VL paring, while in camelid VHHs these residues are partially charged (FERG), which significantly increases VHH solubility and inhibits paring of camelid VL (Soler et al., 2021, Biomolecules, Conrath et al., 2005 J Mol Biol]. Further, residues H37 and H47 are known to interact with the CDR-H3 loop in many VHHs, stabilizing its conformation and thereby contributing to antigen binding affinity. In addition, a significant number of VHHs use framework 2 residues H44, H45 and H47 for antigen binding (Zavrtanik et al., 2018, J Mol Biol). A full humanization of these residues hence frequently results in reduced solubility or aggregation of the VHHs and a reduced or complete loss of binding affinity for the target antigen (van Faassen et al., 2020, Vincke et al., 2009). In consequence, all or at least some of these hallmark residues in framework 2 remain of camelid origin when humanizing VHHs.

[0333] Another approach that may be applied to humanize VHHs is CDR grafting. CDRs of the selected VHHs can be transplanted onto a universal VHH framework that has been partially or fully humanized (Saerens et al., 2009 J Biol Chem, Soler et al., 2021, Vincke et al., 2009 J Biol Chem). CDR grafting has been successfully used in some cases but failed for several others, with VHHs frequently losing their potential to bind to the desired antigen and / or becoming structurally instable with a high tendency to aggregate (van Faassen et al., 2020, FASEB). This is mostly attributed to interactions of CDR3 with specific residues in framework 2 that are important for CDR3 conformation, general VHH stability and overall hydrophobicity, which are impaired by this approach. Sometimes camelid backmutations are introduced into the framework to compensate for these effects (van Faassen et al., 2020, FASEB).

[0334] An alternative strategy to mitigate the need of humanizing the selected VHH sequences is to use fully or partially humanized synthetic VHH libraries instead of camelid immune libraries for VHH discovery (Moutel et al. 2016, eLife; McMahon, 2018, NSMB; Zimmermann et al., 2018, eLife). In many of these libraries the hallmark residues are still of camelid origin for reasons discussed above.

[0335] Other suitable humanizing substitutions are described in WO 09 / 138519 and WO 08 / 020079, as well as Tables A-3 to A-8 from WO 08 / 020079 (which are lists showing possible humanizing substitutions), each of which is incorporated herein by reference in its entirety. Non-limiting examples of such humanizing substitutions include Q108L and A14P. Such humanizing substitutions may also be suitably combined with one or more other mutations as described herein (such as with one or more mutations that reduce binding by pre-existing antibodies).

[0336] ln some embodiments, humanized VHH sequences still retain the residues that are relevant for protein A binding. In some embodiments, the engineering activities during humanization may be appliedto engineer protein A binding properties into a VHH that did previously not interact with protein A (Graille et al., 2000, PNAS).

[0337] Like a "humanized antibody", a "camelized antibody" refers to an antibody having amino acid sequences (typically CDRs) from a donor antibody, e.g., a human antibody, and variable region framework and constant regions, when present, from a camelid antibody. Accordingly, a "camelized VH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been "camelized". Camelized VH may be prepared by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner, for example as described in WO 2008 / 020079. Such "camelizing" substitutions are usually inserted at amino acid positions that form and / or are present at the VH—VL interface, and / or at the so-called Camelidae hallmark residues, e.g., F37, E44, R45 and F47 (see for example WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). In one embodiment, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, or the VH sequence of a human antibody. However, such camelized VH can be obtained in any suitable manner known to a skilled person in the art and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

[0338] The amino acid residues of a single-domain antibody can be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids described in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example FIG. 2 of this publication). The total number of amino acid residues In each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. For example, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. As a result, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain is usually in the range of from 110 to 120, often between 112 and 115. However, smaller and longer sequences may also be suitable for the purposes described herein.

[0339] Determination of CDR regions in a single-domain antibody may be accomplished using different methods, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," Sth Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745." ("Contact" numbering scheme); Lefranc M P et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 January; 27(l):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309(3) :657-70, ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272, ("AbM" numbering scheme), each reference cited herein is incorporated by reference in its entirety.[O34O]The boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular's AbM antibody modeling software.

[0341] ln some embodiments, CDRs can be defined In accordance with any of the Kabat numbering scheme, the Chothia numbering scheme, a combination of Kabat and Chothia, the AbM numbering scheme, and / or the Contact numbering scheme. A VHH typically comprises three CDRs, designated CDR1, CDR2, and CDR3. Table 1-3, below, lists exemplary position boundaries of CDR-H1, CDR-H2, CDR- H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-H1 located before CDR-H1, FR-H2 located between CDR-H1 and CDR-H2, FR- H3 located between CDR-H2 and CDR-H3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop whennumbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.Table 1-3. CDRs definitions according to various numbering schemes.

[0342] Thus, unless otherwise specified, a "CDR" or "complementarity determining region," or Individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) CDR as defined by any of the above-mentioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the VHH, as defined by any of the above-mentioned schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes (see e.g., Table 1-3), although it is understood that a provided antibody can include CDRs as described according to any of the other above-mentioned numbering schemes or other numbering schemes known to a person of ordinary skill In the art.

[0343] ln a single-domain antibody sequence of the present disclosure, the framework sequences may be any suitable framework sequences. For example, the framework sequences may be framework sequences derived from a heavy chain variable domain (e.g., a VH sequence or VHH sequence). In some embodiments, the framework sequences are either framework sequences that have been derived from a VHH sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences (in which said framework sequences may optionally have been partially or fully camelized).

[0344] Antigen-binding fragments (or combinations of fragments) of any of single-domain antibodies described herein, such as fragments that contain one or more CDR sequences, suitably flanked by and / or linked via one or more framework sequences, are also encompassed within the present disclosure.

[0345] lt should be noted, however, that the present disclosure is not limited to the origin of the singledomain antibody (or of the nucleotide sequence used to express it), nor to the way that the singledomain antibody or nucleotide sequence is generated or obtained. Thus, an antigen-binding protein of the present disclosure may comprise naturally occurring sequences (from a suitable species), recombinant sequences, or synthetic or semi-synthetic sequences. Similarly, nucleotide sequences encoding antigen-binding proteins of the present disclosure may comprise naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (for example, sequences that are prepared by PCR or isolated from a library).

[0346] Anti-CD25 antigen-binding proteins (e.g., antibodies such single-domain antibodies) of the present disclosure may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domains as compared to the exemplary antibody sequences provided herein. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The antigen-binding molecules of the present disclosure may comprise antigen-binding domains which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another germline sequence, or to a conservative amino acid substitution of the corresponding germline resldue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigenbinding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VHH domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the correspondingresidue(s) of a different germline sequence (i.e„ a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).

[0347] Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced biological properties (e.g., antagonistic or agonistic effect), reduced immunogenicity, etc. Antigen-binding proteins comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.

[0348] Provided herein are anti-CD25 antigen-binding proteins comprising variants of any of the VHH and / or CDR amino acid sequences disclosed herein having one or more amino acid substitutions. For example, the present disclosure includes anti-CD25 antigen-binding proteins having VHH and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 amino acid substitutions relative to any of the VHH and / or CDR amino acid sequences set forth in Tables 1-1, 1-2, 5, 6 or 7 herein. Amino acid substitutions may be introduced into an antigen-binding protein of interest and the resultant variants can screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or reduced ADCC or CDC.

[0349] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that Influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. In some embodiments, an amino acid substitution is a conservative substitution, meaning exchanging an amino acid with another amino acid of the same class, in some embodiments, amino acid substitutions may also include a non-conservafive substitution, meaning exchanging an amino acid with an amino acid of a different class. Other exemplary amino acid substitutions are shown in Table 1-4.Table 1-4. Exemplary amino acid substitutions

[0350] ln some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations to reduce oxidation levels of oxidation-labile residues such as Met (M). In certain embodiments, it may be desirable to address Met (M) oxidation liability by mutation of a Met (M) residue. In some embodiments, the single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations (e.g., substitution mutations) of a Met residue to reduce oxidation. As a non-limiting example, a Met residue may be substituted in any of the single-domain antibodies described herein with e.g., lie (I), Ala (A), or Leu (L), to reduce oxidation.

