Anti-CD25 Antigen-Binding Proteins and Their Uses

Abstract

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

Description

"ANTI-CD25 ANTIGEN-BINDING PROTEINS AND THEIR USES" CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority over U.S. Provisional Application No. 63 / 452,000, filed March 14, 2023, the disclosure of which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[002] The present application contains a Sequence Listing that was submitted electronically in XML format and is incorporated into the present invention by reference in its entirety. Said XML copy, created on March 11, 2024, is named 260525_000037_SL.xml and is 5,071,660 bytes in size. FIELD OF THE INVENTION

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

[004] 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 that seek to increase or modulate Tregs in autoimmune patients are based on ex vivo expansion of Tregs prior to autologous transfer. However, a major limitation of current strategies is their inability to stabilize the Treg phenotype to ensure long-lasting immunoregulation.

[005] Although Tregs can assist immune homeostasis under normal, healthy conditions, and their activation may be beneficial in the context of autoimmune disease, during proliferative diseases (e.g., cancer), Tregs Petition 870250081421, dated 10 / 09 / 2025, page 12 / 374 2 / 304 can accumulate within the tumor microenvironment where they can hinder antitumor responses generated by immune cell infiltration, effectively protecting cancer cells from immune attack. Tregs are capable of suppressing most types of immune cells including CD4+ and CD8+ T cells, B cells, and antigen-presenting cells (APCs) (e.g., dendritic cells, macrophages, and monocytes), natural killer (NK) cells, and NKT cells. The number of Tregs is higher in tumors and peripheral blood mononuclear cells (PBMCs) of many cancer patients, and high Treg levels may be associated with a poor prognosis, for example, in solid tumors including breast, cervical, renal, melanoma, ovarian, hepatocellular, gastric, and pancreatic cancers.

[006] The cluster of differentiation 25 (CD25), also called the alpha subunit of the interleukin-2 receptor (IL-2Rα or IL2RA), is the alpha chain component of the high-affinity heterotrimeric interleukin-2 (IL-2) receptor, a type I transmembrane protein highly expressed on the surface of most Tregs. IL-2 activation of CD25 may facilitate immunotolerance in Tregs. High expression of CD25 on the cell surface can also occur in malignant cells, for example, in various lymphomas and leukemias.

[007] Consequently, there is a need in the technique to develop molecules that can effectively target and specifically bind to cells that express CD25, for example, Tregs. SUMMARY OF THE INVENTION

[008] As mentioned in the background section above, there is an unmet need in the art to develop molecules that can effectively target and specifically bind to cluster of differentiation 25 (CD25). This application provides compositions and methods to meet these and other related needs. Petition 870250081421, dated 10 / 09 / 2025, p. 13 / 374 3 / 304

[009] In one aspect, the present disclosure provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from a). NAL(G / L / P / Q / W)Y (SEQ ID NO: 31); b). NALR(D / H / N / F) (SEQ ID NO: 34); w) . (K / S / T)TLRY (SEQ ID NO: 36); d). (A / V / S)(K / T)G(R / A / K)(G / H / N / R)SG(S / G)YYP(W / F / L)D(D / E)(Y / V) (SEQ ID NO: 5119); it) . AA(S / T)(D / N / Y / K)(F / V)(L / P)(I / L)A(T / I / A)(T / S / A)IS(A / G)(Y / H)DY (SEQ ID NO: 5208); f) . AAYVYPDYYCS(D / E)YVLL(K / R)YDY (SEQ ID NO: 2263); g) . KIDNEY(P / S)QVP(P / S / T)TRYS (SEQ ID NO: 2265); it h) . AAKRLGP(M / I / A / L)VH(Q / R)YSLEVLTPLFLDEYDY (SEQ ID NO: 4323). [0 10] In some embodiments, the CDR3 comprises a sequence of amino acids selected from at) . NAL(G / L / P / Q / W)Y (SEQ ID NO: 31); b) . NALR(D / H / N / F) (SEQ ID NO: 34); c) . (K / S / T)TLRY (SEQ ID NO: 36); d) . AKGR(H / N)SGSYYPWD(D / E)Y (SEQ ID NO: 39); it) . (A / B)KGR(G / H / N)SGSYYP(W / F)D(D / E)Y (SEQ ID NO: 4430); f) . AA(S / T)(D / N / Y)FL(I / L)ATTIS(A / G)YDY (SEQ ID NO: 41); g) . AAYVYPDYYCS(D / E)YVLL(K / R)YDY (SEQ ID NO: 2263); h) . KIDNEY(P / S)QVP(P / S / T)TRYS (SEQ ID NO: 2265); it i) . AAKRLGPMVH(Q / R)YSLEVLTPLFLDEYDY (SEQ ID NO: 2267).

[011] In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOS: 3, 7, 11, 15, 19, 39, 41, 1237, Petition 870250081421, dated 10 / 09 / 2025, p. 14 / 374 4 / 304 1239, 1271, 1275, 1298, 1301, 1331, 1415, 1419, 1421, 1428, 1432, 1442, 1444, 1445, 1447, 1448, 2244, 2247, 2249, 2250, 2267, 4311 - 4316, 4336, 4340, 4787, 4866, 4875, 4878, 4879, and 4880.

[012] In some embodiments, CDR3 comprises an amino acid sequence selected from the SEQ ID NOs: 3, 7, 11, 15, 19, 1237, 1239, 1271, 1275, 1298, 1301, 1331, 1415, 1419, 1421, 1428, 1432, 1442, 1444, 1445, 1447, 1448, 2244, 2247, 2249, 2250, 4311 - 4316, 4336, 4787, 4866, 4875, 4878, 4879, and 4880.

[013] In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOS: 3, 7, 11, 15, 19, 2244, 2247, 2249, 2250, 4311 - 4316, and 4336.

[014] In some embodiments, the antigen-binding protein described in the present invention may further comprise a CDR1 comprising an amino acid sequence selected from a). GR(K / R / S)FSTLI (SEQ ID NO: 37); b). GFTFS(N / S)YA (SEQ ID NO: 40); w) . 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).

[015] In some embodiments, CDR1 comprises a sequence of amino acids selected from a) . GRSFSTLI (SEQ ID NO: 5); b). GR(S / K)FSTLI (SEQ ID NO: 32); w) . GFTFS(N / S)YA (SEQ ID NO: 40); d). GRTFS(S / W)(F / N / Y)G (SEQ ID NO: 42); e). GFTLDYYA (SEQ ID NO: 2242); and f). G(I / M)P(F / -)(A / -)L(P / V / Y)A (SEQ ID NO: 2266).

[016] In some forms, CDR1 comprises a sequence of Petition 870250081421, dated 10 / 09 / 2025, p. 15 / 374 5 / 304 amino acids selected from the following sequence IDs: 1, 5, 9, 13, 17, 32, 42, 805, 809, 818, 2242, and 2245.

[017] In some embodiments, CDR1 comprises an amino acid sequence selected from the SEQ ID NOs: 1, 5, 9, 13, 17, 32, 2242, and 2245.

[018] In some embodiments, the antigen-binding protein described in the present invention may further comprise a CDR2 comprising an amino acid sequence selected from a). (I / V)(D / E)R(D / G)(D / G)T(A / P / T) (SEQ ID NO: 2241); b). IYSD(G / S)SGT (SEQ ID NO: 4341); w) . 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).

[019] In some embodiments, CDR2 comprises a sequence of amino acids selected from a). (I / V)(D / E)R(D / G)GT(A / P / T) (SEQ ID NO: 33); b). I(D / E)RDGT(T / P) (SEQ ID NO: 35); w) . I(D / E)R(D / G)(D / G)T(P / T) (SEQ ID NO: 38); 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).

[020] In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 18, 33, 35, 38, 942, 946, 959, 967, 992, 1114, 1115, 1116, 1117, 2243, 2246, 2248, and 4335.

[021] In some forms, CDR2 comprises a sequence of Petition 870250081421, dated 10 / 09 / 2025, p. 16 / 374 6 / 304 amino acids selected from the following sequence IDs: 2, 6, 10, 14, 18, 2243, 2246, 2248, and 4335.

[022] In some embodiments, the antigen-binding protein comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 31; ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; iii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 36; iv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 33, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; (v) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 35, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; vi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 38, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 36; vii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 32, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; viii) a CDR1 comprising an amino acid sequence of the SEQ ID Petition 870250081421, dated 10 / 09 / 2025, page 17 / 374 7 / 304 NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; ix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 32, a CDR2 comprising an amino acid sequence from SEQ ID NO: 33, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; x) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 35, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; xi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 40, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 5119; xii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 40, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 4340; xiii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 40, a CDR2 comprising an amino acid sequence with SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 39; xiv) a CDR1 comprising an amino acid sequence with SEQ ID NO: 5209, a CDR2 comprising an amino acid sequence with SEQ ID NO: 5210, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 5208; xv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 42, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 41; Petition 870250081421, dated 10 / 09 / 2025, p. 18 / 374 8 / 304 xvi) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 2263; xvii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2246, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 2265; xviii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 4323; or xix) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 2267.

[023] In some embodiments, the antigen-binding protein comprises i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 1, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 3; ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 6, a CDR3 comprising an amino acid sequence from SEQ ID NO: 7; iii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 9, a CDR2 comprising an amino acid sequence from SEQ ID NO: 10, a CDR3 comprising an amino acid sequence from SEQ ID NO: 11; iv) a CDR1 comprising an amino acid sequence of the SEQ ID Petition 870250081421, dated 10 / 09 / 2025, page 19 / 374 9 / 304 NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, a CDR3 comprising an amino acid sequence of SEQ ID NO: 15; or (v) a CDR1 comprising an amino acid sequence from SEQ ID NO: 17, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 19; vi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2243, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2244; vii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2245, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2246, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2247; viii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2249; ix) a CDR1 comprising an amino acid sequence of the SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 2250; x) a CDR1 comprising an amino acid sequence of the SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4311; xi) a CDR1 comprising an amino acid sequence of SEQ ID Petition 870250081421, dated 10 / 09 / 2025, page 20 / 374 10 / 304 NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4312; xii) a CDR1 comprising an amino acid sequence of the SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4313; xiii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4314; xiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4315; xv) a CDR1 comprising an amino acid sequence of the SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4316 xvi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4875; xvii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1331; xviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: Petition 870250081421, dated 10 / 09 / 2025, page 21 / 374 11 / 304 14, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4787; xix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4866; xx) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4336; xxi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4878; xxii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4879; xxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4880; xxiv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 10, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1239; xxv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 946, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1239; Petition 870250081421, dated 10 / 09 / 2025, page 22 / 374 12 / 304 xxvi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 959, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1237; xxvii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 946, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1237; xxviii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 1, a CDR2 comprising an amino acid sequence from SEQ ID NO: 967, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1271; xxix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 946, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1275; xxx) a CDR1 comprising an amino acid sequence from SEQ ID NO: 9, a CDR2 comprising an amino acid sequence from SEQ ID NO: 10, a CDR3 comprising an amino acid sequence from SEQ ID NO: 11; xxxi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 1, a CDR2 comprising an amino acid sequence from SEQ ID NO: 992, a CDR3 comprising an amino acid sequence from SEQ ID NO: 11; xxxii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 1, a CDR2 comprising an amino acid sequence from SEQ ID NO: 992, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1298; xxxiii) a CDR1 comprising an amino acid sequence of the SEQ ID Petition 870250081421, dated 10 / 09 / 2025, p. 23 / 374 13 / 304 NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 942, a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; xxxiv) a CDR1 comprising an amino acid sequence with SEQ ID NO: 5, a CDR2 comprising an amino acid sequence with SEQ ID NO: 959, a CDR3 comprising an amino acid sequence with SEQ ID NO: 11; xxxv) a CDR1 comprising an amino acid sequence with SEQ ID NO: 1, a CDR2 comprising an amino acid sequence with SEQ ID NO: 942, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1301; xxxvi) a CDR1 comprising an amino acid sequence with SEQ ID NO: 805, a CDR2 comprising an amino acid sequence with SEQ ID NO: 18, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1415; xxxvii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 809, a CDR2 comprising an amino acid sequence with SEQ ID NO: 1114, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1419; xxxviii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 805, a CDR2 comprising an amino acid sequence with SEQ ID NO: 1116, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1421; xxxix) a CDR1 comprising an amino acid sequence with SEQ ID NO: 809, a CDR2 comprising an amino acid sequence with SEQ ID NO: 1117, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1419; xxxx) a CDR1 comprising an amino acid sequence with SEQ ID NO: 818, a CDR2 comprising an amino acid sequence with SEQ ID NO: 1115, a CDR3 comprising an amino acid sequence with SEQ ID NO: Petition 870250081421, dated 10 / 09 / 2025, p. 24 / 374 14 / 304 1428; xxxxi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1432; xxxxii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1442; xxxxiii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1444; xxxxiv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1445; xxxxv) a CDR1 comprising an amino acid sequence with SEQ ID NO: 805, a CDR2 comprising an amino acid sequence with SEQ ID NO: 18, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1447; or xxxxvi) a CDR1 comprising an amino acid sequence with SEQ ID NO: 805, a CDR2 comprising an amino acid sequence with SEQ ID NO: 18, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1448.

[024] In one aspect, the present invention provides an antigen-binding protein that specifically binds to the cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence. Petition 870250081421, dated 10 / 09 / 2025, p. 25 / 374 15 / 304 selected from any of the SEQ ID Nos: 626 - 930, 2831 - 3126, and 4560 - 4670; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID Nos: 931 - 1235, 3127 - 3422, and 4671 - 4780; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID Nos: 1236 - 1540, 3423 - 3718, and 4781 - 4891.

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

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

[027] In some forms, VHH is a camelid VHH.

[028] In some embodiments, VHH comprises an amino acid sequence selected from any of the SEQ ID NOs: 4, 8, 12, 16, 20, 43 - 342, 1541 - 1845, 2251 - 2254, 2268 - 2559, 3719 - 4014, 4337, 4342 - 4451, 4892 - 5002, and 5146 - 5176, or a sequence having at least 75% identity with it.

[029] In some embodiments, VHH comprises an amino acid sequence selected from any of the SEQ ID NOs: 4, 8, 12, 16, 20, 2251 - 2254, 4337, and 5146 - 5176, or a sequence having at least 75% identity with it.

[030] In some modalities, VHH is a humanized VHH.

[031] In some embodiments, the humanized VHH comprises an amino acid sequence selected from any of the SEQ ID NOs: 26 - 30, 343 - 625, 2259 - 2262, 2560 - 2830, 4317 - 4322, 4339, 4452 - 4559, and 5114 5145, or a sequence having at least 75% identity with it.

[032] In some embodiments, the humanized VHH comprises an amino acid sequence selected from any of the SEQ ID NOs: 26 - 30, 2259 - 2262, 4317 - 4322, 4339, and 5114 - 5145, or a sequence having at least Petition 870250081421, dated 10 / 09 / 2025, p. 26 / 374 16 / 304 minus 75% identification with her.

[033] In some forms, the antigen-binding protein binds to human CD25.

[034] In some embodiments, the antigen-binding protein binds to human CD25 with a Kd of less than about 3.5x10-7M.

[035] In some embodiments, the antigen-binding protein binds to human CD25 with a Kd of about 1 x10-10 to about 1 x10-7M.

[036] In some forms, the antigen-binding protein binds to cyno CD25.

[037] In some embodiments, the antigen-binding protein binds to cyno CD25 with a Kd of less than about 1x10-6M.

[038] In some embodiments, the antigen-binding protein binds to cyno CD25 with a Kd of less than about 5x10-7M.

[039] In some embodiments, the antigen-binding protein binds to cyno CD25 with a Kd of about 1x10-8 to about 4x10-7M.

[040] In some forms, the antigen-binding protein binds to the same epitope(s) on CD25 as IL-2.

[041] In some forms, the antigen-binding protein competes with IL-2 for binding to CD25.

[042] In some forms, the antigen-binding protein has an antagonistic effect on CD25 binding.

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

[044] In some forms, the antigen-binding protein does not compete with the binding of CD25 to IL-2.

[045] In another aspect, the present disclosure provides a fusion protein that specifically binds to the cluster of differentiation 25 (CD25), comprising Petition 870250081421, dated 10 / 09 / 2025, p. 27 / 374 17 / 304 one or more of an antigen-binding protein described in the present invention.

[046] In some embodiments, the fusion protein described in the present invention may comprise two antigen-binding proteins described in the present invention.

[047] In some embodiments, the fusion protein described in the present invention may comprise four antigen-binding proteins described in the present invention.

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

[049] In some embodiments, one or more antigen-binding proteins bind to different epitopes on CD25.

[050] In some embodiments, one or more antigen-binding proteins are one or more single-domain antibodies.

[051] In some embodiments, one or more single-domain antibodies are one or more VHHs.

[052] In some embodiments, a fusion protein described in the present invention may further comprise an immunoglobulin Fc region.

[053] In some forms, the Fc region of immunoglobulin is an Fc region of a human immunoglobulin.

[054] In some embodiments, the immunoglobulin Fc region is a human IgG1, IgG2, IgG3 or IgG4 Fc region, or a variant thereof.

[055] In some forms, the immunoglobulin Fc region is a human IgG1 Fc region, or a variant thereof.

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

[057] In some forms, the Fc region of human IgG1 comprises a Petition 870250081421, dated 10 / 09 / 2025, p. 28 / 374 18 / 304 set of selected mutations of 1) . L234A and L235A; 2) . L234A, L235A, and P329A; 3) . D265A, N297A and P329A; and 4) . L234A, L235A, and G237A.

[058] In some embodiments, the immunoglobulin Fc region is a human IgG4 Fc region, or a variant thereof.

[059] In some embodiments, the Fc region of human IgG4 comprises one or more mutations selected from S228P, L235E, L235A, and / or F234A according to EU numbering.

[060] In some forms, the Fc region of human IgG4 comprises a set of selected mutations of 1) . S228P and L235E; 2) . S228P and L235A; 3) . S228P, F234A, and L235E; and 4) . S228P, F234A, and L235A.

[061] In another aspect, the present disclosure provides a conjugate comprising an antigen-binding protein described in the present invention or a fusion protein described in the present invention, wherein the antigen-binding protein or the fusion protein is conjugated to a second moiety.

[062] In some embodiments, the second fraction is selected from a detectable marker, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

[063] In another aspect, the present disclosure provides a polynucleotide molecule encoding an antigen-binding protein described in the present invention or a fusion protein described in the present invention. Petition 870250081421, dated 10 / 09 / 2025, page 29 / 374 19 / 304

[064] In another aspect, the present disclosure provides a recombinant vector comprising a polynucleotide molecule described in the present invention.

[065] In another aspect, the present disclosure provides a host cell comprising a polynucleotide molecule described in the present invention, or an expression vector described in the present invention.

[066] In another aspect, the present disclosure provides a kit comprising an antigen-binding protein described in the present invention, a fusion protein described, a conjugate described in the present invention, a polynucleotide molecule described in the present invention, a recombinant vector described in the present invention, or the host cell described in the present invention, and optionally, instructions and / or packaging therefor.

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

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

[069] In another aspect, the present disclosure provides a method for Petition 870250081421, dated 10 / 09 / 2025, page 30 / 374 20 / 304 targeting a cell expressing CD25 comprising contacting the cell with an antigen-binding protein described in the present invention, a fusion protein described in the present invention, or a conjugate described in the present invention.

[070] In some forms, the cell is a regulatory T cell (Treg).

[071] In some forms, cell contact can occur in vitro.

[072] In some forms, cell contact can occur in vivo.

[073] In some embodiments, a method described in the present invention may further comprise administering the antigen-binding protein, the fusion protein, or the conjugate to an individual who needs it.

[074] In another aspect, the present disclosure provides a method of treating or preventing a disease or disorder in an individual who needs it, and the method may comprise administering to the individual an antigen-binding protein described in the present invention, a fusion protein described in the present invention, or a conjugate described in the present invention.

[075] In some forms, the disease or disorder is an immunological disease, inflammatory disease, cancer, cardiovascular disease, or an infertility and pregnancy-associated disease.

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

[077] In some modalities, 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 dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Petition 870250081421, dated 10 / 09 / 2025, page 31 / 374 21 / 304 Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosus, IgG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disorder, pemphigus vulgaris or related bullous skin disease, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, premature ovarian failure, agammaglobulinemia primary, primary biliary cirrhosis, psoriasis, primary ovarian failure, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis,Scleroderma, Sjögren's syndrome, spondyloarthritis, stiff-man syndrome, type I diabetes, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (granulomatosis with polyangiitis) or other immune vasculitis.

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

[079] In some forms, cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DLE), or acute subcutaneous lupus erythematosus.

[080] In some modalities, 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.

[081] In some modalities, the allergy is selected from food allergy, seasonal allergy, pet allergy, urticaria, hay fever, Petition 870250081421, dated 10 / 09 / 2025, page 32 / 374 22 / 304 allergic conjunctivitis, poison ivy allergy, oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, and chemical allergy.

[082] In some modalities, allograft rejection is selected from skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, and organ graft rejection.

[083] In some modalities, ligament graft rejection is selected from cricothyroid ligament graft rejection, caudal cruciate ligament graft rejection, periodontal ligament graft rejection, suspensory ligament graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, ulnar collateral ligament graft rejection, radial collateral ligament graft rejection, suspensory ligament 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.

[084] In some modalities, organ graft rejection is selected from heart graft rejection, lung graft rejection, kidney graft rejection, liver graft rejection, pancreas graft rejection, bowel graft rejection, and thymus graft rejection.

[085] In some forms, graft-versus-host disease arises from a bone marrow transplant or from one or more blood cells selected from B cells, T cells, basophils, myeloid progenitor cells. Petition 870250081421, dated 10 / 09 / 2025, page 33 / 374 23 / 304 common, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.

[086] In some forms, inflammatory disease is either acute or chronic inflammation.

[087] In some modalities, the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovary syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.

[088] In some modalities, 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 (bone sarcoma), brainstem 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 childhood ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing's sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, Familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST),Germ cell cancer, tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell carcinoma, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, renal cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML). Petition 870250081421, dated 10 / 09 / 2025, page 34 / 374 24 / 304 B-cell prolymphocytic leukemia and hairy cell leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), T-cell chronic lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, Lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH-associated polyposis (or MYH), myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, tumor pancreatic neuroendocrine, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma,Ovarian cancer, fallopian tube and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), Werner syndrome, Wilms' tumor or xeroderma pigmentosum.

[089] In some embodiments, a cardiovascular disease described in the present invention 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 Petition 870250081421, dated 10 / 09 / 2025, page 35 / 374 25 / 304 hypertension.

[090] In some modalities, infertility and pregnancy-related diseases are selected based on recurrent pregnancy loss, pre-eclampsia, premature birth, fetal growth restriction, or intrauterine growth restriction.

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

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

[093] In some modalities, contact with a tissue or organ occurs in vitro.

[094] In some modalities, contact with a tissue or organ occurs in vivo.

[095] In some embodiments, a method disclosed in the present invention may further comprise administering the antigen-binding protein, the fusion protein, or the conjugate to an individual in need thereof.

[096] In another aspect, the present disclosure provides a method for inducing tolerance to a foreign agent and / or preventing or reducing the immune response to a foreign agent in an individual who needs it, and the method may comprise administering to the individual an antigen-binding protein described in Petition 870250081421, dated 10 / 09 / 2025, p. 36 / 374 26 / 304 present invention, a fusion protein described in this invention, or a conjugate described in this invention.

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

[098] In some forms, the viral vector is a DNA or RNA vector.

[099] In some forms, the individual is a mammal.

[0100] In some forms, the mammal is human. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 describes an exemplary general screening strategy for isolating CD25-specific variable heavy chain domain (VHH) antibodies, also referred to in the present invention as V-bodies (Vbs). Ligands for human and rodent CD25 were enriched from VHH immune libraries by two rounds of phage display. BM, bone marrow.

[0102] Figure 2 shows the selection of the VHH immune library for next-generation sequencing (NGS) during the phage display process. Three initial libraries, 12 samples from the first screening round, and 36 samples from the second screening round were sequenced with 20 million, 2 million, and 2 million reads, respectively. Comparison of the V-body enrichment of the initial library with the first and second screening rounds allowed the identification of potential V-body candidates.

[0103] Figure 3 shows a schematic diagram of an exemplary NGS workflow. After phage display, the VHH region of the phage eluate was amplified via polymerase chain reaction (PCR). Unique, sample-specific barcodes were then fused, and NGS was subsequently performed using the Illumina NovaSeq (Genewiz) platform. The raw data were Petition 870250081421, dated 10 / 09 / 2025, page 37 / 374 27 / 304 were demultiplexed and then processed by the NGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions, and VHHs, including complementarity-determining regions (CDRs), were annotated. Based on CDR3 identity, V-body sequences were clustered, thus allowing a detailed analysis of, for example, V-body enrichment during phage display, sequence diversity, CDR3 length distribution, and cluster abundance. Based on these analyses, up to ~300 candidates were selected for DNA synthesis (Twist) and further characterization.

[0104] Figure 4 illustrates the validation of human CD25 (hCD25) binding to V-body at a fixed concentration of 100 nM V-body. The bar histogram shows the mean fluorescence intensity (FMI) of Alexa488 cells positive for V-bodies ODY-46A3 and ODY-47D3 versus an anti-His-only control condition.

[0105] Figure 5 illustrates the binding of V-body to cynomolgus (cCD25) (left panel) and mouse CD25 (mCD25) (right panel) at a fixed concentration of 100 nM V-body. Bar histograms show the mean fluorescence intensity (FMI) of Alexa488-positive cells for the tested bodies ODY-46A3 and ODY47D3 versus a control condition with anti-His only.

[0106] Figures 6A-6B show the binding assays of human CD25 V-bodies at a range of concentrations for the ODY-46A3 and ODY-47D3 V-bodies. The 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 graph in Figure 6A shows the percentage of Alexa488-positive cells for ODY-46A3 and ODY-47D3. The bar graph in Figure 6B shows the mean fluorescence intensity (FMI) of Alexa488-positive cells for ODY-46A3 and ODY-47D3.

[0107] Figure 7 shows a schematic diagram of a configuration Petition 870250081421, dated 10 / 09 / 2025, page 38 / 374 28 / 304 experimental specimen for determining the binding affinities of V-bodies to their respective targets by means of surface plasmon resonance (SPR). The figure depicts “HHHHHH” as SEQ ID NO: 4325.

[0108] Figures 8A-8C represent surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for the anti-CD25 V-bodies ODY-46A3 and ODY-47D3. Fitted binding curves and calculated dissociation constants (KD) are included. Data corresponding to an anti-CD25 IgG control condition (IgG αCD25) are also included (Figure 8C). The figure depicts HHHHHH as SEQ ID NO: 4325.

[0109] Figure 9 shows a summary of the binding affinities of two candidate anti-CD25 Vbodies, human, cynomolgus and mouse. Data corresponding to an anti-CD25 IgG control condition (IgG αCD25) are also included. NB: no binding.

[0110] Figures 10A-10B demonstrate that some humanized anti-CD25 V-bodies targeted the epitope recognized by IL-2. Data are presented for a first experiment 1 (Exp1) and a second experiment 2 (Exp2) performed with V-bodies 46A3 (Figure 10A) and 47D3 (Figure 10B).

