WO2026006708A2 - Protéines de liaison à l'antigène anti-cd25 et leurs utilisations - Google Patents

Protéines de liaison à l'antigène anti-cd25 et leurs utilisations

Info

Publication number
WO2026006708A2
WO2026006708A2 PCT/US2025/035662 US2025035662W WO2026006708A2 WO 2026006708 A2 WO2026006708 A2 WO 2026006708A2 US 2025035662 W US2025035662 W US 2025035662W WO 2026006708 A2 WO2026006708 A2 WO 2026006708A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
antigen
cdr2
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/035662
Other languages
English (en)
Other versions
WO2026006708A3 (fr
WO2026006708A8 (fr
Inventor
Luigi Franchi
Anthony W. Opipari
Laura PREISS
Ferdinand Huber
Paul-Albert KÖNIG
Annegrit SEIFRIED
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Odyssey Therapeutics Inc
Original Assignee
Odyssey Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Odyssey Therapeutics Inc filed Critical Odyssey Therapeutics Inc
Publication of WO2026006708A2 publication Critical patent/WO2026006708A2/fr
Publication of WO2026006708A3 publication Critical patent/WO2026006708A3/fr
Publication of WO2026006708A8 publication Critical patent/WO2026006708A8/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • 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.
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • CD25 cluster of differentiation 25
  • Tregs 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.
  • a major limitation of the current strategies is their inability to stabilize Tregs phenotype to ensure long-lasting immunoregulation.
  • 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.
  • APCs antigen-presenting cells
  • CD25 Cluster of differentiation 25
  • IL-2R ⁇ or IL2RA interleukin-2 receptor subunit alpha
  • IL-2R ⁇ or IL2RA interleukin-2 receptor subunit alpha
  • 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.
  • 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).
  • CDR3 complementarity determining region 3
  • the CDR3 comprises an amino acid sequence selected from SEQ ID Nos: 4314, and 5211-5251.
  • 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).
  • the CDR1 comprises an amino acid sequence SEQ ID NO: 13 or 2242.
  • 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).
  • the CDR2 comprises an amino acid sequence SEQ ID NO: 2248, or 4335.
  • 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/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
  • 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).
  • 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: 5
  • the single-domain antibody is a VHH, a VNAR, or a VH domain.
  • the VHH is a humanized VHH.
  • 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.
  • the antigen-binding protein binds to human CD25.
  • the antigen-binding protein binds to cyno CD25.
  • 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.
  • the antigen-binding protein has an antagonistic effect upon binding to
  • the antigen-binding protein does not bind to the same epitope(s) on CD25 as IL-2.
  • the antigen-binding protein does not compete with binding CD25 with IL-
  • 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.
  • the single-domain antibody comprises one or more modifications at the amino-terminus and/or the carboxy-terminus.
  • 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.
  • the single-domain antibody comprises a substitution of amino acid residue Glu with Asp (EID) at the first position of the amino-terminus.
  • 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-
  • 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.
  • the fusion protein described herein may comprise two antigen-binding proteins described herein.
  • the fusion protein described herein may comprise four antigen-binding proteins described herein.
  • the one or more antigen-binding proteins bind to the same epitope on CD25.
  • the one or more antigen-binding proteins bind to different epitopes on CD25.
  • the one or more antigen-binding proteins are one or more single-domain antibodies.
  • one or more single-domain antibodies are one or more VHHs.
  • a fusion protein described herein may further comprise an immunoglobulin Fc region.
  • the immunoglobulin Fc region is an Fc region of a human immunoglobulin.
  • the immunoglobulin Fc region is an Fc region of human IgG1, lgG2, lgG3 or lgG4, or a variant thereof.
  • the immunoglobulin Fc region is an Fc region of human IgG1, or a variant thereof.
  • 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.
  • the Fc region of human IgG1 comprises a set of mutations selected from 1).L234A and L235A;
  • the Fc region of human IgG1 comprises L234A, L235A, and P329A.
  • the Fc region of human IgG1 comprises M252Y, S254T, and T256E.
  • the immunoglobulin Fc region is an Fc region of human lgG4, or a variant thereof.
  • the Fc region of human lgG4 comprises one or more mutations selected from S228P, L235E, L235A, and/or F234A according to EU numbering.
  • the Fc region of human lgG4 comprises a set of mutations selected from 1). S228P and L235E;
  • 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.
  • 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.
  • the present disclosure provides a host cell comprising a polynucleotide molecule described herein, or an expression vector described herein.
  • 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.
  • the present disclosure provides a pharmaceutical composition
  • 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.
  • 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:
  • 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.
  • the cell is a regulatory T cell (Treg).
  • contacting of a cell may occur in vitro.
  • contacting of a cell may occur in vivo.
  • a method described herein may further comprise administering the antigen-binding protein, the fusion protein, or the conjugate into a subject in need thereof.
  • 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.
  • the disease or disorder is an immunological disease, inflammatory disease, cancer, cardiovascular disease, or an infertility and pregnancy-associated disease.
  • 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.
  • 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.
  • the lupus is systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, neonatal lupus, or drug-induced lupus.
  • SLE systemic lupus erythematosus
  • cutaneous lupus lupus nephritis
  • neonatal lupus or drug-induced lupus.
  • the cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DLE), or subacute cutaneous lupus erythematosus.
  • 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.
  • 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.
  • the allograft rejection is selected from skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, and organ graft rejection.
  • 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.
  • 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.
  • 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.
  • the inflammatory disease is acute or chronic inflammation.
  • the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovarian syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.
  • 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
  • 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.
  • the infertility and pregnancy-associated diseases is selected from recurrent pregnancy loss, pre-eclampsia, preterm labor, fetal growth restriction, or Intrauterine growth restriction.
  • 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.
  • 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.
  • the contacting of a tissue or organ occurs in vitro.
  • the contacting of a tissue or organ occurs in vivo.
  • a method disclosed herein may further comprises administering the antigen-binding protein, the fusion protein, or the conjugate into a subject in need thereof.
  • 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.
  • 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.
  • the viral vector is a DNA or RNA vector.
  • the subject is a mammal.
  • the mammal is human.
  • 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.
  • VHH variable domain of heavy chain
  • Flgure 2 shows VHH immune library selection for next-generation sequencing (NGS) across the phage display process.
  • NGS next-generation sequencing
  • 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.
  • PCR polymerase chain reaction
  • CDRs complementarity determining regions
  • 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.
  • 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.
  • 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.
  • 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 bar histogram in figure SB shows the mean fluorescent intensity (MFI) of Alexa488 positive cells for C-004 and C-006.
  • MFI mean fluorescent intensity
  • 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.
  • 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 (K D ) 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.
  • SPR surface plasmon resonance
  • 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).
  • 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.
  • 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.
  • 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 (K D ) are included.
  • 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 (K D ) are included.
  • 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-
  • 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 ( K D ) are included.
  • 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 (K D ) are included.
  • 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 (K D ) are included.
  • 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.
  • 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.
  • the antigen described herein is CD25, including human, cynomolgus, and/or mouse CD25.
  • 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.
  • 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.
  • an epitope may include non-peptidic moieties on the antigen, such as saccharides, phosphoryl groups, or sulfonyl groups.
  • an antigen-binding protein refers in its broadest sense to a protein that specifically binds an antigen (e.g., CD25).
  • 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.
  • 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.
  • 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.
  • fibronectin components e.g., fibronectin type III domain (FN3)
  • CD25 or “cluster of differentiation 25", or “interleukin 2 receptor alpha chain”, or “interleukin 2 receptor alpha subunit”, or “IL2R ⁇ ”, 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.
  • CD25 is human CD25.
  • CD25 can be expressed by activated lymphocytes (e.g., activated T lymphocytes and/or activated B lymphocytes).
  • 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 IL2R ⁇ , IL2R (beta) ⁇ (also called CD122), and IL2R (gramma) y (also called CD132) can form a high-affinity IL2R.
  • IL2R ⁇ and IL2R ⁇ can form a pseudo-high affinity receptor.
  • 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.
  • 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.
  • 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.
  • 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.
  • CDRs complementarity determining regions
  • 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.
  • 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.
  • single-domain antibody 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.
  • 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.
  • a single-domain antibody as used herein is a naturally occurring singledomain antibody known as heavy chain antibody devoid of light chains.
  • 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.
  • VHH can be derived from antibodies raised in Camelidae species, e.g., camel, llama, dromedary, alpaca and guanaco.
  • a single-domain antibody may be obtained from a Camelidae VH domain.
  • a single-domain antibody may be obtained from human VH by camelization. See Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which being incorporated by reference, for review of single-domain antibodies.
  • 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 (K D ) of about 1x10 -6 M or less (e.g., less than 10 -6 M, less than 5x10 - 7 M, less than 10 -7 M, less than 5x10* M, less than 10 -8 M, less than 5x10 -9 M, less than 10 -9 M, or less than 10 - 10 M).
  • K D dissociation constant
  • an antigen-binding protein e.g., an antibody or an antibody fragment
  • a target antigen e.g., an antibody or an antibody fragment
  • surface plasmon resonance e.g., BIACORE* assays
  • bio-layer interferometry e.g., bio-layer interferometry
  • ligand binding assays e.g., enzyme-linked immunosorbent assay (ELISA)
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescent-activated cell sorting
  • flow cytometry-based binding assays and the like e.g., 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • antigen-binding proteins e.g., antibodies, such as single-domain antibodies
  • operably linked 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.
  • the term "variant”, “derivative” or “derived from” in the context of proteins or polypeptides refer to: (a) a polypeptide that has at least 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FASTA e.g., FA5TA2 and FA5TA3
  • FASTA 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.
  • 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 control molecule/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.
  • 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.
  • composition contemplated herein refers 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.
  • 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.
  • 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.
  • an effective amount refers 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 active ingredient e.g., anti-CD25 antigen-binding protein
  • An effective amount may be an amount that relieves, lessens, or alleviates 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.
  • antigen-binding proteins e.g., antibodies, such as single-domain antibodies
  • CD25 antigen-binding proteins
  • CD25 Cluster of differentiation 25
  • IL-2R ⁇ interleukin-2 receptor subunit alpha
  • IL-2R ⁇ 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 Natl Acad Sci U S A 103, 2788-2793. 2006).
  • the structure of CD25 is stabilized by several intradomain and two interdomain disulfide bonds.
  • 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.
  • 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-2R ⁇ , then engaging subunit ⁇ 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-2R ⁇ 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).
  • CD25/IL- 2R ⁇ makes no direct contact with the other two subunits ⁇ or y.
  • Deglycosylation experiments of the individual subunits were found to impact the complex formation with the ⁇ subunit aggregating, while the subunits ⁇ and ⁇ were still able to bind to IL-2 (Stauber et al.. Proc Natl Acad Sci U S A 103, 2788- 2793. 2006).
  • the glycosylation on CD25/IL-2R ⁇ is not essential for the interaction with IL-2.
  • IL-2- R ⁇ and IL-2R ⁇ are also part of other interleukin receptor complexes while CD25/IL-2R ⁇ is exclusively found in the IL-2 receptor complex (Liao et al., Immunity 38, 13-25. 2013).
  • 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.
  • antigen-binding proteins e.g., antibodies, such as single-domain antibodies
  • 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 of
  • antigen-binding proteins bind to cynomolgus monkey (“cyno”) CD25.
  • cyno CD25 protein is encoded by the cyno interleukin-2 receptor subunit alpha (IL2RA) gene (NCBI Gene ID: 102123605) and has the amino acid sequence of
  • antigen-binding proteins e.g., antibodies, such as single-domain antibodies
  • 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 of
  • 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.
  • antigen-binding proteins of the present disclosure do not have overlapping epitopes with IL-2.
  • antigen-binding proteins of the present disclosure upon binding to CD25 may impair the binding of IL-2 to CD25.
  • antigen-binding proteins of the present disclosure may have overlapping epitopes with IL-2.
  • the antigen-binding proteins may impair IL-2 binding to CD25.
  • the antigen-binding proteins may compete for binding to CD25 with IL-2.
  • 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.
  • 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.
  • antigen binding proteins of the present disclosure 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.
  • 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.