[0351] ln some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications that reduce binding of the single-domain antibodies (e.g., VHH) by preexisting antibodies found In human blood or serum. In some embodiments, single-domain antibodies (e.g., VHHs) of the present disclosure are modified by mutation of amino acid position 11, for example LeullGlu (L11E), LeullLys (L11K), or LeullVal (L11V). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure may comprise a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering). As another example, a single-domain antibody (e.g., VHH) of the present disclosure may comprise an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the single-domain antibody (e.g., VHH). The C-terminus of a VHH is normally VTVSS (SEQ ID NO: 2225). In one embodiment, a singledomain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering). In another embodiment, a single-domain antibody (e.g., VHH) ofthe present disclosure comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering). Accordingly, the C-terminus of a single-domain antibody (e.g., VHH) can be any one of VKVSS (SEQ ID NO: 2226), VQVSS (SEQ ID NO: 2227), VTVKS (SEQ ID NO: 2228), VTVQS (SEQ ID NO: 2229), VKVKS (SEQ ID NO: 2230), VKVQS (SEQ ID NO: 2231), VQVKS (SEQ ID NO: 2232, or VQVQS (SEQ ID NO: 2233). In another embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering), optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) and an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension at the C-terminus of the single-domain antibody (e.g., VHH) (such that the C-terminus of the single-domain antibody (e.g., VHH) for example has the sequence VTVSSA (SEQ ID NO: 2234), VKVSSA (SEQ ID NO: 2235) or VQVSSA (SEQ ID NO: 2236)). In further embodiments, single-domain antibodies (e.g., VHH) of the present disclosure are modified by changes in carboxy-terminal region, for example to a terminal sequence having the sequence GQGTLVTVKPGG (SEQ ID NO: 2237) or GQGTLVTVEPGG (SEQ ID NO: 2238) or modification thereof. Additional modification to reduce binding by pre-existing antibodies in human serum can be found in e.g., W02012 / 175741, WO2015 / 173325, WO2016 / 150845, W02011 / 003622, W02013 / 024059; US 11,426,468, US 10,526,397, which are incorporated herein by reference in their entities.

[0352] ln one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 4326) or VPAG (SEQ ID NO: 4327). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 4326). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-termlnus starting from position 111 according to Chothia the amino acid sequence VPAG (SEQ ID NO: 4327).

[0353] Additional carboxy-terminus modifications to introduce to a single-domain antibody (e.g., VHH) of the present disclosure include those described in in e.g., WO2024 / 238790, which is incorporated herein by reference in its entirety.

[0354] ln some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 8, 12, 16, 20, 26-30, 43-625, 1541-1845, 2251-2254, 2259-2262, 2268-2830, 3719-4014, 4317-4322, 4337, 4339, 4342-4559, 4892-5002,5114-5176, and 5252-5292, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 4326) or VPAG (SEQ ID NO: 4327).

[0355] ln some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 8, 12, 16, 20, 26-30, 2251-2254, 2259- 2262, 4317-4322, 4337, 4339, 5114-5176, and 5252-5292, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 4326) or VPAG (SEQ ID NO: 4327).

[0356] ln some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 26-30, 2259-2262, 4317-4322, 4339, 5114-5145, and 5252-5292, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 4326) or VPAG (SEQ ID NO: 4327).

[0357] ln some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure are modified to enhance binding to staphylococcal protein A (SpA) or streptococcal protein G (SpG). Binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of the antibodies or antibody fragments. The high-affinity interaction of the IgG Fc region with SpA and SpG has been extensively exploited and became the gold standard for monoclonal antibody purification (BJOrck and Kronvall, 1984). Other non-Fc containing antibody fragments, such as VHHs and Fabs do not have the capacity to bind to SpA or SpG via their Fc regions. However, sequence-dependent interaction with SpA has been demonstrated for these non-Fc containing antibody fragments(Graille et al., 2000; Henry et al., 2016). This characteristic circumvents potential use of affinity tags fused to the drug candidate for affinity chromatography that have the disadvantage as being regarded as a sequence liability, as It may impact protein immunogenicity as well as protein structure and stability and could compromise functionality. The interaction of the single-domain antibodies (e.g., VHH) to SpA relies on an alternative binding mode, with a 1-5 μM affinity, which is comparable to the 0.2 -3 μM measured for VH-SpA interactions (To et al., JBC, 2005; Henry et al., Flos One, 2016).[035S]ln some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure have, or are modified to have a SpA-binding motif. For example, The VHH-SpA interface has been mapped to thirteen residues, which cluster within the framework at the back side of the V-body, distant to the CDRs (Graille et al., 2000, Henry et al., 2016). In the absence of a VHH-SpA co-structure, superposition of a SpA-Fab crystal structure and a VHH allows for visualizing the binding mode. Based on a structural and functional analysis, the thirteen residues of the VHH-SpA interface have been characterized to be intolerant to substitutions (residues Gly15, Arg19, Tyr59, Gly65, and Arg66), tolerant to specific substitutions (residues Thr / Lys / Arg57, Thr68, Gln81, Asn82a, and Ser82b) or generally tolerant to a variety of substitutions (residues Serl7, Lys64, and Ser70) (all residue positions refer to Kabat numbering) (Henry et al., Pios One, 2016). Thus, a SpA-blnding motif Included in a single-domain antibody (e.g., VHH) of the present disclosure may include one or more, or all of the thirteen residues.

[0359] ln some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications at N-terminus to prevent formation of a pyroglutamate and product heterogeneity. In one embodiment, the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).

[0360] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 8, 12, 16, 20, 26-30, 43-625, 1541-1845, 2251-2254, 2259-2262, 2268-2830, 3719-4014, 4317-4322, , 4337, 4339, 4342-4559, 4892-5002, 5114-5176, and 5252-5292, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).

[0361] ln some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 8, 12, 16, 20, 26-30, 2251-2254, 2259- 2262, 4317-4322, 4337, 4339, 5114-5176, and 5252-5292, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 9596, at least 9696, at least 97%, at least 98%, or at least 99% identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (E1D).

[0362] ln some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 26-30, 2259-2262, 4317-4322, 4339, 5114-5145, and 5252-5292, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp.Alternative protein scaffolds

[0363] ln some embodiments, anti-CD25 antigen-binding proteins of the present disclosure can adopt an alternative protein scaffold. Such alternative protein scaffold may be a single chain polypeptidic framework, optionally with a reduced size (e.g., less than about 200 amino acids), that contains a highly structured core associated with variable domains of high conformational tolerance allowing insertions, deletions, or other substitutions. Such antigen-binding proteins may be generated by grafting CDRs or variable regions described herein onto a suitable protein scaffold. The structure of alternative scaffolds may vary, but preferably are of human origin for those developed as therapeutics.

[0364] Alternative protein scaffolds of the present disclosure can be based either on a conventional immunoglobulin (Ig) backbone, or are derived from a completely unrelated protein. These variable domains can be modified to create novel binding interfaces toward any targeted antigen. In some embodiments, an alternative protein scaffold of the present disclosure can be derived from Protein A, e.g., the Z-domain thereof (affibodies), lmmE7 (immunity proteins), BPTI / APPI (Kunitz domains), Ras- binding protein AF-6 (PDZ-domains), charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neokarzinostatin, a fibronectin domain (used in "adnectin"), an ankyrin repeat (AR) domain (used in "DARPins"), avidity multimers (also known as "avimers"), or thioredoxin (Skerra, A., Curr. Opin. Biotechnol. 18:295-304 (2005); Kosse et al., Protein Sci. 15:14-27 (2006); Nicaise et al., Protein Sci. 13:1882-1891 (2004); Nygren and Uhlen, Curr. Opin. Struc. Biol. 7:463-469 (1997), all of which are hereby incorporated by reference in their entirety).

[0365] Antlcalins are a suitable type of non-lg based alternative scaffolds for use in the antigen-binding molecules of the present disclosure. Anticalins are a class of engineered ligand-binding proteins that are based on the lipocalin scaffold. Lipocalins are a family of proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have limited sequence homology, but share a common tertiary structure architecture based on eight antiparallel β-barrels. Lipocalins contain four exposed loops built on the rigid β-barrel structure. Exemplary anticalin proteins that are commonly used are about a size of about 180 amino acids and a mass of about 20 kDa.