[0111] Figures 11A-11B demonstrate that the humanized anti-CD25 V-bodies ODY-N1570hu1, ODY-N1572Hu1 and ODY-N1574 are non-competitive ligands. Data are presented for the first experiment 1 (Figure 11A) and the second experiment 2 (Figure 11B).

[0112] Figures 12A-12C represent the SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies ODY83B03Hu1, ODY-83B05Hu1, and ODY-83F07Hu1. Fitted binding curves and calculated dissociation constants (Kd) are included.

[0113] Figures 13A-13C illustrate ligand (IL-2) competition for SPR. Each panel represents an overlay graph of sensorgrams for a single Petition 870250081421, dated 10 / 09 / 2025, page 39 / 374 29 / 304 V-body captured at a separate point. The sensorgrams show competition for IL-2-Fc: association of the human CD25 extracellular domain (CD25-ECD) to the V-body was followed by additional binding by IL2-Fc, indicating an unoccupied epitope (non-overlapping epitopes), or absence of binding by IL2-Fc, indicating epitope blocking (overlapping epitopes), and a buffer control, association and dissociation of human CD25-ECD in the absence of IL2-Fc.

[0114] Figure 14 shows the binding of His-labeled anti-CD25 VHHs to human embryonic kidney cells (HEKs) transfected with human or cyno-CD25, detected by flow cytometry using a fluorescence-labeled secondary anti-His antibody. Binding is expressed as mean fluorescence intensity.

[0115] Figures 15A-15C represent the SPR sensorgrams of VHH binding to human, cynomolgus and mouse CD25 for the anti-CD25 V-bodies ODY-83B05Hu1.8A, ODY-83B05Hu1.8L and ODY-83F07Hu1.8L. Fitted binding curves and calculated dissociation constants (Kd) are included.

[0116] Figures 16A-16C represent the SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies ODY48C10Hu1, ODY-48D11Hu1, ODY-N1769Hu1, ODY-N1783Hu1, ODY-N1808Hu1, ODY-N1810Hu1, ODY-N1811Hu1, ODY-N1812Hu1, and ODY-N1813Hu1. Fitted binding curves and calculated dissociation constants (Kd) are included.

[0117] Figures 17A-17C represent the SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies ODYN2005Hu1, ODY-N2008Hu1, ODY-N2010Hu1, ODY-N2011Hu1, ODY-N2016Hu1, ODY-N2017Hu1, ODY-N2022Hu1, ODY-N2024Hu1, ODY-N2025Hu1, ODYN2026Hu1, and ODY-N2027Hu1. Fitted binding curves and calculated dissociation constants (Kd) are included.

[0118] Figure 18 depicts the SPR sensorgrams of the VHH connection to Petition 870250081421, dated 10 / 09 / 2025, p. 40 / 374 30 / 304 Human, cynomolgus, and mouse CD25 antibodies ODYN1955Hu1, ODY-N1961Hu1, and ODY-N1970Hu1 are used to determine the binding strength of CD25 antibodies. Fitted binding curves and calculated dissociation constants (Kd) are included.

[0119] Figure 19 depicts the SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies ODYN1974Hu1 and ODY-N1978Hu1. Fitted binding curves and calculated dissociation constants (Kd) are included.

[0120] Figures 20A-20B represent the SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies ODYN1998Hu1, ODY-N1999Hu1, ODY-N2000Hu1, ODY-N2001Hu1, ODY-N2002Hu1, and ODY-N2003Hu1. Fitted binding curves and calculated dissociation constants (Kd) are included. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0121] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains. For purposes of interpreting this descriptive report, the following description of terms shall apply and, where appropriate, terms used in the singular shall also include the plural and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that any description of the terms presented conflicts with any document incorporated in the present invention by reference, the description of the term presented below shall prevail.

[0122] As used in the present invention, the term “about”, when used in reference to a particular reported numerical value, means that the value may vary from the reported value by no more than 5%. For example, as used in Petition 870250081421, dated 10 / 09 / 2025, page 41 / 374 31 / 304 of the present invention, the expression "about 100" includes 95 and 105 and all intermediate values ​​(e.g., 96, 97, 98, 99, etc.).

[0123] The term “antigen” encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that can be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T cell receptor. In various embodiments of the present disclosure, the antigen described in the present invention is CD25, including human, cynomolgus, and / or mouse CD25.

[0124] The term epitope can refer to an antigenic determinant on the surface of an antigen to which an antibody molecule binds. A single antigen can have more than one epitope. Thus, different antibodies can bind to different areas on an antigen and can have different biological effects (e.g., agnostic or antagonistic effects). Epitopes can be conformational or linear. A conformational epitope is formed by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include non-peptidic fragments in the antigen, such as saccharides, phosphoryl groups, or sulfonyl groups.

[0125] The term antigen-binding protein refers in its broadest sense to a protein that binds specifically to an antigen (e.g., CD25). In certain embodiments, an antigen-binding protein is an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single-chain antibody (e.g., single-chain fragment variable (scFv)); a diabody; a triabody; a Petition 870250081421, dated 10 / 09 / 2025, p. 42 / 374 32 / 304 tetrabody; a Fab fragment; an F(ab')2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof. The term antigen-binding protein also encompasses, for example, an alternative protein scaffold or artificial scaffold with grafted or CDR-derived CDRs. 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 fully synthetic scaffolds comprising, for example, a biocompatible polymer. In addition, peptide antibody mimetics may be used, as well as scaffolds based on antibody mimetics that utilize fibronectin components (e.g., fibronectin type III domain (FN3)) as a scaffold.

[0126] The term “CD25”, or “cluster of differentiation 25”, or “interleukin 2 receptor alpha chain”, or “interleukin 2 receptor alpha subunit”, or “IL2Rα”, or “IL2RA”, or the like, is used interchangeably in the present invention and may refer to any isoform(s), variant(s), and / or species homolog(s) of CD25 from any source, for example, mammals including primates (e.g., humans and monkeys) and rodents (e.g., rats and mice). The term encompasses naturally occurring variants of CD25 such as, but not limited to, allelic variants and splice variants. The term also encompasses “full-length” or unprocessed CD25 in addition to any form of CD25 that may result from processing such as that which may occur within a cell. In some embodiments, CD25 is human CD25. As one example, but not limited to, CD25 can be expressed by activated lymphocytes (e.g., activated T lymphocytes and / or activated B lymphocytes).Most regulatory T cells (Tregs) can express CD25. A heterodimeric complex comprising IL2Rα, IL2R (beta) β (also called CD122), and IL2R (gramma) γ (also called CD132) can form. Petition 870250081421, dated 10 / 09 / 2025, page 43 / 374 33 / 304 a high-affinity IL2R. IL2Ra and IL2Re can form a pseudo-high-affinity receptor.

[0127] The term “interleukin-2”, or “IL-2”, or “IL2”, or similar, is used interchangeably in the present invention and may refer to any isoform(s), variant(s), and / or species homolog(s) of IL-2 from any source, for example, 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 may result from processing such as that which may occur within a cell.

[0128] The terms “antibody” and “immunoglobulin” or “Ig” are used interchangeably in the present invention, and are used in the broadest sense and encompass, for example, individual monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with polyepitope or monoepitope specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies (e.g., VHH), single-chain antibodies, intracorporeal antibodies, antiidiotypic antibodies (anti-Id), and antigen-binding fragments of antibodies, as described below. An antibody may be human, humanized, camelized, recombinantly produced, chimeric, synthetic, affinity-dematured and / or affinity-matured as well as an antibody from other species, for example mouse, camel, llama, rabbit, etc.In specific embodiments, the specific target antigen that can be bound by an antibody provided in the present invention includes a CD25 polypeptide, CD25 fragment, or CD25 epitope. An “antigen-binding fragment” generally refers to a portion of a... Petition 870250081421, dated 10 / 09 / 2025, p. 44 / 374 34 / 304 heavy and / or light chain polypeptide of antibody 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 antibodies (e.g., VHH), single-chain Fvs (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody, or a chemically modified derivative thereof. In particular, the antibodies provided in the present invention include immunoglobulin molecules and molecules containing immunologically active portion(s) of an immunoglobulin molecule, for example, one or more complementarity-determining regions (CDRs) of an antibody that binds to CD25.Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molecular Biology and Biotechnology: A Comprehensive Reference Table, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178: 497-515 (1989) and in Day, ED, Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990). The antibodies provided in this invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.

[0129] The term “single-domain antibody” or “sdAb” as used in the present invention refers to an antibody or antibody fragment containing a single variable antibody domain that is capable of binding to a specific antigen alone, without the need for another variable antibody domain. The complementarity-determining regions (CDRs) of a single-domain antibody are part of a single variable antibody domain. Examples of single-domain antibodies include, but are not limited to, heavy chain antibodies, antibodies Petition 870250081421, dated 10 / 09 / 2025, page 45 / 374 35 / 304 naturally devoid of light chains, single-domain antibodies derived from conventional four-chain antibodies, modified antibodies, variable domains derived from previously mentioned antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies may be derived from any species including, but not limited to, mouse, human, camel, llama, shark, goat, rabbit, and / or bovine. In some embodiments, a single-domain antibody as used in the present invention is a naturally occurring single-domain antibody known as a light-chain-deprived heavy-chain antibody. For clarity, the variable domain derived from a naturally light-chain-deprived heavy-chain antibody is known in the present invention as a VHH to distinguish it from the conventional VH of four-chain immunoglobulins.Such a VHH molecule can be derived from antibodies created in Camelidae species, for example, camel, llama, dromedary, alpaca, and guanaco. Other species besides Camelidae can produce naturally occurring heavy-chain antibodies lacking a light chain, which are also within the scope of the invention. For example, cartilaginous fish such as sharks can produce immunoglobulin-like structures known as VNAR. In some embodiments, a single-domain antibody can be obtained from a Camelidae VH domain. In some embodiments, a single-domain antibody can be obtained from human VH by camelization. See Saerens et al., Current Opinion in Pharmacology, 2008, 8:600608, disclosure of which is incorporated by reference, for a review of single-domain antibodies.

[0130] The term “specifically binds to” as used in the present invention means that an antigen-binding protein forms a complex with a target antigen that is relatively stable under physiological conditions. Specific binding can be characterized by a dissociation constant (Kd) of about Petition 870250081421, dated 10 / 09 / 2025, p. 46 / 374 36 / 304 1x10-6M or less (e.g., less than 10-6M, less than 5x10-7M, less than 10-7M, less than 5x10-8M, less than 10-8M, less than 5x10-9M, less than 10-9M, or less than 10-10M). Methods for determining the binding affinity of an antigen-binding protein, e.g., an antibody or an antibody fragment, to a target antigen are well known in the state of the art and include, for example, surface plasmonic resonance (e.g., BIACORE® assays), biolayer interferometry, ligand binding assays (e.g., enzyme-linked immunosorbent assay (ELISA)), equilibrium dialysis, fluorescence-activated cell sorting (FACS), or flow cytometry-based binding assays and the like. Specific binding to a particular target antigen from a certain species does not preclude the possibility that the antigen-binding protein may also bind specifically to the analogous target from a different species.For example, specific binding to human CD25 does not preclude the possibility that the antigen-binding protein may also bind specifically to CD25 from cynomolgus (“cyno”) monkeys or mice.

[0131] The term isolate, when used in the context of antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides, and vectors, means that the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides, and vectors are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cell debris and growth medium. An isolated antigen-binding protein may also be at least partially free of expression system components such as biological molecules from a host cell or its growth medium. Generally, the term isolate is not intended to Petition 870250081421, dated 10 / 09 / 2025, page 47 / 374 37 / 304 refers to a complete absence of such biological molecules (for example, minor or insignificant amounts of impurities may remain) or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes antigen-binding proteins (for example, antibodies, such as single-domain antibodies).

[0132] The term “operationally linked” as used in the present invention may 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.

[0133] As used in the present invention, the term “variant”, “derivative” or “derived from” in the context of proteins or polypeptides (for example, antigen-binding proteins or domains thereof) refers 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 with the polypeptide from which it is a variant or derivative; (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 with a nucleotide sequence encoding the polypeptide of which it is a variant or derivative; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and / or substitutions) relative to the polypeptide of which it is a variant or derivative;(d) a polypeptide encoded by nucleic acids can hybridize under high, moderate, or typical stringency hybridization conditions with nucleic acids encoding the polypeptide of which it is a variant or derivative; (e) a polypeptide encoded by a nucleotide sequence that can hybridize under high, moderate, or typical stringency hybridization conditions with a nucleotide sequence encoding a fragment of the polypeptide, of which it is a variant or derivative, of at least 20 contiguous amino acids; Petition 870250081421, dated 10 / 09 / 2025, page 48 / 374 38 / 304 less 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of the polypeptide of which it is a variant or derivative. The terms also encompass a fusion protein or polypeptide comprising the polypeptide of which it is a variant or derivative.

[0134] The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when ideally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there exists nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain examples, encode a polypeptide having the same amino acid sequence or a substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0135] As applied to polypeptides, the term substantial similarity or substantially similar means that two peptide sequences, when ideally aligned, such as by the GAP or BESTFIT programs using standard gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, the residue positions that are not identical differ in conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with chemical properties. Petition 870250081421, dated 10 / 09 / 2025, p. 49 / 374 39 / 304 similar (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated into the present invention by reference.Examples of amino acid groups 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: cysteine ​​and methionine. The preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 14431445, incorporated into the present invention by reference.A moderately conservative substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0136] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software compares similar sequences using similarity measures attributed to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the Petition 870250081421, dated 10 / 09 / 2025, p. 50 / 374 The 40 / 304 GCG software contains programs such as Gap and Bestfit that can be used with standard parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutant of the same type. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA using standard or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentage of sequence identity of the best overlap regions between query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a development sequence with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using standard parameters.See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each incorporated into the present invention by reference.

[0137] The terms “enhance” or “promote”, or “increase”, or “expand”, or “improve” generally refer to the ability of a composition considered in the present invention to produce, induce, or cause a greater physiological response (i.e., downstream effects) compared with the response caused by the 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 killing capacity, among others evident from the art understanding and description in the present invention. In certain modalities, an “increased” or “enhanced” quantity may be a “statistically significant” quantity, 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 intermediate whole numbers and decimal points above 1, Petition 870250081421, dated 10 / 09 / 2025, page 51 / 374 41 / 304 for example, 1.5, 1.6, 1.7, 1.8, etc.) the response produced by the vehicle or a control composition.

[0138] The terms “decrease” or “lower”, or “moderate”, or “reduce”, or “decline”, or “attenuate”, or “suppress” generally refer to the ability of the composition considered in the present invention to produce, induce, or cause a smaller physiological response (i.e., downstream effects) compared to the response caused by the vehicle or a control molecule / composition. In certain modalities, a “decreased” or “reduced” quantity may be a “statistically significant” quantity, 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 intermediate and above integers and decimal points above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) the response (reference response) produced by the vehicle or a control composition.

[0139] The terms “treat” or “treatment” of a state, disorder, or condition include: (1) preventing, delaying, or reducing the incidence and / or probability of the appearance of at least one clinical or subclinical symptom of the development of the state, disorder, or condition in an individual who may be afflicted with or predisposed to the state, disorder, or condition, but who does not yet experience or exhibit clinical or subclinical symptoms of the state, disorder, or condition; or (2) inhibiting the state, disorder, or condition, that is, stopping, reducing, or delaying the development of the disease or a relapse thereof or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, that is, causing the regression of the state, disorder, or condition or at least one of its clinical or subclinical symptoms. The benefit to an individual being treated is statistically significant or at least perceptible to the patient or physician.

[0140] The terms “effective amount” or “therapeutically effective amount” refer to an amount and / or concentration of a composition containing a Petition 870250081421, dated 10 / 09 / 2025, p. 52 / 374 42 / 304 active ingredient (e.g., anti-CD25 antigen-binding protein) that, when administered to a patient alone (i.e., as monotherapy) or in combination with additional therapeutic agents, produces a significant decrease in disease progression, such as by improving or eliminating the symptoms and / or the cause of the disease. An effective amount may be an amount that mitigates, moderates, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents the progression of the disease or disorder, or improves the patient's physical functioning. 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 induce a desired response in the individual.A therapeutically effective dose is also one in which any toxic or harmful effects of the active agent are offset by the therapeutically beneficial effects. A therapeutically effective dose may be administered in one or more doses. A therapeutically effective dose refers to an effective amount, in dosages and for the time periods necessary, to achieve the desired therapeutic and / or prophylactic result.

[0141] The terms “individual” and “patient” are used interchangeably in the present invention to refer to an animal; for example, a mammal. The terms include human and veterinary individuals. In some embodiments, methods of treating mammals, including but not limited to humans, rodents, apes, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The individual may be male or female and may be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric individuals. In some embodiments, an individual may be an individual in need of treatment for a disease or Petition 870250081421, dated 10 / 09 / 2025, page 53 / 374 43 / 304 disorder. In particular modalities, the individual is a human being. Anti-CD25 antigen-binding proteins

[0142] The present disclosure provides antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) that bind to CD25.

[0143] Cluster of differentiation 25 (CD25), also called the alpha subunit of the interleukin-2 receptor (IL-2Rα or IL2RA), is the alpha chain component of the heterotrimeric interleukin-2 receptor complex. IL-2Rα is a type I single-pass 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 necessary to allow CD25 to form a cap-like structure in the IL-2 receptor complex while still being anchored to the membrane. The actual structure and positioning of the loop have not been resolved in any of the available crystal structures. The sushi domains of CD25 form five interlocking beta-sheet sandwiches that are related to each other in a pseudo-double symmetry.The Sushi 1 domain is responsible for the majority of interactions with IL-2 (82%), while the Sushi 2 domain contributes significantly less (Stauber et al., Proc Natl Acad Sci USA 103, 2788-2793. 2006). The CD25 structure is stabilized by several intradomain disulfide bonds and two interdomain disulfide bonds. Furthermore, CD25 carries several glycans with an N-glycosylation located at the C-terminus of the Sushi 2 domain and four O-glycans located in the disordered C-terminal region.

[0144] CD25 interacts with IL-2 in a precise manner. It is postulated that the IL-2 receptor complex forms gradually, beginning with the binding of IL-2 to CD25 / IL-2Ra, then engaging with the β subunit and finally interacting with the γ subunit of the receptor (Stauber et al., Proc Natl Acad Sci USA). Petition 870250081421, dated 10 / 09 / 2025, page 54 / 374 44 / 304 103, 2788-2793. 2006). Interestingly, it has been reported that CD25 can present IL-2 in both cis and trans configurations (Liao et al., Immunity 38, 13-25. 2013; Wuest et al., Nat Med 17, 604-609. 2011), both resulting in the assembly of the IL-2 receptor complex. The CD25 / IL-2Ra complex has the largest interface with IL-2 within the complex, which is reflected in the very high affinity between IL-2 and CD25 (Liao et al., 2013). However, the CD25 / IL-2Ra complex itself does not make direct contact with the other two β or γ subunits. Deglycosylation experiments of the individual subunits were found to impact complex formation with the aggregation of the γ subunit, while the α and β subunits were still able to bind to IL-2 (Stauber et al., Proc Natl Acad Sci USA 103, 2788-2793. 2006). Consequently, glycosylation at CD25 / IL-2Ra is not essential for interaction with IL-2.IL-2-Ry and IL-2Re are also part of other interleukin receptor complexes, while CD25 / IL-2Ra is found exclusively in the IL-2 receptor complex (Liao et al., Immunity 38, 13-25. 2013).

[0145] In addition to the membrane-anchored version of CD25, soluble CD25 has been reported to be found in human serum (Pedersen and Lauritsen, Scand J Immunol 70, 40-43. 2009). This soluble form of CD25 may result from a membrane-anchored protein release event, producing a truncated CD25 with a molecular weight (MW) of ~20kDa.

[0146] In some embodiments, the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) described in the present invention bind to human CD25. In some embodiments, the human CD25 protein is encoded by the human interleukin-2 receptor (IL2RA) alpha subunit gene (NCBI gene ID: 3559) and has the amino acid sequence MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGF RRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQ SPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPA Petition 870250081421, dated 10 / 09 / 2025, p. 55 / 374 45 / 304 ESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTD FQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (UniProtKB Accession No. P01589) (SEQ ID NO: 2210)

[0147] In some embodiments, the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) described in the present invention bind to cynomolgus monkey CD25 (“cyno”). In some embodiments, the cyno CD25 protein is encoded by the cyno interleukin-2 receptor (IL2RA) alpha subunit gene (NCBI Gene ID: 102123605) and has the amino acid sequence of MDPYLLMWGLLTFITVPGCQAELCDDDPPKITHATFKAVAYKEGTMLNCECKRGFR RIKSGSPYMLCTGNSSHSSWDNQCQCTSSAARNTTKQVTPQPEEQKERKTTEMQ SQMQLADQVSLPGHCREPPPWENEATERIYHFVVGQTVYYQCVQGYRALHRGPA ESICKMTHGKTRWTQPQLICTGETEPSQFPGEEEPQASPDGLPESETSRLVTTTDF RIQTEVAATMETFIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKNRRTI (GenBank Accession No. EHH64536.1) (SEQ ID NO: 2211)

[0148] In some embodiments, the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) described in the present invention bind to mouse CD25. In some embodiments, the mouse CD25 protein is encoded by the mouse interleukin 2 receptor alpha subunit gene (Il2ra) (NCBI Gene ID: 16184) and has the amino acid sequence of MEPRLLMLGFLSLTIVPSCRAELCLYDPPEVPNATFKALSYKNGTILNCECKRGFRR LKELVYMRCLGNSWSSNCQCTSNSHDKSRKQVTAQLEHQKEQQTTTDMQKPTQS MHQENLTGHCREPPPWKHEDSKRIYHFVEGQSVHYECIPGYKALQRGPAISICKMK CGKTGWTQPQLTCVDEREHHRFLASEESQGSRNSSPESETSCPITTTDFPQPTETT AMTETFVLTMEYKVAVASCLFLLISILLLSGLTWQHRWRKSRRTI (UniProtKB Accession No. P01590) (SEQ ID NO: 2212) Petition 870250081421, dated 10 / 09 / 2025, page 56 / 374 46 / 304

[0149] In some embodiments, the antigen-binding proteins of the present disclosure after binding to CD25 do not impair the binding of their cognate ligand, interleukin-2 (IL-2), to CD25. In some embodiments, the antigen-binding proteins of the present disclosure do not have epitopes that overlap with IL-2.

[0150] In some embodiments, the antigen-binding proteins of the present disclosure after binding to CD25 may impair the binding of IL-2 to CD25. In some embodiments, the antigen-binding proteins of the present disclosure may have epitopes that overlap with IL-2. In some embodiments, when the antigen-binding proteins have epitopes that overlap with IL-2, the antigen-binding proteins may impair the binding of IL-2 to CD25. In some embodiments, when the antigen-binding proteins have epitopes that overlap with IL-2, the antigen-binding proteins may compete for binding to CD25 with IL-2.

[0151] In several embodiments, the antigen-binding proteins of the present disclosure may have an antagonistic effect (e.g., a blocking effect) after binding to CD25. An antagonistic CD25 ligand may block or diminish CD25 activation and / or attenuate one or more CD25-mediated signal transduction pathways. Antagonistic CD25 ligands may block or diminish CD25 activation by binding to CD25, for example, to induce a conformational change that renders the receptor biologically inactive. For example, antagonistic CD25 ligands may prevent trimerization of an IL-2 receptor complex as may occur due to the interaction between CD25 and its cognate ligand, IL-2, thus impairing CD25-mediated signaling.

[0152] In some embodiments, when the antigen-binding proteins of the present disclosure have epitopes that overlap with IL-2, such antigen-binding proteins may have an antagonistic effect on CD25 binding.

[0153] In several forms, antigen-binding proteins present Petition 870250081421, dated 10 / 09 / 2025, p. 57 / 374 47 / 304 revelation may have an agonistic effect (i.e., a stimulatory effect) upon binding to CD25. An agonistic CD25 ligand may stimulate or enhance CD25 activation and / or reinforce one or more CD25-mediated signal transduction pathways. Agonistic CD25 ligands may stimulate or enhance CD25 activation by binding to CD25, for example, to induce a conformational change that renders the receptor biologically active. For example, agonistic CD25 ligands may promote the trimerization of an IL-2 receptor complex as may occur due to the interaction between CD25 and its cognate ligand, IL-2, thus promoting CD25-mediated signaling.

[0154] In some embodiments, when the antigen-binding proteins of the present disclosure have epitopes overlapping with IL-2, such antigen-binding proteins may have an agonistic effect after binding to CD25.

[0155] In some embodiments, the antigen-binding proteins of the present disclosure bind to human CD25. In some embodiments, the antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present development can bind to human 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 8x10⁻⁸ 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, or less than about 1x10⁻⁹ M, or about 1x10⁻¹⁰ to 1x10⁻⁹ M, 1x10⁻¹⁰ to 5x10⁻⁹ M, about 1x10-10a 1x10-8M, about 1x10-10y 5x10-8M, about 1x10-9y 1x10-8M, about 1x10-9a 5x10-8M, about 1x10-9y 1x10-7M, or about 1x10-8h 1x10-7M.

[0156] In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of about 1.6 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to CD25 Petition 870250081421, dated 10 / 09 / 2025, p. 58 / 374 48 / 304 human with a Kd of approximately 7.6 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 9.4 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 10 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 11 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 12 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 13 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 14 nM. In one embodiment, an antigen-binding protein of the present development binds to human CD25 with a Kd of approximately 17 nM.In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 18 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 19 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 20 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 21 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 22 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 26 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 31 nM.In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 35 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of... Petition 870250081421, dated 10 / 09 / 2025, p. 59 / 374 49 / 304 approximately 49 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 50 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 58 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 61 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 62 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 66 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 73 nM. In one embodiment, an antigen-binding protein of the present development binds to human CD25 with a Kd of approximately 76 nM.In one embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 97 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 102 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 107 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 149 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 241 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to human CD25 with a Kd of approximately 348 nM.

[0157] In some embodiments, the antigen-binding proteins of the present disclosure bind to cynomolgus monkey (“cyno”) CD25. In some embodiments, the antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure can bind to cyno CD25 with a Kd of less than about 1 x10-6M, for example, less than about Petition 870250081421, dated 10 / 09 / 2025, p. 60 / 374 50 / 304 of 5x10-7M, less than about 3x10-7M, less than about 1x10-7M, less than about 8x10-8M, less than about 5x10-8M, less than about 3x10-8M, less than about 1x10-8M, less than about 8x10-9M, less than about 5x10-9M, less than about 3x10-9M, or less than about 1x10-9M, or about 1x10-10a 1x10-9M, 1x10-10a 5x10-9M, about 1x10-10a 1x10-8M, about 1 χ10-10a 5χ10-8M, about 1 χ10-9a 1χ10-8M, about 1 χ10-9a 5χ10-8M, about 1 χ10-9a 1χ10-7M, about 1 χ10-9a 2χ10-7M, about 1χ10-9a 5χ10-7M, about 1χ10-8a 1χ10-7M, about 1χ10-8a 2χ10-7M, about 1 χ10-8a 5χ10-7M, or about 1χ10-8a 1 χ10-6M.