  • 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.
  • antigen binding proteins of the present disclosure 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.
  • antigen-binding proteins of the present disclosure bind to human CD25.
  • antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human CD25 with a K D of less than about 1x10 -6 M, for example, less than about 5x10 -7 M, less than about 3x10 -7 M, less than about 1x10 -7 M, less than about 8x10 -9 M, less than about 5x10 -8 M, less than about 3x10 - 8 M, less than about 1x10 -8 M, less than about 8x10 - 9 M, less than about 5x10 -9 M, less than about 3x10 - 5 M, less than about 1x10 -9 M, about 1x10 -10 to 1x10 - 5 M, about 1x10 -10 to 5x10 -9 M, about 1x10 -10 to 1x10 -8 M, about 1x10 -10 to 5x10 -8 M, about 1x10 -10 to 5x10 -8 M, about
  • an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 1.6 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 7.6 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 9.4 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to human CD25 with a K D of about 10 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 11 nM.
  • an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 12 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 13 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 14 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 17 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 18 nM.
  • an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 19 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 20 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 21 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 22 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 26 nM.
  • an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 31 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 35 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 49 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 50 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 58 nM.
  • an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 61 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 62 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 66 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 73 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 76 nM.
  • an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 97 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 102 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 107 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 149 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 241 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a K D of about 348 nM.
  • antigen-binding proteins of the present disclosure bind to cynomolgus monkey ("cyno") CD25.
  • antigen-binding proteins (e.g., antibodies such as singledomain antibodies) of the present disclosure may bind to cyno CD25 with a K D of less than about 1x10 -6 M, for example, less than about 5x10 -7 M, less than about 3x10 -7 M, less than about 1x10 -7 M, less than about 8x10 -8 M, less than about 5x10 -9 M, less than about 3x10 -8 M, less than about 1x10 -8 M, less than about 8x10 -9 M, less than about 5x10 -9 M, less than about 3x10 -9 M, less than about 1x10 -9 M, about 1x10 -9 M, about 1x10 -9 M, about 1x10 -10 to 1x10 -9 M, about 1x10 -10 to 5x10 -9 M, about 1x10 -10 to 1x10 -8 M,
  • an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 706 pM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of 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.
  • an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 34 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 48 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 49 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 52 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 57 nM.
  • an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 70 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 79 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 97 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 107 nM.
  • an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 112 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 115 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to cyno CD25 with a K D of about 117 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 119 nM.
  • an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 121 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 131 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 136 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to cyno CD25 with a K D of about 142 nM.
  • an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 146 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 148 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 149 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 162 nM.
  • an antigen- binding protein of the present disclosure binds to cyno CD25 with a K D of about 163 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 186 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 191 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 211 nM.
  • an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 235 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to cyno CD25 with a K D of about 283 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 339 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 380 nM.
  • an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 411 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a K D of about 956 nM. In one embodiment, an antigenbinding protein of the present disclosure binds to cyno CD25 with a K D of about 2.1 ⁇ M.
  • antigen-binding proteins of the present disclosure bind to mouse CD25.
  • antigen-binding proteins of the present disclosure may bind to mouse CD25 with a KD of less than about 1x10 -6 M, for example, less than about 5x10 -7 M, less than about 3x10 - 7 M, less than about 1x10 -7 M, less than about 8x10 -6 M, less than about 5x10 -8 M, less than about 3x10 -8 M, less than about 1x10 -8 M, less than about 8x10 -9 M, less than about 5x10 -9 M, less than about 3x10 -9 M, less than about 1x10 -9 M, about 1x10 -10 to 1x10 - 9 M, about 1x10 - 10 to 5x10 -9 M, about 1x10 - 10 to 1x10 -8 M, about 1x10 - 10 to 5x10 s M, about 1x10 -9 to 1x10 -8 M, about 1x10 -9 to 1x10 -8 M,
  • an antigen-binding protein of the present disclosure binds to mouse CD25 with a KD of about 420 nM.
  • 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.
  • SPR surface plasmon resonance
  • BLI bio-layer interferometry
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescent-activated cell sorting
  • 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.
  • nm wavelength shift
  • association and dissociation rates and affinities can be determined.
  • BU can for example be performed using the Octet* Systems.
  • affinities can be measured in Kinetic Exclusion Assay (KinExA) (see e.g., Drake et al. 2004, Anal.
  • 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.
  • the VHH is a camelid VHH, a humanized VHH or, a camelized VH.
  • a single-domain antibody described herein is a VH domain.
  • a single-domain antibody described herein is a naturally occurring VH domain or engineered VH domain.
  • variable domain of an antigen-binding protein e.g., antibody such as a single-domain antibody
  • the variable domain comprises at least three complementarity determining regions (CDRs) which determine its binding specificity.
  • CDRs complementarity determining regions
  • 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.
  • an anti-CD25 antigen-binding protein e.g., antibody such as a singledomain antibody
  • an anti-CD25 antigen-binding protein 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).
  • CDR1 complementarity determining region 1
  • GR(K/R/S)FSTLI SEQID NO: 37
  • GFTFS(N/S)YA SEQ ID NO: 40
  • c complementarity determining region 1
  • 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).
  • 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).
  • an anti-CD25 antigen-binding protein e.g., antibody such as a singledomain antibody
  • 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)
  • GRSFSTLI SEQ ID NO: 5
  • GR(S/K)FSTLI SEQ ID NO: 32
  • GR(K/R/S)FSTLI SEQ ID NO: 37
  • GFTFS(N/S)YA SEQ ID NO: 40
  • 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).
  • 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).
  • an anti-CD25 antigen-binding protein described herein comprises a CDR1 comprising the sequence GFTFS(N/S)YA (SEQ ID NO: 40).
  • an anti-CD25 antigen-binding protein described herein comprises a CDR1 comprising the sequence GFTLDYYA (SEQ ID NO: 2242).
  • an anti-CD25 antigen-binding protein e.g., antibody such as a singledomain antibody
  • a complementarity determining region 2 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).
  • IYSD(G/S)SGT SEQ ID NO: 4341
  • c complementarity determining region 2
  • 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).
  • 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).
  • 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).
  • 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).
  • 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); and h). ISSTDGRT (SEQ ID NO: 2248)
  • 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).
  • an anti-CD25 antigen-binding protein described herein comprises a CDR2 comprising the sequence IYSD(G/S)SGT (SEQ ID NO: 4341).
  • an anti-CD25 antigen-binding protein described herein comprises a CDR2 comprising the sequence ISSTDGRT (SEQ ID NO: 2248).
  • an anti-CD25 antigen-binding protein e.g., antibody such as a singledomain antibody
  • a complementarity determining region 3 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).
  • CDR3 complementarity determining region 3
  • 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.
  • 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/P)(A/W/F/L)(A/D/E)(A /D/E)(A/Y/V); and b).
  • 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).
  • 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).
  • an anti-CD25 antigen-binding protein e.g., antibody such as a singledomain antibody
  • 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)
  • NAL(G/L/P/Q/W)Y SEQ ID NO: 31
  • NALR(D/H/N/F) SEQ ID NO: 34
  • c) NALR(D/H/N/F)
  • (K/S/T)TLRY SEQ ID NO: 36
  • AKGR(H/N)SGSYYPWD(D/E)Y SEQ ID NO: 39
  • e AKGR(H/N)SGSYYPWD(D/E)Y
  • 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.
  • Provided 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.
  • 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 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
  • 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)
  • a CDR3 comprising an amino acid sequence of SEQ
  • 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)
  • a CDR3 comprising an amino acid sequence of SEQ
  • 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 (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).
  • 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 (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).
  • an anti-CD25 antigen-binding protein e.g., antibody such as a singledomain antibody
  • 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)
  • a CDR3 comprising an amino acid sequence of SEQ
  • 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)
  • a CDR3 comprising an amino acid sequence of SEQ ID NO: 34
  • 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)
  • a CDR3 comprising an amino acid sequence of SEQ ID NO: 34
  • 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:
  • 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;
  • 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:
  • anti-CD25 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • 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.
  • an anti-CD25 antigen-binding protein 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.
  • an anti-CD25 antigen-binding protein 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.
  • anti-CD25 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • 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.
  • an anti-CD25 antigen-binding protein 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.
  • an anti-CD25 antigen-binding protein 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.
  • anti-CD25 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • 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.
  • 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-
  • 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,
  • 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:
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1415-1540.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5211-5225.
  • 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.
  • 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.
  • 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.
  • anti-CD25 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • anti-CD25 antigen-binding proteins 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.
  • 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: 5220;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • CDR1 comprising an amino acid sequence of SEQ ID NO: 2242
  • CDR2 comprising an amino acid sequence of SEQ ID NO: 2248
  • CDR3 comprising an amino acid sequence of SEQ ID NO: 5230
  • 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;
  • 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;
  • CDR1 comprising an amino acid sequence of SEQ ID NO: 2242
  • CDR2 comprising an amino acid sequence of SEQ ID NO: 2248
  • CDR3 comprising an amino acid sequence of SEQ ID NO: 4314;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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.
  • 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.
  • anti-CD25 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • anti-CD25 antigen-binding proteins 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.
  • 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-
  • an anti-CD25 antigen-binding protein e.g., antibody such as a singledomain antibody
  • an anti-CD25 antigen-binding protein 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).
  • 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.
  • 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).
  • an anti-CD25 antigen-binding protein e.g., antibody such as a singledomain antibody
  • a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5279.
  • an anti-CD25 antigen-binding protein e.g., antibody such as a singledomain antibody
  • a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 5282.
  • an anti-CD25 antigen-binding protein e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding protein 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.
  • an anti-CD25 antigen-binding protein e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding protein 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.
  • an anti-CD25 antigen-binding protein e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding protein 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 least
  • 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 least
  • 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 least
  • 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 least
  • 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 least
  • 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 least
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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:
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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:
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an anti-CD25 antigen-binding protein e.g., antibody such as a single-domain antibody
  • 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.
  • an anti-CD25 antigen-binding protein e.g., antibody such as a single-domain antibody
  • Single-domain antibodies e.g., antibody such as a single-domain antibody
  • 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.
  • Affinity maturation strategies can be categorized as either targeted/rational approaches or untargeted/random approaches.
  • 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.
  • the resulting library can be screened by employing standard display techniques such as yeast, phage or ribosome display to select for the best binders.
  • 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 ⁇ 10 7 - 10 9 , phage display ⁇ 10 8 -10 10 and ribosome display ⁇ 10 12 -10 13 (Chan and Groves, 2021).
  • yeast display being able to handle library sizes of ⁇ 10 7 - 10 9
  • phage display ⁇ 10 8 -10 10 e display ⁇ 10 8 -10 10
  • ribosome display ⁇ 10 12 -10 13