[0366] DARPins are another suitable non-lg based alternative scaffold that can be used in the antigenbinding molecules of the present disclosure. DARPins are genetically engineered antibody mimeticproteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat (AR) proteins, which usually contain a 33 amino acid protein motif consisting of two α-helices separated by loops, which repeats mediate protein— protein interactions. DARPins can be generated using combinatorial AR libraries constructed based on the 33 amino acid AR motif with seven randomized positions. DARPin libraries can be screened using ribosome display, and library members typically are well produced in Escherichia coli, do not aggregate, and display high thermodynamic stability. Preferably, DARPins contain two to four of these motifs flanked by N- and C- terminal capping motifs to shield hydrophobic regions and allow increased solubility.

[0367] The avimer structure can also be used as a protein backbone to generate a suitable non-lg based alternative scaffold. Avimers typically consist of two or more peptide sequences of 30 to 35 amino acids each, connected by peptide linker. The individual sequences are derived from A-domains of various membrane receptors and have a rigid structure, stabilized by disulfide bridges and calcium. Each A- domain can bind to a certain epitope of the target protein. The combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity.

[0368] Proteins derived from fibronectin III (FN3) domains can also be used to generate a suitable non-lg based alternative scaffold (also known as "monobody"). For example, the tenth fibronectin type III domain (FN10) of human fibronectin corresponds to a β-sandwich with seven β-strands and three connecting loops showing structural homologies to Ig domains without disulfide bridges. In some cases, the connecting loops of FN10, each about 15 to 21 amino acids in length, can be randomized and the domains displayed on both phage and yeast to select for a scaffold with the desirable properties.Adnectins™ is an exemplary scaffold generated using 10thFN3 domains randomized and displayed in this way. Another exemplary scaffold comprising FN3 domains is a Centyrin™. Centryrins™ contain the consensus sequence of FN3 domains of human Tenascin C (TNC), which Is found In the extracellular matrix of various tissues. Centyrin™ scaffolds have loops that have structural homology to antibody variable domains (i.e., CDR1, CDR2 and CDR3), and are small (about 10 kDa), simple, and highly stable single domain proteins that do not contain cysteine, disulfides or glycosylated residues. Centyrin™ possess excellent biophysical properties such as stability to heat, pH, denaturant and organic solvents, reversible unfolding and monodispersity. Another recent exemplary FN3-based scaffold that can be used in the present disclosure is fluctuation-regulated affinity proteins (FLAPs), as described in See et al., 2020. Biotechnology Journal 15(12) :e2000078, which is incorporated herein by reference in its entirety.Fusion Proteins and Conjugates

[0369] ln one aspect, provided herein are fusion proteins and conjugates comprising at least one anti- CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) linked, directly or indirectly, to one or more additional domains or moieties. In some embodiments, the fusion protein or conjugate of the present disclosure comprises a single polypeptide. In other embodiments, the fusion protein or conjugate of the present disclosure comprises more than one polypeptide. In some embodiments, the fusion protein or conjugate of the present disclosure comprises two polypeptides.

[0370] ln some embodiments, the fusion protein or conjugate of the present disclosure comprises at least one anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) described herein. In some embodiments, the fusion protein or conjugate is multivalent. For example, the fusion protein or conjugate of the present disclosure may be at least bivalent, but can also be e.g., trivalent, tetravalent, pentavalent, hexavalent, etc. The terms "bivalent", "trivalent", "tetravalent", "pentavalent", or "hexavalent" all fall under the term "multivalent" and indicate the presence of two, three, four, five or six binding units (e.g., VHHs), respectively.

[0371] ln certain embodiments, the fusion protein or conjugate is multispecific. For example, in some cases, the one or more additional domain or moieties may be one or more additional binding domain that binds to one or more further antigen or protein. The fusion protein or conjugate of the present disclosure may be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc. The terms "bispecific", "trispecific", "tetraspecific", "pentaspecific", etc., all fall under the term "multispecific" and refer to binding to two, three, four, five, etc., different target molecules, respectively.

[0372] When two or more anti-CD25 antigen-binding proteins are Included in a fusion protein or conjugate, the two or more anti-CD25 antigen-binding proteins may comprise the same sequence or may comprise different sequences. In such embodiments, the two or more anti-CD25 antigen-binding proteins may bind to the same epitope on CD25 or different epitopes on CD25. For example, a fusion protein or conjugate of the present disclosure may be biparatopic, e.g., if two VHHs bind two different epitopes on CD25.Fusion or Conjugation to Fc regions

[0373] ln some embodiments, a fusion protein or conjugate of the present disclosure comprises at least one anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) provided herein operably linked to a dimerization domain such as an immunoglobulin Fc region. An immunoglobulin Fc region may be linked indirectly or directly to the at least one anti-CD25 antigen-binding protein (e.g.,antibody such as a single-domain antibody). In some embodiments, a fusion protein or conjugate of the present disclosure comprises one, two, three, four, five, six or more anti-CD25 antigen-binding proteins provided herein operably linked to an Fc region.

[0374] A "Fc region" as used herein refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, an Fc region comprises a hinge, CH2, and CH3. In various embodiments, when an Fc region comprises a hinge, the hinge can mediate dimerization between two Fc-containing polypeptides. In various embodiments, an Fc region included in a fusion protein or conjugate of the present disclosure is a human immunoglobulin Fc region, or is derived from a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is of IgG, IgE, IgM, IgD, IgA or IgY isotype. In some embodiments, the immunoglobulin Fc region is an IgG isotype, such as IgG1, lgG2, IgG3, or lgG4 subclass. The immunoglobulin Fc region may comprise a variant or fragment of a native IgG Fc region.

[0375] A native Fc region typically possesses an effector function, including but not limited to, Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (for example B-cell receptor); and B-cell activation, etc. Such effector functions generally require the Fc region to be combined with a binding domain (for example, an antibody variable domain) and can be assessed using various assays.

[0376] ln some embodiments, a fusion protein or conjugate of the present disclosure can comprise a dimer of Fc regions. In some embodiments, an Fc region mediates dimerization of the CD25-binding units at physiological conditions, such as when expressed from a cell, such that a dimer is formed that doubles the number of CD25 binding units. For example, a fusion polypeptide comprising one VHH domain that binds CD25 and an Fc region is monovalent as a monomer, but the Fc region can mediate dimerization; as a result, the fusion protein Is bivalent (l.e., having two anti-CD25 VHH domains per molecule). Similarly, in some embodiments, two anti-CD25 VHH domains (2x) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is tetravalent (i.e., having four anti-CD25 VHH domains per molecule). In some embodiments, three anti-CD25 VHH domain (3x) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is hexavalent (i.e., having six anti-CD25 VHH domains per molecule).

[0377] ln some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (anti-CD25 VHH)n-Linker-Fc, wherein n can be any integral number (e.g., 1, 2, 3, 4, 5, etc). When n≥2, each anti-CD25 VHH may be optionally operably linked to another anti-CD25 VHH via a linker.

[0378] ln some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (anti-CD25 VHH)n-Linker-Fc- (anti-CD25 VHH)m, wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc). When n≥2 or m≥2, each anti-CD25 VHH may be optionally operably linked to another anti-CD25 VHH via a linker.

[0379] ln some embodiments, a fusion protein or conjugate of the present disclosure is bivalent. In some embodiments, the bivalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CD25 VHH)-Linker-Fc.

[0380] ln some embodiments, a fusion protein or conjugate of the present disclosure is tetravalent. In some embodiments, the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CD25 VHH)-Linker-(anti- CD25 VHH)-Linker-Fc. In some embodiments, the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CD25 VHH)-Linker-Fc-Linker-(anti-CD25 VHH). The multiple linkers used in the fusion protein are not necessarily the same.

[0381] ln some embodiments, a fusion protein or conjugate of the disclosure is hexavalent. In some embodiments, the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CD25 VHH)-Linker-(anti-CD25 VHH)- Linker-(anti-CD25 VHH)-Linker-Fc. In some embodiments, the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CD25 VHH)-Linker-(anti-CD25 VHH)-Linker-Fc-linker-(anti-CD25 VHH). In some embodiments, the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CD25 VHH)-Linker-Fc-Llnker-(anti-CD25 VHH)- Linker-(anti-CD25 VHH). The multiple linkers used in the fusion protein are not necessarily the same.