[0158] In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a Kd of about 706 pM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a Kd of about 793 pM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a Kd of about 1.5 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a Kd of about 73 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a Kd of about 34 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a Kd of about 48 nM. In one embodiment, an antigen-binding protein of the present development binds to cyno CD25 with a Kd of approximately 49 nM.In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 52 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 57 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 70 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of... Petition 870250081421, dated 10 / 09 / 2025, p. 61 / 374 51 / 304 approximately 79 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 97 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 107 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 112 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 115 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 117 nM. In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 119 nM. In one embodiment, an antigen-binding protein of the present development binds to cyno CD25 with a Kd of approximately 121 nM.In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 131 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 136 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 142 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 146 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 148 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 149 nM. In one embodiment, an antigen-binding protein of the present development binds to cyno CD25 with a Kd of approximately 162 nM.In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 163 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 186 nM. In one embodiment... Petition 870250081421, dated 10 / 09 / 2025, p. 62 / 374 In one embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 191 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 211 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 235 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 283 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 339 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cynoCD25 with a Kd of approximately 380 nM. In one embodiment, an antigen-binding protein of the present development binds to cyno CD25 with a Kd of approximately 411 nM.In one embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a Kd of approximately 956 nM. In another embodiment, an antigen-binding protein of the present disclosure binds to cyno CD25 with a Kd of approximately 2.1 μM.

[0159] In some embodiments, the antigen-binding proteins of the present disclosure bind to mouse CD25. In some embodiments, the antigen-binding proteins of the present disclosure can bind to mouse CD25 with a Kd of less than about 1 χ10-6M, for example, less than about 5χ10-7M, less than about 3χ10-7M, less than about 1χ10-7M, less than about 8x10-8M, less than about 5χ10-8M, less than about 3χ10-8M, less than about 1 x10-8M, less than about 8χ10-9M, less than about 5χ10-9M, less than about 3χ10-9M, or less than about 1χ10-9M, or about 1 χ10-10a 1 χ10-9M, 1 χ10-10a 5χ10-9M, about 1χ10-10a 1χ10-8M, about 1χ10-10a 5χ10-8M, about 1χ10-9a 1χ10-8M, about 1χ10-9a 5χ10-8M, about 1χ10-9a 1 χ10-7M, about 1χ10-9a 2χ10-7M, about 1 χ10-9a 5χ10-7M, about 1 χ10-8a 1χ10-7M, about 1 χ10-8 Petition 870250081421, dated 10 / 09 / 2025, p. 63 / 374 53 / 304 to 2χ10-7M, approximately 1 χ10-8 to 5χ10-7M, or approximately 1 χ10-8 to 1χ10-6M. In some embodiments, the antigen-binding proteins of the present development do not bind to mouse CD25.

[0160] In one embodiment, an antigen-binding protein of the present disclosure binds to mouse CD25 with a Kd of approximately 420 nM.

[0161] The binding affinity of a molecular interaction between two molecules can be measured using various techniques, such as surface plasmon resonance (SPR), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescence-activated cell sorting (FACS), or flow cytometry binding assays and the like. Surface plasmon resonance is a biosensor technique that allows the analysis of biospecific interactions in real time by detecting changes in protein concentrations within a biosensor array, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559). SPR can, for example, be performed using the BIACORE® system or the Carterra LSA system.Another biosensor technique that can be used to determine the affinities of biomolecular interactions is biolayer interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. 377: 209-217). Biolayer 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 protein layer immobilized at the tip of the biosensor (signal beam). A change in the number of molecules bound to the biosensor tip causes a change in the interference pattern, reported as a change in wavelength (nm), the magnitude of which is a direct measure of the number of molecules bound to the surface of the biosensor tip. Since the interactions can be measured in real time, Petition 870250081421, dated 10 / 09 / 2025, page 64 / 374 54 / 304 association and dissociation rates and affinities can be determined. BLI can, for example, be performed using Octet® Systems. Alternatively, affinities can be measured in the Kinetic Exclusion Assay (KinExA) (see, for example, Drake et al. 2004, Anal. Biochem., 328: 35-43), which is a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. Equilibrium solutions of an antibody / antigen complex are passed over a column with pre-coated antigen (or antibody) beads, allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is performed with a fluorescently labeled protein that binds to the antibody (or antigen).

[0162] The antigen-binding proteins of the present disclosure may include an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single-chain antibody (e.g., single-chain fragment variable (scFv)); a diabody; a triabody; a tetrabody; a Fab fragment; an F(ab')2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof.

[0163] In some embodiments, an antigen-binding protein that binds to CD25 is a single-domain antibody (also referred to as “sdAb”). The single-domain antibodies of the present disclosure can be derived from numerous sources, including but not limited to VHH, VNAR, or VH domains (naturally occurring or modified VH domains). VHHs can be generated from camelid heavy chain antibodies only and libraries thereof. VNARs can be generated from fish heavy chain antibodies. Petition 870250081421, dated 10 / 09 / 2025, p. 65 / 374 55 / 304 cartilaginous only and libraries thereof. Several methods have been implemented to generate monomeric sdAbs from conventionally heterodimeric VH and VL domains, including interface engineering and selection of specific germline families. In some embodiments, the sdAbs of the present invention are human or humanized.

[0164] In some embodiments, a single-domain antibody described in the present invention is a VHH fragment (also known as a nanobody). VHH fragments are also referred to as “V-bodies” in the present disclosure. In some embodiments, the VHH is a camelid VHH, a humanized VHH, or a camelized VH. In some embodiments, a single-domain antibody described in the present invention is a VH domain. In some embodiments, a single-domain antibody described in the present invention is a naturally occurring VH domain or a modified VH domain.

[0165] The variable domain of an antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises at least three complementarity-determining regions (CDRs) that determine its binding specificity. Preferably, in a variable domain, the CDRs are distributed among the structure regions (FRs). The variable domain typically contains 4 structure regions interspersed with 3 CDR regions, resulting in the following typical antibody variable domain structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs and / or FRs of the single-domain antibody of the present disclosure may be fragments or derivatives of a naturally occurring antibody variable domain or may be synthetic.

[0166] The sequence identifiers corresponding to the exemplary VHH anti-CD25 antibodies provided in the present invention are listed in Table 1-1. Table 1-1 presents the sequence identifiers of the amino acid sequences of the complementarity-determining regions (CDR1, CDR2 and Petition 870250081421, dated 10 / 09 / 2025, p. 66 / 374 56 / 304 CDR3), amino acid sequences and DNA of full-length camelid VHH antibodies, as well as amino acid sequences of corresponding humanized VHH antibodies. Amino acid sequences of additional exemplary anti-CD25 VHH antibodies and corresponding humanized VHH antibodies are provided in Table 1-2. Table 1-1. Sequence identifiers for exemplary VHH anti-CD25 antibodies Antibody ID Group CDR1 CDR2 CDR3 Non-humanized HHV Humanized HHV Amino acid sequence Amino acid sequence Amino acid sequence DNA sequence Amino acid sequence ODYN1570 A 1 2 3 4 21 26 ODYN1572 B 5 6 7 8 22 27 ODYN1574 C 9 10 11 12 23 28 ODY-46A3 D 13 14 15 16 24 29 ODYN1810 D 13 4335 4336 4337 4338 4339 ODY-47D3 E 17 18 19 20 25 30 ODY81A09 F 2242 2243 2244 2251 2255 2259 ODY83B03 2245 2246 2247 2252 2256 2260 ODY83F07 2258 2262 ODY83F07 Hu1.8l H 2242 2248 4311 - - 4317 ODY83B05 Hu1.8l H 2242 2248 4312 - - 4318 ODY83F07 Hu1.8A H 2242 2248 4313 - - 4319 ODY83B05 Hu1.8A H 2242 2248 4314 - - 4320 ODY83F07 Hu1.8L H 2242 2248 4315 - - 4321 ODY83B05 Hu1.8L H 2242 2248 4316 - - 4322 Table 1-2. Sequence identifiers for additional exemplary VHH antibodies and humanized VHH antibodies. Petition 870250081421, dated 10 / 09 / 2025, p. 67 / 374 57 / 304 Cluster Group 0 Non-humanized VHH amino acid sequence Humanized VHH amino acid sequence ODYN1570 A 4 26 ODYN1570 A 43 343 ODYN1570 A 44 344 ODYN1570 A 45 345 ODYN1570 A 46 346 ODYN1570 A 47 347 ODYN1570 A 48 348 ODYN1570 A 49 349 ODYN1570 A 50 350 ODYN1570 A 51 351 ODYN1570 A 52 352 ODYN1570 A 53 353 ODYN1570 A 54 354 ODYN1570 A 55 355 ODYN1570 A 56 356 ODYN1570 A 57 357 ODYN1570 A 58 358 ODYN1570 A 59 359 ODYN1570 A 60 360 ODYN1570 A 61 361 ODYN1570 A 62 362 ODYN1570 A 63 363 ODYN1570 A 64 358 ODYN1570 A 65 364 ODYN1570 A 66 26 ODYN1570 A 67 365 ODYN1570 A 68 366 Petition 870250081421, de 10 / 09 / 2025, pag. 68 / 374 58 / 304 ODYN1570 A 69 367 ODYN1570 A 70 368 ODYN1570 A 71 369 ODYN1570 A 72 370 ODYN1570 A 73 371 ODYN1570 A 74 372 ODYN1572 B 8 27 ODYN1572 B 75 27 ODYN1572 B 76 373 ODYN1572 B 77 374 ODYN1572 B 78 375 ODYN1572 B 79 376 ODYN1572 B 80 377 ODYN1572 B 81 378 ODYN1572 B 82 379 ODYN1572 B 83 380 ODYN1572 B 84 381 ODYN1572 B 85 382 ODYN1572 B 86 383 ODYN1572 B 87 384 ODYN1572 B 88 385 ODYN1572 B 89 386 ODYN1572 B 90 387 ODYN1572 B 91 388 ODYN1572 B 92 389 ODYN1572 B 93 390 ODYN1572 B 94 391 ODYN1572 B 95 392 Petition 870250081421, de 10 / 09 / 2025, pag. 69 / 374 59 / 304 ODYN1572 B 96 393 ODYN1572 B 97 394 ODYN1572 B 98 395 ODYN1572 B 99 396 ODYN1572 B 100 397 ODYN1574 C 12 28 ODYN1574 C 101 398 ODYN1574 C 102 399 ODYN1574 C 103 400 ODYN1574 C 104 401 ODYN1574 C 105 402 ODY46A3 D 106 403 ODY46A3 D 107 404 ODY46A3 D 108 405 ODY46A3 D 109 406 ODY46A3 D 16 29 ODY46A3 D 110 407 ODY46A3 D 111 408 ODY46A3 D 112 409 ODY46A3 D 113 410 ODY46A3 D 114 411 ODY46A3 D 115 412 ODY46A3 D 116 413 ODY46A3 D 117 414 ODY46A3 D 118 415 ODY46A3 D 119 416 ODY46A3 D 120 417 ODY46A3 D 121 418 Petição 870250081421, de 10 / 09 / 2025, pág. 70 / 374 60 / 304 ODY46A3 D 122 419 ODY46A3 D 123 420 ODY46A3 D 124 421 ODY46A3 D 125 422 ODY46A3 D 126 423 ODY46A3 D 127 424 ODY46A3 D 128 425 ODY46A3 D 129 426 ODY46A3 D 130 427 ODY46A3 D 131 428 ODY46A3 D 132 429 ODY46A3 D 133 430 ODY46A3 D 134 431 ODY46A3 D 135 432 ODY46A3 D 136 404 ODY46A3 D 137 433 ODY46A3 D 138 434 ODY46A3 D 139 435 ODY46A3 D 140 436 ODY46A3 D 141 437 ODY46A3 D 142 438 ODY46A3 D 143 439 ODY46A3 D 144 440 ODY46A3 D 145 441 ODY46A3 D 146 442 ODY46A3 D 147 443 ODY46A3 D 148 444 ODY46A3 D 149 445 Petition: 870250081421, on September 10, 2025, page. 71 / 374 61 / 304 ODY46A3 D 150 446 ODY46A3 D 151 447 ODY46A3 D 152 448 ODY46A3 D 153 449 ODY46A3 D 154 450 ODY46A3 D 155 451 ODY46A3 D 156 452 ODY46A3 D 157 453 ODY46A3 D 158 454 ODY46A3 D 159 455 ODY46A3 D 160 456 ODY46A3 D 161 457 ODY46A3 D 162 458 ODY46A3 D 163 459 ODY46A3 D 164 460 ODY46A3 D 165 461 ODY46A3 D 166 462 ODY46A3 D 167 463 ODY46A3 D 168 464 ODY46A3 D 169 465 ODY46A3 D 170 466 ODY46A3 D 171 467 ODY46A3 D 172 468 ODY46A3 D 173 469 ODY46A3 D 174 470 ODY46A3 D 175 471 ODY46A3 D 176 472 ODY46A3 D 177 473 Petition: 870250081421, on September 10, 2025, page. 72 / 374 62 / 304 ODY46A3 D 178 474 ODY46A3 D 179 475 ODY46A3 D 180 476 ODY46A3 D 181 477 ODY46A3 D 182 478 ODY46A3 D 183 479 ODY46A3 D 184 480 ODY46A3 D 185 481 ODY46A3 D 186 482 ODY46A3 D 187 483 ODY46A3 D 188 484 ODY46A3 D 189 485 ODY46A3 D 190 486 ODY46A3 D 191 487 ODY46A3 D 192 488 ODY46A3 D 193 489 ODY46A3 D 194 490 ODY46A3 D 195 491 ODY46A3 D 196 447 ODY46A3 D 197 492 ODY46A3 D 198 493 ODY46A3 D 199 494 ODY46A3 D 200 495 ODY46A3 D 201 496 ODY46A3 D 202 497 ODY46A3 D 203 498 ODY46A3 D 204 499 ODY46A3 D 205 500 Petition: 870250081421, on September 10, 2025, page. 73 / 374 63 / 304 ODY46A3 D 206 501 ODY46A3 D 207 502 ODY46A3 D 208 503 ODY46A3 D 209 504 ODY46A3 D 210 505 ODY46A3 D 211 506 ODY46A3 D 212 507 ODY46A3 D 213 508 ODY46A3 D 214 509 ODY46A3 D 215 510 ODY46A3 D 216 511 ODY46A3 D 217 512 ODY46A3 D 4342 4452 ODY46A3 D 4343 4453 ODY46A3 D 4344 4454 ODY46A3 D 4345 4455 ODY46A3 D 4346 4456 ODY46A3 D 4347 4457 ODY46A3 D 4348 4458 ODY46A3 D 4349 4459 ODY46A3 D 4350 4460 ODY46A3 D 4351 4461 ODY46A3 D 4352 4462 ODY46A3 D 4353 4463 ODY46A3 D 4354 4464 ODY46A3 D 4355 4465 ODY46A3 D 4356 4466 ODY46A3 D 4357 4467 Petition: 870250081421, on September 10, 2025, page. 74 / 374 64 / 304 ODY46A3 D 4358 4468 ODY46A3 D 4359 4469 ODY46A3 D 4360 4470 ODY46A3 D 4361 4471 ODY46A3 D 4362 4472 ODY46A3 D 4363 4473 ODY46A3 D 4364 4474 ODY46A3 D 4365 4475 ODY46A3 D 4366 4476 ODY46A3 D 4367 4477 ODY46A3 D 4368 4478 ODY46A3 D 4369 4479 ODY46A3 D 4370 4480 ODY46A3D 4371 4481 ODY46A3 D 4372 4482 ODY46A3 D 4373 4483 ODY46A3 D 4374 4484 ODY46A3 D 4375 4460 ODY46A3 D 4376 4485 ODY46A3 D 4377 4486 ODY46A3 D 4378 4487 ODY46A3 D 4379 4488 ODY46A3 D 4380 4489 ODY46A3 D 4381 4490 ODY46A3 D 4382 4491 ODY46A3 D 4383 4492 ODY46A3 D 4384 4493 ODY46A3 D 4385 4494 Petition: 870250081421, on September 10, 2025, page. 75 / 374 65 / 304 ODY46A3 D 4386 4495 ODY46A3 D 4387 4496 ODY46A3 D 4388 4497 ​​ODY46A3 D 4389 4498 ODY46A3 D 4390 4485 ODY46A3 D 4391 4499 ODY46A3 D 4392 4500 ODY46A3 D 4393 4501 ODY46A3 D 4394 4502 ODY46A3 D 4395 4503 ODY46A3 D 4396 4504 ODY46A3 D 4397 4505 ODY46A3 D 4398 4506 ODY46A3D 4399 4507 ODY46A3 D 4400 4508 ODY46A3 D 4401 4509 ODY46A3 D 4402 4510 ODY46A3 D 4403 4511 ODY46A3 D 4404 4512 ODY46A3 D 4405 4513 ODY46A3 D 4406 4514 ODY46A3 D 4407 4515 ODY46A3 D 4408 4516 ODY46A3 D 4409 4517 ODY46A3 D 4410 4518 ODY46A3 D 4411 4519 ODY46A3 D 4412 4520 ODY46A3 D 4413 4521 Petition: 870250081421, on September 10, 2025, page. 76 / 374 66 / 304 ODY46A3 D 4414 4522 ODY46A3 D 4415 4523 ODY46A3 D 4416 4524 ODY46A3 D 4417 4525 ODY46A3 D 4418 4526 ODY46A3 D 4419 4527 ODY46A3 D 4420 4528 ODY46A3 D 4421 4529 ODY46A3 D 4422 4530 ODY46A3 D 4423 4531 ODY46A3 D 4424 4532 ODY46A3 D 4425 4533 ODY46A3 D 4426 4534 ODY46A3D 4427 4535 ODY46A3 D 4428 4536 ODY46A3 D 4429 4537 ODY46A3 D 4430 4538 ODY46A3 D 4431 4539 ODY46A3 D 4432 4540 ODY46A3 D 4433 4541 ODY46A3 D 4434 4542 ODY46A3 D 4435 4543 ODY46A3 D 4436 4544 ODY46A3 D 4437 4545 ODY46A3 D 4337 4339 ODY46A3 D 4438 4546 ODY46A3 D 4439 4547 ODY46A3 D 4440 4548 Petition: 870250081421, on September 10, 2025, page. 77 / 374 67 / 304 ODY46A3 D4441D4549 4555 ODY46A3 D 4448 4556 ODY46A3 D 4449 4557 ODY46A3 D 4450 4558 ODY46A3 D 4451 4559 ODY47D3 E 218 513 ODY47D3 E 20 30 ODY47D3 E 219 514 ODY47D3 E 220 515 ODY47D3 E 221 516 ODY47D3 E 222 517 ODY47D3 E 223 518 ODY47D3 E 224 519 ODY47D3 E 225 520 ODY47D3 E 226 521 ODY47D3 E 227 522 ODY47D3 E 228 523 ODY47D3 AND 229 524 ODY47D3 AND 230 525 ODY47D3 AND 231 526 ODY47D3 AND 232 527 ODY47D3 AND 233 528 Petition 870250081421, dated 10 / 09 / 2025, page 78 / 374 68 / 304 ODY47D3 E 234 529 ODY47D3 E 235 530 ODY47D3 E 236 531 ODY47D3 E 237 532 ODY47D3 E 238 533 ODY47D3 E 239 534 ODY47D3 E 240 535 ODY47D3 E 241 536 ODY47D3 E 242 537 ODY47D3 E 243 538 ODY47D3 E 244 539 ODY47D3 E 245 540 ODY47D3 E 246 541 ODY47D3 E 247 542 ODY47D3 E 248 528 ODY47D3 E 249 543 ODY47D3 E 250 544 ODY47D3 E 251 545 ODY47D3 E 252 546 ODY47D3 E 253 547 ODY47D3 E 254 548 ODY47D3 E 255 531 ODY47D3 E 256 549 ODY47D3 E 257 550 ODY47D3 E 258 551 ODY47D3 E 259 552 ODY47D3 E 260 553 ODY47D3 E 261 554 Petition 870250081421, dated 10 / 09 / 2025, page 79 / 374 69 / 304 ODY47D3 E 262 555 ODY47D3 E 263 556 ODY47D3 E 264 557 ODY47D3 E 265 515 ODY47D3 E 266 558 ODY47D3 E 267 559 ODY47D3 E 268 560 ODY47D3 E 269 561 ODY47D3 E 270 562 ODY47D3 E 271 563 ODY47D3 E 272 564 ODY47D3 E 273 565 ODY47D3 E 274 566 ODY47D3 E 275 528 ODY47D3 E 276 567 ODY47D3 E 277 568 ODY47D3 E 278 569 ODY47D3 E 279 570 ODY47D3 E 280 571 ODY47D3 E 281 572 ODY47D3 E 282 573 ODY47D3 E 283 574 ODY47D3 E 284 575 ODY47D3 E 285 576 ODY47D3 E 286 515 ODY47D3 E 287 30 ODY47D3 E 288 30 ODY47D3 E 289 30 Petition 870250081421, dated 10 / 09 / 2025, page 80 / 374 70 / 304 ODY47D3 E 290 577 ODY47D3 E 291 578 ODY47D3 E 292 579 ODY47D3 E 293 549 ODY47D3 E 294 515 ODY47D3 E 295 580 ODY47D3 E 296 581 ODY47D3 E 297 582 ODY47D3 E 298 583 ODY47D3 E 299 584 ODY47D3 E 300 585 ODY47D3 E 301 586 ODY47D3 E 302 587 ODY47D3 E 303 588 ODY47D3 E 304 589 ODY47D3 E 305 590 ODY47D3 E 306 591 ODY47D3 E 307 592 ODY47D3 E 308 593 ODY47D3 E 309 594 ODY47D3 E 310 595 ODY47D3 E 311 596 ODY47D3 E 312 597 ODY47D3 E 313 598 ODY47D3 E 314 599 ODY47D3 E 315 600 ODY47D3 E 316 601 ODY47D3 E 317 602 Petition 870250081421, dated 10 / 09 / 2025, page 81 / 374 71 / 304 ODY47D3 E 318 603 ODY47D3 E 319 604 ODY47D3 E 320 605 ODY47D3 E 322 606 ODY47D3 E 322 607 ODY47D3 E 323 E 323 E 4608 ODY47D3 E 325 610 ODY47D3 E 326 611 ODY47D3 E 327 612 ODY47D3 E 329 613 ODY47D3 E 329 565 ODY47D3 E 310 E 534614 ODY47D3 E 332 616 ODY47D3 E 333 517 ODY47D3 E 334 617 ODY47D3 E 335 618 ODY47D3 E 336 619 ODY47D3 E 31ODY 43720 ODY47D3 E 339 622 ODY47D3 E 340 623 ODY47D3 E 341 624 ODY47D3 E 342 625 ODY81A09 F 2268 22509 ODY81A09 F 22609 F 22609 2561 Petition 870250081421, dated 10 / 09 / 2025, p. 82 / 374 72 / 304 ODY81A09 F 2271 2562 ODY81A09 F 2272 2563 ODY81A09 F 2251 2259 ODY81A09 F 2273 2564 ODY81A09 F 2274 2565 ODY81A09 F 2275 2566 ODY81A09 F 2276 2567 ODY81A09 F 2277 2568 ODY81A09 F 2278 2569 ODY81A09 F 2279 2570 ODY81A09 F 2280 2259 ODY81A09 F 2281 2571 ODY81A09 F 2282 2572 ODY81A09 F 2283 2573 ODY81A09 F 2284 2574 ODY81A09 F 2285 2575 ODY81A09 F 2286 2576 ODY81A09 F 2287 2577 ODY81A09 F 2288 2578 ODY81A09 F 2289 2579 ODY81A09 F 2290 2575 ODY81A09 F 2291 2580 ODY81A09 F 2292 2581 ODY81A09 F 2293 2582 ODY81A09 F 2294 2583 ODY81A09 F 2295 2572 ODY81A09 F 2296 2584 ODY81A09 F 2297 2585 Petição 870250081421, de 10 / 09 / 2025, pág. 83 / 374 73 / 304 ODY81A09 F 2298 2586 ODY81A09 F 2299 2587 ODY81A09 F 2300 2588 ODY81A09 F 2301 2589 ODY81A09 F 2302 2590 ODY81A09 F 2303 2591 ODY81A09 F 2304 2592 ODY81A09 F 2305 2593 ODY81A09 F 2306 2594 ODY81A09 F 2307 2595 ODY81A09 F 2308 2596 ODY81A09 F 2309 2597 ODY81A09 F 2310 2598 ODY81A09 F 2311 2599 ODY81A09 F 2312 2600 ODY81A09 F 2313 2601 ODY81A09 F 2314 2602 ODY81A09 F 2315 2603 ODY81A09 F 2316 2604 ODY81A09 F 2317 2605 ODY81A09 F 2318 2606 ODY81A09 F 2319 2607 ODY81A09 F 2320 2608 ODY81A09 F 2321 2609 ODY81A09 F 2322 2603 ODY81A09 F 2323 2610 ODY81A09 F 2324 2611 ODY81A09 F 2325 2612 Petição 870250081421, de 10 / 09 / 2025, pág. 84 / 374 74 / 304 ODY81A09 F 2326 2613 ODY81A09 F 2327 2614 ODY81A09 F 2328 2615 ODY81A09 F 2329 2616 ODY81A09 F 2330 2617 ODY81A09 F 2331 2618 ODY81A09 F 2332 2619 ODY81A09 F 2333 2620 ODY81A09 F 2334 2621 ODY81A09 F 2335 2622 ODY81A09 F 2336 2623 ODY81A09 F 2337 2624 ODY81A09 F 2338 2625 ODY81A09 F 2339 2626 ODY81A09 F 2340 2627 ODY81A09 F 2341 2628 ODY81A09 F 2342 2629 ODY81A09 F 2343 2630 ODY81A09 F 2344 2631 ODY81A09 F 2345 2632 ODY81A09 F 2346 2633 ODY81A09 F 2347 2634 ODY81A09 F 2348 2635 ODY81A09 F 2349 2636 ODY81A09 F 2350 2637 ODY81A09 F 2351 2638 ODY81A09 F 2352 2639 ODY81A09 F 2353 2640 Petição 870250081421, de 10 / 09 / 2025, pág. 85 / 374 75 / 304 ODY81A09 F 2354 2641 ODY81A09 F 2355 2642 ODY81A09 F 2356 2643 ODY81A09 F 2357 2644 ODY81A09 F 2358 2645 ODY81A09 F 2359 2646 ODY81A09 F 2360 2647 ODY81A09 F 2361 2648 ODY81A09 F 2362 2649 ODY81A09 F 2363 2650 ODY81A09 F 2364 2651 ODY81A09 F 2365 2652 ODY81A09 F 2366 2653 ODY81A09 F 2367 2654 ODY81A09 F 2368 2655 ODY81A09 F 2369 2656 ODY81A09 F 2370 2657 ODY81A09 F 2371 2658 ODY81A09 F 2372 2659 ODY81A09 F 2373 2660 ODY81A09 F 2374 2661 ODY81A09 F 2375 2662 ODY81A09 F 2376 2663 ODY81A09 F 2377 2664 ODY81A09 F 2378 2665 ODY81A09 F 2379 2666 ODY81A09 F 2380 2667 ODY81A09 F 2381 2668 Petição 870250081421, de 10 / 09 / 2025, pág. 86 / 374 76 / 304 ODY81A09 F 2382 2669 ODY81A09 F 2383 2670 ODY81A09 F 2384 2671 ODY81A09 F 2385 2672 ODY81A09 F 2386 2673 ODY81A09 F 2387 2674 ODY81A09 F 2388 2675 ODY81A09 F 2389 2676 ODY81A09 F 2390 2677 ODY81A09 F 2391 2678 ODY81A09 F 2392 2679 ODY81A09 F 2393 2680 ODY81A09 F 2394 2681 ODY81A09 F 2395 2682 ODY81A09 F 2396 2683 ODY81A09 F 2397 2684 ODY81A09 F 2398 2685 ODY81A09 F 2399 2686 ODY81A09 F 2400 2687 ODY81A09 F 2401 2688 ODY81A09 F 2402 2689 ODY81A09 F 2403 2690 ODY81A09 F 2404 2691 ODY81A09 F 2405 2692 ODY81A09 F 2406 2693 ODY81A09 F 2407 2694 ODY81A09 F 2408 2695 ODY81A09 F 2409 2696 Petição 870250081421, de 10 / 09 / 2025, pág. 87 / 374 77 / 304 ODY81A09 F 2410 2697 ODY81A09 F 2411 2698 ODY81A09 F 2412 2699 ODY81A09 F 2413 2700 ODY81A09 F 2414 2701 ODY81A09 F 2415 2702 ODY81A09 F 2416 2703 ODY81A09 F 2417 2704 ODY81A09 F 2418 2705 ODY81A09 F 2419 2706 ODY81A09 F 2420 2707 ODY81A09 F 2421 2708 ODY81A09 F 2422 2709 ODY81A09 F 2423 2710 ODY81A09 F 2424 2711 ODY81A09 F 2425 2712 ODY81A09 F 2426 2713 ODY81A09 F 2427 2714 ODY81A09 F 2428 2715 ODY81A09 F 2429 2716 ODY81A09 F 2430 2717 ODY81A09 F 2431 2718 ODY81A09 F 2432 2719 ODY81A09 F 2433 2720 ODY81A09 F 2434 2618 ODY81A09 F 2435 2721 ODY81A09 F 2436 2722 ODY81A09 F 2437 2723 Petição 870250081421, de 10 / 09 / 2025, pág. 88 / 374 78 / 304 ODY81A09 F 2438 2724 ODY81A09 F 2439 2725 ODY81A09 F 2440 2726 ODY81A09 F 2441 2727 ODY81A09 F 2442 2728 ODY81A09 F 2443 2729 ODY81A09 F 2444 2730 ODY81A09F 2445 2731 ODY81A09 F 2446 2732 ODY81A09 F 2447 2733 ODY81A09 F 2448 2734 ODY81A09 F 2449 2735 ODY81A09 ODY81A09 F 2451 2737 ODY81A09 F 2452 2738 ODY81A09 F 2453 2739 ODY81A09 F 2454 2740 ODY81A09 F 2455 2741 ODY81A09 F 2456 2742 ODY83B03 G 2252 2260 ODY83B03 G 2457 2743 ODY83B03 G 2458 2744 ODY83B03 G 2459 2745 ODY83B03 G 2460 2746 ODY83B03 G 2461 2747 ODY83B03 G 2462 2748 ODY83B03 G 2463 2749 ODY83B03 G 2464 2750 Petition 870250081421, dated 10 / 09 / 2025, page 89 / 374 79 / 304 ODY83B03 G 2465 2751 ODY83B03 G 2466 2752 ODY83B03 G 2467 2753 ODY83B03 G 2468 2754 ODY83B03 G 2469 2755 ODY83B03 G 2470 2756 ODY83B03 G 2471 2757 ODY83B03 G 2472 2758 ODY83B03 G 2473 2759 ODY83B03 G 2474 2760 ODY83B03 G 2475 2744 ODY83B03 G 2476 2761 ODY83B03 G 2477 2762 ODY83B03 G 2478 2763 ODY83B03 G 2479 2764 ODY83B03 G 2480 2765 ODY83B03 G 2481 2766 ODY83B03 G 2482 2767 ODY83B03 G 2483 2768 ODY83B03 G 2484 2769 ODY83B03 G 2485 2770 ODY83B03 G 2486 2771 ODY83B03 G 2487 2765 ODY83B03 G 2488 2772 ODY83B03 G 2489 2773 ODY83B03 G 2490 2774 ODY83B03 G 2491 2775 ODY83B03 G 2492 2776 Petition 870250081421, dated 10 / 09 / 2025, page 90 / 374 80 / 304 ODY83B03 G 2493 2777 ODY83B03 G 2494 2778 ODY83B03 G 2495 2779 ODY83B03 G 2496 2780 ODY83B03 G 2497 2781 ODY83B03 G 2498 2769 ODY83B03 G ODY83B03 G 2500 2782 ODY83B03 ODY83B03 G 2506 2787 ODY83B03 G 2507 2788 ODY83B03 G 2508 2771 ODY83B03 G 2509 2789 ODY83B03 G 2510 2784 ODY83B03 G 2511 2790 ODY83B03 G 2512 2791 ODY83B03 G 2513 2792 ODY83B03 G 2514 2777 ODY83B03 G 2515 2759 ODY83B03 G 2516 2793 ODY83B03 G 2517 2759 ODY83B03 G 2518 2794 ODY83B03 G 2519 2795 ODY83B03 G 2520 2796 Petition 870250081421, dated 10 / 09 / 2025, page 91 / 374 81 / 304 ODY83B03 G 2521 2797 ODY83B03 G 2522 2798 ODY83B03 G 2523 2799 ODY83B03 G 2524 2800 ODY83B03 G 2525 2801 ODY83B03 G 2526 2802 ODY83B03 G 2527 2803 ODY83B03 G 2528 2804 ODY83B03 G 2529 2805 ODY83B03 G 2530 2796 ODY83B03 G 2531 2806 ODY83B03 G 2532 2807 ODY83B03 G 2533 2808 ODY83B03 G 2534 2809 ODY83B03 G 2535 2810 ODY83B03 G 2536 2811 ODY83B03 G 2537 2768 ODY83B03 G 2538 2812 ODY83B03 G 2539 2813 ODY83B03 G 2540 2814 ODY83B03 G 2541 2815 ODY83B03 G 2542 2816 ODY83B03 G 2543 2759 ODY83B03 G 2544 2795 ODY83B03 G 2545 2817 ODY83B03 G 2546 2818 ODY83B03 G 2547 2819 ODY83B03 G 2548 2820 Petition 870250081421, dated 10 / 09 / 2025, page 92 / 374 82 / 304 ODY83B03 G 2549 2821 ODY83B03 G 2550 2822 ODY83B03 G 2551 2766 ODY83B03 G 2552 2823 ODY83B03 G 2553 2824 ODY83B03 G 2554 2825 ODY83B03 2555 2826 ODY83B03 G 2556 2827 ODY83B03 G 2557 2828 ODY83B03 G 2558 2829 ODY83B03 G 2559 2830