  • the selected affinity matured clones may be further evaluated by a developability assessment to test for undesired properties, such as unspecific binding to off-targets or VHH instability.
  • a set of selected residues within the CDRs of a VHH may be mutated (Tiller et al virgin 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.
  • 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.
  • 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).
  • CDR shuffling or swapping is applied for VHH affinity maturation, such as described in Zupancic et al., 2021.
  • 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 and reassembled 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.
  • 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.
  • AHEAD autonomous hypermutation yeast surface display
  • 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.
  • 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.
  • Single-domain antibodies can also be derived from conventional antibodies.
  • 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 display techniques for identification of the most specific antigens.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • all or at least some of these hallmark residues in framework 2 remain of camelid origin when humanizing VHHs.
  • CDR grafting 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).
  • humanized VHH sequences still retain the residues that are relevant for protein A binding.
  • the engineering activities during humanization may be applied to engineer protein A binding properties into a VHH that did previously not interact with protein A (Graille et al., 2000, PNAS).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification.
  • the Kabat scheme is based on structural alignments
  • 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.
  • 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.
  • 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 when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
  • CDR complementary metal-oxide-semiconductor
  • CDR-H1, CDR-H2, CDR-H3 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.
  • a particular CDR e.g., a CDR-H3
  • a CDR-H3 contains the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence
  • 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.
  • 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.
  • the framework sequences may be any suitable framework sequences.
  • the framework sequences may be framework sequences derived from a heavy chain variable domain (e.g., a VH sequence or VHH sequence).
  • 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).
  • 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.
  • 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.
  • 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).
  • Anti-CD25 antigen-binding proteins e.g., antibodies such single-domain antibodies
  • Anti-CD25 antigen-binding proteins 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").
  • 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.
  • 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.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e quilt a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • A alanine
  • the C-terminus of a VHH is normally VTVSS (SEQ ID NO: 2225).
  • a singledomain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering).
  • a single-domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering).
  • the C-terminus of a single-domain antibody 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).
  • VTVSSA SEQ ID NO: 2234
  • VKVSSA SEQ ID NO: 2235
  • VQVSSA SEQ ID NO: 2236
  • single-domain antibodies e.g., VHH
  • single-domain antibodies 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.
  • 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).
  • 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).
  • 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).
  • a single-domain antibody e.g., VHH
  • 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).
  • a single-domain antibody e.g., VHH
  • VHH single-domain antibody
  • a single-domain antibody 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).
  • single-domain antibodies e.g., VHH
  • VHH single-domain antibodies
  • SpA staphylococcal protein A
  • SpG streptococcal protein G
  • 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.
  • single-domain antibodies e.g., VHH
  • VHH single-domain antibodies
  • 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).
  • superposition of a SpA-Fab crystal structure and a VHH allows for visualizing the binding mode.
  • 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.
  • single-domain antibodies e.g., VHH
  • single-domain antibodies comprise one or more modifications at N-terminus to prevent formation of a pyroglutamate and product heterogeneity.
  • the amino acid residue Glu at the first position of the single-domain antibody e.g., VHH
  • Asp EID
  • a single-domain antibody e.g., VHH
  • E1D Asp
  • a single-domain antibody e.g., VHH
  • 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.
  • 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.
  • Ig immunoglobulin
  • 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.
  • Protein A e.g., the Z-domain thereof (affibodies), lmmE7 (immunity proteins), BPTI/APPI (Kunitz domains), Ras- binding protein AF-6 (PDZ-domains), char
  • Anticalins 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 ⁇ -barrels. Lipocalins contain four exposed loops built on the rigid ⁇ -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.
  • 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 mimetic proteins 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 ⁇ -helices separated by loops, which repeats mediate protein— protein interactions.
  • AR ankyrin repeat
  • 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.
  • DARPins contain two to four of these motifs flanked by N- and C- terminal capping motifs to shield hydrophobic regions and allow increased solubility.
  • fibronectin III domains can also be used to generate a suitable non-lg based alternative scaffold (also known as "monobody").
  • FN10 tenth fibronectin type III domain
  • 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.
  • AdnectinsTM is an exemplary scaffold generated using 10 th FN3 domains randomized and displayed in this way.
  • Another exemplary scaffold comprising FN3 domains is a CentyrinTM.
  • CentryrinsTM contain the consensus sequence of FN3 domains of human Tenascin C (TNC), which Is found In the extracellular matrix of various tissues.
  • CentyrinTM 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.
  • CentyrinTM 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.
  • FLAPs fluctuation-regulated affinity proteins
  • 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.
  • the fusion protein or conjugate of the present disclosure comprises a single polypeptide.
  • the fusion protein or conjugate of the present disclosure comprises more than one polypeptide.
  • the fusion protein or conjugate of the present disclosure comprises two polypeptides.
  • 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.
  • the fusion protein or conjugate is multivalent.
  • 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.
  • the fusion protein or conjugate is multispecific.
  • 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 two or more anti-CD25 antigen-binding proteins may comprise the same sequence or may comprise different sequences.
  • the two or more anti-CD25 antigen-binding proteins may bind to the same epitope on CD25 or different epitopes on CD25.
  • a fusion protein or conjugate of the present disclosure may be biparatopic, e.g., if two VHHs bind two different epitopes on CD25.
  • 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).
  • 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.
  • an Fc region refers to a portion of a heavy chain constant region comprising CH2 and CH3.
  • an Fc region comprises a hinge, CH2, and CH3.
  • the hinge can mediate dimerization between two Fc-containing polypeptides.
  • 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.
  • the immunoglobulin Fc region is of IgG, IgE, IgM, IgD, IgA or IgY isotype.
  • 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.
  • 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.
  • 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.
  • a fusion protein or conjugate of the present disclosure can comprise a dimer of Fc regions.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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).
  • n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc).
  • each anti-CD25 VHH may be optionally operably linked to another anti-CD25 VHH via a linker.
  • a fusion protein or conjugate of the present disclosure is bivalent.
  • 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.
  • a fusion protein or conjugate of the present disclosure is tetravalent.
  • 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.
  • 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.
  • a fusion protein or conjugate of the disclosure is hexavalent.
  • 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.
  • 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).
  • 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.
  • 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.
  • the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization.
  • a heterodimerization domain can be incorporated into the fusion protein such that the construct is a heterodimeric fusion protein.
  • the first and second Fc regions may be of the same IgG isotype such as, e.g., IgG1/IgG1, lgG2/lgG2, lgG4/lgG4.
  • the first and second Fc regions may be of different IgG isotypes such as, e.g., lgG1/lgG2, lgG1/lgG4, lgG2/lgG4, etc.
  • the Fc region included in a fusion protein or conjugate of the present disclosure can be mutated or modified.
  • the mutations include one or more amino acid substitutions to reduce an effector function of the Fc region.
  • mutations to Fc regions to alter, such as reduce, effector function are known, including any as described below.
  • 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).
  • the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • 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.
  • 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.
  • 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.
  • nonradioactive assay methods may be employed, such as ACTITM non-radioactive cytotoxicity assay for flow cytometry or CytoTox96TM non-radioactive cytotoxicity assay.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • 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 be carried 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).
  • 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)).
  • mutations that enhance ADCC include modification at Ser239 and Ile332, for example Ser239Asp and He332Glu (S239D, 1332E).
  • mutations that enhance CDC include modifications at Lys326 and Glu333.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • Asp265 Kabat Numbering
  • 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).
  • Pro329A Pro329Ala
  • Pro329Gly P329G
  • 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).
  • 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).
  • 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).
  • the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding.
  • the human IgG1 Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A).
  • the human IgG1 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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 one or more of the Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding.
  • 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.
  • such Fc polypeptides do not engage FcyRs and thus are referred to as "effector silent" or "effector null.”
  • Fc deletion of these three amino acids reduces the complement protein Clq binding.
  • 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.
  • 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
  • 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 P329A also known as "LALAPA” variant
  • the immunoglobulin Fc region of the fusion protein is a variant of human
  • IgG1 Fc region having an amino acid sequence:
  • IgG1 D265A, N297A and P329A also known as "DANAPA” variant
  • 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)
  • 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)
  • 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)
  • 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)
  • 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)
  • 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)
  • 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)
  • 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)
  • the human IgG Fc region is modified to enhance FcRn binding.
  • 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.
  • the Fc region lacks or has reduced fucose attached to the N-linked glycan- chain at N297.
  • fucosylation 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.
  • 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.
  • 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.
  • the Fc domain included in a fusion protein or conjugate is derived from a human Fc domain and comprises mutations to induce heterodimerization.
  • mutations include those referred to as "knob" and "hole” mutations.
  • 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).
  • the "knob" Fc domain comprises the mutation T366W.
  • 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).
  • 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.
  • one member of a heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding.
  • 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.
  • 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 Fc domain does not.
  • the hole-R mutation improves purification of the heterodimer over homodimeric hole Fc domains that may be present.
  • the human IgG Fc region is modified to prevent dimerization.
  • 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.
  • the immunoglobulin Fc region of the fusion protein is of human lgG3 isotype, or a variant thereof.
  • 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).
  • the human lgG3 Fc region Is modified at amino acid 435 to extend the half-life, e.g., Arg435His (R435H).
  • the human lgG3 Fc region lacks Lys447 (ELI index of Kabat et al 1991).
  • the immunoglobulin Fc region of the fusion protein is of human lgG4 isotype, or a variant thereof.
  • the human lgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E).
  • 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).
  • the human lgG4 Fc region is lacks Lys447 (EU index of Kabat et al 1991).
  • 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).
  • 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).
  • 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)
  • 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)
  • the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence:
  • 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)
  • 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)
  • the fusion protein or conjugate contains an immunoglobulin hinge region.
  • the hinge region serves as a linker to connect one or more CD25 binding units (e.g., VHHs) to the Fc region.
  • 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.
  • 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 to serine, e.g., Cys220Ser (C220S).
  • the fusion protein contains a truncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 2223).
  • 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).
  • S228P Ser228Pro
  • ESKYGPPCPPC SEQ ID NO: 2224
  • a fusion protein or conjugate of the present disclosure may comprise sequences other than an Fc region to achieve multimerization (e.g., dimerization).
  • multimerization e.g., dimerization
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to human serum albumin.
  • the binding moiety is a single-domain antibody (e.g., VHH).
  • 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 incorporated herein by reference is its entirety, can be used in the fusion protein or conjugate of the present disclosure.
  • Anti-CD25 antigen-binding proteins 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.
  • a conjugate of the present disclosure comprises a label, which can generate a detectable signal.
  • a label can be used for research or diagnostic purposes, such as for the in vivo detection of cancer.
  • the label is capable of producing, either directly or indirectly, a detectable signal.