[0382] ln some embodiments, the CH3 domain of the Fc region can be used as homodimerization domain, such that the resulting fusion protein may be formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization. For example, a heterodimerization domain can be incorporated into the fusion protein such that the construct is a heterodimeric fusion protein.

[0383] When a dimer of Fc regions is used in a fusion protein or conjugate of the present disclosure, the first and second Fc regions may be of the same IgG isotype such as, e.g., IgG1 / IgG1, lgG2 / lgG2,lgG4 / lgG4. Alternatively, the first and second Fc regions may be of different IgG isotypes such as, e.g., lgG1 / lgG2, lgG1 / lgG4, lgG2 / lgG4, etc.

[0384] ln some embodiments, the Fc region included in a fusion protein or conjugate of the present disclosure can be mutated or modified. In some embodiments, the mutations include one or more amino acid substitutions to reduce an effector function of the Fc region. Various examples of mutations to Fc regions to alter, such as reduce, effector function are known, including any as described below. In general, the numbering of the residues in an immunoglobulin heavy chain or portion thereof, such as an Fc region, is according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, Sth Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0385] ln some embodiments, the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Non-limiting examples of amino acid modifications that can alter ADCC and / or CDC are described in Alegre et al, 1992 J Immunol, 148: 3461-3468; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Shields et al., 2001 JBC, 276(9): 6591-6604; Lazar et al., 2006 PNAS, 103(11): 4005-4010; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Moore et al., 2010 mAbs, 2(2): 181-189; and Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11, each of which is incorporated herein by reference in its entirety.

[0386] ln some embodiments, an Fc region included in a fusion protein or conjugate of the present disclosure exhibits reduced effector functions (such as CDC and ADCC). Various in vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the fusion protein construct and / or cleaved components thereof lack FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC are NK cells which express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in e.g., US 5,500,362; US 5,821,337; Hellstrom. et al., Proc. Natl Acad. Sci. USA 83:7059-7063 (1986); and Hellstrom et al., Proc. Nat'l Acad. Sci.USA 82:1499-1502 (1985); Bruggemann. et al., J. Exp. Med. 166:1351-1361 (1987). Alternatively, nonradioactive assay methods may be employed, such as ACTI™ non-radioactive cytotoxicity assay for flow cytometry or CytoTox96™ non-radioactive cytotoxicity assay. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Natl Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also becarried out to confirm that the fusion protein construct or cleaved components thereof is unable to bind Clq and hence lacks CDC activity (see, e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano- Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al.. Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, 5. B. et al., Inti. Immunol. 18(12):1759-1769 (2006)).

[0387] Examples of mutations that enhance ADCC include modification at Ser239 and Ile332, for example Ser239Asp and He332Glu (S239D, 1332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions, for example Lys326Ala and / or Glu333Ala (K326A and E333A) using the Kabat numbering system.

[0388] ln some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) orAla327 (A327) or Pro329 (P329). For example, Leu 234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), 5er298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A) or Pro239Gly (P329G), Asn325Glu (N325E) orAla327Ser (A327S). In some embodiments, modifications within the Fc region reduce binding to Fc-receptor-gamma receptors (FcyRs) while have minimal impact on binding to the neonatal Fc receptor (FcRn).

[0389] ln some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein Is modified at amino acid Leu235 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329,e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A / L235A / P329A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Asp265 (Kabat Numbering) to alter Fc receptor interactions, e.g Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat Numbering) to alter Fc receptor interactions, e.g., Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, 265 and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding. For example, wherein Gly235 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A). In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0390] ln some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Gly, Leu235Ser, Gly236Arg (L234G / L235S / G236R). In some embodiments, the Fc region of the fusion protein Is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Thr, Gly236Arg (L234S / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Val, Gly236Arg (L234S / L235V / G236R). In some embodiments, the Fc region of the fusion protein Is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Gln, Gly236Arg (L234T / L235Q / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Thr, Gly236Arg (L234T / L235T / G236R). In some embodiments, the Fc region of the fusion protein fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Thr, Leu235Thr, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 252, 254, and 256, e.g., Met252Tyr, Ser254Thr, Thr256Glu (M252Y / S254T / T256E).

[0391] ln some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (1235). In some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions Glu233 (E233), Leu234 (1234), or Leu235 (1235) and is modified at oneor more of the Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding. For example, an Fc region included in a CD25 binding polypeptide is derived from a human Fc domain, and comprises a three amino acid deletion in the lower hinge corresponding to IgG1 E233, 1234, and 1235. In some embodiments, such Fc polypeptides do not engage FcyRs and thus are referred to as "effector silent" or "effector null." For example, Fc deletion of these three amino acids reduces the complement protein Clq binding. In some embodiments, a polypeptide with an Fc region with Fc deletion of these three amino acids retains binding to FcRn and therefore has extended half-life and transcytosis associated with FcRn mediated recycling.

[0392] ln one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence:IgG1 L234A, L235A (also known as "LALA" variant) (mutations bolded in the sequence below)DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2213).

[0393] ln one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgG1 Fc region, having an amino acid sequence:IgG1 L234A, L235A. and P329A (also known as "LALAPA" variant) (mutations bolded in the sequence below)DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTaVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2214).

[0394] ln one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgG1 Fc region, having an amino acid sequence:IgG1 D265A, N297A and P329A (also known as "DANAPA" variant) (mutations bolded in the sequence below)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST YRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL5LSPG (SEQ ID NO: 2215).

[0395] ln one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence:IgG1 L234A, L235A, and G237A (also known as "LALAGA" variant) (mutations bolded in the sequence below)DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2216).

[0396] ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234G / L235S / G236R (mutations bolded in the sequence below)DKTHTCPPCPAPEGSRGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:4328).

[0397] ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence:IgG1 L234S / L235T / G236R (mutations bolded in the sequence below)DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:4329).

[0398] ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234S / L235V / G236R (mutations bolded In the sequence below)DKTHTCPPCPAPESVRGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:4330).

[0399] ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234T / L235Q / G236R (mutations bolded in the sequence below)DKTHTCPPCPAPETQRGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:4331).

[0400] ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234T / L235T / G236R (mutations bolded in the sequence below)DKTHTCPPCPAPETTRGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:4332).

[0401] ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234A / L235A / P329G (mutations bolded in the sequence below)DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:4333)

[0402] ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 M252Y / S254T / T256E (mutations bolded in the sequence below)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:4334).

[0403] ln some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T2S6E, respectively) (Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159), or Met252lle, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively) (EU Index of Kabat et al 1991 Sequences of Proteins of Immunological Interest), Asn434Ala (N434A), Asn434Trp (N434W), Thr256Asp, Thr307Gln (T256D / T307Q), Thr256Asp, Thr307Trp (T256D / T307W), Met252Tyr, Thr256Asp (M252Y / T256D), Thr307Gln, Gln311Val, Ala378Val (T307Q / Q3HV / A378V), Thr256Asp, His286Asp,Thr307Arg, Gln311Val, Ala378Val (T256D / H286D / T307R / Q311V / A378V), or Leu309Asp, Gln311His, Asn434Ser (L309D / Q31 1H / N434S) (see, Ko et al., BioDrugs (2021) 35:147-157).

[0404] ln some embodiments, the Fc region lacks or has reduced fucose attached to the N-linked glycan- chain at N297. There are numerous ways to prevent fucosylation, including but not limited to production in a FUT8 deficient cell line; addition inhibitors to the mammalian cell culture media, for example Castanospermine; and metabolic engineering of the production cell line.

[0405] ln some embodiments, the Fc domain included in a fusion protein or conjugate of the present disclosure is derived from a human Fc domain and comprises mutations M252Y and M428V. In some embodiments, the mutated or modified Fc polypeptide includes the following mutations: M252Y and M428L using the Kabat numbering system. In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5), while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn mediated recycling and extended half-life.