[0167] In some embodiments, an antiCD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from (the amino acids listed in a pair of square brackets represent the possible amino acids at the particular position, and “-” indicates that an amino acid residue is missing at the particular position) a). GR(K / R / S)FSTLI (SEQ ID NO: 37); b). GFTFS(N / S)YA (SEQ ID NO: 40); w) . 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).

[0168] In some embodiments, when an anti-CD25 antigen-binding protein described in the present invention may comprise a complementarity-determining region 1 (CDR1) comprising the sequence GR(K / R / S)FSTLI (SEQ ID NO: 37), the CDR1 may comprise, for example, the sequence GR(S / K)FSTLI (SEQ ID NO: 32). Petition 870250081421, dated 10 / 09 / 2025, page 93 / 374 83 / 304

[0169] In some embodiments, when an anti-CD25 antigen-binding protein described in the present invention may comprise a complementarity-determining region 1 (CDR1) comprising the sequence GR(K / R / S)FSTLI (SEQ ID NO: 37), the CDR1 may comprise, for example, the sequence GRSFSTLI (SEQ ID NO: 5).

[0170] In some embodiments, an antiCD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from (the amino acids listed in a pair of square brackets represent the possible amino acids at the particular position, and “-” indicates that an amino acid residue is missing at the particular position) a) . GRSFSTLI (SEQ ID NO: 5); b). GR(S / K)FSTLI (SEQ ID NO: 32); w) . GFTFS(N / S)YA (SEQ ID NO: 40); d). GRTFS(S / W)(F / N / Y)G (SEQ ID NO: 42); e). GFTLDYYA (SEQ ID NO: 2242); and f). G(I / M)P(F / -)(A / -)L(P / V / Y)A (SEQ ID NO: 2266)..

[0171] In some embodiments, an antiCD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from (the amino acids listed in a pair of square brackets represent the possible amino acids at the particular position) a). (I / V)(D / E)R(D / G)(D / G)T(A / P / T) (SEQ ID NO: 2241) b). IYSD(G / S)SGT (SEQ ID NO: 4341); w) . IS(Q / R / G)(S / G)GGRT (SEQ ID NO: 5210); Petition 870250081421, dated 10 / 09 / 2025, p. 94 / 374 84 / 304 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).

[0172] In some embodiments, where an anti-CD25 antigen-binding protein described in the present invention may comprise a complementarity-determining region 2 (CDR2) comprising the sequence (I / V)(D / E)R(D / G)(D / G)T(A / P / T) (SEQ ID NO: 2241), CDR2 may comprise, for example, the sequence (I / V)(D / E)R(D / G)GT(A / P / T) (SEQ ID NO: 33).

[0173] In some embodiments, where an anti-CD25 antigen-binding protein described in the present invention may comprise a complementarity-determining region 2 (CDR2) comprising the sequence (I / V)(D / E)R(D / G)(D / G)T(A / P / T) (SEQ ID NO: 2241), the CDR2 may comprise, for example, the sequence I(D / E)RDGT(T / P) (SEQ ID NO: 35).

[0174] In some embodiments, where an anti-CD25 antigen-binding protein described in the present invention may comprise a complementarity-determining region 2 (CDR2) comprising the sequence (I / V)(D / E)R(D / G)(D / G)T(A / P / T) (SEQ ID NO: 2241), the CDR2 may comprise, for example, the sequence I(D / E)R(D / G)(D / G)T(P / T) (SEQ ID NO: 38).

[0175] In some embodiments, an antiCD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from (the amino acids listed in a pair of square brackets represent the possible amino acids at the particular position) a). (I / V)(D / E)R(D / G)GT(A / P / T) (SEQ ID NO: 33); b). I(D / E)RDGT(T / P) (SEQ ID NO: 35); w) . I(D / E)R(D / G)(D / G)T(P / T) (SEQ ID NO: 38) Petition 870250081421, dated 10 / 09 / 2025, p. 95 / 374 85 / 304 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)

[0176] In some embodiments, an antiCD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from (the amino acids listed in a pair of square 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); w) . (K / S / T)TLRY (SEQ ID NO: 36); d). (A / V / S)(K / T)G(R / A / K)(G / H / N / R)SG(S / G)YYP(W / F / L)D(D / E)(Y / V) (SEQ ID NO: 5119); and e). AA(S / T)(D / N / Y / K)(F / V)(L / P)(I / L)A(T / I / A)(T / S / A)IS(A / G)(Y / H)DY (SEQ ID NO: 5208); f). AAYVYPDYYCS(D / E)YVLL(K / R)YDY (SEQ ID NO: 2263); g). NIYR(P / S)QVP(P / S / T)TRYS (SEQ ID NO: 2265); and h). AAKRLGP(M / I / A / L)VH(Q / R)YSLEVLTPLFLDEYDY (SEQ ID NO: 4323). [0 177] In some embodiments, an antiCD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from (the amino acids listed in a pair of square brackets represent the possible amino acids at the particular position) Petition 870250081421, dated 10 / 09 / 2025, p. 96 / 374 86 / 304 a). NAL(G / L / P / Q / W)Y (SEQ ID NO: 31); b). NALR(D / H / N / F) (SEQ ID NO: 34); w) . (K / S / T)TLRY (SEQ ID NO: 36); d). AKGR(H / N)SGSYYPWD(D / E)Y (SEQ ID NO: 39); e). (A / V)KGR(G / H / N)SGSYYP(W / F)D(D / E)Y (SEQ ID NO: 4430); f). AA(S / T)(D / N / Y)FL(I / L)ATTIS(A / G)YDY (SEQ ID NO: 41); g). AAYVYPDYYCS(D / E)YVLL(K / R)YDY (SEQ ID NO: 2263); h). NIYR(P / S)QVP(P / S / T)TRYS (SEQ ID NO: 2265); and i). AAKRLGPMVH(Q / R)YSLEVLTPLFLDEYDY (SEQ ID NO: 2267). [0 178] Anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) comprising amino acid sequences selected from any of the CDR1, CDR2, and CDR3 amino acid sequences described above are provided in the present invention. In certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; ii i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 36; iv) a CDR1 comprising an amino acid sequence of SEQ ID Petition 870250081421, dated 10 / 09 / 2025, page 97 / 374 87 / 304 NO: 40, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 39; (v) a CDR1 comprising an amino acid sequence from SEQ ID NO: 42, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 41; vi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 2263; vi i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2246, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 2265; or vi ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 2267.

[0179] In certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 33, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 35, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; iii) a CDR1 comprising an amino acid sequence of the SEQ ID Petition 870250081421, dated 10 / 09 / 2025, p. 98 / 374 88 / 304 NO: 37, a CDR2 comprising an amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 36; iv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 40, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 39; (v) a CDR1 comprising an amino acid sequence from SEQ ID NO: 42, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 41; vi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 2263; vii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2246, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 2265; or viii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 2267.

[0180] In certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 32, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: Petition 870250081421, dated 10 / 09 / 2025, page 99 / 374 89 / 304 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; iii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 36; iv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 40, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 39; (v) a CDR1 comprising an amino acid sequence from SEQ ID NO: 42, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 41; vi) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 2263; vii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2246, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 2265; or viii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 2267.

[0181] In certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises Petition 870250081421, dated 10 / 09 / 2025, p. 100 / 374 90 / 304 i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4311; ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4312; iii) a CDR1 comprising an amino acid sequence of the SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4313; iv) a CDR1 comprising an amino acid sequence of the SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4314; (v) a CDR1 comprising an amino acid sequence of the SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4315; or vi) a CDR1 comprising an amino acid sequence of SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4316.

[0182] In certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises Petition 870250081421, dated 10 / 09 / 2025, page 101 / 374 91 / 304 i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; iii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 36; iv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 33, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; (v) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 35, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; vi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 38, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 36; vii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 32, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; viii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; Petition 870250081421, dated 10 / 09 / 2025, page 102 / 374 92 / 304 ix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 32, a CDR2 comprising an amino acid sequence from SEQ ID NO: 33, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; x) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 35, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; xi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 40, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 5119; xii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 40, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 4340; xiii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 40, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 39; xiv) a CDR1 comprising an amino acid sequence with SEQ ID NO: 5209, a CDR2 comprising an amino acid sequence with SEQ ID NO: 5210, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 5208; xv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 42, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 41; xvi) a CDR1 comprising an amino acid sequence with SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence with SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence with SEQ ID Petition 870250081421, dated 10 / 09 / 2025, p. 103 / 374 93 / 304 NO: 2263; xvii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2246, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 2265; xviii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 4323; or xix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 2267.

[0183] Anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) are provided in the present invention comprising a CDR1 comprising an amino acid sequence selected from any of the CDR1 amino acid sequences listed in Table 1-1, Table 5, or Table 6 or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0184] In some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos: 1, 5, 9, 13, 17, 626 - 930, 2242, 2245, 2831 - 3126, and 4560 - 4670, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0185] In some embodiments, an anti-antigen binding protein Petition 870250081421, dated 10 / 09 / 2025, p. 104 / 374 94 / 304 CD25 (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos: 1, 5, 9, 13, 17, 32, 42, 805, 809, 818, 2242, and 2245, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0186] Anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a CDR2 comprising an amino acid sequence selected from any of the CDR2 amino acid sequences listed in Table 1-1, Table 5, or Table 6, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity, are provided in the present invention.

[0187] In some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence selected from the 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.

[0188] In some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence selected from the 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.

[0189] Anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a CDR3 comprising an amino acid sequence are provided in the present invention. Petition 870250081421, dated 10 / 09 / 2025, page 105 / 374 95 / 304 selected from any of the CDR3 amino acid sequences listed in Table 1-1, Table 5, or Table 6, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0190] In some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence selected from the SEQ ID NOs: 3, 7, 11, 15, 19, 1236 - 1540, 2244, 2247, 2249, 2250, 3423 - 3718, 4311 4316, 4336, and 4781 - 4891 or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0191] In some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence selected from the SEQ ID NOs: 3, 7, 11, 15, 19, 1237, 1239, 1271, 1275, 1298, 1301, 1331, 1415, 1419, 1421, 1428, 1432, 1442, 1444, 1445, 1447, 1448, 2244, 2247, 2249, 2250, 4311, 4316, 4336, 4787, 4866, 4875, 4878, 4879, and 4880 or a similar sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0192] In some embodiments, an antiCD25 antigen-binding protein comprises a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 626 - 930, 2831 - 3126, and 4560 - 4670; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 931 - 1235, 3127 - 3422, and 4671 - 4780; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1236 - 1540, 3423 - 3718, and 4781 - 4891.

[0193] In some embodiments, an anti-antigen binding protein Petition 870250081421, dated 10 / 09 / 2025, p. 106 / 374 96 / 304 CD25 comprises a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 626 - 930, 2831 - 3126, and 4560 - 4670; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 931 - 1235, 3127 - 3422, and 4671 - 4780; and a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1236 - 1540, 3423 - 3718, and 4781 - 4891.

[0194] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 626 - 658; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 931 - 963; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1236 - 1268.

[0195] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 626 - 658; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 931 - 963; and a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1236 - 1268.

[0196] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 659 - 685; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 964 - 990; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1269 - 1295. Petition 870250081421, dated 10 / 09 / 2025, p. 107 / 374 97 / 304

[0197] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 659 - 685; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 964 - 990; and a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1269 - 1295.

[0198] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 686 - 691; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 991 - 996; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1296 - 1301.

[0199] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 686 - 691; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 991 - 996; and a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1296 - 1301.

[0200] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 692 - 804 and 4560 - 4670; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 997 - 1109 and 4671 - 4780; and / or a CDR3 Petition 870250081421, dated 10 / 09 / 2025, p. 108 / 374 98 / 304 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1302 - 1414 and 4781 - 4891.

[0201] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 692 - 804 and 4560 - 4670; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 997 - 1109 and 4671 - 4780; and a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1302 - 1414 and 4781 - 4891.

[0202] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 805 - 930; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1110 - 1235; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1415 - 1540.

[0203] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 805 - 930; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1110 - 1235; and a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 1415 - 1540.

[0204] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence. Petition 870250081421, dated 10 / 09 / 2025, p. 109 / 374 99 / 304 selected from any of the SEQ ID NOs: 2831 - 3020; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3127 - 3316; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3423 - 3612.

[0205] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 2831 - 3020; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3127 - 3316; and a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3423 - 3612.

[0206] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3021 - 3124; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3317 - 3420; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3613 - 3716.

[0207] In one aspect, the present invention provides an antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3021 - 3124; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3317 - 3420; and a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3613 - 3716.

[0208] In one aspect, the present invention provides an antigen-binding protein that specifically binds to the cluster of differentiation 25 (CD25), Petition 870250081421, dated 10 / 09 / 2025, p. 110 / 374 100 / 304 comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3125 - 3126; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3421 - 3422; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3717 - 3718.

[0209] In one aspect, the present invention provides an antigen-binding protein that specifically binds to the cluster of differentiation 25 (CD25), comprising a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3125 - 3126; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3421 - 3422; and a CDR3 comprising an amino acid sequence selected from any of the SEQ ID NOs: 3717 - 3718.

[0210] Anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained in any of the exemplary anti-CD25 HHV antibodies listed in Table 1-1, Table 1-2, Table 5, or Table 6 are provided in the present invention. In certain embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 1, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 3; ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 6, a CDR3 comprising an amino acid sequence from SEQ ID NO: 7; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 9, a CDR2 comprising an amino acid sequence of SEQ ID NO: Petition 870250081421, dated 10 / 09 / 2025, page 111 / 374 101 / 304 10, a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; iv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 15; (v) a CDR1 comprising an amino acid sequence from SEQ ID NO: 17, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 19; vi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2243, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2244; vii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2245, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2246, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2247; viii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2249; ix) a CDR1 comprising an amino acid sequence of the SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 2250; x) a CDR1 comprising an amino acid sequence of the SEQID NO: 2242, a CDR2 comprising an amino acid sequence of SEQID NO: 2248, a CDR3 comprising an amino acid sequence of SEQID NO: 4311; Petition 870250081421, dated 10 / 09 / 2025, page 112 / 374 102 / 304 xi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4312; xii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4313; xiii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4314; xiv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4315; xv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4316; xvi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4875; xvii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1331; xviii) a CDR1 comprising an amino acid sequence of the SEQ ID Petition 870250081421, dated 10 / 09 / 2025, page 113 / 374 103 / 304 NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 14, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4787; xix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4866; xx) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4336; xxi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4878; xxii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4879; xxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence of SEQ ID NO: 4880; xxiv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 10, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1239; xxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 946, a CDR3 comprising an amino acid sequence of SEQ ID NO: Petition 870250081421, dated 10 / 09 / 2025, page 114 / 374 104 / 304 1239; xxvi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 959, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1237; xxvii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 946, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1237; xxviii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 1, a CDR2 comprising an amino acid sequence from SEQ ID NO: 967, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1271; xxix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 946, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1275; xxx) a CDR1 comprising an amino acid sequence with SEQ ID NO: 9, a CDR2 comprising an amino acid sequence with SEQ ID NO: 10, a CDR3 comprising an amino acid sequence with SEQ ID NO: 11; xxxi) a CDR1 comprising an amino acid sequence with SEQ ID NO: 1, a CDR2 comprising an amino acid sequence with SEQ ID NO: 992, a CDR3 comprising an amino acid sequence with SEQ ID NO: 11; xxxii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 1, a CDR2 comprising an amino acid sequence with SEQ ID NO: 992, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1298; Petition 870250081421, dated 10 / 09 / 2025, p. 115 / 374 105 / 304 xxxiii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 1, a CDR2 comprising an amino acid sequence with SEQ ID NO: 942, a CDR3 comprising an amino acid sequence with SEQ ID NO: 11; xxxiv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 959, a CDR3 comprising an amino acid sequence from SEQ ID NO: 11; xxxv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 1, a CDR2 comprising an amino acid sequence from SEQ ID NO: 942, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1301; xxxvi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1415; xxxvii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 809, a CDR2 comprising an amino acid sequence from SEQ ID NO: 1114, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1419; xxxviii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 805, a CDR2 comprising an amino acid sequence with SEQ ID NO: 1116, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1421; xxxix) a CDR1 comprising an amino acid sequence with SEQ ID NO: 809, a CDR2 comprising an amino acid sequence with SEQ ID NO: 1117, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1419; xxxx) a CDR1 comprising an amino acid sequence with SEQ ID NO: 818, a CDR2 comprising an amino acid sequence with SEQ ID NO: Petition 870250081421, dated 10 / 09 / 2025, p. 116 / 374 106 / 304 1115, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1428; xxxxi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1432; xxxxii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1442; xxxxiii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1444; xxxxiv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1445; xxxxv) a CDR1 comprising an amino acid sequence with SEQ ID NO: 805, a CDR2 comprising an amino acid sequence with SEQ ID NO: 18, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1447; or xxxxvi) a CDR1 comprising an amino acid sequence with SEQ ID NO: 805, a CDR2 comprising an amino acid sequence with SEQ ID NO: 18, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1448.

[0211] In a related embodiment, the present invention provides anti-CD25 antigen-binding proteins (for example, antibodies such as Petition 870250081421, dated 10 / 09 / 2025, p. 117 / 374 107 / 304 single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained in 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, or Table 6. For example, antibodies, or antigen-binding fragments thereof, comprising the CDR1-CDR2-CDR3 amino acid sequence set contained in a VHH amino acid sequence selected from the SEQ ID NOs: 4, 8, 12, 16, 20, 26-30, 43-625, 1541-1845, 2251-2254, 2259-2262, 2268-2830, 3719-4014, are provided in the present invention. 4317 - 4322, 4337, 4339, 4342 - 4559, 4892 - 5002, and 5114 5176.

[0212] In some embodiments, an antiCD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure may include a) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4; b) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 8; c) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 12; d) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 16; (e) a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 20; f) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2251; g) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2252; Petition 870250081421, dated 10 / 09 / 2025, page 118 / 374 108 / 304 h) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2253; i) a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2254; or j) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4337. [0 213] In some embodiments, an antiCD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure may include a) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 26; b) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 27; c) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 28; d) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 29; (e) a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 30; f) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2259; g) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2260; h) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2261; i) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2262; Petition 870250081421, dated 10 / 09 / 2025, page 119 / 374 109 / 304 j) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4317; k) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4318; l) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4319; m) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4320; n) a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4321; o) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4322; or p) . a variable domain comprising a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4339.

[0214] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure may include an amino acid sequence of VHH selected from the SEQ ID NOs: 4, 8, 12, 16, 20, 43 - 342, 1541 - 1845, 2251 - 2254, 2268 - 2559, 3719 - 4014, 4337, 4342 - 4451, 4892 - 5002, and 5146 - 5176, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity of sequence.

[0215] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure may include a VHH amino acid sequence selected from the SEQ ID NOs: 4, 8, 12, 16, 20, 2251 - 2254, 4337, and 5146 Petition 870250081421, dated 10 / 09 / 2025, p. 120 / 374 110 / 304 - 5176, or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0216] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure may include a humanized VHH amino acid sequence selected from the SEQ ID NOs: 26 - 30, 343 - 625, 2259 2262, 2560 - 2830, 4317 - 4322, 4339, 5114 - 5145, and 4452 - 4559, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0217] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure may include a humanized VHH amino acid sequence selected from the SEQ ID NOs: 26 - 30, 2259 - 2262, 4317 4322, 4339, and 5114 - 5145, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0218] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0219] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence Petition 870250081421, dated 10 / 09 / 2025, p. 121 / 374 111 / 304 of SEQ ID NO: 27, or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0220] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0221] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0222] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0223] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity. Petition 870250081421, dated 10 / 09 / 2025, p. 122 / 374 112 / 304

[0224] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0225] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0226] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0227] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0228] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence Petition 870250081421, dated 10 / 09 / 2025, p. 123 / 374 113 / 304 of SEQ ID NO: 4318, or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0229] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0230] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0231] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0232] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity. Petition 870250081421, dated 10 / 09 / 2025, p. 124 / 374 114 / 304

[0233] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0234] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5114, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0235] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5115, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0236] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 5116, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0237] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence Petition 870250081421, dated 10 / 09 / 2025, p. 125 / 374 115 / 304 of SEQ ID NO: 5117, or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0238] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0239] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0240] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0241] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity. Petition 870250081421, dated 10 / 09 / 2025, p. 126 / 374 116 / 304

[0242] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0243] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0244] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0245] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0246] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence Petition 870250081421, dated 10 / 09 / 2025, p. 127 / 374 117 / 304 of SEQ ID NO: 5127, or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0247] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0248] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0249] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0250] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity. Petition 870250081421, dated 10 / 09 / 2025, p. 128 / 374 118 / 304

[0251] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0252] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0253] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0254] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0255] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence Petition 870250081421, dated 10 / 09 / 2025, p. 129 / 374 119 / 304 of SEQ ID NO: 5136, or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0256] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0257] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0258] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0259] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity. Petition 870250081421, dated 10 / 09 / 2025, p. 130 / 374 120 / 304

[0260] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0261] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0262] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0263] In an embodiment provided in the present invention, 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 95%, at least 98%, or at least 99% sequence identity.