  • 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 ⁇ - galactosidase, alkaline phosphatase, or horseradish peroxidase); an imaging agent; or a metal ion.
  • 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 ⁇ - galactosidase, alkaline phosphatase, or horseradish peroxid
  • 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.
  • NMR nuclear magnetic resonance
  • Zirconium-89 may also be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011/056983).
  • Anti-CD25 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • another moiety such as an epitope tag, e.g., for the purpose of purification or detection.
  • epitope tag e.g., for the purpose of purification or detection.
  • 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.
  • Conjugates containing 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.
  • 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.
  • solid phase resins include agarose beads, which are compatible with purifications in a
  • 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.
  • 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).
  • a drug conjugates described herein may allow targeted delivery of a drug moiety to a target tissue (e.g., tumors).
  • a conjugate of the present disclosure comprises a toxin.
  • 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.
  • the toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.
  • anti-CD25 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • CNS central nervous system
  • 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.
  • an anti-CD25 antigen-binding protein e.g., antibody such as singledomain antibody
  • a moiety e.g., an antibody
  • Tf R transferrin 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.
  • an anti-CD25 antigen-binding protein e.g., antibody such as single-domain antibody
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • an anti-CD25 antigen-binding protein e.g., antibody such as singledomain antibody
  • one or more second moieties e.g., about 1 to about 20 moieties per molecule, optionally via a linker.
  • 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 or more 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.
  • 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.
  • MC 6- maleimidocaproyl
  • MP maleimidopropanoyl
  • val-cit valine-citrulline
  • ala-phe alaninephenylalanine
  • 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.
  • Conjugates of an anti-CD25 antigen-binding protein e.g., antibody such as single-domain antibody
  • second moiety e.g., cytotoxic agent
  • 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
  • SPDP N-succinimi
  • Conjugates of the present disclosure can be prepared by a variety of methods.
  • 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.
  • Nucleophilic groups on proteins including antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups (e.g., lysine), (iii) side chain thiol groups (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.
  • active esters such as NHS esters, HOBt esters, haloformates, and acid halides
  • alkyl and benzyl halides such as haloacetamides
  • aldehydes, ketones, carboxyl, and maleimide 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, hal
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 the fusion protein.
  • the fusion protein can be recovered from the cell culture and purified using techniques known in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GS-linker glycines and serines
  • GS-linker a monomer or multimer of GnS (SEQ ID NO: 2195) or SG n (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.
  • the linker is or comprises a monomer or multimer of repeat of G 4 S (SEQ ID NO: 2151), e.g., (GGGGS)n (SEQ ID NO: 2197).
  • 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) (SEQ ID NO: 2199), six consecutive glycines (6Gly) (SEQ ID NO: 2200), seven consecutive glycines (7Gly) (SEQ ID NO:
  • a GS-linker used herein comprises an amino acid sequence selected from
  • 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.
  • 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.
  • the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via a "rigid" peptide linker.
  • a rigid peptide linker comprises PAPAPAPAPAPAPAPAPAP (SEQ ID NO:
  • a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 1
  • a rigid peptide linker comprises A(EAAAK) n A (SEQ ID NO: 2209), where n is any integer, e.g., 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10.
  • a polynucleotide molecule may be used totransform/transfect a host cell or host organism, e.g., for expression and/or production of a polypeptide.
  • Suitable hosts or host cells for production of an anti- CD25 polypeptides described herein include any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
  • a host or host cell comprising a polynucleotide molecule encoding an anti-CD25 antigen-binding protein polypeptide or fusion protein described herein is also encompassed by the present disclosure.
  • Polynucleotides encoding the two or more polypeptide chains (when present and differ from one another) of an anti-CD25 antigen-binding protein or fusion protein of the present disclosure can be inserted into separate vectors, or, optionally, Incorporated into the same expression vector.
  • the recombinant expression vectors of the invention may include regulatory sequences that control the expression of genes encoding the polypeptide chain(s) in a host cell.
  • the design of the expression vector, including the selection of regulatory sequences, may depend on the choice of the host cell to be transformed and/or the desired level of protein expression.
  • suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • viral elements include those described in, e.g., U.S. Pat. Nos. 5, 168,062; 4,510,245; and 4,968,615; the disclosures of each of which are incorporated herein by reference.
  • Recombinant expression vectors of the present disclosure may carry additional sequences, such as sequences that regulate replication of the vector In host cells (e.g., origins of replication) and selectable marker genes.
  • a selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., US 4,399,216; US 4,634,665; and US 5,179,017; the disclosure of each of which is incorporated herein by reference in its entirety).
  • the selectable marker gene confers resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin, or cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced.
  • Suitable selectable marker genes can include the dihydrofolate reductase (DHFR) gene (for use in DHFR deficient host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • Vectors of the present disclosure may further include sequence elements that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5' and 3' untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the
  • viral vectors can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell).
  • Viral vectors are particularly useful for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents to Induce gene Integration.
  • Suitable viral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses (AAV) such as AAV2, AAV8, AAV9), negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpes virus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), baculovirus, coronavirus, and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canary
  • viruses useful for delivering polynucleotides encoding polypeptides of the present disclosure include, for example Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus.
  • retroviruses include, but are not limited to, avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M.1996. Fundamental Virology, DMKDN Fields, PM Howley, ed.
  • compositions and methods of the present disclosure include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline sarcoma virus, feline leukemia virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.
  • the present disclosure also provides host cells or host organisms that comprise the polynucleotides or vectors encoding the anti-CD25 antigen-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or other relevant polypeptides described herein.
  • Suitable host cells or host organisms can be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • Host cells can also include cells transfected in vivo with a polynucleotide(s) or vector provided herein.
  • Example eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris); plant cells; and insect cells.
  • mammalian cells include, but are not limited to, NSO cells, PER.C6* cells (Crucell), COS cells, SP2/0 cells, and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells.
  • Exemplary prokaryotic cells include bacterial cells such as Escherichia coli.
  • the present disclosure also provides methods of producing the anti-CD25 antigen-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or conjugates described herein.
  • anti-CD25 antigen-binding proteins e.g., antibodies including single-domain antibodies
  • fusion proteins e.g., fusion proteins, or conjugates described herein.
  • a method may comprise transforming/transfecting a host cell or host organism with a polynucleotide encoding an anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or other relevant polypeptide(s) described herein, expressing the anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or other relevant polypeptide(s) in the host, optionally followed by one or more isolation and/or purification steps.
  • an anti-CD25 antigen-binding protein e.g., antibody such as single-domain antibody
  • fusion protein e.g., fusion protein, or other relevant polypeptide(s) described herein
  • recombinant expression vectors encoding one or more polypeptide(s) of an anti-CD25 antigen-binding protein e.g., antibody such as single-domain antibody
  • fusion protein, or conjugate of the present disclosure are introduced into mammalian host cells
  • the host cells are cultured for a period of time sufficient to allow for expression of the protein(s) or polypeptide(s) in the host cells or secretion of the protein(s) or polypeptide(s) into the culture medium in which the host cells are grown.
  • Protein(s) or polypeptide(s) can be recovered from the culture medium using standard protein purification methods.
  • Host cells can also be used to produce portions of intact antibodies, such as VHH domains.
  • a protein or polypeptide of the present disclosure has been produced by recombinant expression, it can be purified by any method known in the art for purification of a protein or polypeptide, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for CD25 after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the proteins or polypeptides of the present disclosure can be fused to heterologous polypeptide sequences described herein (e.g., His-tag) or otherwise known in the art to facilitate purification or to produce therapeutic conjugates below). Once isolated, a protein or polypeptide of the present disclosure can, if desired, be further purified, e.g., by high performance liquid chromatography, or by gel filtration chromatography, such as on a SuperdexTM column.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for CD25 after Protein
  • the present disclosure also provides a composition comprising anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or conjugate of the present technology, at least one polynucleotide molecule encoding the same, at least one vector comprising such a polynucleotide molecule, or at least one host cell comprising the polynucleotide molecule or vector.
  • the composition may be a pharmaceutical composition.
  • the composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be Incorporated into the compositions.
  • suitable formulations include, but are not limited to, solutions, suspensions, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al.
  • a pharmaceutical composition of the present disclosure may be formulated according to its intended route of administration.
  • suitable routes of administration include, e.g., intravenous, subcutaneous, intratumoral, oral (e.g., buccal, sublingual), intranasal, inhalation, intraocular, intramuscular, intradermal, transdermal (i.e., topical), intraperitoneal, transmucosal, vaginal, and rectal administration, or injection to the CNS/brain (e.g., intraspinal, intracerebral, or intrathecal administration).
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; fixed oils; chelating agents such as ethylenedlaminetetraacetlc acid (EDTA); buffers such as phosphates, acetates, or citrates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of plastic or glass.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include, for example, physiological saline, bacteriostatic water, Cremophor EL*, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition is preferably sterile and has a proper fluidity. In most embodiments, the composition is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the contamination by microorganisms can be achieved by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Step injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation include vacuum drying and/or freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions may include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, capsules, or liquid forms. Formulation In tablet and liquid forms may be used for protease Insensitive VHHs.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemlc administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • compounds of the present disclosure may be formulated to facilitate crossing of the blood-brain barrier.
  • anti-CD25 antigen-binding proteins e.g., antibody such as single-domain antibody
  • fusion proteins, or conjugates of the present disclosure may be encapsulated into brain targeted liposomes, lipid nanoparticles, lipid microparticles, or lipid microcapsules for brain delivery.
  • Example liposomes delivery systems are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.
  • the active compounds are prepared with carriers that can protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,811, which is incorporated herein by reference in its entirety.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure is dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in diagnostic kits with instructions for use.
  • the pharmaceutical compositions are administered In an amount effective for treatment or prophylaxis of the specific indication.
  • the therapeutically effective amount is typically dependent on the weight of the subject being treated, the physical or health condition of the subject, the extensiveness of the condition to be treated, or the age of the subject being treated.
  • the pharmaceutical composition may be administered in an amount in the range of about 50 pg/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 100 pg/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 100 pg/kg body weight to about 20 mg/kg body weight per dose.
  • the pharmaceutical composition may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.
  • the frequency and the duration of the treatment can be adjusted.
  • Effective dosages and schedules for administering a pharmaceutical composition of the present disclosure may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • interspecies scaling of dosages can be performed using well- known methods in the art (e.g., Mordent! et al., 1991, Phdomalnaceut. Res. 8:1351).
  • the pharmaceutical composition may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered In an amount In the range of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 200 mg per dose.
  • the antigen-binding proteins of the present disclosure are administered as a viral vector (e.g., an AAV)
  • dose ranges and frequency of administration of the viral vector described herein can vary depending on the nature of the viral vector, and the medical condition, as well as parameters of a specific patient and the route of administration used.
  • viral vector compositions can be administered to a subject at a dose ranging from about 1xio 5 plaque forming units (pfu) to about 1x10 15 pfu, depending on mode of administration, the route of administration, the nature of the disease and condition of the subject.
  • the viral vector compositions can be administered at a dose ranging from about 1x10 8 pfu to about 1x10 15 pfu, or from about 1x10 10 pfu to about 1x10 15 pfu, or from about 1x10 8 pfu to about 1x10 12 pfo.
  • a more accurate dose can also depend on the subject in which it is being administered. For example, a lower dose may be required if the subject is Juvenile, and a higher dose may be required If the subject Is an adult human subject. In certain embodiments, a more accurate dose can depend on the weight of the subject.
  • a juvenile human subject can receive from about 1x10 8 pfu to about 1x10 10 pfu, while an adult human subject can receive a dose from about 1x10 10 pfu to about 1x10 12 pfu.
  • Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, intraspinal, intracerebral, intrathecal and oral routes.
  • the composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • a pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida.
  • a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.
  • the Injectable preparations may include dosage forms for Intravenous, subcutaneous, intracutaneous, intramuscular, intratumoral, intraperitoneal, intraspinal, intracerebral, and intrathecal injections, drip infusions, etc. in one embodiment, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • the amount of the antigen-binding proteins described herein may be about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, the antigen-binding proteins described herein may be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.
  • the pharmaceutical composition may be administered as needed to a subject.
  • an effective dose of the pharmaceutical composition is administered to a subject one or more times.
  • an effective dose of the pharmaceutical composition is administered to the subject once a month, less than once a month, such as, for example, every two months, every three months, or every six months.
  • an effective dose of the pharmaceutical composition is administered more than once a month, such as, for example, every two weeks, every week, twice per week, three times per week, daily, or multiple times per day.
  • An effective dose of the pharmaceutical composition is administered to the subject at least once.
  • the effective dose of the pharmaceutical composition may be administered multiple times, including for periods of at least a month, at least six months, or at least a year.
  • the pharmaceutical composition is administered to a subject as needed to alleviate one or more symptoms of a condition.
  • a pharmaceutical composition of the present disclosure may be administered to a subject at levels lower than that required to achieve the desired therapeutic effect and the dosage may be gradually increased until the desired effect is achieved.
  • a pharmaceutical composition of the present disclosure may be administered at a high dose and subsequently administered progressively lower doses until a therapeutic effect is achieved.
  • a suitable daily dose of an antigen-binding protein of the invention is an amount of the antibody which is the lowest dose effective to produce a therapeutic effect.
  • compositions of the present disclosure may optionally include more than one active agent.
  • compositions of the present disclosure may contain an anti-CD25 antigenbinding protein conjugated to, admixed with, or administered separately from another pharmaceutically active molecule, e.g., Treg cell, or an additional agent that is useful for induction of Treg cell expansion.
  • an anti-CD25 antigen-binding protein may be admixed with one or more additional active agents, such as IL-2 or TNFa, to treat an immunological disease, e.g., a disorder described herein.
  • compositions of the present disclosure may be formulated for coadministration or sequential administration with one or more additional active agents that can be used to attenuate CD8+ T-cell growth.
  • additional active agents that can be used to attenuate cytotoxic T-cell proliferation and that can be conjugated to, admixed with, or administered separately from an anti-CD25 antigen-binding protein of the present disclosure include cytotoxic agents, e.g., those described herein.
  • a method of using anti-CD25 antigen-binding proteins, fusion proteins, or conjugates of the present disclosure to effectively target a CD25-expressing cell such as a regulatory T (Treg) (e.g., CD4+, CD25+, FOXP3+ Treg cells).
  • a regulatory T e.g., CD4+, CD25+, FOXP3+ Treg cells.
  • the methods may comprise contacting the cell (e.g., Treg) with an anti-CD25 antigen-binding protein, fusion protein or conjugate described herein.
  • the methods may be carried out in vitro or in vivo.
  • the methods can further comprise administering the anti-CD25 antigen-binding protein, fusion protein or conjugate described herein into a subject.
  • Tregs are a subset of T cells that play a crucial role in peripheral self-tolerance and the prevention of autoimmunity.
  • Tregs have been identified as a CD4 subset that specifically express CD25, the high affinity IL-2 receptor alpha chain (Sakaguchi et al., 1995).
  • FOXP3 transcription factor was identified as CD4 Treg's master regulator (Hori et al., 2003).
  • FOXP3 deficiency leads to systemic autoimmunity in both mouse and human in which it causes the Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome due to Tregs deficiency and unregulated effector T cell function (Bennett et al., 2001).
  • CD4 Tregs can differentiate during T cell development (thymic "tTregs”) or in the periphery (peripheral "pTregs”) under non-inflammatory T cell receptor stimulation (Wing et al., 2019).
  • CD4 Tregs regulate immune response through various mechanisms including the secretion of regulatory cytokines (e.g., IL-10, IL-35, TGF- ⁇ ), IL-2 scavenging, adenosine production, direct cytotoxicity and dendritic cell regulation (Vlgnali et al., 2008).
  • regulatory cytokines e.g., IL-10, IL-35, TGF- ⁇
  • IL-2 scavenging adenosine production
  • direct cytotoxicity and dendritic cell regulation Vlgnali et al., 2008.
  • regulatory T cells or “Treg” as used herein are meant to encompass all the above-described subsets of regulatory T cells.
  • tTregs have enhanced affinity for MHC Il-presented self-antigen peptide and have a TCR repertoire that is non-overlapping with effector CD4 T cells (Fazilleau et al., 2007; Hsieh et al., 2006; Pacholczyk et al., 2006). Therefore, self-antigen recognition in the periphery can induce tTregs activation (Moran et al., 2011).
  • Tregs can suppress effector cells that have different antigen specificity through bystander suppression (Thornton and Shevach, 2000; Yeh et al., 2017; Yu et al., 2005) by regulating antigen presenting cells or soluble factors.
  • Tregs retain some plasticity and can lose FOXP3 expression.
  • These so called “ex-Tregs” have increased level of FOXP3 promoter methylation and lower FOXP3 expression compared to Tregs and can acquire effector function (Zhou et al., 2009).
  • the demethylation of FOXP3 promoter particularly in the "Treg-speclflc demethylated region” (TSDR) (Huehn et al., 2009), stabilizes gene expression.
  • TSDR Tet-speclflc demethylated region
  • human Tregs exposed to IL-2 + inflammatory cytokines have been shown to lose FOXP3 expression while upregulating RORg and IL-17, a feature associated with TH17 cells.
  • Treg fragility Instability of the Treg phenotype in the presence of inflammatory cytokines can be referred to as "Treg fragility” and is of crucial relevance for therapeutic purpose in autoimmune diseases. Indeed, to induce a long-lasting therapeutic benefit, it is important to stabilize the phenotype and function of Tregs and prevent their conversion to pathogenic cells that further contribute to disease.
  • anti-CD25 antigen-binding proteins e.g., antibody such as single-domain antibody
  • fusion proteins conjugates, polynucleotide molecules, vectors, and/or host cells described herein, or pharmaceutical compositions thereof
  • an anti- CD25 antigen-binding protein e.g., antibody such as single-domain antibody
  • a fusion protein e.g., a conjugate, a polynucleotide molecule, a vector, or a host cell for use as a medicament.
  • a (prophylactic and/or therapeutic) method of treating a disease or disorder comprising administering, to a subject in need thereof, a pharmaceutically active amount of an antkCD25 antigen-binding protein (e.g., antibody such as single-domain antibody), a fusion protein, a conjugate, a polynucleotide molecule, a vector, or a host cell described herein.
  • an antkCD25 antigen-binding protein e.g., antibody such as single-domain antibody
  • a fusion protein e.g., a fusion protein, a conjugate, a polynucleotide molecule, a vector, or a host cell described herein.
  • the diseases or disorder that can be treated with the compositions and methods described herein include, but are not limited to, immunological diseases (e.g., autoimmune diseases), inflammatory diseases, cancers, cardiovascular diseases (e.g., 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, hypertension) infertility and pregnancy-associated diseases (e.g., recurrent pregnancy loss, pre-eclampsia, preterm labor, fetal growth restriction, intrauterine growth restriction).
  • immunological diseases e.g., autoimmune diseases
  • inflammatory diseases e.g., cancers
  • cardiovascular diseases e.g., atherosclerosis, heart failure, left heart failure with reduced ejection fraction, left heart failure with preserved ejection fraction, right ventricular failure, congestive heart failure, restrictive
  • immunological diseases that can be treated with the compositions and methods described herein include, but are not limited to, autoimmune diseases, allergies, asthma, neurological diseases, metabolic diseases (e.g., diabetes), macular diseases (e.g., macular degeneration), muscular atrophy, diseases related to miscarriage, vascular diseases (e.g., atherosclerosis), diseases related to bone loss (e.g., bone loss as a result of menopause or osteoporosis), blood disorders (e.g., hemophilia), musculoskeletal disease, diseases related to growth receptor expression or activity, obesity, graft- versus-host disease (GVHD), or allograft rejections.
  • autoimmune diseases e.g., allergies, asthma, neurological diseases, metabolic diseases (e.g., diabetes), macular diseases (e.g., macular degeneration), muscular atrophy, diseases related to miscarriage, vascular diseases (e.g., atherosclerosis), diseases related to bone loss (e.g., bone loss as a result of menopause
  • the compositions and methods described herein are used to treat an autoimmune disease.
  • 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.
  • the compositions and methods described herein are used to treat lupus.
  • the lupus is systemic lupus erythematosus (SLE), cutaneous lupus (including acute cutaneous lupus, chronic cutaneous lupus erythematosus, or discoid lupus erythematosus (DLE) and subacute cutaneous lupus erythematosus), lupus nephritis, neonatal lupus, or drug-induced lupus.
  • the autoimmune disease is atopic dermatitis, psoriasis, systemic lupus erythematosus, or arthritis.
  • the compositions and methods described herein are used to treat allergy.
  • the allergy is an allergic conjunctivitis, chemical allergy, cosmetic allergy, drug allergy, dust allergy, food allergy, hayfever, hives, mold allergy, pet allergy, poison ivy allergy oak allergy, or seasonal allergy.
  • the compositions and methods described herein are used to treat a neurological condition.
  • the neurological condition is 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, or stroke.
  • ALS amyotrophic lateral sclerosis
  • compositions and methods described herein are used to treat a graft rejection.
  • anti-CD25 antigen-binding proteins of the present disclosure may treat graft rejections, e.g., by binding CD25 receptors on the surface of autoreactive CD8+ T-cells that bind antigens presented on the surface of the graft and inducing apoptosis in these CD8+ T-cells, or by inducing the expansion of Treg cells that may subsequently eliminate autoreactive CD8+ T-cells.
  • graft rejections that can be treated with the compositions and methods described herein include, without limitation, skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection (e.g., anterior cruciate ligament graft rejection, anterior sacroiliac ligament graft rejection, caudal cruciate ligament graft rejection, cranial cruciate ligament graft rejection, cricothyroid ligament graft rejection, dorsal radiocarpal ligament graft rejection, inferior pubic ligament graft rejection, lateral collateral ligament graft rejection, medial collateral ligament graft rejection, palmar radiocarpal ligament graft rejection, patellar ligament graft rejection, periodontal ligament graft rejection, posterior cruciate ligament graft rejection, posterior sacroiliac ligament graft rejection, radial collateral ligament graft rejection, sacrospinous ligament graft rejection, sacrotuberous ligament graft rejection, superior pubic ligament graft rejection, suspensory ligament of the breast
  • the compositions and methods described herein are used to treat a graft- versus-host disease.
  • the graft-versus-host disease arises from a bone marrow transplant or one or more blood cells such as 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.
  • the compositions and methods described herein are used to treat an inflammatory disease.
  • the inflammatory disease may be acute or chronic inflammation.
  • the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovarian syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.
  • the compositions and methods described herein are used to treat a cancer.
  • the cancer is an 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
  • anti-CD25 antigen-binding proteins of the present disclosure can also be used to treat a patient in need of organ repair or regeneration, e.g., by inducing the proliferation of cells within a damaged tissue or organ. While not wishing to be bound by any theory, it is contemplated that agonistic CD25 antibodies may stimulate organ repair or regeneration, e.g., by binding CD25 on the surface of cells within damaged tissue to induce TRAF2/3- and/or NF-xB-mediated cell proliferation.
  • Additional diseases that can be treated with the compositions and methods of the present disclosure include genetic diseases with an immunological phenotype. Exemplary genetic diseases with an immunological phenotype are described in, e.g., Table 52 of Tangye et al., Journal of Clinical Immunology volume 42, pagesl473-1507 (2022), which is incorporated herein by reference in its entirety.
  • patients receiving an anti-CD25 treatment of the present disclosure can be monitored for their responsiveness to the treatment.
  • a physician may monitor the response of a mammalian subject (e.g., a human) to treatment with anti-CD25 antigen-binding proteins of the present disclosure by analyzing the quantity of IFNy secreted by CD8+ T-cells within a particular patient.
  • a composition of the present disclosure may be capable of reducing IFNy secretion by between 1% and 100% (e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%).
  • a physician may monitor the responsiveness of a subject (e.g., a human) to treatment with a composition of the present disclosure by analyzing the Treg cell population in the lymph of a particular subject. For example, a physician may withdraw a sample of blood from a mammalian subject (e.g., a human) and determine the quantity or density of a population of Treg cells (e.g., CD4+- CD25+ FOXP3+ Treg cells or CD17+ Treg cells) using established procedures, such as FACS analysis.
  • a mammalian subject e.g., a human
  • Treg cells e.g., CD4+- CD25+ FOXP3+ Treg cells or CD17+ Treg cells
  • high counts of Treg cells can be indicative of efficacious therapy, while lower Treg cell counts may indicate that the patient is to be prescribed or administered higher dosages of the anti-CD25 antigen-binding protein of the present disclosure until, e.g., an ideal Treg cell count is achieved.
  • a physician of skill in the art may monitor the effect of treatment by administration of a composition of the present disclosure to a subject suffering from an immunological disorder, such as an autoimmune disease described herein, by analyzing the quantity of autoreactive CD8+ T-cells within a lymph sample isolated from the patient.
  • Anti-CD25 antigen-binding proteins of the invention may attenuate the proliferation of autoreactive T-cells, e.g., by binding CD25 at the surface of an autoreactive T-cell and inducing apoptosis, and/or by stimulating the expansion of Treg cells that subsequently eliminate autoreactive T lymphocytes.
  • Treatment with anti-CD25 antigen-binding proteins may lead to reduced quantities of autoreactive T-cells within the lymph isolated from a patient receiving treatment, and a rapid decline in the population of autoreactive T-cells in a lymph sample isolated from such a patient may indicate effective treatment.
  • a physician may prescribe the patient higher doses of the antibody or an antigen-binding fragment thereof or may administer the anti-CD25 antigen-binding proteins with higher frequency, e.g., multiple times per day, week, or month.
  • Anti-CD25 antigen-binding proteins described herein may be administered as a monotherapy or in combination with one or more additional therapeutic agents.