[0406] ln some embodiments, the Fc domain included in a fusion protein or conjugate is derived from a human Fc domain and comprises mutations to induce heterodimerization. In some embodiments, such mutations include those referred to as "knob" and "hole" mutations. For example, having an amino acid modification within the CH3 domain at Thr366, which when replaced with a bulkier amino acid, e.g., Try (T366W), is able to preferentially pair with a second CH3 domain having amino acid modifications to less bulky amino acids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala and Vai, respectively (T366S / L368A / Y407V). In some embodiments, the "knob" Fc domain comprises the mutation T366W. In some embodiments, the "hole" Fc domain comprises mutations T366S, L368A, and Y407V. Heterodimerization via CH3 modifications can be further stabilized by the introduction of a disulfide bond, for example by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposite CH3 domains (Reviewed In Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, Fc domains used for heterodimerization comprise additional mutations, such as the mutation S354C on a first member of a heterodimeric Fc pair that forms an asymmetric disulfide with a corresponding mutation Y349C on the second member of a heterodimeric Fc pair. In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding. In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc domain comprises the modification H435R or H435K (referred to as "hole-R" in some instances when the modification is H435R), while the knob Fcdomain does not. In some instances, the hole-R mutation improves purification of the heterodimer over homodimeric hole Fc domains that may be present.

[0407] ln some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion proteins of the present disclosure are monomeric. For example, modification at residue Thr366 to a charged residue, e.g., Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0408] ln some embodiments, the immunoglobulin Fc region of the fusion protein is of human lgG3 isotype, or a variant thereof. In one embodiment, the lgG3 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human lgG3 Fc region Is modified at amino acid 435 to extend the half-life, e.g., Arg435His (R435H). In some embodiments, the human lgG3 Fc region lacks Lys447 (ELI index of Kabat et al 1991).

[0409] ln some embodiments, the immunoglobulin Fc region of the fusion protein is of human lgG4 isotype, or a variant thereof. In one embodiment, the human lgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the human lgG4 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human lgG4 Fc region is lacks Lys447 (EU index of Kabat et al 1991).

[0410] ln some embodiments, the lgG4 Fc region of the fusion protein is altered at amino acids at 228 and 235, e.g., Ser228Pro, Leu235Glu or Leu235Ala (S228P / L235E or S228P / L235A). In some embodiments, the lgG4 Fc region of the fusion protein is altered at amino acids at 228, 234 and 235, e.g., Ser228Pro, Phe234Ala, Leu235Glu or Leu235Ala (S228P / F234A / L235E or S228P / F234A / L235A). In some embodiments, the lgG4 Fc region of the fusion protein Is altered at amino acids at 228, 235, and 329, e.g., Ser228Pro, Leu235Glu and P329G (S228P / L235E / P329G).

[0411] ln one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKnPPVLDSDGSFFLYSRL7VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2217)

[0412] ln one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence:lgG4 S228P. L235A (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAICTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2218)

[0413] ln one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence:|gG4 S228P, F234A, L235E (mutations bolded in the sequence below)ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2219)

[0414] ln one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, F234A, L235A (mutations bolded in the sequence below)ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYICTTPPVLDSDGSFFLYSRL7VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ IDNO: 2220)

[0415] ln one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 P329G, S228P, L235E (mutations bolded in the sequence below)ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEIVITKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2221)

[0416] Addltlonal lgG4 heavy chain modifications suitable for use In the fusion proteins or conjugates of the present disclosure include those described in Tables 1 and 2 of Dumet et al., mAbs, 11:8, 1341-1350, which Is Incorporated herein by reference in its entirety.

[0417] ln some embodiments, the fusion protein or conjugate contains an immunoglobulin hinge region. In some embodiments, the hinge region serves as a linker to connect one or more CD25 binding units (e.g., VHHs) to the Fc region. In other embodiments, the fusion protein can comprise a linker in addition to the hinge region to connect the one or more CD25 binding units (e.g., VHHs) to the Fc region. The hinge region can be selected from any of the human IgG subclasses. For example, the fusion protein may contain a modified IgG1 hinge having the sequence of EPKSSDKTHTCPPC (SEQ ID NO: 2222), wherein the Cys220 that typically forms a disulfide bond with the C-terminal cysteine of the light chain is mutated toserine, e.g., Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 2223).

[0418] ln some embodiments, the fusion protein or conjugate has a modified hinge from lgG4, which is modified to prevent or reduce strand exchange, e.g., Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 2224).

[0419] ln alternative embodiments, a fusion protein or conjugate of the present disclosure may comprise sequences other than an Fc region to achieve multimerization (e.g., dimerization). For example, an amino acid sequence containing at least one cysteine residue may be included to facilitate dimerization of two polypeptides by formation of a disulfide bond between the two polypeptides. In some embodiments, such multimerizlng domain may comprise one or more cysteine residues, or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0420] Fc mutations suitable for use in the fusion proteins disclosed herein are also discussed in, e.g., Wilkinson et al., Fc-engineered antibodies with immune effector functions completely abolished. PLoS One. 2021; WO2021234402A2; US 8,969,526; EP3692065B1; and US 7,083,784, each of which is incorporated herein by reference.Fusion or Conjugation to Half-Life Extension Moleties

[0421] ln some embodiments, a fusion protein or conjugate of the present disclosure may comprise one or more other moieties which provide the fusion protein or conjugate with increased ( / n v / vo) half-life. In vivo half-life extension means, that the fusion protein or conjugate has an increased half-life in a mammal, such as a human subject, after administration.

[0422] Non-limiting examples of half-life extension moieties suitable for use in the present disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

[0423] ln some embodiments, a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG. In one embodiment, a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to human serum albumin. In one embodiment, the binding moiety is a single-domain antibody (e.g., VHH).

[0424] For example and without limitation, albumin binders that are described in, e.g., WO 04 / 041865, WO 06 / 122787, W02012 / 175400, WO 2012 / 175741, WO2015 / 173325, W02017 / 080850, WO2017 / 085172, WO2018 / 104444, W02018 / 134235, WO2018 / 134234, each of which is incorporatedherein by reference is its entirety, can be used in the fusion protein or conjugate of the present disclosure.Fusion or Conjugation to Other Moieties

[0425] Anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) provided herein may be operably linked, directly or Indirectly, to a second moiety, such as but not limited to, a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytotoxic agent, a small molecule drug, a chemotherapeutic agent, a therapeutic agent, a diagnostic agent, or a combination thereof.

[0426] ln some embodiments, a conjugate of the present disclosure comprises a label, which can generate a detectable signal. Such conjugates can be used for research or diagnostic purposes, such as for the in vivo detection of cancer. Preferably, the label is capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope (such as 3H, 14C, 32P, 35S, 1231, 1251, 1311); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound (such as fluorescein isothiocyanate, rhodamine or luciferin); an enzyme (such as β- galactosidase, alkaline phosphatase, or horseradish peroxidase); an imaging agent; or a metal ion. In some embodiments, the label is a radioactive atom for scintigraphic studies, for example 99Tc or 1231, or a spin label for nuclear magnetic resonance (NMR) imaging, such as zlrconium-89, iodine-123, iodine- 131, lndlum-111, fluorlne-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or Iron. Zirconium-89 may also be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011 / 056983).

[0427] Anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may be conjugated to another moiety, such as an epitope tag, e.g., for the purpose of purification or detection. Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is commonly employed in protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound. Non-limiting examples of epitope tag molecules that can be conjugated to anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure, e.g., for the purposes of molecular recognition include a poly-histidine tag (His-tag), a myc-tag, human influenza hemagglutinin (HA) tag, a FLAG-tag, maltose-binding protein, glutathione-S-transferase, biotin, and streptavidin. Conjugatescontaining the epitopes presented by these molecules are capable of being recognized by complementary molecules such as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. For example, one can purify an anti-CD25 antigen-binding protein of the present disclosure that has been conjugated to an epitope tag from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc.) by treating the mixture with a solid phase resin containing a complementary molecule that can selectively recognize and bind the epitope tag of the CD25 antibody or fragment thereof. Examples of solid phase resins include agarose beads, which are compatible with purifications in aqueous solution.

[0428] ln some embodiments, a conjugate of the present disclosure may comprise one or more anti- CD25 VHH domains described herein conjugated to a therapeutic agent, which can be cytotoxic, cytostatic or otherwise provides some therapeutic benefit. In some embodiments, the cytotoxic agent is a drug, a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate). Such conjugates may be applicable to, e.g., the treatment or prevention of a disease associated with autoreactive cytotoxic T-cell activity. In some embodiments, antibody drug conjugates described herein may allow targeted delivery of a drug moiety to a target tissue (e.g., tumors).