[0264] In an embodiment provided in the present invention, an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence Petition 870250081421, dated 10 / 09 / 2025, p. 131 / 374 121 / 304 of SEQ ID NO: 5145, or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0265] In some embodiments, the present disclosure also provides an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) that competes for binding to CD25 with any of the exemplary VHH anti-CD25 antibodies listed in Table 1-1, Table 1-2, Table 5, or Table 6.

[0266] In some embodiments, the present disclosure also provides an anti-CD25 antigen-binding protein (e.g., antibody such as a single-domain antibody) that binds to the same epitope on CD25 as any of the exemplary VHH anti-CD25 antibodies listed in Table 1-1, Table 1-2, Table 5, or Table 6. Single-domain antibodies

[0267] A single-domain antibody (e.g., VHH) can be obtained by immunizing dromedaries, camels, llamas, alpacas, or sharks with the desired antigen and subsequently isolating the mRNA encoding the heavy chain antibodies. Antigens can be purified from natural sources or during recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of these antigens. By reverse transcription and polymerase chain reaction (PCR), a genetic library of single-domain antibodies containing several million clones can be produced. Screening techniques, such as phage display, yeast display, and ribosome display, help identify clones that bind to the antigen. Methods for generating heavy chain antibody fragments are described, for example, in WO 94 / 04678; Hamers-Casterman et al. 1993; Muyldermans et al. 2001; and Arbabi Ghahroudi, M.et al. (1997). FEBS Letters 414 (3):. Petition 870250081421, dated 10 / 09 / 2025, page 132 / 374 122 / 304 521-526, each of which is incorporated into the present invention by reference in its entirety.

[0268] A different method may utilize gene libraries from animals that have not been previously immunized. These naive libraries generally contain only antibodies with low affinity for the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step. See, for example, Saerens, D.; et al. (2008). “Single-domain antibodies as building blocks for novel therapeutics”. Current Opinion in Pharmacology 8 (5): 600-608.

[0269] Affinity maturation strategies can be categorized as targeted / rational or untargeted / random approaches. For targeted approaches, information about the VHH of interest is required, such as critical points for affinity maturation or structural information about the VHH:antigen complex, while for untargeted approaches, no prior information is required. Targeted approaches that can be applied to 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 exchange, and autonomous yeast surface display by hypermutation, the latter two being new, emerging, and very time-efficient techniques.Most of these strategies have in common that, after applying a specific randomization strategy to generate a mutational library, the resulting library can be screened using standard display techniques, such as yeast, phage, or ribosome display, to select the best ligands. The choice of display system is often guided by the size of the library to be displayed, with yeast display being able to handle library sizes of ~10⁷ to 10⁹, phage display of ~10⁸ to 10¹⁰, and ribosome display of ~10¹⁰. Petition 870250081421, dated 10 / 09 / 2025, page 133 / 374 123 / 304 ~1012a 1013 (Chan and Groves, 2021). Notably, during affinity maturation, the number of highly interactive residues, such as aromatic amino acids, generally increases in the CDR regions. Selected clones with affinity maturation can be further evaluated by a developmental assessment to test for undesirable properties such as nonspecific binding to unidentified targets or VHH instability.

[0270] For targeted in vitro mutagenesis, a set of selected residues within the CDRs of a VHH can be mutated (Tiller et al., 2017; Yau et al., 2005). Pre-selection of these residues can be performed using alanine scanning to identify critical residues for mutation or using structural data from the antigen:VHH complex to identify positions to be mutated. These sites can then be subjected to saturation mutagenesis to replace a specific site with all possible amino acids or specific amino acid substitutions, producing several smaller libraries. After mutagenesis, ligands can be screened to select the best matured candidate. Typically, several rounds of targeted mutagenesis are performed with separate sub-libraries to obtain combinations of individual mutations that cooperatively result in higher binding affinity.

[0271] Computer-assisted / in silico methods are frequently used to guide targeted in vitro mutagenesis. Using homology modeling of the target:VHH complex or docking, it is possible to identify critical points for mutations that are then subjected to in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et al., 2019; Inoue et al., 2013; Mahajan et al., 2018). Furthermore, in silico methods can search all designed variants in a virtual library (~1040 members) in a relatively short period to identify a viable number of promising candidates for experimental testing. These techniques can be especially valuable if structural data on the interaction are available. Petition 870250081421, dated 10 / 09 / 2025, page 134 / 374 124 / 304 target drugs are available.

[0272] Non-targeted / random affinity maturation strategies that can be applied to affinity-matured VHHs include in vitro random mutagenesis, CDR shuffling / swapping, and in vivo affinity maturation via yeast display. For in vitro random mutagenesis, the entire VHH sequence or only the CDRs are randomly mutated (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 proofreading activity and PCR conditions that further increase the polymerase error rate. This technique can be applied without extensive structural knowledge or information about the importance of residues contributing to the antigen:VHH interaction. The resulting mutational library can then be displayed to select the best matured candidate.This technique can also be combined with NGS sequencing of display elutions to obtain an in-depth reading of all candidates obtained, allowing the identification of less abundant but still promising clones (Chen et al., 2021).

[0273] In some embodiments, CDR shuffling or swapping is applied for the affinity maturation of VHHs, as described in Zupancic et al., 2021. For CDR swapping, enriched libraries can be used as input material for a PCR reaction to individually amplify the CDR of the VHHs. The PCR products can then be mixed and reassembled using overlapping PCR to generate the entire plasmid for additional display rounds in order to select the best-matured ligand. A limitation of this approach is that it can only be used for VHHs that comprise the same structure as the synthetic libraries.

[0274] In some embodiments, in vivo affinity maturation via yeast display is applied for VHH affinity maturation, such as Petition 870250081421, dated 10 / 09 / 2025, page 135 / 374 125 / 304 described in Wellner et al., 2021. The method is based on an autonomous yeast hypermutation surface display (AHEAD), which mimics somatic hypermutation during VHH selection using engineered yeast strains. The yeast's error-prone orthogonal DNA replication system can generate new variants during plasmid replication by randomly introducing mutations. The new variants can then be displayed and selected using the yeast surface display to identify the best ligands. This allows for the production of high-affinity clones in a very short time (about 2 weeks), which is significantly faster than classical affinity maturation procedures. The method can be applied using synthetic or immunological libraries using unenriched libraries, enriched libraries, or a subset of pre-selected clones.

[0275] If ligands with medium affinity are needed, as is the case with anti-CD25 V-bodies, and the affinity of the identified candidates needs to be reduced, very similar techniques can be applied. For example, mutations aimed at reducing affinity can be introduced using the same targeted or untargeted approaches described for affinity maturation. Subsequent selection can be adapted accordingly. If larger libraries are generated that need to be screened using a display technique, the selection strategy can be adapted to enrich medium-affinity ligands, excluding high-affinity candidates. This could be, for example, a phage display preprocessing with low antigen concentration to remove all higher-affinity candidates, followed by a high-antigen selection to obtain medium-affinity VHHs. For libraries up to 1.For 000 candidates, a kinetic characterization of deviation rate can be used to obtain immediate information about the kinetic behavior of the candidates.

[0276] After identifying the most potent clones, their DNA sequence Petition 870250081421, dated 10 / 09 / 2025, page 136 / 374 The 126 / 304 design can be optimized, for example, to improve its stability against enzymes. Another objective is humanization, in order to prevent immunological reactions of the human organism against the antibody. Humanization can be achieved based on homology between camelid VHH fragments and human VH, described in more detail below. Finally, the optimized single-domain antibody can be translated and expressed in suitable organisms, such as E. coli or Saccharomyces cerevisiae.

[0277] Single-domain antibodies can also be derived from conventional antibodies. In some embodiments, single-domain antibodies can be produced from conventional murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or non-immunized donors and display techniques for identifying the most specific antigens. However, the binding region of a conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate due to their lipophilicity. Monomerization can be achieved by replacing lipophilic amino acids with hydrophilic ones. (See, for example, Borrebaeck, CAK; Ohlin, M. (2002). “Antibody evolution beyond Nature”. Nature Biotechnology 20 (12): 1189-90.) If affinity can be maintained after monomerization, single-domain antibodies can similarly be produced in E. coli, S. cerevisiae, or other suitable organisms.

[0278] A “humanized antibody” refers to a chimeric, genetically modified antibody in which amino acid sequences (typically CDRs) of an antibody (donor antibody), for example, a camelid antibody, are grafted onto a human antibody (acceptor antibody). Thus, a humanized antibody typically comprises CDRs from a donor antibody and a variable region structure and constant regions, when present, from a human antibody. Consequently, a “humanized VHH” comprises CDRs that correspond to Petition 870250081421, dated 10 / 09 / 2025, p. 137 / 374 127 / 304 CDRs of a naturally occurring VHH domain (e.g., a camelid VHH), but which has been “humanized”. The humanized VHH can be prepared by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly in the structural sequences) with one or more amino acid residues occurring at the corresponding position(s) in a VH domain of a conventional 4-chain human antibody. Such humanized VHHs can be obtained in any suitable manner known to a person skilled in the art and are thus not strictly limited to the methods described in the present invention.

[0279] Humanization of VHHs can be achieved using CDR recapping or grafting. Recapping strategies have been described, for example, in Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et al., 2018; Vincke et al., 2009 J Biol Chem, and CDR grafting strategies have been described, for example, in ben Abderrazek et al., 2011; van Faassen et al., 2020 FASEB; Li et al., 2018; Vaneycken et al., 2010; Vincke et al., 2009 J Biol Chem; and Yu et al., 2017, each of which is incorporated into the present invention by reference in its entirety.

[0280] To humanize a camelid VHH using a recapping approach, a human germline reference that is most similar to the sequence of the selected camelid germline VHH can be identified. Most camelid VHHs isolated in the literature belong to camelid IGHV3 subfamily 2 (Nguyen et al., 2000, EMBO J) with DP-47 / VH3-23 of the IGHV3 family commonly used as a human reference. The structure of the camelid VHH can then be compared to the human reference sequence. Surface-exposed residues are replaced with their human counterpart, as their contribution to protein stability is presumed to be quite low. Buried residues, however, remain of camelid origin, as they likely contribute to the overall stability of the VHH. Humanization of Petition 870250081421, dated 10 / 09 / 2025, page 138 / 374 128 / 304 regions of structure 1, 3, and 4 generally do not affect the physicochemical properties of VHHs, while a general humanization of structure 2 would significantly increase local hydrophobicity. The residues H37, H44, H45, and H47 (Chothia numbering) in structure 2, the so-called tetrad or trademark residues, have a rather hydrophobic nature in human VHs (VGLW), as they are partially buried and involved in VH / VL pairing, while in camelid VHHs these residues are partially charged (FERG), which significantly increases VHH solubility and inhibits camelid VL pairing (Soler et al., 2021, Biomolecules, Conrath et al., 2005 J Mol Biol). Furthermore, it is known that residues H37 and H47 interact with the CDR-H3 loop in many VHHs, stabilizing its conformation and thus contributing to antigen-binding affinity.Furthermore, a significant number of HHVs utilize the H44, H45, and H47 residues of structure 2 for antigen binding (Zavrtanik et al., 2018, J Mol Biol). Complete humanization of these residues often results in reduced solubility or aggregation of HHVs and a reduced or complete loss of target antigen binding affinity (van Faassen et al., 2020, Vincke et al., 2009). Consequently, all or at least some of these characteristic residues of structure 2 remain of camelid origin during HHV humanization.

[0281] Another approach that can be applied to humanize HHVs is CDR grafting. CDRs from selected HHVs can be transplanted into a universal HHV scaffold 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 used successfully in some cases, but has failed in several others, with HHVs frequently losing their potential to bind to the desired antigen and / or becoming structurally unstable with a high tendency to aggregate (van Faassen et al., 2020, FASEB). This is mainly attributed to the interactions of CDR3 with specific residues in the 2 scaffold that are important for the Petition 870250081421, dated 10 / 09 / 2025, page 139 / 374 129 / 304 conformation of CDR3, overall stability of VHH, and overall hydrophobicity, which are impaired by this approach. Sometimes, camelid retromutations are introduced into the structure to compensate for these effects (van Faassen et al., 2020, FASEB).

[0282] An alternative strategy to mitigate the need to humanize selected VHH sequences is to use fully or partially humanized synthetic VHH libraries instead of camelid-immune libraries for VHH discovery (Moutel et al. 2016, eLife; McMahon, 2018, NSMB; Zimmermann et al., 2018, eLife). In many of these libraries, the characteristic residues are still of camelid origin, for the reasons discussed above.

[0283] Other suitable humanizing substitutions are described in documents WO 09 / 138519 and WO 08 / 020079, as well as in Tables A-3 to A-8 of document WO 08 / 020079 (which are lists showing possible humanizing substitutions), each of which is incorporated into the present invention by reference in its entirety. Non-limiting examples of such humanizing substitutions include Q108L and A14P. Such humanizing substitutions may also be suitably combined with one or more other mutations, as described herein (such as with one or more mutations that reduce binding by pre-existing antibodies).

[0284] In some embodiments, humanized VHH sequences still retain residues that are relevant for protein A binding. In some embodiments, engineering activities during humanization can be applied to engineer protein A binding properties in a VHH that previously did not interact with protein A (Graille et al., 2000, PNAS).

[0285] Like a “humanized antibody,” a “camelized antibody” refers to an antibody with amino acid sequences (typically CDRs) from a donor antibody, for example, a human antibody, and variable region structure. Petition 870250081421, dated 10 / 09 / 2025, p. 140 / 374 130 / 304 and constant regions, when present, of a camelid antibody. Consequently, a “camelized VH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but which has been “camelized”. Camelized VH can be prepared by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain of a conventional 4-chain antibody with one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be accomplished in a manner, for example, as described in WO 2008 / 020079.Such camelizing substitutions are generally inserted at amino acid positions that form and / or are present at the VH-VL interface and / or at so-called Camelidae characteristic residues, for example, F37, E44, R45, and F47 (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). In one embodiment, the VH sequence used as a starting material or starting point for generating or designing the camelized VH is a mammalian VH sequence or a human antibody VH sequence. However, such camelized VH can be obtained in any suitable manner known to a person skilled in the art and is thus not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VH domain as a starting material.

[0286] The amino acid residues of a single-domain antibody can be numbered according to the general numbering for VH domains provided by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to the Camelid VHH domains described in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see, for example, FIG. 2 of this publication). The total number of amino acid residues in each of the CDRs may vary and may not Petition 870250081421, dated 10 / 09 / 2025, p. 141 / 374 131 / 304 corresponds to the total number of amino acid residues indicated by Kabat numbering. For example, one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering. As a result, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain is generally in the range of 110 to 120, frequently between 112 and 115. However, shorter and longer sequences may also be suitable for the purposes described in the present invention.

[0287] The determination of CDR regions in a single-domain antibody can be performed using different methods, including those described by Kabat et al. (1991), “Sequences of proteins of immunological interest”, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (numbering scheme of “Kabat”); Al-Lazikani et al., (1997) JMB 273,927-948 (numbering scheme of “Chothia”); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography”, J. Mol. Biol. 262, 732-745. (numbering scheme of “Contact”); Lefranc MP et al., “Unique IMGT number for immunoglobulin receptor variable domains and T and similar domains of the Ig superfamily”, Dev Comp Immunol, 2003 Jan; scheme for immunoglobulin variable domains: an automated modeling and analysis tool”, J Mol Biol, 2001 Jun.8; 309(3):657-70, (numbering scheme of “Aho”); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm”, PNAS, 1989, 86(23):9268-9272, (numbering scheme of “AbM”), each reference cited in the present invention is incorporated by reference in its entirety.

[0288] The limits of a given CDR or structure (FR) may vary Petition 870250081421, dated 10 / 09 / 2025, page 142 / 374 132 / 304 depending on the identification scheme used. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common sequence lengths of the antibody region, with insertions accommodated by insertion letters, for example, 30a, and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (indels) in different positions, resulting in differential numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a middle ground between the Kabat and Chothia definitions, based on that used by Oxford Molecular's AbM antibody modeling software.

[0289] In some embodiments, CDRs may be defined according to 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 limits of CDR-H1, CDR-H2, and CDR-H3, as identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using 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 on.Note that, as the Kabat numbering scheme shown places inserts at H35A and H35B, the end of the Chothia CDR-H1 handle, when numbered using the Kabat numbering convention shown, varies between H32 and H34, depending on the handle length. Table 1-3. Definitions of CDRs according to various schemes of Petition 870250081421, dated 10 / 09 / 2025, page 143 / 374 133 / 304 numbering. CDR Kabat Chothia AbM Contact CDR-H1 (Kabat1 numbering) H31 - H35B H26 H32...34 H26 - H35B H30 - H35B CDR-H1 (Chothia2 numbering) H31 - H35 H26 - H32 H26 - H35 H30 - H35 CDR-H2 H50 - H65 H52 - H56 H50 - H58 H47 - H58 CDR-H3 H95 - H102 H95 - H102 H95 - H102 H93 - H101 1Kabat et al. (1991), “Sequences of proteins of immunological interest”, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD; 2Al-Lazikani etal., (1997) JMB 273, 927 - 948

[0290] Thus, unless otherwise specified, a “CDR” or “complementary determinant region”, or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, shall be understood as encompassing one (or the specific CDR) as defined by any of the schemes mentioned above. For example, when it is stated that a specific CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given amino acid sequence of VHH, it is understood that such CDR possesses a sequence of the corresponding CDR (e.g., CDR-H3) within VHH, as defined by any of the schemes mentioned above. In some embodiments, specific CDR sequences are specified.Exemplary CDR sequences of supplied antibodies are described using various numbering schemes (see, for example, Table 1-3), although it is understood that a supplied antibody may include CDRs as described according to any of the other numbering schemes mentioned above or other numbering schemes known to a person skilled in the art.

[0291] In a single-domain antibody sequence of the present Petition 870250081421, dated 10 / 09 / 2025, page 144 / 374 134 / 304 disclosure, the structure sequences can be any suitable structure sequences. For example, the structure sequences can be structure sequences derived from a variable heavy chain domain (e.g., a VH sequence or a VHH sequence). In some embodiments, the structure sequences are structure sequences derived from a VHH sequence (in which said structure sequences may have been optionally partially or fully humanized) or are conventional VH sequences (in which said structure sequences may have been optionally partially or fully camelized).

[0292] Antigen-binding fragments (or combinations of fragments) of any of the single-domain antibodies described herein, such as fragments containing one or more CDR sequences, suitably flanked by and / or linked via one or more structure sequences, are also covered by this disclosure.

[0293] It should be noted, however, that the present disclosure is not limited to the origin of the single-domain antibody (or the nucleotide sequence used to express it), nor to the manner in which the single-domain antibody or nucleotide sequence is generated or obtained. Thus, an antigen-binding protein of the present disclosure may comprise naturally occurring sequences (from a suitable species), recombinant sequences, or synthetic or semi-synthetic sequences. Similarly, the nucleotide sequences encoding the antigen-binding proteins of the present disclosure may comprise naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (e.g., sequences prepared by PCR or isolated from a library).

[0294] Anti-CD25 antigen-binding proteins (e.g., single-domain antibodies) of the present disclosure may comprise one or more Petition 870250081421, dated 10 / 09 / 2025, p. 145 / 374 135 / 304 amino acid substitutions, insertions, and / or deletions in the structure and / or CDR regions of the variable domains of the heavy chain, compared to the exemplary antibody sequences provided here. Such mutations can be easily determined by comparing the amino acid sequences revealed here with germline sequences available, for example, in public antibody sequence databases.The antigen-binding molecules of the present disclosure may comprise antigen-binding domains derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more structure 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 residue(s) (such sequence changes are collectively referred to herein as germline mutations). A practitioner with average knowledge in the field, starting from the variable region sequences of the heavy chain disclosed herein, can easily produce numerous antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof.In certain embodiments, all structure 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, for example, only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more structure and / or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e.,...). Petition 870250081421, dated 10 / 09 / 2025, p. 146 / 374 136 / 304 a germline sequence different from the germline sequence from which the antigen-binding domain was originally derived).

[0295] Furthermore, antigen-binding domains can contain any combination of two or more germline mutations within the structure and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a specific germline sequence, while certain other residues that differ from the original germline sequence are retained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations can be easily tested for one or more desired properties, such as enhanced 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 manner are generally covered by the present disclosure.

[0296] Anti-CD25 antigen-binding proteins comprising variants of any of the VHH and / or CDR amino acid sequences disclosed in the present invention having one or more amino acid substitutions are provided in the present invention. For example, the present disclosure includes anti-CD25 antigen-binding proteins containing VHH and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, 3 or less, 2 or 1 amino acid substitutions relative to any of the VHH and / or CDR amino acid sequences presented in Tables 1, 1-2, 5 or 6 in the present invention. Amino acid substitutions may be introduced into an antigen-binding protein of interest and the resulting variants may be screened for a desired activity, for example, retained / enhanced antigen binding, Petition 870250081421, dated 10 / 09 / 2025, p. 147 / 374 137 / 304 decreased immunogenicity or reduced ADCC or CDC.

[0297] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. In some embodiments, an amino acid substitution is a conservative substitution, meaning the exchange of one amino acid for another amino acid of the same class. In some embodiments, amino acid substitutions may also include a non-conservative substitution, meaning the exchange of one amino acid for an amino acid of a different class. Other exemplary amino acid substitutions are shown in Table 1-4. Table 1-4. Exemplary amino acid substitutions Original residue Exemplary replacements Ala (A) Val; Read; Ile Arg(R)Lys; Gln; Asn Asn(N)Gln; His; Asp, Lys; Arg Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly(G) Wing His (H) Asn; Gln; Lys; Arg lle(l) Leu; Val; Met; Allah; Phe; Norleucine Leu (L) Norleucine; lie; Val; Met; Allah; PheLys(K)Arg; Gln; Asn Met (M) Leu; Phe; Ile Phe (F) Trp; Read; Val; Ile; Allah; Tyr Pro (P) Ala Ser (S) Thr Thr (T) Val; Ser Trp (W) Tyr; PheTyr(Y)Trp; Phe; Thr; Ser Val (V) Ile; Read; Met; Phe; Allah; Norleucine

[0298] In some embodiments, the present disclosure single-domain antibodies (e.g., VHH) may comprise one or more mutations. Petition 870250081421, dated 10 / 09 / 2025, page 148 / 374 138 / 304 to reduce the oxidation levels of oxidation-labile residues, such as Met (M). In certain embodiments, it may be desirable to address the oxidation susceptibility of Met (M) by mutating a Met (M) residue. In some embodiments, the single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations (e.g., substitution mutations) of a Met residue to reduce oxidation. As a non-limiting example, a Met residue may be substituted in any of the single-domain antibodies described in this invention by, for example, Ile (I), Ala (A), or Leu (L), to reduce oxidation.

[0299] In some embodiments, the single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications that reduce the binding of the single-domain antibodies (e.g., VHH) to pre-existing antibodies found in human blood or serum. In some embodiments, the single-domain antibodies (e.g., VHHs) of the present disclosure are modified by mutation at amino acid position 11, for example, Leu11Glu (L11E), Leu11Lys (L11K), or Leu11Val (L11V). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure may comprise a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering).As another example, a single-domain antibody (e.g., VHH) of the present disclosure may comprise a 1 to 5 amino acid extension (of natural occurrence), such as a single alanine (A) extension, at the C-terminal end of the single-domain antibody (e.g., VHH). The C-terminal end of a VHH is typically VTVSS (SEQ ID NO: 2225). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering). In another embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or... Petition 870250081421, dated 10 / 09 / 2025, page 149 / 374 139 / 304 glutamine (Q) at position 112 (according to Kabat numbering). Consequently, the C-terminal of a single-domain antibody (e.g., VHH) can be any of VKVSS (SEQ ID NO: 2226), VQVSS (SEQ ID NO: 2227), VTVKS (SEQ ID NO: 2228), VTVQS (SEQ ID NO: 2229), VKVKS (SEQ ID NO: 2230), VKVQS (SEQ ID NO: 2231), VQVKS (SEQ ID NO: 2232) or VQVQS (SEQ ID NO: 2233).In another embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering), optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering), and an extension of 1 to 5 amino acids (naturally occurring), such as a single alanine (A) extension at the C-terminus of the single-domain antibody (e.g., VHH) (such that the C-terminus of the single-domain antibody (e.g., VHH), for example, has the sequence VTVSSA (SEQ ID NO: 2234), VKVSSA (SEQ ID NO: 2235), or VQVSSA (SEQ ID NO: 2236)). In other embodiments, the single-domain antibodies (e.g., VHH) of the present disclosure are modified by alterations in the carboxy-terminal region, for example, to a terminal sequence with the sequence GQGTLVTVKPGG (SEQ ID NO: 2237) or GQGTLVTVEPGG (SEQ ID NO: 2238) or a modification thereof.Further modifications to reduce binding by pre-existing antibodies in human serum can be found in, for example, WO2012 / 175741, WO2015 / 173325, WO2016 / 150845, WO2011 / 003622, WO2013 / 024059; US 11,426,468, US 10,526,397, which are incorporated into the present invention by reference in their entirety.

[0300] In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises, at the carboxy terminus, from position 111 according to Chothia, the amino acid sequence VAGG (SEQ ID NO: 4326) or VPAG (SEQ ID NO: 4327). In one embodiment, a single-domain antibody (e.g., VHH) Petition 870250081421, dated 10 / 09 / 2025, p. 150 / 374 140 / 304 example, VHH) of the present disclosure comprises, at the carboxy terminus, from position 111 according to Chothia, the amino acid sequence VAGG (SEQ ID NO: 4326). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises, at the carboxy terminus, from position 111 according to Chothia, the amino acid sequence VPAG (SEQ ID NO: 4327).