  • anti-CD25 antigen-binding proteins of the present disclosure may also be admixed, conjugated, or administered with, or administered separately from, another agent that promotes Treg cell proliferation.
  • Additional agents that can be used to promote Treg cell expansion include, e.g., IL-2 and TNF ⁇ , the cognate ligand for CD25.
  • compositions of the invention may be formulated for coadministration or sequential administration with one or more additional active agents that can be used to inhibit CD8+ T-cell growth.
  • additional active agents that can be used to inhibit cytotoxic T-cell proliferation and that can be conjugated to, admixed with, or administered separately from an anti-CD25 antigen-binding protein of the present disclosure include cytotoxic agents, e.g., those described herein.
  • cytotoxic agents that can be conjugated to, admixed with, or administered separately from anti-CD25 antigen-binding protein of the present disclosure include, but not limited to, 13-cis retinoic acid, 14-hydroxy-retro-retinol, 2-chloro-2'-deoxyadenosine, 2-Chloro-2 , -arabino-fluoro-2'- deoxyadenosine, 2-chlorodeoxyadenosine, 2-chlorodeoxyadenosine (2-Cda), 2 -deoxycoformycin, 3- methyl TTNEB, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, 9-cis retinoic acid, aclarubicin, acodazole hydrochloride, acronine, adozelesin, adozelesin, adriamycin, aldesleukin, all-trans retinoi
  • ⁇ ество antigen-binding protein of the present disclosure include, but are not limited to, 2' deoxycoformycin (DCF), 1,25 dihydroxyvitamin D3, 5-ethynyluracil, 9-dioxamycin, abiraterone, acylfulvene, adecypenol, ALL-TK antagonists, ambamustine, amidox, amifostlne, aminolevulinic acid, amrubicin, anagrelide, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, antiandrogen, prostatic carcinoma, anti-dorsalizing morphogenetic protein-1, antiestrogen, antineoplaston, antisense oligonucleotides, aphldlcolln glycinate, apoptosis gene modulators, apoptosis regulators,
  • DCF 2' deoxycoformycin
  • anti-CD25 antigen-binding proteins of the present disclosure may be admixed, conjugated, or administered with, or administered separately from, an anti-inflammatory agent.
  • anti-inflammatory agents useful In conjunction with the compositions and methods of the invention include steroids, colchicine, hydroxychloroquine, sulfasalazine, dapsone, methotrexate, mycophenolate mofetil, azathioprine, cyclosporine, sirolimus, everolimus, azathioprine, leflunomide, mycophenolate, inhibitors of IL-1/l L-2/1 L-4/IL5/I L-6/1 L-13/1 L-17/1 L- 23/TNF/complement/BAFF/interferon/JAK/CD28/lgE/lntegrins/T cell costimulation pathway or B-cell depleting agents.
  • anti-CD25 antigen-binding proteins of the present disclosure may be admixed, conjugated, or administered with, or administered separately from, an immunotherapy agent.
  • immunotherapy agents useful in conjunction with the compositions and methods of the invention include an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-Ll agent, an anti-PD-L2 agent, a TN Fa cross-linking agent, a TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti- CD40 agent, an anti-4-lBB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILRl agent, an anti-TRAILR2 agent, an anti-TWEAKR agent, an anti-TLIA agent, an anti-LIGHT agent, an anti-BTLA agent, an anti-LAG3 agent, an anti-Siglecs agent, an anti-ICOS ligand agent, an anti-B7-H3 antibody; an anti-CTLA-4 agent, an anti-PD
  • H4 agent an anti-VISTA agent; an anti-TMIGD2 agent; an anti-BTNL2 agent; an anti-CD48 agent; an anti ⁇
  • the immunotherapy agent described herein may be, for example, an antibody, a small molecule, or a chimeric antigen receptor.
  • anti-CD25 antigen-binding protein of the present disclosure can also be admixed with, co-administered with, or administered separately from Bacillus Calmette-Guerin (BCG), a bacterial strain that has been used to treat a variety of immunological disorders, such as type I diabetes, multiple sclerosis, scleroderma, Sjogren's disease, systemic lupus erythematosus, Grave's disease, hypothyroidism, Crohn's disease, colititis, an autoimmune skin disease, and rheumatoid arthritis, among others.
  • BCG Bacillus Calmette-Guerin
  • anti-CD25 antigen-binding protein of the present disclosure may be Included In a therapeutic regimen in combination with BCG for the treatment of an immunological disorder (e.g., one of those described above, such as type I diabetes or rheumatoid arthritis).
  • the anti-CD25 antigenbinding protein may be co-administered with BCG, e.g., by an injection route described herein.
  • the anti-CD25 antigen-binding protein may be administered separately from a BCG- containing composition.
  • BCG to treat immunological disorders has been described, e.g., in US 6,660,487; and US 6,599,710; the disclosures of each of which are incorporated herein by reference in its entirety.
  • PBMCs Peripheral blood mononuclear cells
  • Conventional and heavy chain IgH cDNA fragments were amplified by polymerase chain reaction (PCR) using primers annealing to the IgH leader sequence region and the CH2 region.
  • the resulting amplicons represented the VHH and VH cDNAs, respectively.
  • the VHH fragment was isolated and used as template for a nested PCR to introduce appropriate endonuclease recognition sites for cloning into the pQBl phagemid in frame with gene III.
  • Libraries were transformed into electrocompetent £ co// TGI cells. In total, six libraries were built, with 95.5% to 100% VHH insert frequency and maximum library sizes between 4.2x10 8 and 2.4x10 9 . Phage for phage display was prepared following standard protocols.
  • This panning regimen was implemented to identify binders that crossreacted with human and mouse CD25. Antigen concentration in the second panning round was reduced by a factor of 10 and 100 to favor the retention of strong binders. High affinity CD25 bindings were enabled to drive cell specificity.
  • Panning substrates were commercially purchased (see Table 3). The panning substrates were immobilized either by direct coating on enzyme-linked immunoassay (ELISA) plates or by binding of biotinylated antigen on neutravidin-coated ELISA plate. Glycerol stocks were prepared from all outputs and are stored at -80*C.
  • Panning outputs were analyzed by random clone picking/periplasmic extract (PE)-ELISA/Sanger sequencing (QVQ) and next-generation sequencing (NGS; Genewiz/PipeBio).
  • rescued outputs of the first and second panning rounds were plated out and 460 random single clones (equal numbers of colonies from each condition) were selected to create masterplates (96-well format). From the masterplates, expression cultures in deep-well plates were inoculated to produce periplasmic extracts containing monoclonal VHH. Periplasmic extracts were used to determine binding of individual VHHs to human, mouse and cynomolgus antigen by ELISA. For conditions where the panning substrate was biotinylated and captured by neutravidin, background binders were identified by ELISA with neutravidin. All masterplates were sequenced by the Sanger method.
  • each library was sequenced with a total of 20 million reads, compared to the first and second round of panning with 2 million reads each.
  • This strategy allowed for covering sufficient sequence space in the libraries, as well as in the panning eluates.
  • a spike-in of 30% of a standard PhiX reference genome control into the sequencing reaction helped to provide a technical quality control for assessing sequencing accuracy.
  • the NGS raw data contained multiplexed sequencing reads, which were de-multiplexed based on the sample-specific barcodes. The de-multiplexed data containing unmerged sequencing reads were then processed by employing an NGS analysis platform.
  • V-body sequences were clustered, allowing for a detailed analysis of V- body enrichment during phage display, sequence diversity, CDR3 length distribution and cluster abundance.
  • Identified V-bodies which can be classified into eight distinct clusters, as follows: C-001 (group A), C-002 (group B), and C-003 (group C), C-004/C-005 (group D), C-006 (group E), C-007 (group F), C-008 (group G), and C-009/C-010 (group H).
  • Table 4-1 to Table 4-24 display the amino acid frequency distribution at each amino acid (AA) position (IMGT) for CDR1, CDR2 and CDR3 for the eight clusters.
  • Table 5 provides 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 VHH domain for the identified V-bodies.
  • HEK293 cells were transfected with plasmids encoding for respective antigens. After 48 to 72 hours, binding was measured by incubation of His-tagged V-bodies with cells at various fixed concentrations, followed by washing and detection with Alexa488 fluorophore-labelled anti-His antibodies.
  • HEK293T cells were transiently transfected with a plasmid encoding human CD25 (hCD25; hCD25_pcDNA3.4.dna). After 48 hours, HEK293T cells were harvested and incubated with 100 nM purified His-tagged (myc-his tag) VHHs. VHH binding was then detected using an Alexa488-labeled anti-His tag antibody and measured by flow cytometry (iQue).
  • HEK293T cells were transiently transfected with a plasmid encoding cynomolgus CD25 (cCD25; cCD25_pcDNA3.4.dna) (top panel) or mouse CD25 (mCD25; mCD25_pcDNA3.4.dna) (bottom panel). After 48 hours, HEK293T cells were harvested and incubated with 100 nM purified His-tagged (myc-his tag) VHHs. VHH binding was then detected using an Alexa488- labeled anti-His tag antibody and measured by flow cytometry (iQue).
  • Flgures 6A-6B show testing of human CD25 V-body binding across a range of concentrations for C- 004 and C-006.
  • V-bodies were tested at the following molar concentrations: 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.
  • HEK293T cells were transiently transfected with a plasmid encoding human CD25 (hCD25; hCD25_pcDNA3.4.dna).
  • HEK293T cells were harvested and incubated with increasing molar concentrations of purified His-tagged (myc-his tag) VHHs, including a control VHH against an irrelevant antigen. VHH binding was then detected using an Alexa488-labeled anti-His tag antibody and measured by flow cytometry (iQue).
  • the bar histogram in Figure 6A shows the percentage of Alexa488 positive cells for C-004 and C-006.
  • the bar histogram in Figure 6B shows the mean fluorescent intensity of Alexa488 positive cells for C-004 and C-006.
  • HEK293T cells were transiently transfected with a plasmid encoding human CD25 (hCD25; hCD25_pcDNA3.4.dna) or cynomolgus CD25 (cCD25; cCD25_pcDNA3.4.dna). After 48 hours, HEK293T cells were harvested and incubated with 100 nM purified His-tagged (myc-his tag) VHHs. VHH binding was then detected using an Alexa488-labeled anti- His tag antibody and measured by flow cytometry (iQue). The bar histogram in Figure 14 shows the mean fluorescent intensity of Alexa488 positive cells for C-007, C-008, C-009, and C-010.
  • Figure 9 shows a summary of binding affinities of two anti-CD25 V-bodies: C-004 and C-006. Data corresponding to an anti-CD25 IgG control condition are also shown.
  • the interaction with human, cynomolgus, and mouse CD25 (extracellular domain) (V-body coupling concentration: 0.2 ⁇ M) was also separately measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25°C) using eight different antigen concentrations (3-fold serial dilutions, starting at from 500 nM) for V-body candidate anti-CD25 clone C-004, and applying an inverse setup in single channel mode. Resulting sensorgrams (see, e.g., Figure 8A) were analyzed and equilibrium-binding affinities (K D S) were calculated using Carterra's data analysis software.
  • Human CD25-ECD was injected (500 nM) under physiological conditions (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25°C) followed by human IL2-Fc (1000 nM).
  • V-body C-008Hu1 was identified as a ligand competitive binder.
  • C-010Hu1 and C-009Hu1 were identified as non-competitive binders.
  • VHH variants A number of anti-CD25 VHH antibodies were generated by alanine scanning mutagenesis to fine tune the binding affinity with CD25.
  • the list of VHH variants is provided in Table 7.
  • “A#” or “G#” indicates the residue at position "#” in the CDR3 of the parental VHH antibody has been mutated to the amino acid residue alanine or glycine, respectively.
  • “L8” indicates that the residue at the 8 th position in the CDR3 of the parental VHH antibody has been mutated to the amino acid residue leucine.
  • Figures 21A-21B show monovalent VHH binding without anti-His antibody pre-incubatlon. To increase binding sensitivity, His-tagged VHHs were pre-incubated with anti-His antibody at a 2:1 ratio to form complexes of 2 VHHs to 1 anti-His antibody.
  • Figures 21C-21D show bivalent VHH binding with anti ⁇
  • SEQ ID NO: 4 C-001 full-length VHH, non-humanized VHH, amino acid sequence EVQLVESGGGLVQAGGSLRLSCAASGRKFSTLIMAWYRQAPGKQRELVATIERDGTTTYADSVEGRFFISRDNAKNTVT LQMNNLEPEDSATYYCNALQYWGQGTQVTVSS
  • VHH full-length VHH, non-humanized VHH, amino acid sequence EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKGRNSGSYYPWDDYWGQGTQVTVSS
  • VHH DNA nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCnGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC TGGCAGAAGCTTCAGTACCCTTATTATGGCCTGGTACCGCCAGGCTCCAGGGGAGCAGCGCGAGTTGGTCGCGAC TATTGAGAGGGACGGTACGCCAACCTATACAGACTCCGTGAAGGGCCGATTCTTCATCTCCAGAGACAACGCCAA GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGAGGACACAGCCATCTATTACTGTAATGCCCTCCGGTTC TGGGGCCAGGGGACCCAGGTCACCGTCCTCA
  • VHH DNA nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC TGGATTCACCTTCAGTAACTATGCCATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG TATTTATAGTGATGGTAGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAATACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTGCAAAAGGGAG GAATAGTGGTAGTTACTATCCCTGGGATGACTACTGGCCAGGGGACCCAGGTCACCGTCCTCA
  • SEQ ID NO: 26 C-001 humanized VHH, amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGRKFSTUMAWYRQAPGKQRELVATIERDGTnYADSVKGRFTISRDNAKNTVY LQMNSLRPEDTAVYYCNALQYWGQGTQVTVSS
  • SEQ ID NO: 39 Group D, CDR3 consensus, amino acid sequence AKGR(H/N)SGSYYPWD(D/E)Y
  • SEQ ID NO: 41 Group E, CDR3 consensus, amino acid sequence AA(S/T)(D/N/Y)FL(lA)AniS(A/G)YDY
  • SEQ ID NO: 2242 Group F, CDR1 consensus; amino acid sequence GFTLDYYA
  • SEQ ID NO: 2251 C-007 Full-length VHH;
  • Group F non-humanized VHH sequence, amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISRDGDSTNYGDSVKGRFTISRDNAKNTV YLQMNSLEPEDTAVYYCAAYVYPDYYCSEYVLLKYDYWGQGTQVTVSS
  • Group G non-humanized VHH sequence, amino acid sequence EVQLVESGGGLVQAGGSLRLSCAASGMPLVAMGWYRQAPGKQRELVASISSGGNTGYAEFVKGRFTISRDNAKKMV YLQMNSVKPEDTGVYYCNIYRSQVPPTRYSWGQGTQVTVSS
  • SEQ ID NO: 2253 C-009 Full-length VHH;
  • Group H non-humanized VHH sequence, amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVD LQLNSLKPEDTALYYCAAKRLGPMVHQYSLEVLTPLFLDEYDYWGQGTQVTVSS
  • SEQ ID NO: 2254 C-010 Full-length VHH;
  • Group H non-humanized VHH sequence, amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVD
  • SEQ ID NO: 2258 C-010 Full-length VHH DNA, nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC TGGATTCACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTTATC CATTAGTAGTACGGATGGCAGGACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATTTCCAGAGACAACCC CAAGAACACGGTCGATCTGCAATTGAACAGCCTGAAACCTGAGGACACAGCCCTTTATTACTGTGCAGCAAAACG ATTAGGTCCAATGGTTCATCGGTATTCTCTTGAAGTCCTTACACCACTATTTCTAGATGAGTATGACTACTGGGGCC AGGGGACCCAGGTCACCGTCCTCA
  • SEQ ID NO: 2259 C-007 Humanized VHH; Group F, humanized VHH sequence, amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISRDGDSTNYADSVKGRFTISRDNAKNTV YLQMNSLRPEDTAVYYCAAYVYPDYYCSEYVLLKYDYWGQGTQVTVSS
  • SEQ ID NO: 2260 C-008 Humanized VHH; Group G, humanized VHH sequence, amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGMPLVAMGWYRQAPGKQRELVASISSGGNTGYADSVKGRFTISRDNAKKTVY LQMNSVRPEDTGVYYCNIYRSQVPPTRYSWGQGTQVTVSS
  • SEQ ID NO: 2261 C-009 Humanized VHH; Group H, humanized VHH sequence, amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY LQLNSLRPEDTAVYYCAAKRLGPMVHQYSLEVLTPLFLDEYDYWGQGTQVTVSS
  • SEQ ID NO: 2262 C-010 Humanized VHH; Group H, humanized VHH sequence, amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
  • SEQ ID NO: 2266 Group G, CDR1 consensus, amino acid sequence G(I/M)P(F/-)(A/-)L(P/V/Y)A, wherein can be absent
  • SEQ ID NO: 4318 C-010Hu1.l8, Group H, Humanized VHH sequence, amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY LQLNSLRPEDTAVYYCAAKRLGPIVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
  • SEQ ID NO: 4319 C-009Hu1.A8 Group H, Humanized VHH sequence, amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY LQLNSLRPEDTAVYYCAAKRLGPAVHQYSLEVLTPLFLDEYDYWGQGTQVTVSS SEQ ID NO: 4320 C-010Hu1.A8, Group H, Humanized VHH sequence, amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
  • SEQ ID NO: 4340 Group D_CDR3 consensus amino acid sequence (A/V)KGR(G/H/N)SGSYYP(W/F)D(D/E)Y