[0429] ln some embodiments, a conjugate of the present disclosure comprises a toxin. In some embodiments, the toxin includes, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Scl. USA 93:8618-8623 (1996)), and calicheamlcln (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al.. Cancer Res. 53:3336-3342 (1993)). The toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0430] ln some embodiments, anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may be fused or conjugated to one or more moieties that facilitate delivery to the central nervous system (CNS) / brain. The moiety that can facilitate delivery of an anti- CD25 antigen-binding protein to the central nervous system (CNS) / brain can be for example, a peptide, a polypeptide, small molecule, a lipid, or a synthetic polymer. Various approaches to deliver singledomain antibodies into the brain are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.

[0431] As a non-limiting example, an anti-CD25 antigen-binding protein (e.g., antibody such as singledomain antibody) of the present disclosure may be fused or conjugated to a moiety (e.g., an antibody) that binds to the transferrin receptor (Tf R) or insulin receptor. The transferrin receptor (TfR) is highly expressed by brain capillary endothelial cells (BCECs) forming the blood-brain barrier (BBB) and has been utilized as a target for brain drug delivery. Monoclonal antibodies binding to the TfR, such as clone Ri7, have been shown to internalize into BCECs in vivo. As another example, an anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody) of the present disclosure may be conjugated to hydrophobic fatty acid moieties, such as C18 fatty acid (stearic acid), C16 fatty acid (palmitic acid) or C8 fatty acid (octanoic acid) moieties; or amphiphilic block copolymer moieties, such as poly(ethylene oxlde)-poly(propylene oxide)-poly(ethylene oxide) (pluronlcs or poloxamers) or poly(2-oxasolines). Various fatty acid moieties and block copolymer moieties that can be utilized for brain delivery of proteins are described in, e.g., Yi and Kabanov, J Drug Target. 2013; 21(10): 940-955, which is incorporated herein by reference in its entirety.

[0432] Example methods for attaching a moiety, such as a label, to a binding protein include those described in Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. And Cytochem. 30:407 (1982); Wensel and Meares, Elsevier, N.Y. (1983); and Colcher et al., Meth. Enzymol., 121 :802-16 (1986). Additional suitable methods for preparing the conjugates of the present disclosure include those described in, e.g., WO 2009 / 067800, WO 2011 / 133886, and US2014322129, incorporated by reference herein in their entirety.

[0433] ln some embodiments, the attachment between an anti-CD25 antigen-binding protein and a second moiety can be covalent or non-covalent, e.g., via a biotin-streptavidin non-covalent interaction. In some embodiments, a second moiety can be attached to an anti-CD25 antigen-binding protein using any of various molecular biological or chemical conjugation and linkage methods known In the art and described below. In some embodiments, linkers such as peptide linkers, cleavable linkers, non-cleavable linkers or linkers that aid in the conjugation reaction, can be used to link or conjugate a second moiety to an anti-CD25 antigen-binding protein described herein.

[0434] ln some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as singledomain antibody) is conjugated to one or more second moieties, e.g., about 1 to about 20 moieties per molecule, optionally via a linker. In some embodiments, the one or more second moieties can be the same or different. The linker may be composed of one or more linker components. For covalent attachment of an antibody and the second moiety, the linker typically has two reactive functional groups, i.e., bivalency in a reactive sense. Bivalent linker reagents which are useful to attach two ormore functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups have been described in, e.g., Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242.

[0435] ln some embodiments, a linker used in a conjugate of the present disclosure may include 6- maleimidocaproyl ("MC), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit”), a alaninephenylalanine ("ala-phe”), p-aminobenzyloxycarbonyl ("PAB"), N-Succinimidyl 4-(2- pyridylthio)pentanoate ("SPP"), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate ("SMCC"), or N-Succinimidyl(4-iodo-acetyl)aminobenzoate ("STAB"), or a combination thereof.

[0436] ln some embodiments, a linker used in a conjugate of the present disclosure may comprise amino acid residues. Exemplary amino acid linker components Include a dlpeptide, a trlpeptlde, a tetrapeptide or a pentapeptide. Exemplary dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues used in an amino acid linker component may include naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.

[0437] Conjugates of an anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody) and second moiety (e.g., cytotoxic agent) can be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldit hiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p- azldobenzoyl) hexanediamlne), bls-diazonium derivatives (such as bls-(p-dlazonlumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-d'rfluoro-2,4-dinitrobenzene).

[0438] Conjugates of the present disclosure can be prepared by a variety of methods. For example, the conjugation method may include: (1) reaction of a nucleophilic group of a VHH domain with a bivalent linker reagent, to form VHH-Linker, via a covalent bond, followed by reaction with a drug moiety; or (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form drug-linker, via a covalent bond, followed by reaction with the nucleophilic group of a VHH domain.

[0439] Nucleophilic groups on proteins including antibodies (e.g., VHH domains), include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups (e.g., lysine), (iii) side chain thiolgroups (e.g., cysteine), and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Additional nucleophilic groups can be introduced into proteins (e.g., antibodies such as VHH domains) through the reaction of lysines with 2- iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into a protein (e.g., antibody such as a VHH domain) by introducing one, two, three, four, or more cysteine residues.

[0440] Conjugates, such as antibody drug conjugates, may also be produced by modification of an antibody, such as a VHH domain, to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may lead with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium metaperiodate, resulting in production of an aldehyde in place of the first amino acid. Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[0441] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thlosemlcarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBi esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[0442] Alternatively, a fusion protein containing a VHH domain and a polypeptidic agent may be made, e.g., by recombinant DNA techniques or peptide synthesis. A DNA sequence may be engineered to comprise respective regions encoding the two portions of the fusion protein either adjacent to one another or separated by a region encoding a linker peptide which does not impair the desired properties of the fusion protein. The DNA sequence can be then transfected into a host cell that expresses thefusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art.Linkers

[0443] ln some embodiments, the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via peptide linkers. A peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects a peptide linker ranges from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length.

[0444] ln some embodiments, a peptide linker, e.g., a peptide linker separating two VHH domains or an VHH domain and a heavy chain constant region, is at least 5 amino acids, at least 6 amino acids or at least 7 amino acids in length and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids or up to 60 amino acids in length.

[0445] ln some embodiments, the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length. In other embodiments of the foregoing, the linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids In length. In yet other embodiments of the foregoing, the linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids in length.

[0446] ln some embodiments, charged (e.g., charged hydrophilic linkers) and / or flexible linkers are used. Examples of flexible linkers that can be used in the fusion proteins of the disclosure include those disclosed by Chen et al, 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et a / ., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or comprise repeats of glycines and serines (termed "GS-linker" herein), e.g., a monomer or multimer of GnS (SEQ ID NO: 2195) or SGn(SEQ ID NO: 2196), where n is an integer from 1 to 10, e.g., 12, 3, 4, 5, 6, or 7, 8, 9 or 10. In one embodiment, the linker is or comprises a monomer or multimer of repeat of G4S (SEQ ID NO: 2151), e.g., (GGGGS)n (SEQ ID NO: 2197).

[0447] Polyglycine linkers can suitably be used in the fusion proteins of the disclosure. In some embodiments, a peptide linker used herein comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 2198), five consecutive glycines (5Gly) (SEQID NO: 2199), six consecutive glycines (6Gly) (SEQ ID NO: 2200), seven consecutive glycines (7Gly) (SEQID NO: 2201), eight consecutive glycines (8Gly) (SEQ ID NO: 2202), or nine consecutive glycines (9Gly)(SEQ ID NO: 2203).

[0448] ln some embodiments, a GS-linker used herein comprises an amino acid sequence selected fromGGSGGS, i.e., (GGS)i(SEQ ID NO: 2204); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 2205); GGSGGSGGSGGS, i.e„ (GGS)4(SEQ ID NO: 2206); and GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 2207). In some embodiments, the fusion proteins can include a combination of a GS-linker and a glycine linker.

[0449] ln one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGS (SEQ ID NO: 2152) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGS (SEQ ID NO: 2208) linker.In one embodiment, a VHH and an Fc region are linked via a GGGGSESKYGPPCPSCP (SEQ ID NO: 2190) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGS (SEQ ID NO: 2151) linker.

[0450] ln some embodiments, the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via a "rigid" peptide linker. Such peptidic linker may comprise a prolinerich peptide. In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO:2191). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQID NO: 2194). In one embodiment, a rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 2209), where n is any integer, e.g., 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10.