[0301] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any of the following SEQ ID Nos: 4, 8, 12, 16, 20, 26-30, 43-625, 1541-1845, 2251-2254, 2259-2262, 2268-2830, 3719-4014, 4317-4322, 4337, 4339, 4342-4559, 4892-5002, and 5114-5176, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, by less than 98%, or at least 99% sequence identity with it, wherein the amino acid sequence at the carboxy terminus of position 111 according to Chothia comprises VAGG (SEQ ID NO: 4326) or VPAG (SEQ ID NO: 4327).

[0302] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any of the SEQ ID NOs: 4, 8, 12, 16, 20, 26-30, 2251-2254, 2259-2262, 4317-4322, 4337, 4339, and 5114-5176, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity therewith, wherein the amino acid sequence at the carboxy terminus of position 111 according to Chothia comprises VAGG (SEQ ID NO: 4326) or VPAG (SEQ ID NO: 4327).

[0303] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any of the SEQ ID NOs: 26 - 30, 2259 - 2262, 4317 - 4322, 4339, and 5114 - 5145, or a sequence having at least 70%, at least 75%, Petition 870250081421, dated 10 / 09 / 2025, p. 151 / 374 141 / 304 at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with it, wherein the amino acid sequence at the carboxy terminus from position 111 according to Chothia comprises VAGG (SEQ ID NO: 4326) or VPAG (SEQ ID NO: 4327).

[0304] In some embodiments, the present disclosure's single-domain antibodies (e.g., VHH) are modified to enhance binding to staphylococcal protein A (SpA) or streptococcal protein G (SpG). The binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of antibodies or antibody fragments. The high-affinity interaction of the IgG Fc region with SpA and SpG has been extensively explored and has become the gold standard for the purification of monoclonal antibodies (Bjorck and Kronvall, 1984). Other non-Fc-containing antibody fragments, such as VHHs and Fabs, lack the ability to bind to SpA or SpG through their Fc regions. However, sequence-dependent interaction with SpA has been demonstrated for these non-Fc-containing antibody fragments (Graille et al., 2000; Henry et al., 2016).This feature avoids the potential use of affinity markers fused to the drug candidate for affinity chromatography, which have the disadvantage of being considered a sequence liability, as they can impact the immunogenicity of the protein, as well as the protein structure and stability, and may compromise functionality. The interaction of single-domain antibodies (e.g., VHH) with SpA relies on an alternative binding mode, with an affinity of 1 to 5 μM, which is comparable to the 0.2 to 3 μM measured for VHSpA interactions (To et al., JBC, 2005; Henry et al., Plos One, 2016).

[0305] In some embodiments, the present disclosure’s single-domain antibodies (e.g., VHH) possess, or are modified to possess, an SpA-binding motif. For example, the VHH-SpA interface has been mapped to thirteen residues, which cluster within the structure in the posterior part of the V-body, distant Petition 870250081421, dated 10 / 09 / 2025, p. 152 / 374 142 / 304 of the CDRs (Graille et al., 2000, Henry et al., 2016). In the absence of a VHH-SpA costructure, the superposition of an SpA-Fab crystal structure and a VHH allows visualization of the bonding mode. Based on a structural and functional analysis, the thirteen residues of the VHH-SpA interface were characterized as intolerant to substitutions (residues Gly15, Arg19, Tyr59, Gly65 and Arg66), tolerant to specific substitutions (residues Thr / Lys / Arg57, Thr68, Gln81, Asn82a and Ser82b) or generally tolerant to a variety of substitutions (residues Ser17, Lys64 and Ser70) (all residue positions refer to Kabat numbering) (Henry et al., Plos One, 2016). Thus, an SpA-binding motif included in a single-domain antibody (e.g., VHH) of the present disclosure may include one or more, or all thirteen residues.

[0306] In some embodiments, the single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more N-terminus modifications to avoid pyroglutamate formation and product heterogeneity. In one embodiment, the Glu amino acid residue in the first position of the single-domain antibody (e.g., VHH) is replaced by Asp (E1 D).

[0307] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any of the following SEQ ID Nos: 4, 8, 12, 16, 20, 26-30, 43-625, 1541-1845, 2251-2254, 2259-2262, 2268-2830, 3719-4014, 4317-4322, 4337, 4339, 4342-4559, 4892-5002, and 5114-5176, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with it, in which the Glu amino acid residue at the first position of the single-domain antibody (e.g., VHH) is replaced by Asp (E1D).

[0308] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence Petition 870250081421, dated 10 / 09 / 2025, p. 153 / 374 143 / 304 selected from any of the SEQ ID NOs: 4, 8, 12, 16, 20, 26 - 30, 2251 - 2254, 2259 - 2262, 4317 - 4322, 4337, 4339, and 5114 - 5176, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with it, wherein the Glu amino acid residue in the first position of the single-domain antibody (e.g., VHH) is replaced by Asp (E1 D).

[0309] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any of the SEQ ID NOs: 26 - 30, 2259 - 2262, 4317 - 4322, 4339, and 5114 - 5145, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity therewith, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced by Asp. Alternative protein scaffolds

[0310] In some embodiments, the antiCD25 antigen-binding proteins of the present disclosure may adopt an alternative protein scaffold. Such an alternative protein scaffold may be a single-chain polypeptide structure, optionally with a reduced size (e.g., less than about 200 amino acids), containing 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 in the present invention onto a suitable protein scaffold. The structure of alternative scaffolds may vary, but preferably is of human origin for those developed as therapeutic agents.

[0311] Alternative proteins of the present disclosure may be based on a conventional immunoglobulin (Ig) structure or derived from a protein Petition 870250081421, dated 10 / 09 / 2025, p. 154 / 374 144 / 304 completely different. These variable domains can be modified to create new binding interfaces for any target antigen. In some embodiments, an alternative protein structure of the present disclosure may be derived from Protein A, for example, its Z domain (afficorpos), ImmE7 (immunity proteins), BPTI / APPI (Kunitz domains), Ras-binding protein AF-6 (PDZ domains), caribdotoxin (scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neocarzinostatin, a fibronectin domain (used in “adnectin”), an ankyrin (AR) repeat domain (used in “DARPins”), avidity multimers (also known as “avimers”) or thioredoxin (Skerra, A., Curr. Opin. Biotechnol. 18:295-304 (2005); Hosse et al., Protein Sci. 15:14-27 (2006); Nicaise et al., Protein Sci. 13:1882-1891 (2004); Nygren and Uhlen, Curr. Opinion. Struc. Biol.7:463-469 (1997), all of which are incorporated into the present invention by reference in their entirety).

[0312] Antichalins are a suitable type of alternative non-Ig-based scaffold for use in the antigen-binding molecules of the present invention. Antichalins are a class of ligand-binding proteins designed based on the lipocalin scaffold. Lipocalins are a family of proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have limited sequence homology but share a common tertiary structure architecture based on eight antiparallel β-barrels. Lipocalins contain four exposed loops built into the rigid β-barrel structure. Commonly used exemplary antichalin proteins have a size of approximately 180 amino acids and a mass of approximately 20 kDa.

[0313] DARPins are another suitable alternative structure, not based on Ig, that can be used in the antigen-binding molecules of the present disclosure. DARPins are genetically modified antibody-mimetic proteins that typically exhibit highly specific and high-affinity binding to the target protein. Petition 870250081421, dated 10 / 09 / 2025, page 155 / 374 145 / 304 They are derived from natural ankyrin repeat (AR) proteins, which typically contain a 33-amino acid protein motif consisting of two α-helices separated by loops, whose repeats mediate protein-protein interactions. DARPins can be generated using combinatorial AR libraries constructed based on the 33-amino acid AR motif with seven random positions. DARPin libraries can be screened using ribosome display, and library members are typically well-produced in Escherichia coli, do not aggregate, and exhibit high thermodynamic stability. Preferably, DARPins contain two to four of these motifs flanked by N- and C-terminal cover motifs to protect hydrophobic regions and allow for greater solubility.

[0314] The avimer structure can also be used as a protein scaffold to generate a suitable alternative scaffold, not based on Ig. Avimers typically consist of two or more peptide sequences of 30 to 35 amino acids each, connected by a peptide linker. The individual sequences are derived from A domains of various membrane receptors and have a rigid structure, stabilized by disulfide and calcium bridges. Each A domain can bind to a specific epitope of the target protein. The combination of domains that bind to different epitopes of the same protein increases the affinity for that protein, an effect known as avidity.

[0315] Proteins derived from fibronectin III (FN3) domains can also be used to generate a suitable alternative, non-Ig-based scaffold (also known as a monobody). For example, the tenth fibronectin type III (FN10) domain of human fibronectin corresponds to a β-sandwich with seven β-strands and three connecting loops, exhibiting structural homology with Ig domains without disulfide bridges. In some cases, the connecting loops of FN10, each approximately 15 to 21 amino acids long, can be randomized and Petition 870250081421, dated 10 / 09 / 2025, page 156 / 374 146 / 304 of the domains displayed in both phage and yeast were used to select a scaffold with the desired properties. Adnectins™ is an exemplary scaffold generated using 10 randomized fn3 domains and displayed in this way. Another exemplary scaffold comprising FN3 domains is Centyrin™. Centyrins™ contain the consensus sequence of FN3 domains from human Tenascin C (TNC), which is found in the extracellular matrix of various tissues. Centyrin™ scaffolds have loops that have structural homology with variable antibody domains (i.e., CDR1, CDR2, and CDR3) and are small (approximately 10 kDa), simple, and highly stable single-domain proteins that do not contain cysteine, disulfides, or glycosylated residues. Centyrin™ possesses excellent biophysical properties such as stability to heat, pH, denaturing 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 into the present invention by reference in its entirety. Fusion Proteins and Conjugates

[0316] In one aspect, the present invention provides fusion proteins and conjugates comprising at least one anti-CD25 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) linked, directly or indirectly, to one or more additional domains or fragments. In some embodiments, the fusion protein or conjugate of the present disclosure comprises a single polypeptide. In other embodiments, the fusion protein or conjugate of the present disclosure comprises more than one polypeptide. In some embodiments, the fusion protein or conjugate of the present disclosure comprises two polypeptides.

[0317] In some embodiments, the fusion protein or conjugate of the present disclosure comprises at least one anti-antigen-binding protein. Petition 870250081421, dated 10 / 09 / 2025, page 157 / 374 147 / 304 CD25 (for example, an antibody, such as a single-domain antibody) described in the present invention. In some embodiments, the fusion protein or conjugate is multivalent. For example, the fusion protein or conjugate of the present disclosure may be at least bivalent, but may also be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc. The terms bivalent, trivalent, tetravalent, pentavalent, or hexavalent fall under the term multivalent and indicate the presence of two, three, four, five, or six binding units (for example, VHHs), respectively.

[0318] In certain embodiments, the fusion protein or conjugate is multispecific. For example, in some cases, one or more additional domains or fragments may be one or more additional binding domains that bind to one or more additional antigens or proteins. The fusion protein or conjugate of the present disclosure may be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc. The terms bispecific, trispecific, tetraspecific, pentaspecific, etc., all fall under the term multispecific and refer to binding to two, three, four, five, etc., different target molecules, respectively.

[0319] When two or more anti-CD25 antigen-binding proteins are included in a fusion protein or conjugate, the two or more anti-CD25 antigen-binding proteins may comprise the same sequence or different sequences. In such embodiments, the two or more anti-CD25 antigen-binding proteins may bind to the same epitope on CD25 or to different epitopes on CD25. For example, a fusion protein or conjugate of the present disclosure may be biparatopic, for example, if two VHHs bind to two different epitopes on CD25. Fusion or Conjugation with Fc regions

[0320] In some embodiments, a fusion protein or conjugate of the present invention comprises at least one anti-antigen binding protein. Petition 870250081421, dated 10 / 09 / 2025, p. 158 / 374 148 / 304 CD25 (e.g., an antibody, such as a single-domain antibody) provided herein, operationally linked to a dimerization domain, such as an immunoglobulin Fc region. An immunoglobulin Fc region may be linked indirectly or directly to at least one anti-CD25 antigen-binding protein (e.g., an antibody, such as a single-domain antibody). In some embodiments, a fusion protein or conjugate of the present invention comprises one, two, three, four, five, six, or more anti-CD25 antigen-binding proteins provided in the present invention operationally linked to an Fc region.

[0321] An “Fc region” as used in the present invention refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, an Fc region comprises a hinge, CH2 and CH3. In several embodiments, when an Fc region comprises a hinge, it may mediate dimerization between two Fc-containing polypeptides. In several embodiments, an Fc region included in a fusion protein or conjugate of the present disclosure is a human immunoglobulin Fc region or is derived from a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is of the IgG, IgE, IgM, IgD, IgA, or IgY isotype. In some embodiments, the immunoglobulin Fc region is an IgG isotype, such as the IgG1, IgG2, IgG3, or IgG4 subclass. The immunoglobulin Fc region may comprise a variant or fragment of a native IgG Fc region.

[0322] A native Fc region typically has 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; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation, etc. These effector functions generally require the Fc region to be combined with a binding domain (e.g., a variable domain of Petition 870250081421, dated 10 / 09 / 2025, page 159 / 374 149 / 304 antibody) and can be evaluated using various assays.

[0323] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise a dimer of Fc regions. In some embodiments, an Fc region mediates the dimerization of CD25-binding units under physiological conditions, such as when expressed from a cell, so that a dimer is formed that doubles the number of CD25-binding units. For example, a fusion polypeptide comprising a CD25-binding VHH domain and an Fc region is monovalent as a monomer, but the Fc region may mediate dimerization; as a result, the fusion protein is bivalent (i.e., it has two anti-CD25 VHH domains per molecule). Similarly, in some embodiments, two anti-CD25 VHH domains (2x) are fused to an IgG Fc region and, as a result of dimerization, the fusion protein is tetravalent (i.e., it has four anti-CD25 VHH domains per molecule).In some embodiments, three anti-CD25 VHH domains (3x) are fused to an Fc region of IgG and, as a result of dimerization, the fusion protein is hexavalent (i.e., having six anti-CD25 VHH domains per molecule).

[0324] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (VHH anti-CD25)n-Fc-Ligand, wherein n may be any integer (e.g., 1, 2, 3, 4, 5, etc.). When n>2, each VHH anti-CD25 may optionally be operationally linked to another VHH anti-CD25 via a ligand.

[0325] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (VHH anti-CD25)n-Ligand-Fc-(VHH anti-CD25)m, wherein n may independently be any integer (e.g., 1, 2, 3, 4, 5, etc.). When n>2 or m>2, each VHH anti-CD25 may be Petition 870250081421, dated 10 / 09 / 2025, pp. 160 / 374 150 / 304 optionally operationally linked to another anti-CD25 VHH via a linker.

[0326] In some embodiments, a fusion protein or conjugate of the present disclosure is bivalent. In some embodiments, the bivalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each with the following structure: (anti-CD25 VHH)-Ligand-Fc.

[0327] In some embodiments, a fusion protein or conjugate of the present disclosure is tetravalent. In some embodiments, the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each with the following structure: (anti-CD25 VHH)-Ligand-(anti-CD25 VHH)-Fc-Ligand. In some embodiments, the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each with the following structure: (anti-CD25 VHH)-Fc-Ligand-(anti-CD25 VHH). The multiple ligands used in the fusion protein are not necessarily the same.

[0328] In some embodiments, a fusion protein or revelation conjugate is hexavalent. In some embodiments, the hexavalent fusion protein or revelation conjugate comprises two polypeptide chains, each with the following structure: (anti-CD25 VHH)-Ligand-(anti-CD25 VHH)-Ligand-(anti-CD25 VHH)-Ligand-Fc. In some embodiments, the hexavalent fusion protein or revelation conjugate comprises two polypeptide chains, each with the following structure: (anti-CD25 VHH)-Ligand-(anti-CD25 VHH)-Ligand-Fc-ligand(anti-CD25 VHH). In some embodiments, the hexavalent fusion protein or revelation conjugate comprises two polypeptide chains, each polypeptide chain with the following structure: (anti-CD25 VHH)-Ligand-Fc-Ligand-(anti-CD25 VHH)-Ligand-(anti-CD25 VHH). The multiple ligands used in the fusion protein are not necessarily the same.

[0329] In some embodiments, the CH3 domain of the Fc region can be used Petition 870250081421, dated 10 / 09 / 2025, page 161 / 374 151 / 304 as a homodimerization domain, so that the resulting fusion protein can be formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to allow heterodimerization. For example, a heterodimerization domain can be incorporated into the fusion protein, so that the construct is a heterodimeric fusion protein.

[0330] When an Fc region dimer is used in a fusion protein or conjugate of the present disclosure, the first and second Fc regions may be of the same IgG isotype, such as, for example, IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second Fc regions may be of different IgG isotypes, such as, for example, IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0331] In some embodiments, the Fc region included in a fusion protein or conjugate of the present disclosure may be mutated or modified. In some embodiments, the mutations include one or more amino acid substitutions to reduce an effector function of the Fc region. Several examples of mutations in Fc regions to alter, such as reduce, effector function are known, including any described below. In general, the numbering of 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, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0332] In some embodiments, the Fc region of human IgG is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Non-limiting examples of amino acid modifications that may 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, Petition 870250081421, dated 10 / 09 / 2025, pp. 162 / 374 152 / 304 103(11): 4005-4010; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Moore et al., 2010 mAbs, 2(2): 181-189; and Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11, each of which is incorporated into the present invention by reference in its entirety.

[0333] In some embodiments, an Fc region included in a presently disclosed fusion protein or conjugate exhibits reduced effector functions (such as CDC and ADCC). Several in vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the fusion protein construct and / or its cleaved components do not bind to FcyR (therefore, likely lack ADCC activity) but retain the ability to bind to FcRn. The primary cells to mediate ADCC are NK cells, which express only FcyRIII, while monocytes express FcyRI, FcyRII, and FcyRIII. Non-limiting examples of in vitro assays to assess the ADCC activity of a molecule of interest are described, for example, in US 5,500,362; US 5,821,337; Hellstrom. et al., Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986); and Hellstrom et al., Proc. Nat'l Acad.Sci. USA 82:1499-1502 (1985); Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987). Alternatively, non-radioactive assay methods can be employed, such as the ACTI™ non-radioactive cytotoxicity assay for flow cytometry or the CytoTox96™ non-radioactive cytotoxicity assay. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and Natural Killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm the protein construct. Petition 870250081421, dated 10 / 09 / 2025, pp. 163 / 374 153 / 304 fusion or its cleaved components are unable to bind to C1q and therefore do not show CDC activity (see, for example, C1q and C3c binding ELISA in documents WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay can be performed (see, for example, GazzanoSantoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the state of the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0334] Examples of mutations that enhance ADCC include modifications in Ser239 and Ile332, for example, Ser239Asp and Ile332Glu (S239D, 1332E). Examples of mutations that enhance CDC include modifications in Lys326 and Glu333. In some forms, the Fc region is modified in one or both positions, for example, Lys326Ala and / or Glu333Ala (K326A and E333A) using the Kabat numbering system.

[0335] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327) or Pro329 (P329). For example, Leu 234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A) or Pro239Gly (P329G), Asn325Glu (N325E) or Ala327Ser (A327S). In some modalities, modifications in the Fc region reduce binding to gamma Fc receptors (FcyRs), but have minimal impact on binding to the neonatal Fc receptor (FcRn).

[0336] In some embodiments, the Fc region of human IgG1 is modified at the amino acid Asn297 (Kabat numbering) to prevent glycosylation of the protein. Petition 870250081421, dated 10 / 09 / 2025, pp. 164 / 374 154 / 304 fusion, for example, Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein is modified at the amino acid Leu235 (Kabat numbering) to alter interactions with the Fc receptor, for example, Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at the amino acid Leu234 (Kabat numbering) to alter interactions with the Fc receptor, for example, Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at the amino acid Leu234 (Kabat numbering) to alter interactions with the Fc receptor, for example, Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234 and 235, for example, Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A).In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 297, for example, Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, for example, Leu234Ala, Leu235Ala, Pro239Ala (L234A / L235A / P329A). In some embodiments, the Fc region of the fusion protein is modified at the amino acid Asp265 (Kabat numbering) to alter interactions with the Fc receptor, for example, Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat numbering) to alter interactions with the Fc receptor, for example, Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 265 and 329, for example, Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G).In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 265, for example, Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, for example, Leu234Ala, Leu235Ala, Pro329Gly. Petition 870250081421, dated 10 / 09 / 2025, pp. 165 / 374 155 / 304 (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, 265, and 329, for example, Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce binding to the Fc receptor. For example, where Gly235 is deleted from the fusion protein. In some embodiments, the Fc region of human IgG1 is modified at amino acid Gly236 to increase interaction with CD32A, for example, Gly236Ala (G236A). In some forms, the Fc region of human IgG1 lacks Lys447 (EU index of Kabat et al 1991 Sequences of proteins of immunological interest).

[0337] In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Gly, Leu235Ser, Gly236Arg (L234G / L235S / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Ser, Leu235Thr, Gly236Arg (L234S / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Ser, Leu235Val, Gly236Arg (L234S / L235V / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Thr, Leu235Gln, Gly236Arg (L234T / L235Q / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Thr, Leu235Thr, Gly236Arg (L234T / L235T / G236R).In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, for example, Leu234Thr, Leu235Thr, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 252, 254, and 256, for example, Met252Tyr, Ser254Thr, Thr256Glu (M252Y / S254T / T256E).

[0338] In some embodiments, the Fc region of the fusion protein lacks an amino acid at one or more of the following positions to reduce binding to Petition 870250081421, dated 10 / 09 / 2025, pp. 166 / 374 156 / 304 Fc receptor: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In some embodiments, the Fc region of the fusion protein lacks an amino acid at one or more of the following positions: Glu233 (E233), Leu234 (L234), or Leu235 (L235) and is modified in one or more of the following domains: Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce binding to the Fc receptor. For example, an Fc region included in a CD25-binding polypeptide is derived from a human Fc domain and comprises a three-amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235. In some embodiments, such Fc polypeptides do not bind to FcyRs and are therefore called silent effectors or null effectors. For example, Fc deletion of these three amino acids reduces binding to the C1q complement protein.In some embodiments, a polypeptide with an Fc region with Fc deletion of these three amino acids retains the FcRn binding and therefore exhibits prolonged half-life and transcytosis associated with FcRn-mediated recycling.

[0339] In one embodiment, the Fc region of the immunoglobulin fusion protein is a variant of the Fc region of human IgG1, having an amino acid sequence: IgG1 L234A, L235A (also known as the “LALA” variant) (mutations in bold in the sequence below) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 2213).

[0340] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region, having an amino acid sequence: Petition 870250081421, dated 10 / 09 / 2025, pp. 167 / 374 157 / 304 IgG1 L234A, L235A, and P329A (also known as the “LALAPA” variant) (mutations in bold in the sequence below) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 2214).

[0341] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region, having an amino acid sequence: IgG1 D265A, N297A and P329A (also known as the “DANAPA” variant) (mutations in bold in the sequence below) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 2215).

[0342] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region, having an amino acid sequence: IgG1 L234A, L235A, and G237A (also known as the “LALAGA” variant) (mutations in bold in the sequence below) DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 2216). Petition 870250081421, dated 10 / 09 / 2025, pp. 168 / 374 158 / 304

[0343] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having an amino acid sequence: IgG1 L234G / L235S / G236R (mutations in bold in the sequence below) DKTHTCPPCPAPEGSRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 4328).

[0344] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having an amino acid sequence: IgG1 L234S / L235T / G236R (mutations in bold in the sequence below) DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 4329).

[0345] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having an amino acid sequence: IgG1 L234S / L235V / G236R (mutations in bold in the sequence below) DKTHTCPPCPAPESVRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 4330). Petition 870250081421, dated 10 / 09 / 2025, page 169 / 374 159 / 304

[0346] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having an amino acid sequence: IgG1 L234T / L235Q / G236R (mutations in bold in the sequence below) DKTHTCPPCPAPETQRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 4331).

[0347] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having an amino acid sequence: IgG1 L234T / L235T / G236R (mutations in bold in the sequence below) DKTHTCPPCPAPETTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 4332).

[0348] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having an amino acid sequence: IgG1 L234A / L235A / P329G (mutations in bold in the sequence below) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 4333) Petition 870250081421, dated 10 / 09 / 2025, pp. 170 / 374 160 / 304

[0349] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having an amino acid sequence: IgG1 M252Y / S254T / T256E (mutations in bold in the sequence below) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 4334).

[0350] In some forms, the Fc region of human IgG is modified to enhance binding to FcRn. Examples of Fc mutations that enhance FcRn binding are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (numbering Kabat, Dall'Acqua et al 2006, J. Biol Chem Vol. 281 (33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159), or Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively) (EU index of Kabat et al 1991 Protein sequences of immunological interest).

[0351] In some embodiments, the Fc region either lacks or has reduced fucose attached to the N-linked glycan chain at N297. There are numerous ways to prevent fucosylation, including, but not limited to, production in a FUT8-deficient cell line; the addition of inhibitors to the culture medium of mammalian cells, for example, Castanospermine; and metabolic engineering of the production cell line.

[0352] In some embodiments, the Fc domain included in a fusion protein or conjugate of the present disclosure is derived from a human Fc domain and comprises the M252Y and M428V mutations. In some embodiments, the mutated or modified Fc polypeptide includes the following mutations: M252Y and M428L, utilizing the Petition 870250081421, dated 10 / 09 / 2025, pp. 171 / 374 161 / 304 Kabat numbering system. In some embodiments, such mutations increase FcRn binding at the acidic pH of the endosome (close to 6.5), while losing detectable binding at neutral pH (around 7.2), allowing greater FcRn-mediated recycling and prolonged half-life.

[0353] In some embodiments, the Fc domain included in a fusion protein or conjugate is derived from a human Fc domain and comprises mutations to induce heterodimerization. In some embodiments, such mutations include those termed knob and hole mutations. For example, having an amino acid modification within the CH3 domain at Thr366, which when replaced by a bulkier amino acid, e.g., Try (T366W), is able to preferentially pair with a second CH3 domain with amino acid modifications to less bulky amino acids at the Thr366, Leu368, and Tyr407 positions, e.g., Ser, Ala, and Val, respectively (T366S / L368A / Y407V). In some embodiments, the Fc knob domain comprises the T366W mutation. In some embodiments, the Fc hole domain comprises the T366S, L368A, and Y407V mutations.Heterodimerization via CH3 modifications can be further stabilized by the introduction of a disulfide bond, for example, by altering Ser354 to Cys (S354C) and Y349 to Cys (Y349C) in opposing CH3 domains (Reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, the Fc domains used for heterodimerization comprise additional mutations, such as the S354C mutation in a first member of a heterodimeric Fc pair forming an asymmetric disulfide with a corresponding Y349C mutation in the second member of a heterodimeric Fc pair. In some embodiments, a member of a heterodimeric Fc pair comprises the H435R or H435K modification to prevent binding to protein A while maintaining binding to FcRn. In some embodiments, one member of a heterodimeric Fc pair comprises the H435R or H435K modification, while the second member of the heterodimeric Fc pair does not. Petition 870250081421, dated 10 / 09 / 2025, pp. 172 / 374 162 / 304 modified in H435. In several embodiments, the hole-type Fc domain comprises the H435R or H435K modification (referred to as “hole-R” in some cases when the modification is H435R), while the knob Fc domain does not. In some cases, the hole-R mutation improves heterodimer purification compared to homodimeric hole Fc domains that may be present.