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des protéines de liaison à l'antigène (par exemple, des anticorps tels que des anticorps à domaine unique) qui se lient spécifiquement à la classe de différenciation 25 (CD25). L'invention concerne également des protéines de fusion et des conjugués comprenant les protéines de liaison à l'antigène, des polynucléotides et des vecteurs recombinants codant pour les protéines de liaison à l'antigène, ainsi que des cellules hôtes et des procédés de préparation des protéines de liaison à l'antigène. L'invention concerne en outre des compositions pharmaceutiques comprenant les protéines de liaison à l'antigène.
PCT/US2025/035662 2024-06-27 2025-06-27 Protéines de liaison à l'antigène anti-cd25 et leurs utilisations Pending WO2026006708A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463664893P 2024-06-27 2024-06-27
US63/664,893 2024-06-27

Publications (3)

Publication Number Publication Date
WO2026006708A2 true WO2026006708A2 (fr) 2026-01-02
WO2026006708A3 WO2026006708A3 (fr) 2026-03-05
WO2026006708A8 WO2026006708A8 (fr) 2026-04-09

Family

ID=96806194

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/035662 Pending WO2026006708A2 (fr) 2024-06-27 2025-06-27 Protéines de liaison à l'antigène anti-cd25 et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2026006708A2 (fr)

Citations (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4510245A (en) 1982-11-18 1985-04-09 Chiron Corporation Adenovirus promoter system
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4968615A (en) 1985-12-18 1990-11-06 Ciba-Geigy Corporation Deoxyribonucleic acid segment from a virus
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
WO1994004678A1 (fr) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulines exemptes de chaines legeres
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US6599710B1 (en) 1999-03-10 2003-07-29 The General Hospital Corporation Treatment of autoimmune disease
EP1391213A1 (fr) 2002-08-21 2004-02-25 Boehringer Ingelheim International GmbH Compositions et méthodes pour le traitement du cancer en utilisant un conjugué d'un anticorps contre le CD44 avec un maytansinoide et des agents chimiothérapeutiques
WO2004041865A2 (fr) 2002-11-08 2004-05-21 Ablynx N.V. Anticorps a domaine unique stabilises
WO2005100402A1 (fr) 2004-04-13 2005-10-27 F.Hoffmann-La Roche Ag Anticorps anti-p-selectine
WO2006029879A2 (fr) 2004-09-17 2006-03-23 F.Hoffmann-La Roche Ag Anticorps anti-ox40l
US7083784B2 (en) 2000-12-12 2006-08-01 Medimmune, Inc. Molecules with extended half-lives, compositions and uses thereof
WO2006122787A1 (fr) 2005-05-18 2006-11-23 Ablynx Nv Proteines de liaison a l'albumine serique
WO2008020079A1 (fr) 2006-08-18 2008-02-21 Ablynx N.V. Séquences d'acides aminés dirigées contre l'il-6r et polypeptides les contenant utilisés pour le traitement de maladies et de troubles associés au signal médié par il-6
WO2009067800A1 (fr) 2007-11-27 2009-06-04 Viventia Biotech Inc. Anticorps dirigés contre un épitope associé au cancer de variant de nfkbib et ses utilisations
WO2009138519A1 (fr) 2008-05-16 2009-11-19 Ablynx Nv Séquences d'acides aminés dirigées contre cxcr4 et autres gpcr et composés renfermant ces dernières
WO2011003622A1 (fr) 2009-07-10 2011-01-13 Ablynx N.V. Procédé pour la production de domaines variables
WO2011056983A1 (fr) 2009-11-05 2011-05-12 Genentech, Inc. Conjugués d'anticorps modifiés par cystéine, radiomarqués par le zirconium
WO2011133886A2 (fr) 2010-04-23 2011-10-27 Genentech, Inc. Production de protéines hétéromultimères
WO2012175741A2 (fr) 2011-06-23 2012-12-27 Ablynx Nv Techniques permettant de prédire, détecter et réduire une interférence protéinique spécifique dans des dosages impliquant des domaines variables uniques d'immunoglobuline
WO2012175400A1 (fr) 2011-06-23 2012-12-27 Ablynx Nv Protéines se liant à la sérumalbumine
WO2013024059A2 (fr) 2011-08-17 2013-02-21 Glaxo Group Limited Protéines et peptides modifiés
US20140322129A1 (en) 2013-03-14 2014-10-30 Genentech, Inc. Anti-b7-h4 antibodies and immunoconjugates
US8969526B2 (en) 2011-03-29 2015-03-03 Roche Glycart Ag Antibody Fc variants
WO2015173325A2 (fr) 2014-05-16 2015-11-19 Ablynx Nv Domaines variables d'immunoglobulines améliorés
WO2016150845A1 (fr) 2015-03-20 2016-09-29 Ablynx Nv Domaines variables uniques d'immunoglobuline glycosylés
WO2017080850A1 (fr) 2015-11-13 2017-05-18 Ablynx Nv Domaines variables d'immunoglobuline de liaison améliorée à l'albumine sérique
WO2017085172A2 (fr) 2015-11-18 2017-05-26 Ablynx Nv Liants d'albumine sérique améliorés
WO2018104444A1 (fr) 2016-12-07 2018-06-14 Ablynx Nv Domaines variables uniques d'immunoglobuline se liant à l'albumine sérique améliorée
WO2018134234A1 (fr) 2017-01-17 2018-07-26 Ablynx Nv Liants d'albumine sérique améliorés
WO2018134235A1 (fr) 2017-01-17 2018-07-26 Ablynx Nv Liants d'albumine sérique améliorés
US10526397B2 (en) 2015-01-21 2020-01-07 Inhibrx, Inc. Non-immunogenic single domain antibodies
WO2021234402A2 (fr) 2020-05-21 2021-11-25 Mabsolve Limited Régions fc d'imunoglobuline modifiée
EP3692065B1 (fr) 2017-10-02 2022-02-16 Cilag GmbH International Mutants fc igg1 avec fonctions d'effecteur supprimées
US11426468B2 (en) 2014-12-19 2022-08-30 Ablynx N.V. Cysteine linked nanobody dimers
WO2024238790A1 (fr) 2023-05-17 2024-11-21 Odyssey Therapeutics, Inc. Anticorps à domaine unique modifiés

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103333248B (zh) * 2013-06-07 2014-07-09 东南大学 一种cd25纳米抗体、其编码序列及应用
US10752691B2 (en) * 2018-03-13 2020-08-25 Tusk Therapeutics Ltd. Anti-CD25 antibody agents
WO2022150791A2 (fr) * 2021-01-11 2022-07-14 Synthekine, Inc. Compositions et procédés se rapportant à la liaison au récepteur il2
CN118240080A (zh) * 2022-12-22 2024-06-25 深圳市福元生物科技有限公司 一种cd25单域抗体、人源化抗体、及制备方法
TW202444757A (zh) * 2023-03-14 2024-11-16 美商奧迪希治療公司 抗cd25抗原結合蛋白及其用途

Patent Citations (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US4510245A (en) 1982-11-18 1985-04-09 Chiron Corporation Adenovirus promoter system
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US4968615A (en) 1985-12-18 1990-11-06 Ciba-Geigy Corporation Deoxyribonucleic acid segment from a virus
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1994004678A1 (fr) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulines exemptes de chaines legeres
US6599710B1 (en) 1999-03-10 2003-07-29 The General Hospital Corporation Treatment of autoimmune disease
US6660487B2 (en) 1999-03-10 2003-12-09 The General Hospital Corporation Treatment of autoimmune disease
US7083784B2 (en) 2000-12-12 2006-08-01 Medimmune, Inc. Molecules with extended half-lives, compositions and uses thereof
EP1391213A1 (fr) 2002-08-21 2004-02-25 Boehringer Ingelheim International GmbH Compositions et méthodes pour le traitement du cancer en utilisant un conjugué d'un anticorps contre le CD44 avec un maytansinoide et des agents chimiothérapeutiques
WO2004041865A2 (fr) 2002-11-08 2004-05-21 Ablynx N.V. Anticorps a domaine unique stabilises
WO2005100402A1 (fr) 2004-04-13 2005-10-27 F.Hoffmann-La Roche Ag Anticorps anti-p-selectine
WO2006029879A2 (fr) 2004-09-17 2006-03-23 F.Hoffmann-La Roche Ag Anticorps anti-ox40l
WO2006122787A1 (fr) 2005-05-18 2006-11-23 Ablynx Nv Proteines de liaison a l'albumine serique
WO2008020079A1 (fr) 2006-08-18 2008-02-21 Ablynx N.V. Séquences d'acides aminés dirigées contre l'il-6r et polypeptides les contenant utilisés pour le traitement de maladies et de troubles associés au signal médié par il-6
WO2009067800A1 (fr) 2007-11-27 2009-06-04 Viventia Biotech Inc. Anticorps dirigés contre un épitope associé au cancer de variant de nfkbib et ses utilisations
WO2009138519A1 (fr) 2008-05-16 2009-11-19 Ablynx Nv Séquences d'acides aminés dirigées contre cxcr4 et autres gpcr et composés renfermant ces dernières
WO2011003622A1 (fr) 2009-07-10 2011-01-13 Ablynx N.V. Procédé pour la production de domaines variables
WO2011056983A1 (fr) 2009-11-05 2011-05-12 Genentech, Inc. Conjugués d'anticorps modifiés par cystéine, radiomarqués par le zirconium
WO2011133886A2 (fr) 2010-04-23 2011-10-27 Genentech, Inc. Production de protéines hétéromultimères
US8969526B2 (en) 2011-03-29 2015-03-03 Roche Glycart Ag Antibody Fc variants
WO2012175741A2 (fr) 2011-06-23 2012-12-27 Ablynx Nv Techniques permettant de prédire, détecter et réduire une interférence protéinique spécifique dans des dosages impliquant des domaines variables uniques d'immunoglobuline
WO2012175400A1 (fr) 2011-06-23 2012-12-27 Ablynx Nv Protéines se liant à la sérumalbumine
WO2013024059A2 (fr) 2011-08-17 2013-02-21 Glaxo Group Limited Protéines et peptides modifiés
US20140322129A1 (en) 2013-03-14 2014-10-30 Genentech, Inc. Anti-b7-h4 antibodies and immunoconjugates
WO2015173325A2 (fr) 2014-05-16 2015-11-19 Ablynx Nv Domaines variables d'immunoglobulines améliorés
US11426468B2 (en) 2014-12-19 2022-08-30 Ablynx N.V. Cysteine linked nanobody dimers
US10526397B2 (en) 2015-01-21 2020-01-07 Inhibrx, Inc. Non-immunogenic single domain antibodies
WO2016150845A1 (fr) 2015-03-20 2016-09-29 Ablynx Nv Domaines variables uniques d'immunoglobuline glycosylés
WO2017080850A1 (fr) 2015-11-13 2017-05-18 Ablynx Nv Domaines variables d'immunoglobuline de liaison améliorée à l'albumine sérique
WO2017085172A2 (fr) 2015-11-18 2017-05-26 Ablynx Nv Liants d'albumine sérique améliorés
WO2018104444A1 (fr) 2016-12-07 2018-06-14 Ablynx Nv Domaines variables uniques d'immunoglobuline se liant à l'albumine sérique améliorée
WO2018134234A1 (fr) 2017-01-17 2018-07-26 Ablynx Nv Liants d'albumine sérique améliorés
WO2018134235A1 (fr) 2017-01-17 2018-07-26 Ablynx Nv Liants d'albumine sérique améliorés
EP3692065B1 (fr) 2017-10-02 2022-02-16 Cilag GmbH International Mutants fc igg1 avec fonctions d'effecteur supprimées
WO2021234402A2 (fr) 2020-05-21 2021-11-25 Mabsolve Limited Régions fc d'imunoglobuline modifiée
WO2024238790A1 (fr) 2023-05-17 2024-11-21 Odyssey Therapeutics, Inc. Anticorps à domaine unique modifiés