[0451] Other exemplary peptide linkers that can be used in the fusion proteins described herein are shown in Table 2.Table 2. Exemplary Peptide Linker SequencesSignal Sequences

[0452] ln some embodiments, the fusion protein described herein may further comprise a signal sequence at its N-terminus. Signal sequences may be present in the precursor molecule of the fusion protein and may be removed after the protein is secreted from the host cell during production. In some embodiments, the signal sequence is MAVMAPRTL...

Claims

Claims 1. An antigen-binding protein that specifically binds cluster of differentiation 25 (CD25), comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence a).(A / V / S)(A / K / T)(A / G)(A / R / K)(A / G / H / N / R)(A / S)(A / G)(A / S / G)(A / Y)(A / Y)(A / P)(A / W / F / L)(A / D / E)(A / D / E)(A / Y / V); or b). (A / G)(A / G)(A / K)(A / R)(A / L)(A / G)(A / P)(M / I / A / L)(A / V)(A / H)(A / R / Q)(A / Y)(A / S)(A / L)(A / E)(A / V) (A / L)(A / T)(A / P)(A / L)(A / F)(A / L)(A / D)(A / E)(A / Y)(A / D)(A / Y).

2. The antigen-binding protein of claim 1, wherein the CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 4314 and 5211-5251.

3. The antigen-binding protein of claim 1 or 2, further comprising a CDR1 comprising an amino acid sequence a). GFTFS(N / S)YA (SEQ ID NO: 40); or b). GFTLDYYA (SEQ ID NO: 2242).

4. The antigen-binding protein of claim 3, wherein the CDR1 comprises an amino acid sequence SEQ ID NO: 13 or 2242.

5. The antigen-binding protein of any one of claims 1-4, further comprising a CDR2 comprising an amino acid sequence a). IYSD(G / S)SGT (SEQ ID NO: 4341); or b). ISSTDGRT (SEQ ID NO: 2248).

6. The antigen-binding protein of claim 5, wherein the CDR2 comprises an amino acid sequence SEQ IDNO: 2248 or 4335.

7. The antigen-binding protein of any one of claims 1, 3, and 5, wherein the antigen-binding protein comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence of(A / V / S)(A / K / T)(A / G)(A / R / K)(A / G / H / N / R)(A / S)(A / G)(A / S / G)(A / Y)(A / Y)(A / P)(A / W / F / L)(A / D / E)(A / D / E)( A / Y / V); or ii). a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of (A / G)(A / G)(A / K)(A / R)(A / L)(A / G)(A / P)(M / I / A / L)(A / V)(A / H)(A / R / Q)(A / Y)(A / S)(A / L)(A / E)(A / V) (A / L)(A / T)(A / P)(A / L)(A / F)(A / L)(A / D)(A / E)(A / Y)(A / D)(A / Y).

8. The antigen-binding protein of any one of claims 1-7, wherein the antigen-binding protein comprises i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5211; ii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5212; iii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5213; iv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5214; v)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5215; vi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5216; vii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5217; viii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5218; ix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5219; x)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5220; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5221; xii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5222;xiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5223; x'rv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5224; xv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5225; xvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5226; xvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5227; xviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5228; xix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5229; xx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5230; xxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5231; xxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5232; xxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5233; xxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5234; xxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5235;xxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5236; xxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5237; xxviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5238; xxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5239; xxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5240; xxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5241; xxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5242; xxxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5243; xxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5244; xxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5245;xxxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5246; xxxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5247; xxxviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5248; xxxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO:5249; xl)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5250; or xli)a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5251.

9. The antigen-binding protein of any one of claims 1-8, wherein the antigen-binding protein is a single-domain antibody.

10. The antigen-binding protein of claim 9, wherein the single-domain antibody "is a VHH, a VNAR, or a VH domain.

11. The antigen-binding protein of claim 10, wherein the VHH is a humanized VHH.

12. The antigen-binding protein of claim 11, wherein the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5252-5292, or a sequence having at least 75% identity thereto.

13. The antigen-binding protein of any one of claims 1-12, wherein the antigen-binding protein binds to human CD25.

14. The antigen-binding protein of any one of claims 1-12, wherein the antigen-binding protein binds to cyno CD25.

15. The antigen-binding protein of any one of claims 1-14, wherein the antigen-binding protein binds to the same epitope(s) on CD25 as IL-2.

16. The antigen-binding protein of any one of claims 1-15, wherein the antigen-binding protein competes for binding to CD25 with IL-2.

17. The antigen-binding protein of claim 15 or 16, wherein the antigen-binding protein has an antagonistic effect upon binding to CD25.

18. The antigen-binding protein of any one of claims 1-14, wherein the antigen-binding protein does not bind to the same epitopefs) on CD25 as IL-2.

19. The antigen-binding protein of any one of claims 1-14 and 18, wherein the antigen-binding protein does not compete for binding to CD25 with IL-2.

20. The antigen-binding protein of any one of claims 1-19, wherein the antigen-binding protein comprises one or more modifications that reduce binding of said antigen-binding protein by preexisting antibodies found in human blood or serum.

21. The antigen-binding protein of any one of claims 9-20, wherein the single-domain antibody comprises one or more modifications at the amino-terminus and / or the carboxy-terminus.

22. The antigen-binding protein of claim 21, wherein the single-domain antibody comprises the amino acid sequence VPAG (SEQ ID NO: 4327) or VAGG (SEQ ID NO: 4326) at the carboxy-terminus starting from position 111 according to Chothia.

23. The antigen-binding protein of claim 21 or 22, wherein the single-domain antibody comprises a substitution of amino acid residue Glu with Asp (EID) at the first position of the amino-terminus.

24. An antigen-binding protein that specifically binds cluster of differentiation 25 (CD25), comprising a means for binding an epitope within human CD25 bound by an antibody selected from C-005Hu1.A1, C-005Hu1.A2, C-005Hu1.A3, C-005Hu1.A4, C-005Hu1.AS, C-005Hu1.A6, C-005Hu1.A7, C-005Hu1.A8, C-005Hu1.A9, C-005Hu1.A10, C-005Hu1.A11, C-005Hu1.A12, C-005Hu1.A13, C-005Hu1.A14, C-005Hu1.A15, C-010Hu1.L8.G1, C-010Hu1.L8.G2, C-010Hu1.L8.A3, C-010Hu1.L8.A4, C-010Hu1.L8.A5, C-010Hu1.L8.A6, C-010Hu1.L8.A7, C-010Hu1.A8, C-010Hu1.L8.A9, C-010Hu1.L8.A10,C-010Hu1.L8.A11, C-010Hu1.L8.A12, C-010Hu1.L8.A13, C-010Hu1.L8.A14, C-010Hu1.L8.A15, C-010Hu1.L8.A16, C-010Hu1.L8.A17, C-010Hu1.L8.A18, C-010Hu1.L8.A19, C-010Hu1.L8.A20, C-010Hu1.L8.A21, C-010Hu1.L8.A22, C-010Hu1.L8.A23, C-010Hu1.L8.A24, C-010Hu1.L8.A25, C-010Hu1.L8.A26, and C-010Hu1.L8.A27.

25. A fusion protein that specifically binds cluster of differentiation 25 (CD25), comprising one or more of said antigen-binding proteins of any one of claims 1-24.

26. The fusion protein of claim 25, which comprises two said antigen-binding proteins.

27. The fusion protein of claim 25, which comprises four said antigen-binding proteins.

28. The fusion protein of any one of claims 25-27, wherein the one or more antigen-binding proteins bind to the same epitope on CD25.

29. The fusion protein of any one of claims 25-27, wherein the one or more antigen-binding proteins bind to different epitopes on CD25.

30. The fusion protein of any one of claims 25-29, wherein the one or more antigen-binding proteins are one or more single-domain antibodies.

31. The fusion protein of claim 30, wherein one or more single-domain antibodies are one or moreVHHs.

32. The fusion protein of any one of claims 25-31, which further comprises an immunoglobulin Fc region.

33. The fusion protein of claim 32, wherein the immunoglobulin Fc region is an Fc region of a human immunoglobulin.

34. The fusion protein of claim 33, wherein the immunoglobulin Fc region is an Fc region of human IgG1, lgG2, lgG3 or lgG4, or a variant thereof.