[0354] In some embodiments, the Fc region of human IgG is modified to prevent dimerization. In these embodiments, the present development fusion proteins are monomeric. For example, modification of the Thr366 residue to a charged residue, e.g., Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3CH3 dimerization.

[0355] In some embodiments, the Fc region of the immunoglobulin fusion protein is of the human IgG3 isotype, or a variant thereof. In one embodiment, the Fc region of IgG3 is modified at the amino acid Asn297 (Kabat numbering) to prevent glycosylation of the antibody, for example, Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of human IgG3 is modified at amino acid 435 to extend the half-life, for example, Arg435His (R435H). In some embodiments, the Fc region of human IgG3 lacks Lys447 (EU index of Kabat et al 1991).

[0356] In some embodiments, the Fc region of the immunoglobulin fusion protein is of the human IgG4 isotype, or a variant thereof. In one embodiment, the Fc region of human IgG4 is modified at amino acid 235 to alter interactions with the Fc receptor, for example, Leu235Glu (L235E). In some embodiments, the Fc region of human IgG4 is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the antibody, for example, Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of human IgG4 lacks Lys447 (EU index of Kabat et al 1991). Petition 870250081421, dated 10 / 09 / 2025, pp. 173 / 374 163 / 304

[0357] In some embodiments, the Fc region of IgG4 fusion protein is altered at amino acids 228 and 235, for example, Ser228Pro, Leu235Glu or Leu235Ala (S228P / L235E or S228P / L235A). In some embodiments, the Fc region of IgG4 fusion protein is altered at amino acids 228, 234 and 235, for example, Ser228Pro, Phe234Ala, Leu235Glu or Leu235Ala (S228P / F234A / L235E or S228P / F234A / L235A). In some forms, the IgG4 Fc region of the fusion protein is altered at amino acids 228, 235, and 329, for example, Ser228Pro, Leu235Glu, and P329G (S228P / L235E / P329G).

[0358] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, L235E (mutations in bold in the sequence below) ESKYGPPCPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 2217)

[0359] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, L235A (mutations in bold in the sequence below) ESKYGPPCPCPAPEFAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 2218)

[0360] In one embodiment, the Fc region of the immunoglobulin protein Petition 870250081421, dated 10 / 09 / 2025, pp. 174 / 374 164 / 304 fusion is a variant of the Fc region of human IgG4, having an amino acid sequence: IgG4 S228P, F234A, L235E (mutations in bold in the sequence below) ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 2219)

[0361] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, F234A, L235A (mutations in bold in the sequence below) ESKYGPPCPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 2220)

[0362] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region, having an amino acid sequence: IgG4 P329G, S228P, L235E (mutations in bold in the sequence below) ESKYGPPCPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL GSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 2221)

[0363] Additional IgG4 heavy chain modifications suitable for Petition 870250081421, dated 10 / 09 / 2025, pp. 175 / 374 165 / 304 Use in fusion proteins or conjugates of the present disclosure includes those described in Tables 1 and 2 of Dumet et al., mAbs, 11:8, 1341-1350, which is incorporated into the present invention by reference in its entirety.

[0364] In some embodiments, the fusion protein or conjugate contains an immunoglobulin hinge region. In some embodiments, the hinge region serves as a linker to connect one or more CD25-binding units (e.g., VHHs) to the Fc region. In other embodiments, the fusion protein may comprise a linker in addition to the hinge region to connect one or more CD25-binding units (e.g., VHHs) to the Fc region. The hinge region may be selected from any of the human IgG subclasses. For example, the fusion protein may contain an IgG1 hinge modified with the sequence EPKSSDKTHTCPPC (SEQ ID NO: 2222), in which Cys220, which typically forms a disulfide bond with the C-terminal cysteine ​​of the light chain, is mutated to serine, e.g., Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge with a DKTHTCPPC sequence (SEQ ID NO: 2223).

[0365] In some embodiments, the fusion protein or conjugate has a modified IgG4 hinge, which is modified to prevent or reduce strand switching, for example, Ser228Pro (S228P), having the sequence ESKYGPPCPCC (SEQ ID NO: 2224).

[0366] In alternative embodiments, a fusion protein or conjugate of the present disclosure may comprise different sequences of an Fc region to achieve multimerization (e.g., dimerization). For example, an amino acid sequence containing at least one cysteine ​​residue may be included to facilitate the dimerization of two polypeptides by forming a disulfide bond between the two polypeptides. In some embodiments, such a multimerizing domain may comprise one or more cysteine ​​residues, or a short peptide containing Petition 870250081421, dated 10 / 09 / 2025, pp. 176 / 374 166 / 304 cysteine. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled spiral motif.

[0367] Fc mutations suitable for use in the fusion proteins disclosed in the present invention are also discussed in, for example, Wilkinson et al., Fc-engineered antibodies with immunoeffector functions completely abolished. PLoS One. 2021; WO2021234402A2; US 8,969,526; EP3692065B1; and US 7,083,784, each of which is incorporated into the present invention by reference. Fusion or Conjugation with Half-Life Extension Fractions

[0368] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise one or more other fractions that impart to the fusion protein or conjugate an extended half-life (in vivo). Extended half-life in vivo means that the fusion protein or conjugate has an increased half-life in a mammal, such as a human, after administration.

[0369] Non-limiting examples of half-life extension fractions suitable for use in the present disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc moiety, and small proteins or peptides that can bind to serum proteins.

[0370] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to serum albumin, such as human serum albumin, or to a serum immunoglobulin, such as IgG. In one embodiment, a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to human serum albumin. In one embodiment, the binding moiety is a single-domain antibody (e.g., VHH).

[0371] For example, and without limitation, albumin ligands described, by Petition 870250081421, dated 10 / 09 / 2025, page 177 / 374 167 / 304 example, in documents WO 04 / 041865, WO 06 / 122787, WO2012 / 175400, WO 2012 / 175741, WO2015 / 173325, WO2017 / 080850, WO2017 / 085172, WO2018 / 104444, WO2018 / 134235, WO2018 / 134234, each of which is incorporated in the present invention by reference in its entirety, can be used in the fusion protein or conjugate of the present disclosure. Fusion or Conjugation with Other Fractions

[0372] Anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) provided herein may be operationally linked, directly or indirectly, to a second moiety, such as, but not limited to, a detectable marker, 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.

[0373] In some embodiments, a conjugate of the present disclosure comprises a marker, which can generate a detectable signal. Such conjugates can be used for research or diagnostic purposes, such as for in vivo cancer detection. Preferably, the marker is capable of producing, directly or indirectly, a detectable signal. For example, the marker may be radiopaque or a radioisotope (such as 3H, 14C, 32P, 35S, 123I, 125I, 131I); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound (such as fluorescein isothiocyanate, rhodamine, or luciferin); an enzyme (such as β-galactosidase, alkaline phosphatase, or horseradish peroxidase); an imaging agent; or a metal ion.In some applications, the tracer is a radioactive atom for scintigraphic studies, for example, 99Tc or 123I, or a spin tracer for nuclear magnetic resonance (NMR) imaging, such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. Zirconium-89 can also be complexed with various metal chelating agents and conjugated to... Petition 870250081421, dated 10 / 09 / 2025, page 178 / 374 168 / 304 antibodies, for example, for PET images (WO 2011 / 056983).

[0374] Anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure can be conjugated to another moiety, such as an epitope tag, for purification or detection purposes. Examples of such molecules useful in protein purification include those that have 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 to the immobilized compound.Non-limiting examples of epitope marker molecules that can be conjugated to anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure, for example, for molecular recognition purposes, include a polyhistidine marker (His marker), a myc marker, a human influenza hemagglutinin (HA) marker, a FLAG marker, a 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.For example, it is possible to purify an anti-CD25 antigen-binding protein of the present invention, conjugated to an epitope tag, from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc.) by treating the mixture with a solid-phase resin containing a complementary molecule capable of selectively recognizing and binding to the CD25 antibody epitope tag or a fragment thereof. Examples of solid-phase resins include agarose beads, compatible with purifications in aqueous solution. Petition 870250081421, dated 10 / 09 / 2025, pp. 179 / 374 169 / 304

[0375] In some embodiments, a conjugate of the present disclosure may comprise one or more anti-CD25 VHH domains described in the present invention, conjugated to a therapeutic agent, which may be cytotoxic, cytostatic, or otherwise provide some therapeutic benefit. In some embodiments, the cytotoxic agent is a drug, a chemotherapeutic agent, a growth inhibitor, a toxin (for example, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (for example, a radioconjugate). Such conjugates may be applicable, for example, to the treatment or prevention of a disease associated with the activity of autoreactive cytotoxic T cells. In some embodiments, the antibody-drug conjugates described in the present invention may allow targeted delivery of a fraction of the drug to a target tissue (for example, tumors).

[0376] In some embodiments, a conjugate of the present disclosure comprises a toxin. In some embodiments, the toxin includes, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:10251028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calichamycin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al. al., Cancer Answer: Toxins can exert their cytotoxic and cytostatic effects through mechanisms including binding to tubulin, binding to DNA, or inhibition of topoisomerase.

[0377] In some embodiments, anti-CD25 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may be fused or conjugated to one or more fractions that facilitate administration to the central nervous system (CNS) / brain. The fraction that may facilitate Petition 870250081421, dated 10 / 09 / 2025, page 180 / 374 170 / 304 The administration of an anti-CD25 antigen-binding protein to the central nervous system (CNS) / brain may be, for example, a peptide, a polypeptide, a small molecule, a lipid, or a synthetic polymer. Several approaches to the administration of single-domain antibodies to the brain are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated into the present invention by reference in its entirety.

[0378] As a non-limiting example, an anti-CD25 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) of the present development can be fused or conjugated to a moiety (e.g., an antibody) that binds to the transferrin receptor (TfR) or the insulin receptor. The transferrin receptor (TfR) is highly expressed by brain capillary endothelial cells (BCECs) that form the blood-brain barrier (BBB) ​​and has been used as a target for drug delivery in the brain. Monoclonal antibodies that bind to TfR, such as the Ri7 clone, have been shown to internalize into BCECs in vivo.As another example, an anti-CD25 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) of the present disclosure can be conjugated to hydrophobic fatty acid fractions, such as C18 (stearic acid), C16 (palmitic acid), or C8 (octanoic acid) fatty acid fractions; or amphiphilic block copolymer fractions, such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (pluronic acids or poloxamers) or poly(2-oxasolins). Various fatty acid fractions and block copolymer fractions that can be used for protein delivery to the brain are described, for example, in Yi and Kabanov, J Drug Target. 2013; 21(10): 940-955, which is incorporated into the present invention by reference in its entirety.

[0379] Example methods for linking a fraction, such as a marker, 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. Petition 870250081421, dated 10 / 09 / 2025, p. 181 / 374 171 / 304 40:219 (1981); Nygren, J. Histochem. E Cytochem. 30:407 (1982); Wensel and Meares, Elsevier, NY (1983); and Colcher et al., Meth. Enzymol., 121:802-16 (1986). Additional suitable methods for preparing the conjugates of the present disclosure include those described, for example, in documents WO 2009 / 067800, WO 2011 / 133886, and US2014322129, which are incorporated by reference in the present invention in its entirety.

[0380] In some embodiments, the linkage between an anti-CD25 antigen-binding protein and a second moiety may be covalent or non-covalent, for example, via a non-covalent biotin-streptavidin interaction. In some embodiments, a second moiety may be linked to an anti-CD25 antigen-binding protein using any of several known molecular or chemical biological conjugation and linkage methods described below. In some embodiments, ligands, such as peptide ligands, cleavable ligands, non-cleavable ligands, or ligands that assist in the conjugation reaction, may be used to link or conjugate a second moiety to an anti-CD25 antigen-binding protein described in the present invention.

[0381] In some embodiments, an anti-CD25 antigen-binding protein (e.g., antibody such as single-domain antibody) is conjugated to one or more secondary fragments, for example, about 1 to about 20 fragments per molecule, optionally via a ligand. In some embodiments, one or more secondary fragments may be the same or different. The ligand may be composed of one or more linker components. For the covalent linkage of an antibody and the second fragment, the linker typically has two reactive functional groups, i.e., bivalence in a reactive sense. Bivalent linker reagents that are useful for linking two or more functional or biologically active fragments such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups have been described, for example, in Hermanson, GT (1996) Bioconjugate Petition 870250081421, dated 10 / 09 / 2025, p. 182 / 374 172 / 304 Techniques; Academic Press: New York, pp 234-242.

[0382] In some embodiments, a ligand used in a conjugate of the present disclosure may include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (“SMCC”), or N-succinimidyl(4-iodoacetyl)aminobenzoate (“STAB”), or a combination thereof.

[0383] In some embodiments, a linker used in a conjugate of the present disclosure may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, 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 amino acid analogs of non-naturally occurring amino acids, such as citrulline. The amino acid linker components may be designed and optimized in their selectivity for enzymatic cleavage by specific enzymes, for example, a tumor-associated protease, cathepsin B, C, and D, or a plasmin protease.

[0384] Conjugates of an anti-CD25 antigen-binding protein (e.g., antibody, such as single-domain antibody) and a second moiety (e.g., cytotoxic agent) can be manufactured using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3-(2-pyridylthiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p Petition 870250081421, dated 10 / 09 / 2025, pp. 183 / 374 173 / 304 azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), di-isocyanates (such as 2,6-diisocyanate toluene) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0385] The conjugates of the present disclosure can be prepared by a variety of methods. For example, the conjugation method may include: (1) reacting a nucleophilic group of a VHH domain with a bivalent ligand reagent to form the VHH ligand via a covalent bond followed by reaction with a drug moiety; or (2) reacting a nucleophilic group of a drug moiety with a bivalent ligand reagent to form the drug ligand via a covalent bond followed by reaction with the nucleophilic group of a VHH domain.

[0386] Nucleophilic groups in proteins, including antibodies (e.g., VHH domains), include, but are not limited to: (i) N-terminal amine groups, (ii) side-chain amine groups (e.g., lysine), (iii) side-chain thiol groups (e.g., cysteine), and (iv) hydroxyl or amino sugar groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups in binding and reacting moieties, 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 groups, and maleimide. Additional nucleophilic groups can be introduced into proteins (e.g., antibodies, such as VHH domains) by reacting lysines with 2-iminothiolane (Traut's reagent), resulting in the conversion of an amine to a thiol.Reactive thiol groups can be introduced into a protein (e.g., antibodies, as a VHH domain) by the introduction of one, two, three, four, or more cysteine ​​residues.

[0387] Conjugates, such as antibody-drug conjugates, can also be produced by modifying an antibody, such as a VHH domain, to introduce Petition 870250081421, dated 10 / 09 / 2025, pp. 184 / 374 174 / 304 electrophilic groups, which can react with nucleophilic substituents in the binding reagent or drug. The sugars of glycosylated antibodies can be oxidized, for example, with periodate oxidizing reagents, to form aldehyde or ketone groups that can bind to the amine group of the binding reagents or drug groups. The resulting imine-Schiff base groups can form a stable bond or can be reduced, for example, by borohydride reagents to form stable amine linkages. In one embodiment, the reaction of the carbohydrate portion of a glycosylated antibody with galactose oxidase or sodium meta-periodate can produce carbonyl groups (aldehyde and ketone) in the protein that can react with appropriate groups in the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in the production of an aldehyde in place of the first amino acid.This aldehyde can react with a fraction of the drug or a nucleophilic ligand.

[0388] Similarly, nucleophilic groups in a drug fraction include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate and aryl-hydrazide groups capable of reacting to form covalent bonds with electrophilic groups in binding fractions and binding 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 groups and maleimide.

[0389] Alternatively, a fusion protein containing a VHH domain and a polypeptide agent can be produced, for example, by recombinant DNA techniques or peptide synthesis. A DNA sequence can be designed to comprise respective regions encoding the two portions of the fusion protein, adjacent to each other or separated by a region encoding a linker peptide that does not impair the desired properties of the fusion protein. A Petition 870250081421, dated 10 / 09 / 2025, pp. 185 / 374 The 175 / 304 DNA sequence can then be transfected into a host cell expressing the fusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the state of the art. Binders

[0390] In some embodiments, one or more polypeptides of the fusion proteins of the present description are operationally linked by means of peptide linkers. A peptide linker may range from 2 to 60 or more amino acids and, in certain aspects, a peptide linker ranges from 3 to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, from 10 to 60 amino acids, from 12 to 20 amino acids, from 20 to 50 amino acids or from 25 to 35 amino acids in length.

[0391] In some embodiments, a peptide linker, for example, a peptide linker that separates two VHH domains or a VHH domain and a constant region of the heavy chain, has at least 5 amino acids, at least 6 amino acids or at least 7 amino acids in length and, optionally, has up to 30 amino acids, up to 40 amino acids, up to 50 amino acids or up to 60 amino acids in length.

[0392] In some embodiments, the ligand varies from 5 to 50 amino acids in length, for example, it varies from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length. In other embodiments, the ligand varies from 6 to 50 amino acids in length, for example, it varies from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length. In other embodiments of the above, the ligand varies from 7 to 50 amino acids in length, for example, it varies 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.

[0393] In some embodiments, charged (e.g., charged hydrophilic ligands) and / or flexible ligands are used. Examples of ligands Petition 870250081421, dated 10 / 09 / 2025, pp. 186 / 374 176 / 304 flexible proteins that can be used in the fusion proteins of the present invention include those disclosed by Chen et al, 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible ligands are or comprise repeats of glycines and serines (referred to herein as “GS ligand”), for example, a monomer or multimer of GnS (SEQ ID NO: 2195) or SGn (SEQ ID NO: 2196), where n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, or 7, 8, 9, or 10. In one embodiment, the ligand is or comprises a repeating monomer or multimer of G4S (SEQ ID NO: 2151), for example, (GGGGS)n (SEQ ID NO: 2197).

[0394] Polyglycine linkers can be suitably used in the fusion proteins of the present invention. In some embodiments, a peptide linker used herein comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 2198), five consecutive glycines (5Gly) (SEQ ID NO: 2199), six consecutive glycines (6Gly) (SEQ ID NO: 2200), seven consecutive glycines (7Gly) (SEQ ID NO: 2201), eight consecutive glycines (8Gly) (SEQ ID NO: 2202) or nine consecutive glycines (9Gly) (SEQ ID NO: 2203).

[0395] In some embodiments, a GS linker used in the present invention comprises an amino acid sequence selected from GGSGGS, i.e., (GGS)2 (SEQ ID NO: 2204); GGSGGSGGS, i.e., (GGS)a (SEQ ID NO: 2205); GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 2206); and GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 2207). In some embodiments, the fusion protein may include a combination of a GS linker and a glycine linker.

[0396] In one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGS linker (SEQ ID NO: 2152). In one embodiment, two or more VHHs are linked via a GGGGSGGGGS linker. Petition 870250081421, dated 10 / 09 / 2025, pp. 187 / 374 177 / 304 (SEQ ID NO: 2208). In one embodiment, a VHH and an Fc region are linked via a GGGGSESKYGPPCPSCP linker (SEQ ID NO: 2190). In another embodiment, a VHH and an Fc region are linked via a GGGGS linker (SEQ ID NO: 2151).

[0397] In some embodiments, one or more polypeptides of the fusion proteins of the present disclosure are operationally linked by means of a “rigid” peptide linker. Such a peptide linker may comprise a proline-rich peptide. In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO: 2191). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 2194). In one embodiment, a rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 2209), where n is any integer, for example, 1, 2, 3, 4, 5, 6, or 7, 8, 9, or 10.

[0398] Other exemplary peptide ligands that can be used in the fusion proteins described in the present invention are shown in Table 2. Table 2. Exemplary Peptide Ligand Sequences Ligand Amino acid sequence SEQ ID NO G4S GGGGS 2151 (G4S)3 GGGGSGGGGSGGGGS 2152 Ligand 3 GGSEGKSSGSGSESKSTGGS 2153 Ligand 4 GGGSGGGS 2154 Ligand 3 GGGSGGGSGGGS 2155 Ligand 4 GGGSGGGSGGGSGGGS 2156 Ligand 5 GGGSGGGSGGGSGGGSGGGS 2157 Ligand 6 GGGGSGGGGSGGGGSGGGGS 2158 Ligand 7 GGGGSGGGGSGGGGSGGGGSGGGGS 2159 Ligand 8 IRPRAIGGSKPRVA 2160 Petition 870250081421, dated 10 / 09 / 2025, pp. 188 / 374 178 / 304 Ligament 9 GKGGSGKGGSGKGGS 2161 Ligament 10 GGKGSGGKGSGGKGS 2162 Ligament 11 GGGKSGGGKSGGGKS 2163 Ligament 12 GKGKSGKGKSGKGKS 2164 Ligament 13 GGGKSGGKGSGKGGS 2165 Ligament 14 GKPGSGKPGSGKPGS 2166 Ligament 15 GKPGSGKPGSGKPGSGKPGS 2167 Ligament 16 GKGKSGKGKSGKGKSGKGKS 2168 Ligament 17 STAGDTHLGGEDFD 2169 Ligament 18 GEGGSGEGGGSGEGGS 2170 Ligament 19 GGEGSGGEGGSGGEGS 2171 Ligament 20 GEGESGEGESGEGES 2172 Ligament 21 GGGESGGEGGSGEGGS 2173 Ligament 22 GEGESGEGESGEGESGEGES 2174 Ligante 23 GSTSGSGKPGSGEGSTKG 2175 Ligante 24 PRGASKSGSASQTGSAPGS 2176 Ligante 25 GTAAAGAGAAGGAAAGAAG 2177 Ligante 26 GTSGSSGSGSGGSGSGGGG 2178 Ligante 27 GSGS 2179 Ligante 28 APAPAPAPAP 2180 Ligante 29 APAPAPAPAPAPAPAPAPAP 2181 Ligament 30 AEAAAKEAAAKEAAAAAKEAAAAAKEAAAAKAAA 2182 Ligament (GGG GS)6 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 2183 Petition 870250081421, de 10 / 09 / 2025, pág. 189 / 374 179 / 304 Linker (GGG GS)7 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 2184 G1 hinge EPKSCDKTHTCPPCP 2185 9GS-G1 hinge GGGGSGGGSEPKSCDKTHTCPPCP 2186 Llama upper long hinge region EPKTPKPQPAAA 2187 Lift hinge G3 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPP PCPRCPEPKSCDTPPPCPRCP 2188 Hinge G4 ESKYGPPCPSCP 2189 Hinge G4SG4 GGGGSESKYGPPCPSCP 2190 Proline - linker 1 PAPAPAPAPAPAPAPAP 2191 Proline - linker 2 GGGGSPAPAPAPAPAPAPAPAP 2192 Proline -ligand 3 PAPAPAPAPAPAPAPAPGGGGS 2193 Proline -ligand 4 GGGGSPAPAPAPAPAPAPAPAPGGGGS 2194 Signal Sequences

[0399] In some embodiments, the fusion protein described in the present invention may further comprise a signal sequence at its N-terminus. The signal sequences may be present in the precursor molecule of the fusion protein and may be removed after the protein is secreted from the host cell during production. In some embodiments, the signal sequence is Petition 870250081421, dated 10 / 09 / 2025, pp. 190 / 374 180 / 304 MAVMAPRTLVLLLSGALALTQTWA (SEQ ID NO: 2239) or a fragment or variant thereof. In some embodiments, the signal sequence is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 2240), or a fragment or variant thereof. Polynucleotide molecules

[0400] In another aspect, polynucleotide molecules encoding anti-CD25 antigen-binding proteins (e.g., antibodies including single-domain antibodies) or fusion proteins described in the present invention are provided in the present invention. Polynucleotide molecules encoding polypeptide portion(s) of a conjugate of the present disclosure are also covered by the present disclosure.

[0401] In some embodiments, a polynucleotide molecule of the present disclosure encodes an anti-CD25 VHH amino acid sequence selected from the SEQ ID NOs: 4, 8, 12, 16, 20, 26 - 30, 43 - 625, 1541 - 1845, 2251 - 2254, 2259 - 2262, 2268 - 2830, 3719 - 4014, 4317 - 4322, 4337, 4339, 4342, 4559, 4892 - 5002, and 5114 - 5176, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0402] In some embodiments, a polynucleotide molecule of the present disclosure encodes an anti-CD25 VHH amino acid sequence selected from the SEQ ID NOs: 4, 8, 12, 16, 20, 43 - 342, 1541 - 1845, 2251 2254, 2268 - 2559, and 3719 - 4014, 4337, 4342 - 4451, 4892 - 5002, and 5146 - 5176, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0403] In some embodiments, a polynucleotide molecule of the present disclosure encoding an anti-CD25 VHH comprises the sequence Petition 870250081421, dated 10 / 09 / 2025, pp. 191 / 374 181 / 304 nucleotide of any of the SEQ ID NOs: 21 - 25, 1846 - 2150, 2255 - 2258, 4015 - 4310, 4338, and 5003 - 5113, or a similar sequence of the same having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0404] In an embodiment provided in the present invention, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence selected from the SEQ ID NOs: 26 - 30, 343 - 625, 2259 2262, 2560 - 2830, 4317 - 4322, 4339, 5114 - 5145, and 4452 - 4559, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0405] A polynucleotide molecule can be used to transform / transfect a host cell or host organism, for example, for the expression and / or production of a polypeptide. Suitable hosts or host cells for the production of an anti-CD25 polypeptide described in the present invention 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 in the present invention is also covered by the present disclosure.

[0406] A polynucleotide molecule may be, for example, DNA, RNA, or a hybrid thereof, and may also comprise (for example, chemically) modified nucleotides, such as locked nucleic acids (LNA) or peptide nucleic acids (PNA). In some embodiments, the polynucleotide is single-stranded. In other embodiments, the polynucleotide is double-stranded. In one embodiment, the polynucleotide is in the form of double-stranded DNA (for example, Petition 870250081421, dated 10 / 09 / 2025, page 192 / 374 182 / 304 plasmid). In some embodiments, the polynucleotide is in the form of single-stranded RNA (e.g., mRNA).

[0407] Techniques for generating polynucleotides may include, for example, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combination of two or more natural and / or synthetic sequences (or two or more parts thereof); introduction of mutations leading to the expression of a truncated expression product; introduction of one or more restriction sites (e.g., to create cassettes and / or regions that can be easily digested and / or ligated using suitable restriction enzymes); and / or the introduction of mutations via a PCR reaction using one or more mismatched primers. Alternatively, the polynucleotides of the present disclosure may be isolated from a suitable natural source. Polynucleotide sequences encoding natural (poly)peptides may, for example, be subjected to site-directed mutagenesis to generate a polynucleotide molecule encoding a sequence-variant polypeptide. Vectors

[0408] Vectors comprising polynucleotide molecules encoding anti-CD25 antigen-binding proteins (e.g., antibodies, including single-domain antibodies), fusion proteins, or other relevant polypeptides of the present invention are also provided in the present invention. A vector as used in the present invention is a suitable vehicle for transporting genetic material into a host cell. A vector may include a nucleic acid vector, such as a plasmid or mRNA, or nucleic acids incorporated into a larger structure, such as a liposome or viral vector.