Non-Patent Citations (82)

* Cited by examiner, † Cited by third party
Title
"Wensel and Meares", 1983, ELSEVIER
ABDICHE ET AL., ANAL. BIOCHEM., vol. 377, 2008, pages 209 - 217
AI-LAZIKANI ET AL., JMB, vol. 273, 1997, pages 927 - 948
ALEGRE ET AL., J IMMUNOL, vol. 148, 1992, pages 3461 - 3468
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 402
ALTSCHUL, J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ARBABI GHAHROUDI, M., FEBS LETTERS, vol. 414, no. 3, 1997, pages 521 - 526
BORREBAECK, C. A. KOHLIN, M.: "Antibody evolution beyond Nature", NATURE BIOTECHNOLOGY, vol. 20, no. 12, 2002, pages 1189 - 90, XP037103813, DOI: 10.1038/nbt1202-1189
BRUGGEMANN. ET AL., J. EXP. MED., vol. 166, 1987, pages 1351 - 1361
CHEN, ADV DRUG DELIV REV, vol. 65, no. 10, 2013, pages 1357 - 1369
CLYNES ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 95, 1998, pages 652 - 656
COLCHER ET AL., METH. ENZYMOL., vol. 121, 1986, pages 802 - 16
CONRATH ET AL., J MOL BIOL, 2005
CRAGG, M. S. ET AL., BLOOD, vol. 101, 2003, pages 1045 - 1052
CRAGG, M. S.M. J. GLENNIE, BLOOD, vol. 103, 2004, pages 2738 - 2743
DALL'ACQUA ET AL., J. BIOL CHEM, vol. 281, no. 33, 2006, pages 23514 - 23524
DAVID ET AL., BIOCHEMISTRY, vol. 13, 1974, pages 1014
DRAKE ET AL., ANAL. BIOCHEM., vol. 328, 2004, pages 35 - 43
DUMET ET AL., MABS, vol. 11, no. 8, pages 1341 - 1350
GAZZANO-SANTORO ET AL., J. IMMUNOL. METHODS, vol. 202, 1996, pages 163
GONNET ET AL., SCIENCE, vol. 256, 1992, pages 1443 - 1445
GRAILLE ET AL., PNAS, 2000
HELLSTROM ET AL., PROC. NAT'L ACAD. SCI., vol. 82, 1985, pages 1499 - 1502
HELLSTROM. ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 83, 1986, pages 7059 - 7063
HENRY ET AL., PLOS ONE, 2016
HERMANSON, G. T.: "Bioconjugate Techniques;", 1996, PHILADELPHIA, LIPPINCOTT-RAVEN PUBLISHERS, pages: 763 - 843
HINMAN ET AL., CANCER RES., vol. 53, 1993, pages 3336 - 3342
HONEGGER APLICKTHUN A: "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J MOL BIOL, vol. 309, no. 3, 8 June 2001 (2001-06-08), pages 657 - 70, XP004626893, DOI: 10.1006/jmbi.2001.4662
HOSSE ET AL., PROTEIN SCI., vol. 15, 2006, pages 14 - 27
HUNTER ET AL., NATURE, vol. 144, 1962, pages 945
HUSTON ET AL., CELL BIOPHYSICS, vol. 22, 1993, pages 189 - 224
IDUSOGIE ET AL., J IMMUNOL, vol. 166, no. 4, 2001, pages 2571 - 5
J. MOL. BIOL., vol. 262, pages 732 - 745
JOURNAL OF IMMUNOLOGICAL METHODS, vol. 248, 2001, pages 7 - 15
KANEKONIWA, BIODRUGS, vol. 25, no. 1, 2011, pages 1 - 11
KLEIN, PROTEIN ENGINEERING, DESIGN & SELECTION, vol. 27, no. 10, 2014, pages 325 - 330
KO ET AL., BIODRUGS, vol. 35, 2021, pages 147 - 157
LANGER, SCIENCE, vol. 249, 1990, pages 1527 - 1533
LAZAR ET AL., PNAS, vol. 103, no. 11, 2006, pages 4005 - 4010
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, vol. 27, no. 1, January 2003 (2003-01-01), pages 55 - 77, XP055585227, DOI: 10.1016/S0145-305X(02)00039-3
LIAO ET AL., IMMUNITY, vol. 38, 2013, pages 13 - 25
LIU ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 8618 - 8623
LODE ET AL., CANCER RES., vol. 58, 1998, pages 2928
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MAHONEY ET AL., CANCER IMMUNOTHERAPY, vol. 14, 2015, pages 561 - 584
MANDLER ET AL., BIOCONJUGATE CHEM., vol. 13, 2002, pages 786 - 791
MANDLER ET AL., BIOORGANIC & MED. CHEM. LETTERS, vol. 10, 2000, pages 1025 - 1028
MANDLER ET AL., J. NAT. CANCER INST., vol. 92, no. 19, 2000, pages 1573 - 1581
MARTIN ET AL.: "Modeling antibody hypervariable loops: a combined algorithm", PNAS, vol. 86, no. 23, 1989, pages 9268 - 9272, XP000165667, DOI: 10.1073/pnas.86.23.9268
MOORE ET AL., R IABS, vol. 2, no. 2, 2010, pages 181 - 189
MORDENTI ET AL., PHDOMAINACEUT. RES., vol. 8, 1991, pages 1351
NATSUME, CANCER RES, vol. 68, no. 10, 2008, pages 3863 - 72
NGUYEN ET AL., EMBO J, 2000
NICAISE ET AL., PROTEIN SCI., vol. 13, 2004, pages 1882 - 1891
NYGREN, J., HISTOCHEM. AND CYTOCHEM, vol. 30, 1982, pages 407
NYGRENUHLEN, CURR. OPIN. STRUC. BIOL., vol. 7, 1997, pages 463 - 469
OBER ET AL., INTERN. IMMUNOLOGY, vol. 13, 2001, pages 1551 - 1559
PAIN ET AL., J. IRNMUNOL. METH, vol. 40, 1981, pages 219
PEARSON, METHODS MOL. BIOL., vol. 24, 1994, pages 307 - 331
PEDERSENLAURITSEN, SCAND J IMMUNOL, vol. 70, 2009, pages 40 - 43
PETKOVA, S. B. ET AL., INT'L. IMMUNOL., vol. 18, no. 12, 2006, pages 1759 - 1769
PLÜCKTHUNSKERRA, METH. ENZYMOL., vol. 178, 1989, pages 497 - 515
POTHIN ET AL., PHARMACEUTICS, vol. 12, no. 10, 2020, pages 937
POWELL ET AL.: "Compendium of excipients for parenteral formulations", J PHDOMAIN SCI TECHNOL, vol. 52, 1998, pages 238 - 311, XP009119027
RIECHMANNMUYLDERMANS, J. IMMUNOL. METHODS, vol. 240, no. 1-2, 2000, pages 185 - 195
SAERENS ET AL., J BIOL CHEM, 2009
SAERENS, D.; ET AL.: "Single-domain antibodies as building blocks for novel therapeutics", CURRENT OPINION IN PHARMACOLOGY, vol. 8, no. 5, 2008, pages 600 - 608
SEE ET AL., BIOTECHNOLOGY JOURNAL, vol. 15, no. 12, 2020, pages e2000078
SHIELDS ET AL., I18C, vol. 276, no. 9, 2001, pages 6591 - 6604
SKERRA, A, CURR. OPIN. BIOTECHNOL., vol. 18, 2005, pages 295 - 304
STAUBER ET AL., PROC NATL ACAD SCI U 5 A, vol. 103, 2006, pages 2788 - 2793
STAUBER ET AL., PROC NATL ACAD SCI U S A, vol. 103, 2006, pages 2788 - 2793
STAVENHAGEN ET AL., ADVAN. ENZYME REGUL., vol. 48, 2008, pages 152 - 164
STAVENHAGEN ET AL., CANCER RES, vol. 67, no. 18, 2007, pages 8882 - 8890
TANGYE ET AL., JOURNAL OF CLINICAL IMMUNOLOGY, vol. 42, 2022, pages 1473 - 1507
TO ET AL., JBC, 2005
WANG ET AL., SCIENCE, vol. 310, 2005, pages 1159 - 1163
WILKINSON ET AL.: "Fc-engineered antibodies with immune effector functions completely abolished.", PLOS ONE, 2021
WU ET AL., J. BIOL. CHEM., vol. 262, 1987, pages 4429 - 4432
WUEST ET AL., NAT MED, vol. 17, pages 604 - 609
ZALEVSKY ET AL., NATURE BIOTECH, vol. 28, no. 2, 2010, pages 157 - 159
ZAVRTANIK ET AL., J MOL BIOI, 2018

Also Published As

Publication number Publication date
WO2026006708A3 (fr) 2026-03-05
WO2026006708A8 (fr) 2026-04-09

Similar Documents

Publication Publication Date Title
JP7587656B2 (ja) Ｐｄ－ｌ１及びｃｄ１３７に結合する抗体分子
US10233258B2 (en) Bispecific binding proteins that bind CD40 and mesothelin
JP2023051968A (ja) 多重特異的ＮＫｐ４６結合タンパク質
KR20230146578A (ko) Bcma, gprc5d, 및 cd3을 표적화하는 삼중특이적 항체
WO2022256563A1 (fr) Anticorps anti-ccr8 et leurs utilisations
EP3929212A1 (fr) Fragment fc modifié, anticorps le comprenant et son application
TW202204386A (zh) 經遮蔽之il-12細胞激素及其裂解產物
CN114507285A (zh) Vista抗原结合分子
TW201420600A (zh) 抗－間皮素結合蛋白質
JP6792552B2 (ja) Ｃｃｒ６と結合する抗体およびその使用
TW202306988A (zh) Ｉｌ—２１多肽及靶向構築體
AU2024234615A1 (en) Anti-cd25 antigen-binding proteins and uses thereof
EP4648792A2 (fr) Protéines de liaison à l'antigène anti-tnfr2 et leurs utilisations
KR20250130296A (ko) 항-ccr8 항체 및 이의 용도
WO2026006708A2 (fr) Protéines de liaison à l'antigène anti-cd25 et leurs utilisations
WO2026006809A1 (fr) Molécules multispécifiques se liant à tnfr2 et cd25 et leurs utilisations
WO2025217240A1 (fr) Protéines de liaison à l'antigène anti-tnfr2 et leurs utilisations
WO2025080751A2 (fr) Protéines de liaison à l'antigène anti-cdh17 et leurs utilisations
KR20260032566A (ko) 항-trailr2 항원-결합 단백질 및 그의 용도
TWI833227B (zh) 靶向pd-l1和cd73的特異性結合蛋白及其應用
JP2026071208A (ja) Ｂｃｍａに結合する多重特異性抗体
WO2025255558A2 (fr) Protéines de liaison à l'antigène anti-lymphopoïétine stromale thymique (tslp) et leurs utilisations
CN117881701A (zh) 抗cll-1抗体及其用途
HK40050345B (en) Antibody molecules that bind pd-l1 and cd137
HK40050345A (en) Antibody molecules that bind pd-l1 and cd137

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25756326

Country of ref document: EP

Kind code of ref document: A2