35. The fusion protein of claim 34, wherein the immunoglobulin Fc region is an Fc region of human IgG1, or a variant thereof.

36. The fusion protein of claim 35, wherein the Fc region of human IgG1 comprises one or more mutations selected from L234A, L235A, G237A, M252Y, S254T, T256E, D265A, N297A, and / or P329A according to EU numbering.

37. The fusion protein of claim 36, wherein the Fc region of human IgG1 comprises a set of mutations selected from1).L234A and L235A;2).L234A, L235A, and P329A;3).D265A, N297A and P329A;4).L234A, L235A, and G237A;and5).M252Y, S254T,and T256E.

38. The fusion protein of claim 37, wherein the Fc region of human IgG1 comprises M252Y, S254T, and T256E.

39. The fusion protein of claim 34, wherein the immunoglobulin Fc region is an Fc region of human lgG4, or a variant thereof.

40. The fusion protein of claim 39, wherein the Fc region of human lgG4 comprises one or more mutations selected from S228P, L235E, L235A, and / or F234A according to EU numbering.

41. The fusion protein of claim 40, wherein the Fc region of human lgG4 comprises a set of mutations selected from1). S228P and L235E;2].S228P and L235A;3].S228P, F234A, and L235E; and4).S228P, F234A, and L235A.

42. A conjugate comprising the antigen-binding protein of any one of claims 1-24 or the fusion protein of any one of claims 25-41, wherein the antigen-binding protein or fusion protein is conjugated to a second moiety.

43. The conjugate of claim 42, wherein the second moiety is selected from a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

44. A polynucleotide molecule encoding the antigen-binding protein of any one of claims 1-24 or the fusion protein of any one of claims 25-41.

45. The polynucleotide molecule of claim 44, wherein the polynucleotide molecule comprises a nucleotide sequence selected from SEQ ID Nos: 5295-5336.

46. A recombinant vector comprising the polynucleotide molecule of claim 44 or 45.

47. A host cell comprising the polynucleotide molecule of claim 44 or 45, or the expression vector of claim 46.

48. A kit comprising the antigen-binding protein of any one of claims 1-24, the fusion protein of any one of claims 25-41, the conjugate of claim 42 or 43, the polynucleotide molecule of claim 44 or 45, the recombinant vector of claim 46, or the host cell of claim 47, and optionally, instructions and / or packaging for the same.

49. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-24, the fusion protein of any one of claims 25-41, the conjugate of claim 42 or 43, the polynucleotide molecule of claim 44 or 45, or the recombinant vector of claim 46, and a pharmaceutically acceptable carrier and / or excipient.

50. A method for preparing an antigen-binding protein or a fusion protein that specifically binds cluster of differentiation 25 (CD25), comprising the steps of:(a) culturing the host cell of claim 47 in a culture medium under conditions suitable for expression of the antigen-binding protein or fusion protein, and(b) Isolating the antigen-binding protein or fusion protein from the host cell and / or culture medium.

51. A method for targeting a cell expressing CD25 comprising contacting the cell with the antigenbinding protein of claims 1-24, the fusion protein of any one of claims 25-41, or the conjugate of claim 42 or 43.

52. The method of claim 51, wherein the cell is a regulatory T cell (Treg).

53. The method of claim 51 or 52, wherein said contacting occurs in vitro.

54. The method of claim 51 or 52, wherein said contacting occurs in vivo.

55. The method of claim 54, wherein the method further comprises administering the antigen-binding protein, the fusion protein, or the conjugate Into a subject In need thereof.

56. A method of treating or preventing a disease or disorder in a subject in need thereof, said method comprising administering to the subject an effective amount of the antigen-binding protein of claims 1-24, the fusion protein of any one of claims 25-41, or the conjugate of claim 42 or 43.

57. The method of claim 56, wherein the disease or disorder is an immunological disease, inflammatory disease, cancer, cardiovascular disease, or an infertility and pregnancy-associated disease.

58. The method of claim 57, wherein the immunological disease is selected from an autoimmune disease, a neurological condition, an allergy, asthma, macular degeneration, muscular atrophy, a disease related to miscarriage, atherosclerosis, bone loss, a musculoskeletal disease, obesity, a graft- versus-host disease, and an allograft rejection.

59. The method of claim 58, wherein the autoimmune disease is selected from lupus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic Inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpastures disease, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosis, lgG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disease, pemphigus vulgaris or related blistering skin disease, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, premature ovarian failure, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, primary ovarian insufficiency, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, stiff-man syndrome, type I diabetes, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (Granulomatosis with polyangiitis) or other Immune vasculitis.

60. The method of claim 59, wherein the lupus is systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, neonatal lupus, or drug-induced lupus.

61. The method of claim 60, wherein the cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DLE), or subacute cutaneous lupus erythematosus.

62. The method of claim 58, wherein the neurological condition is selected from a brain tumor, a brain metastasis, a spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease, and stroke.

63. The method of claim 58, wherein the allergy is selected from food allergy, seasonal allergy, pet allergy, hives, hayfever, allergic conjunctivitis, poison ivy allergy oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, and chemical allergy.

64. The method of claim 58, wherein the allograft rejection is selected from skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, and organ graft rejection.

65. The method of claim 58, wherein the ligament graft rejection is selected from cricothyroid ligament graft rejection, caudal cruciate ligament graft rejection, periodontal ligament graft rejection, suspensory ligament of the lens graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, ulnar collateral ligament graft rejection, radial collateral ligament graft rejection, suspensory ligament of the breast graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection, lateral collateral ligament graft rejection, posterior cruciate ligament graft rejection, medial collateral ligament graft rejection, cranial cruciate ligament graft rejection, and patellar ligament graft rejection.

66. The method of claim 58, wherein the organ graft rejection Is selected from heart graft rejection, lung graft rejection, kidney graft rejection, liver graft rejection, pancreas graft rejection, intestine graft rejection, and thymus graft rejection.

67. The method of claim 58, wherein the graft-versus-host disease arises from a bone marrow transplant or one or more blood cells selected from B-cells, T-cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.

68. The method of claim 57, wherein the inflammatory disease is acute or chronic inflammation.

69. The method of claim 57, wherein the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovarian syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.

70. The method of claim 57, wherein the cancer is selected from adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, Inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (All), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia and hairy cell leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, hodgkln lymphoma, non- hodgkin lymphoma, lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostatecancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma), Werner syndrome, Wilms tumor, or xeroderma pigmentosum.

71. The method of claim 57, wherein the cardiovascular disease is selected from atherosclerosis, heart failure, left heart failure with reduced ejection fraction, left heart failure with preserved ejection fraction, right ventricular failure, congestive heart failure, restrictive cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, idiopathic cardiomyopathy, and hypertension.

72. The method of claim 57, wherein the infertility and pregnancy-associated diseases is selected from recurrent pregnancy loss, pre-eclampsia, preterm labor, fetal growth restriction, or intrauterine growth restriction.

73. A method of regenerating a tissue or organ comprising one or more CD25+ cells, said method comprising contacting the tissue or organ with an effective amount of the antigen-binding protein of claims 1-24, the fusion protein of any one of claims 25-41, or the conjugate of claim 42 or43.

74. The method of claim 73, wherein said tissue or organ is selected from pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerve, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structures, embryo, and testes tissue.

75. The method of claim 73 or 74, wherein said contacting occurs in vitro.

76. The method of claim 73 or 74, wherein said contacting occurs in vivo.

77. The method of claim 76, wherein the method further comprises administering the antigen-binding protein, the fusion protein, or the conjugate into a subject in need thereof.

78. A method for inducing tolerance to a foreign agent and / or preventing or reducing immune response to a foreign agent in a subject in need thereof, said method comprising administering to the subject an effective amount of the antigen-binding protein of claims 1-24, the fusion protein of any one of claims 25-41, or the conjugate of any one of claim 42 or 43.

79. The method of claim 78, wherein the foreign agent is a therapeutic protein or peptide, a viral vector, a bacterial vector, a fungal vector, a biochemical vector, a lipid, carbohydrate, a nucleic acid, a sperm, an oocyte, or an embryo.

80. The method of claim 79, wherein the viral vector is a DNA or RNA vector.

81. The method of any one of claims 55-72 and 77-80, wherein the subject is a mammal.

82. The method of claim 81, wherein the mammal is human.