[0409] A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters, enhancers, terminators) that regulate the expression of a polypeptide of Petition 870250081421, dated 10 / 09 / 2025, pp. 193 / 374 183 / 304 of interest and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, such as β-galactosidase). For DNA-based vectors, this usually includes the presence of elements for transcription (e.g., a promoter and a polyA signal) and translation (e.g., the Kozak sequence). In some embodiments, the vector is an expression vector, i.e., a vector suitable for expressing a polypeptide or encoded construct under appropriate conditions in a host cell.

[0410] To express an anti-CD25 antigen-binding protein or fusion protein (or fragments thereof) of the present disclosure, polynucleotides encoding partial or complete polypeptide chains, for example, obtained as described above (e.g., VHH, VHH-Fc), can be inserted into expression vectors so that the genes are operationally linked to one or more transcriptional and translational control sequences. The expression vector and the expression control sequences are chosen to be compatible with the host cell of expression used. Polynucleotides encoding the two or more polypeptide chains (when present and different from each other) 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.

[0411] In addition to the polynucleotides encoding the polypeptide chain(s) of an antigen-binding protein or anti-CD25 fusion protein, 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 host cell to be transformed and / or the desired level of protein expression. For example, regulatory sequences Petition 870250081421, dated 10 / 09 / 2025, pp. 194 / 374 184 / 304 suitable for expression in mammalian host cells 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., adenovirus major late promoter (AdMLP)), and polyome. Additional examples of viral regulatory elements and their sequences include those described, for example, in U.S. Pat. Nos. 5,168,062; 4,510,245; and 4,968,615; the disclosures of each of which are incorporated into the present invention by reference.

[0412] The recombinant expression vectors of the present disclosure may contain additional sequences, such as sequences that regulate vector replication in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US 4,399,216; US 4,634,665; and US 5,179,017; the disclosure of each of which is incorporated in the present invention by reference in its entirety). For example, typically the selectable marker gene confers resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseotricin, or to 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 may include the dihydrofolate reductase (DHFR) gene (for use in DHFR-deficient host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0413] The vectors of the present disclosure may also include sequence elements that increase the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, an internal ribosomal entry site (IRES), and a polyadenylation signal site. Petition 870250081421, dated 10 / 09 / 2025, pp. 195 / 374 185 / 304 to target efficient transcription of the gene carried in the expression vector.

[0414] Viral vectors can be used for the efficient delivery of exogenous genes to the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral vectors are particularly useful for gene delivery because the polynucleotides contained in these 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 additional proteins or reagents to induce gene integration.Examples of suitable viral vectors include retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses (AAVs) such as AAV2, AAV8, AAV9), negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded D...

Claims

CLAIMS 1. Antigen-binding protein that binds specifically to cluster of differentiation 25 (CD25), CHARACTERIZED in that it comprises a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from a) . NAL(G / L / P / Q / W)Y (SEQ ID NO: 31); b) . NALR(D / H / N / F) (SEQ ID NO: 34); c) . (K / S / T)TLRY (SEQ ID NO: 36); d) . (A / V / S)(K / T)G(R / A / K)(G / H / N / R)SG(S / G)YYP(W / F / L)D(D / E)(Y / V) (SEQ ID NO: 5119); e) . AA(S / T)(D / N / Y / K)(F / V)(L / P)(I / L)A(T / I / A)(T / S / A)IS(A / G)(Y / H)DY (SEQ ID NO: 5208); f). AAYVYPDYYCS(D / E)YVLL(K / R)YDY (SEQ ID NO: 2263); g). NIYR(P / S)QVP(P / S / T)TRYS (SEQ ID NO: 2265); eh). AAKRLGP(M / I / A / L)VH(Q / R)YSLEVLTPLFLDEYDY (SEQ ID NO: 4323).

2. Antigen-binding protein, according to claim 2, CHARACTERIZED in that CDR3 comprises an amino acid sequence selected from the SEQ ID NOs: 3, 7, 11, 15, 19, 39, 41, 1237, 1239, 1271, 1275, 1298, 1301, 1331, 1415, 1419, 1421, 1428, 1432,1442, 1444, 1445, 1447, 1448, 2244, 2247, 2249, 2250, 2267, 4311 - 4316, 4336, 4340, 4787, 4866, 4875, 4878, 4879, and 4880.

3. Antigen-binding protein, according to claim 1 or 2, CHARACTERIZED in that it further comprises a CDR1 comprising an amino acid sequence selected from a) . GR(K / R / S)FSTLI (SEQ ID NO: 37); b) . GFTFS(N / S)YA (SEQ ID NO: 40); c) . GRTF(A / S)(S / W / D)(F / N / Y)G (SEQ ID NO: 5209); Petition 870250081421, dated 10 / 09 / 2025, p. 316 / 374 2 / 23 d) . GFTLDYYA (SEQ ID NO: 2242); ee) . G(I / M)P(F / -)(A / -)L(P / V / Y)A (SEQ ID NO: 2266).

4. Antigen-binding protein according to claim 3, CHARACTERIZED in that CDR1 comprises an amino acid sequence selected from the SEQ ID NOs: 1, 5, 9, 13, 17, 32, 42, 805, 809, 818, 2242, and 2245.

5. Antigen-binding protein according to any one of claims 1 to 4,CHARACTERIZED by the fact that it further comprises a CDR2 comprising an amino acid sequence selected from a) . (I / V)(D / E)R(D / G)(D / G)T(A / P / T) (SEQ ID NO: 2241); b) . IYSD(G / S)SGT (SEQ ID NO: 4341); c) . IS(Q / R / G)(S / G)GGRT (SEQ ID NO: 5210); d) . IS(R / S)(D / S)G(D / G)ST (SEQ ID NO: 2264); e) . ISSGGNT (SEQ ID NO: 2246); f) . ISSTDGRT (SEQ ID NO: 2248).

6. Antigen-binding protein according to claim 5, characterized in that CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 18, 33, 35, 38, 942, 946, 959, 967, 992, 1114, 1115, 1116, 1117, 2243, 2246, 2248, and 4335.

7. Antigen-binding protein according to any one of claims 1, 3, and 5, characterized in that the antigen-binding protein comprises i) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241,and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and Petition 870250081421, dated 10 / 09 / 2025, page 317 / 374 3 / 23 a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; iii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 36; iv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 33, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; v) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 35,and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; vi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 37, a CDR2 comprising an amino acid sequence from SEQ ID NO: 38, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 36; vii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 32, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 31; viii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2241, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 34; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 33,and a CDR3 comprising an amino acid sequence of SEQ ID NO: 31; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 35, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; xi) 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 Petition 870250081421, dated 10 / 09 / 2025, p. 318 / 374 4 / 23 a CDR3 comprising an amino acid sequence of SEQ ID NO: 5119; xii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 40, a CDR2 comprising an amino acid sequence with SEQ ID NO: 4341, and a CDR3 comprising an amino acid sequence with SEQ ID NO: 4340; xiii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 40, a CDR2 comprising an amino acid sequence with SEQ ID NO: 14,and a CDR3 comprising an amino acid sequence from SEQ ID NO: 39; xiv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5209, a CDR2 comprising an amino acid sequence from SEQ ID NO: 5210, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 5208; xv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 42, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 41; xvi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2264, and a CDR3 comprising an amino acid sequence from SEQ ID NO: 2263; xvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2266, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2246,and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2265; xviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4323; or xix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2248, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2267.

8. Antigen-binding protein, according to any of the Petition 870250081421, dated 10 / 09 / 2025, p. 319 / 374 5 / 23 claims 1 to 7, CHARACTERIZED in that the antigen-binding protein comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2,and a CDR3 comprising an amino acid sequence from SEQ ID NO: 3; ii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 6, a CDR3 comprising an amino acid sequence from SEQ ID NO: 7; iii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 9, a CDR2 comprising an amino acid sequence from SEQ ID NO: 10, a CDR3 comprising an amino acid sequence from SEQ ID NO: 11; iv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 15; v) a CDR1 comprising an amino acid sequence from SEQ ID NO: 17, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 19; vi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242,a CDR2 comprising an amino acid sequence from SEQ ID NO: 2243, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2244; vii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2245, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2246, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2247; viii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2249; ix) a CDR1 comprising an amino acid sequence from SEQ ID NO: Petition 870250081421, dated 10 / 09 / 2025, page. 320 / 374 6 / 23 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 2250; x) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242,a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4311; xi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4312; xii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4313; xiii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4314; xiv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248,a CDR3 comprising an amino acid sequence from SEQ ID NO: 4315; xv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 2242, a CDR2 comprising an amino acid sequence from SEQ ID NO: 2248, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4316; xvi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4875; xvii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1331; xviii) a CDR1 comprising an amino acid sequence from SEQ ID Petition 870250081421, dated 10 / 09 / 2025, page 321 / 374 7 / 23 NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14,a CDR3 comprising an amino acid sequence from SEQ ID NO: 4787; xix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 14, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4866; xx) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4336; xxi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4878; xxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4335,a CDR3 comprising an amino acid sequence from SEQ ID NO: 4879; xxiii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 13, a CDR2 comprising an amino acid sequence from SEQ ID NO: 4335, a CDR3 comprising an amino acid sequence from SEQ ID NO: 4880; xxiv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 10, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1239; xxv) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 946, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1239; xxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 959,a CDR3 comprising an amino acid sequence from SEQ ID NO: 1237; xxvii) a CDR1 comprising an amino acid sequence from SEQ ID Petition 870250081421, dated 10 / 09 / 2025, page 322 / 374 8 / 23 NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 946, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1237; xxviii) a CDR1 comprising an amino acid sequence from SEQ ID NO: 1, a CDR2 comprising an amino acid sequence from SEQ ID NO: 967, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1271; xxix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 5, a CDR2 comprising an amino acid sequence from SEQ ID NO: 946, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1275; xxx) a CDR1 comprising an amino acid sequence from SEQ ID NO: 9, a CDR2 comprising an amino acid sequence from SEQ ID NO: 10,a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; xxxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 992, a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; xxxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 992, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1298; xxxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 942, a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; xxxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 959,a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; xxxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 942, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1301; xxxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1415; xxxvii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 809, a CDR2 comprising an amino acid sequence with SEQ ID NO: 1114, a CDR3 comprising an amino acid sequence with SEQ ID NO: 1419; xxxviii) a CDR1 comprising an amino acid sequence with SEQ ID NO: 805, a CDR2 comprising an amino acid sequence with SEQ ID NO: 1116,a CDR3 comprising an amino acid sequence from SEQ ID NO: 1421; xxxix) a CDR1 comprising an amino acid sequence from SEQ ID NO: 809, a CDR2 comprising an amino acid sequence from SEQ ID NO: 1117, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1419; xxxx) a CDR1 comprising an amino acid sequence from SEQ ID NO: 818, a CDR2 comprising an amino acid sequence from SEQ ID NO: 1115, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1428; xxxxi) a CDR1 comprising an amino acid sequence from SEQ ID NO: 805, a CDR2 comprising an amino acid sequence from SEQ ID NO: 18, a CDR3 comprising an amino acid sequence from SEQ ID NO: 1432; xxxxi i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18,a CDR3 comprising an amino acid sequence of SEQ ID NO: 1442; xxxxii i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1444; xxxxi v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1445; xxxx v) a CDR1 comprising an amino acid sequence of SEQ ID Petition 870250081421, dated 10 / 09 / 2025, p. 324 / 374 10 / 23 NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, a CDR3 comprising an amino acid sequence of SEQ ID NO: 1447; or xxxxv i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 805, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18,a CDR3 comprising an amino acid sequence from SEQ ID NO: 1448.

9. Antigen-binding protein that specifically binds to cluster of differentiation 25 (CD25), CHARACTERIZED in that it comprises a CDR1 comprising an amino acid sequence selected from any of the SEQ ID NOs: 626 - 930, 2831 - 3126, and 4560 - 4670; a CDR2 comprising an amino acid sequence selected from any of the SEQ ID NOs: 931 - 1235, 3127 - 3422, and 4671 - 4780; and / or a CDR3 comprising an amino acid sequence selected from any of the SEQ ID Nos: 1236 - 1540, 3423 - 3718, and 4781 - 4891.

10. Antigen-binding protein according to any of claims 1 to 9, CHARACTERIZED in that the antigen-binding protein is a single-domain antibody.

11. Antigen-binding protein according to claim 10, CHARACTERIZED in that the single-domain antibody is a VHH, VNAR domain,or VH.

12. Antigen-binding protein according to claim 11, CHARACTERIZED in that the VHH is a camelid VHH.

13. Antigen-binding protein according to claim 12, CHARACTERIZED in that the VHH comprises an amino acid sequence selected from any of the SEQ ID NOs: 4, 8, 12, 16, 20, 43 - 342, 1541 - 1845, 2251 - 2254, 2268 - 2559, 3719 - 4014, 4337, 4342 - 4451, 4892 - 5002, and 5146 - 5176, or a sequence having at least 75% identity therewith. Petition 870250081421, dated 10 / 09 / 2025, pp. 325 / 374 11 / 23 14. Antigen-binding protein, according to claim 12 or 13, CHARACTERIZED in that VHH comprises an amino acid sequence selected from any of the SEQ ID NOs: 4, 8, 12, 16, 20, 2251 - 2254, 4337, and 5146 - 5176, or a sequence having at least 75% identity with it.

15. Antigen-binding protein, according to claim 11,CHARACTERIZED in that the VHH is a humanized VHH.

16. Antigen-binding protein, according to claim 15, CHARACTERIZED in that the humanized VHH comprises an amino acid sequence selected from any of the SEQ ID NOs: 26 - 30, 343 - 625, 2259 - 2262, 2560 - 2830, 4317 - 4322, 4339, 4452 - 4559, and 5114 - 5145, or a sequence having at least 75% identity with it.

17. Antigen-binding protein, according to claim 16, CHARACTERIZED in that the humanized VHH comprises an amino acid sequence selected from any of the SEQ ID NOs: 26-30, 2259-2262, 4317-4322, 4339, and 5114-5145, or a sequence having at least 75% identity therewith.

18. Antigen-binding protein, according to any one of claims 1 to 17, CHARACTERIZED in that the antigen-binding protein binds to human CD25.

19. Antigen-binding protein, according to claim 18,CHARACTERIZED in that the antigen-binding protein binds to human CD25 with a Kd of less than about 3.5 χ10-7 M.

20. Antigen-binding protein, according to claim 19, CHARACTERIZED in that the antigen-binding protein binds to human CD25 with a Kd of about 1 χ10-10 to about 1 χ10-7 M.

21. Antigen-binding protein, according to any one of claims 1 to 20, CHARACTERIZED in that the antigen-binding protein binds to cyno CD25.

22. Antigen-binding protein according to claim 21, CHARACTERIZED in that the antigen-binding protein binds to cynoCD25 with a Kd of less than about 1χ10-6 M.

23. Antigen-binding protein according to claim 22,23. Antigen-binding protein, characterized in that the antigen-binding protein binds to cynoCD25 with a Kd of about 1χ10-8 to about 4χ10-7 M.

24. Antigen-binding protein, according to any one of claims 1 to 23, characterized in that the antigen-binding protein binds to the same epitope(s) on CD25 as IL-2.

25. Antigen-binding protein, according to any one of claims 1 to 24, characterized in that the antigen-binding protein competes for binding to CD25 with IL-2.

26. Antigen-binding protein, according to claim 24 or 25, characterized in that the antigen-binding protein has an antagonistic effect on binding to CD25.

27. Antigen-binding protein, according to any one of claims 1 to 23, CHARACTERIZED in that the antigen-binding protein does not bind to the same epitope(s) on CD25 as IL-2.

28. Antigen-binding protein,28. Fusion protein according to any one of claims 1 to 23 and 27, characterized in that the antigen-binding protein does not compete for binding to CD25 with IL-2.

29. Fusion protein that specifically binds to cluster of differentiation 25 (CD25), characterized in that it comprises one or more of said antigen-binding proteins as defined in any one of claims 1 to 28.

30. Fusion protein according to claim 29, characterized in that it comprises two of said antigen-binding proteins.

31. Fusion protein according to claim 29, characterized in that it comprises four of said antigen-binding proteins.

32. Fusion protein, according to any one of claims 29 to 31, CHARACTERIZED in that one or more antigen-binding proteins bind to the same epitope on CD25.

33. Fusion protein,33. Fusion protein, according to any one of claims 29 to 31, characterized in that one or more antigen-binding proteins bind to different epitopes on CD25.

34. Fusion protein, according to any one of claims 29 to 33, characterized in that one or more antigen-binding proteins are one or more single-domain antibodies.

35. Fusion protein, according to claim 34, characterized in that one or more single-domain antibodies are one or more VHHs.

36. Fusion protein, according to any one of claims 29 to 35, characterized in that it further comprises an immunoglobulin Fc region.

37. Fusion protein, according to claim 36, characterized in that the immunoglobulin Fc region is an Fc region of a human immunoglobulin.

38. Fusion protein, according to claim 37, CHARACTERIZED in that the immunoglobulin Fc region is an IgG1, IgG2 Fc region,Human IgG3 or IgG4, or a variant thereof.

39. Fusion protein, according to claim 38, CHARACTERIZED in that the immunoglobulin Fc region is a human IgG1 Fc region, or a variant thereof.

40. Fusion protein, according to claim 39, CHARACTERIZED in that the human IgG1 Fc region comprises one or more mutations selected from L234A, L235A, G237A, D265A, N297A, and / or P329A according to EU numbering.

41. Fusion protein, according to claim 40, CHARACTERIZED in that the Fc region of human IgG1 comprises a set of mutations selected from 1) L234A and L235A; 2) . L234A, L235A, and P329A; 3) . D265A, N297A and P329A; and 4) L234A, L235A, and G237A.

42. Fusion protein according to claim 38, characterized in that the immunoglobulin Fc region is a human IgG4 Fc region, or a variant thereof.

43. Fusion protein according to claim 42, characterized in that the human IgG4 Fc region comprises one or more mutations selected from S228P, L235E, L235A, and / or F234A according to EU numbering.

44. Fusion protein according to claim 43, characterized in that the human IgG4 Fc region comprises a set of mutations selected from 1) S228P and L235E; 2) S228P and L235A; 3) S228P, F234A, and L235E; and 4) . S228P, F234A, and L235A.

45. Conjugate, CHARACTERIZED in that it comprises the antigen-binding protein as defined in any one of claims 1 to 28 or the fusion protein as defined in any one of claims 29 to 44, in Petition 870250081421, dated 10 / 09 / 2025,p. 329 / 374 15 / 23 that the antigen-binding protein or fusion protein is conjugated to a second fraction.

46. Conjugate, according to claim 45, CHARACTERIZED in that the second fraction is selected from a detectable marker, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

47. Polynucleotide molecule, CHARACTERIZED in that it encodes the antigen-binding protein as defined in any one of claims 1 to 28 or the fusion protein as defined in any one of claims 29 to 44.

48. Recombinant vector, CHARACTERIZED in that it comprises the polynucleotide molecule as defined in claim 47.

49. Host cell, CHARACTERIZED in that it comprises the polynucleotide molecule as defined in claim 47.or the expression vector as defined in claim 48.

50. Kit, CHARACTERIZED in that it comprises the antigen-binding protein as defined in any one of claims 1 to 28, the fusion protein as defined in any one of claims 29 to 44, the conjugate as defined in claim 45 or 46, the polynucleotide molecule as defined in claim 47, the recombinant vector as defined in claim 48, or the host cell as defined in claim 49, and optionally, instructions and / or packaging therefor.

51. Pharmaceutical composition, CHARACTERIZED in that it comprises the antigen-binding protein as defined in any one of claims 1 to 28, the fusion protein as defined in any one of claims 29 to 44, the conjugate as defined in claim 45 or 46, the molecule of Petition 870250081421, dated 10 / 09 / 2025,pg. 330 / 374 16 / 23 polynucleotide as defined in claim 47, or the recombinant vector as defined in claim 48, and a pharmaceutically acceptable carrier and / or excipient.

52. Method for preparing an antigen-binding protein or a fusion protein that specifically binds to the cluster of differentiation 25 (CD25), CHARACTERIZED in that it comprises the steps of: (a) culturing the host cell as defined in claim 49 in a culture medium under conditions suitable for the expression of the antigen-binding protein or fusion protein, and (b) isolating the antigen-binding protein or fusion protein from the host cell and / or culture medium.

53. Method for targeting a cell expressing CD25, CHARACTERIZED in that it comprises contacting the cell with the antigen-binding protein as defined in claims 1 to 28, the fusion protein as defined in any one of claims 29 to 44,or the conjugate as defined in claim 45 or 46.

54. Method according to claim 53, CHARACTERIZED in that the cell is a regulatory T cell (Treg).

55. Method according to claim 53 or 54, CHARACTERIZED in that said contact occurs in vitro.

56. Method according to claim 53 or 54, CHARACTERIZED in that said contact occurs in vivo.

57. Method according to claim 56, CHARACTERIZED in that the method further comprises administering the antigen-binding protein, the fusion protein, or the conjugate to an individual in need thereof.

58. Method of treating or preventing a disease or disorder in an individual in need thereof, said method CHARACTERIZED in that Petition 870250081421, dated 10 / 09 / 2025, p. 331 / 374 17 / 23 comprising administering to the individual the antigen-binding protein as defined in claims 1 to 28,the fusion protein as defined in any one of claims 29 to 44, or the conjugate as defined in claim 45 or 46.

59. Method according to claim 58, CHARACTERIZED in that the disease or disorder is an immunological disease, inflammatory disease, cancer, cardiovascular disease, or an infertility and pregnancy-associated disease.

60. Method according to claim 59, CHARACTERIZED in that the immunological disease is selected from an autoimmune disease, a neurological condition, an allergy, asthma, macular degeneration, muscular atrophy, a disease related to spontaneous abortion, atherosclerosis, bone loss, a musculoskeletal disease, obesity, a graft-versus-host disease, and an allograft rejection.

61. Method according to claim 60, CHARACTERIZED in that 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 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, Goodpasture's disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosus, IgG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis spectrum disorder Optic nerve injury, pemphigus vulgaris or related bullous skin disease, pernicious anemia, polyarteritis nodosa, polychondritis, syndromes. Petition 870250081421, dated 10 / 09 / 2025.pg. 332 / 374 18 / 23 polyglandular, polymyalgia rheumatica, polymyositis and dermatomyositis, premature ovarian failure, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, primary ovarian failure, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthritis, stiff man syndrome, type I diabetes, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (granulomatosis with polyangiitis) or other immunological vasculitis.

62. Method according to claim 61, CHARACTERIZED in that the lupus is systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, neonatal lupus, or drug-induced lupus.

63. Method according to claim 62, CHARACTERIZED in that the cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DLE),or acute subcutaneous lupus erythematosus.

64. Method according to claim 60, CHARACTERIZED in that 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.

65. Method according to claim 60, CHARACTERIZED in that the allergy is selected from food allergy, seasonal allergy, pet allergy, urticaria, hay fever, allergic conjunctivitis, poison ivy allergy, oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, and chemical allergy.

66. Method according to claim 60, CHARACTERIZED in that the allograft rejection is selected from skin graft rejection, bone graft rejection, vascular tissue graft rejection,Rejection of the graft, Petition 870250081421, dated 10 / 09 / 2025, page 333 / 374 19 / 23, ligament, and organ graft rejection.

67. Method according to claim 60, CHARACTERIZED in that the ligament graft rejection is selected from the following: cricothyroid ligament graft rejection, caudal cruciate ligament graft rejection, periodontal ligament graft rejection, suspensory ligament graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, ulnar collateral ligament graft rejection, radial collateral ligament graft rejection, suspensory ligament 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,Rejection of the superior pubic ligament graft, rejection of the anterior cruciate ligament graft, rejection of the lateral collateral ligament graft, rejection of the posterior cruciate ligament graft, rejection of the medial collateral ligament graft, rejection of the cranial cruciate ligament graft, and rejection of the patellar ligament graft.

68. Method according to claim 60, characterized in that 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.

69. Method according to claim 60, characterized in that graft-versus-host disease arises from a bone marrow transplant or from 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. Petition 870250081421, dated 10 / 09 / 2025, pp. 334 / 374 20 / 23 70. Method, according to claim 59, CHARACTERIZED in that the inflammatory disease is acute or chronic inflammation.

71. Method, according to claim 59, CHARACTERIZED in that the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovary syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.

72. Method according to claim 59, characterized in that 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 (bone sarcoma), brainstem 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's sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis, and renal cell carcinoma. hereditary mixed polyposis syndromeHereditary pancreatitis, hereditary papillary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, renal cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia and hairy cell leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic lymphocytic T-cell leukemia, eosinophilic leukemia, Petition 870250081421, dated 10 / 09 / 2025, p. 335 / 374 21 / 23 Li-Fraumeni syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer,Nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, complex of Tuberous sclerosis, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), Werner syndrome.Wilms' tumor or xeroderma pigmentosum.

73. Method, according to claim 59, CHARACTERIZED in that cardiovascular disease is selected from atherosclerosis, heart failure, left heart failure with reduced ejection fraction, left heart failure with preserved ejection fraction, right ventricular failure, congestive heart failure, restrictive cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, idiopathic cardiomyopathy, and hypertension.

74. Method, according to claim 59, CHARACTERIZED in that infertility and pregnancy-related diseases are selected from recurrent pregnancy loss, pre-eclampsia, preterm labor, fetal growth restriction, or intrauterine growth restriction.

75. A method of regenerating a tissue or organ comprising one or more CD25+ cells.The method described is characterized in that it comprises contacting the tissue or organ with an effective amount of the antigen-binding protein as defined in claims 1 to 28, the fusion protein as defined in any one of claims 29 to 44, or the conjugate as defined in claim 45 or 46.

76. Method according to claim 75, characterized in that the tissue or organ described is selected from pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerve, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal tract, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structures, embryo, and testicular tissue.

77. Method, according to claim 75 or 76, CHARACTERIZED in that said contact occurs in vitro.

78. Method,According to claim 75 or 76, CHARACTERIZED in that said contact occurs in vivo.

79. Method, according to claim 78, CHARACTERIZED in that the method further comprises administering the antigen-binding protein, the fusion protein, or the conjugate to an individual in need thereof.

80. Method for inducing tolerance to a foreign agent and / or preventing or reducing the immune response to a foreign agent in an individual in need thereof, said method CHARACTERIZED in that it comprises administering to the individual the antigen-binding protein as defined in claims 1 to 870250081421, dated 10 / 09 / 2025, p. 337 / 374 23 / 23 28, the fusion protein as defined in any one of claims 29-44, or the conjugate as defined in claim 45 or 46.

81. Method according to claim 80, CHARACTERIZED in that the foreign agent is a therapeutic protein or peptide,a viral vector, a bacterial vector, a fungal vector, a biochemical vector, a lipid, a carbohydrate, a nucleic acid, a spermatozoon, an oocyte, or an embryo.

82. Method according to claim 81, CHARACTERIZED in that the viral vector is a DNA or RNA vector.

83. Method according to any one of claims 57 to 74 and 79 to 82, CHARACTERIZED in that the individual is a mammal.

84. Method according to claim 83, CHARACTERIZED in that the mammal is human.

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